CA3105541C - Cross-linked pyrrolobenzodiazepine dimer (pbd) derivative and its conjugates - Google Patents

Abstract

A novel cross-linked cytotoxic agents, pyrrolobenzo-diazepine dimer (PBD) derivatives, and their conjugates to a cell-binding molecule, a method for preparation of the conjugates and the therapeutic use of the conjugates.

Description

DEMANDE OU BREVET VOLUMINEUX LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND PLUS D’UN TOME. CECI EST LE TOME 1 DE 2 CONTENANT LES PAGES 1 À 179 NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE VOLUME THIS IS VOLUME 1 OF 2 CONTAINING PAGES 1 TO 179 NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME : NOTE POUR LE TOME / VOLUME NOTE: 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 CROSS-LINKED PYRROLOBENZODIAZEPINE DIMER (PBD) DERIVATIVE AND ITS CONJUGATES FIELD OF THE INVENTION The present invention relates to novel cross-linked cytotoxic agents, pyrrolobenzodiazepine dimer (PBD) derivatives, and their conjugates to a cell-binding molecule, a method for preparation of the conjugates and the therapeutic use of the conjugates. BACKGROUND OF THE INVENTION Antibody-drug conjugates (ADCs) have great promise as anti-cancer drugs, with four ADCs have been approved by FDA and over 100 others in clinical investigation by using several different classes of cytotoxic payloads such as maytansines (Zhao, Robert Y, et al 2011 J. Med. Chern. 54, 3606; Widdison, W.; et al 2006 J. Med. Chem., 49, 4392-4408); auristatins and its parent compound dolastatin (Doronina, S. O. et al, Nat. Biotechnol. 2003, 21 (7), 778-784; Maderna, A., 2014 J. Med. Chem., 57(24), 10527—10543); calicheamycins (Ricart, A. D. 2011 Clin Cancer Res 2011;17:6417-6427; Ricart A. D, et al Nat Clin Pract Oncol. 2007;4:245-255), duocarmycins and its analog CC-1065 (Elgersma, R. C. 2015 Mol. Pharmaceutics, 12 (6), 1813-1835; Zhao, Robert Y. et al, 2012 J. Med. Chem 55, 766-782), tubulysins (Li, J. Y. et al, 2016 Cancer Cell. 29(1):117-29; Tumey, L. N. et al 2016 ACS Med Chem Lett. 7(ll):977-982; Huang Y.Y., Zhao, Robert Y. et al, Med Chem. #44, 249th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO2014009774), Eribulins (US20170252458); camptothecin and its analog SN-38 (Cardillo. T. M, et al, 2015 Bioconjug Chem. 26(5):919-31; Cardillo. T. M, et al, 2011 Clin Cancer Res. 17(10): 3157-69; Doi T, et al, 2017 Lancet Oncol. 18(11):1512-1522; PCT/JP2013/006069; US20160333112); amanitin (Moldenhauer G, et al, J. Natl. Cancer Inst. 2012; 104(8):622-34; Zhao, Robert Y. et al, PCT/IB2016/052246); vinblastine (Laguzza, B. C. et al, 1989 J. Med. Chem., 32, 548-555; Starling, J. J., et al 1992 Bioconj. Chem., 3, 315-322); doxorubicin (Yang, H. M. and Reisfeld, R. A., 1988 Proc. Natl. Acad. Sci. USA, 85, 1189-93; Sapra, P., et al 2005 Clin Cancer Res 11, 5257-64; Geretti, E. et al, 2015 Mol Cancer Ther 14, 2060-71); Cryptophycin (Verma, V. A. et al, 2015 Bioorg. Med. Chem. Lett., 25 (4), 864-8); Eribulin (Furuuchi, K. et al, poster MORAb-202, 8th World ADC, Sept., 19, 2017; US2017252458); and pyrrolobenzodiazepine dimers (PBDs) (Mantaj, J. et al 2016 Angew. Chem. Int. Ed. 55, 2-29; D. Antonow and D. Thurston, Chem. Rev. 2011, 111, 2815-2864). In recent years, there has been increasing applications of the PBD class of cytotoxic payloads as ADC therapeutics (Kung Sutherland, M. S., et al 2013 Blood 122, 1455-63; Jeffrey, S. C., 2013, Bioconjug Chem. 1256-63); Saunders, L. R. et al 2015 Sci Transi Med., 7(302):302ral36; Flynn, M. J. et al 2016 Mol Cancer Ther.,15(11), 2709-272;. Zhao, Robert Y., WO2015028850; GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Donnell, A. F. et al 2017 Bioorg Med Chem Lett 27, 5267-5271) due to PBDs’ extream potency against most of the US National Cancer Institute’s (NCI’s) NCI 60-tumor-cell line panels along with unique mechanism of antitumor antibiotics activity (Mantaj, J. et al 2016 Angew. Chem. Int. Ed. 55, 2-29). 5 As shown in structures below, all PBDs are usually conjugated to an antibody with one conditionally stable linker, because it is normally believed that linkers of ADCs must be stable while the ADC is circulating in the blood to limit off-target toxicity, but allow for release of the drug once it is inside the target cancer cells (Alain Beck 2017 Nature Reviews Drug Discovery, doi:10.1038/nrd.2016.268). Unfortunately the most advanced in clinic evaluation of Vadastuxi- 10 mab talirine (CD33 antibody-PBD conjugate having a conditionally stable linker) (Stein, E. M., et al, 2018 Blood 131, 387-96) experienced a set-back because of liver toxicity that apparently killed four patients in the early stage trials (www.xconomy.com/seattle/2017/06/19). malennidocaproyl SGD-1910 । | SGD-la82 The SGD-1882 conjugate, studied by Seattle Genetics, O O | SG3199 SG3249 conjugate| | The SG3249 conjugate studied by ADC Therapeutics Ltd and by Medlmmune/AstraZeneca. The D-212 conjugate studied by Cellerant Therapeutics, 2 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Indolinobenzodiazepine dimer conjugate An IGN conjugate, studied by ImmunoGen, Inc., A Tomaymysin dimer (a PBD) conjugates for Sanofi, A PBD dimer conjugate, studied by Hangzhou DAC Biotech Co., Ltd, Linker-—mAb A PBD dimer conjugate, studied by Hangzhou DAC Biotech Co., Ltd, 10 The SG-3199 conjugate, studied by Stemcentrx/Abbvie and by ADC Therapeutics Ltd. 3 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 O O The SG-3199 conjugate, studied by Allozyne, Inc. The SG2057 conjugate, used by Gcncntcch Inc. O O A PBD conjugate via a traceless linker by Genentech Inc 10 Here we are disclosing the conjugation of PBD dimer derivatives through a dual cross linker that attached both N10 positions of the PBD dimer, thus DNA alkylation sites of the imine warheads are in the forms of prodrugs wherein the conjugate linkers have to be decomposed prior to converting back the potency. Indeed, the application of the dual cross-linking prodrug strategy of PBD conjugation demonstrated much wider thereapeutic windows than the single 15 linkage of PBD conjugates both in vitro and in vivo. It therefore may have much potentially better antitumor antibiotic activities for PBD conjugates in clinic applications. 4 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 SUMMARY OF THE INVENTION The first embodiment of this invention is to disclose a dual linkage of conjugation of PBDs via attachement of both N10 positions of pyrrolo[2,l-c][l,4]benzodiazepine derivatives to a cell binding molecule, as shown in Formula (I), for targeted treatment of cell proliferation. or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers; wherein: represents an optional single bond or can be absent; = represents an optional single bond or a double bond; V and V’, the same or different, are independently selected from the group consisting of H, OH, -NHOH; OR5(an ether); OCOR5 (an ester); OCOOR5(a carbonate); NR5R5’, NR5COR5’, or NRsNRs’NRs” (an amine); OCONR5R5’ (a carbamate); NR5(C=NH)NR5’R5’’ ( a guanidinum); NRsCONRs’Rs” (a urea); OCSNHR5 (a thiocarbamate); -SH (a thiol); -SR5 (a sulfide ); SOR5 a sulphoxide (a sulphoxide ); SOOR5 (a sulfone); SO3, HSO3, HSO2, or a salt of HSO3', SOs2- or - HSO2' (a sulphite); OSO3 (a bisulphite); NR5SOOR5’ (a sulfonamide); H2S2O5 or a salt of S2O52’(a metabisulfite); PO3SH3, PO2S2H2, POS3H2, PS4H2 or a salt of PO3S3’, PO2S23', POS3 ' , PS?'(mono- ,di-, tri-, and tetra-thiophosphatc); (RsO^OSRs’ (a thiophosphate ester); HS2O3 or a salt of S2O32'(a thiosulfate); HS2O4 or a salt of S2O42' (a dithionite); P(=S)(OR5)(S)(OH) (a phosphorodithioate) or a salt thereof form with a cation; -NR5OR5’ (a hydroxylamine derivative); RsC(=O)NOH (a hydroxamic acid ) or a salt formed with a cation; HOCH2SO2', or its salts (a formaldehyde sulfoxylate ); NR5COR5 (an amide); N3 (an azido); CN (a cyano); X (a halo); CtRsXRs’XRs”) (a trialkyl), OP(O)(OR5)(NHR5’) or OP(O)(NHR5)(NHR5) (a phosphoramidate (phosphoramidic acid), or PlRsXRyXRs”) triarylphosphonium; Aa (an amino acid), or NRsCO(Aa)t (a peptide ), wherein Aa is an amino acid or a polypeptide containing between t =1- 100 amino acid units; an aminoacidderived group, such as an a-, 0-, y-, or co-aminoacid, or an unnatural aminoacid; where R5, R5’ and Rs” are described below; 5 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 1, m, q, 1’, m’ and q’ are independently a number of 0, 1, 2. 3, 4, or 5; n is l~30; X, X’, Y and Y’ the same or different, independently, represent N, O, S, an alkyl, such as CH2 or CHR5, an alkene, such as =CH- or =CRs-, an ether, such as C(ORs )H-.; Z and Z’ the same or different, independently, represent N, CH, CR5, COH, CNHi,CNHR5 or COR5. or Z and Z’ link together with -COR5OC-. R5 is independently selected from Ci~C8 alkyl and aryl; Ri, R2, R3, R4, Ri’, R2’, R3’, and R4’ are the same or different and independently chosen from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, -(OCH2CH2)tR5 ( a polyethylene glycol unit), halogen, NH(C=NH)NH2 (a guanidinium), -OR5, -NR5R5', -NO2, -NCO, -NR5COR5', -SR5, -SOR5(a sulfoxide), -SO2Rs (a sulfone), —SOfM+ or -SO3H (a sulfonate), -OSO3'M+ or OSO3H (a sulfate), -SO2NR5R5’ (a sulfonamide), CN (a cyano), N3 (an azido), —COR5, —OCOR5, - OCONR5R5', CF3, OR5, Aryl, heterocycle, or P^jRsRs’Rs” and the linking group (L”) with the reactive group or a cell binding agent bonded thereto when Q, Q’ and T are absent; R5, R5’ and R5” are independently selected from H, Ci~Cg of alkyl, alkenyl, alkinyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts; In addition, Ri and R2 join together, or Rf and R2’ join together form a =O (ketone), =S, =NR, -C(=O)R, or a double bond containing group =CRsR5’; and Ri and R2join together, or Rf and R2’ join together, or R3 and R4 join together or R3’ and R4’ join together form a C3-C12 aromatic, heterocyclic, or heteroaryl ring; G is -CH2-, O, -N(Rs)-, S, -P(O)(OR5)-, -P(O)(N R5R5 )-, are defined above; ; wherein Z and Z’ U and U’ are independently C(O), C(O)O, C(O)NH, C(O)N(R5), C(=NH), C(=NH)O, C(=NH)NH, C(=NH)N(R5), -C=N-, C(=S), C(O)S, C(S)NH, C(S)N(R5), S(O), S(O)O, S(O)NH, S(O)(OR5), S(O)(N(R5)), S(O2), S(O2)O, P(O)(OR5), P(O)(OR5)O, P(O)(NH2), P(O)(NR5R5 ), P(O)(OR5)NH-, P(O)(OR5)NR5’-, P(O) (N(R5R5 ) (N(R5), P(S)(OR5), P(S)(OR5)O, P(S)(NH2), P(S)(NR5R5 ), P(S)(OR5)NH-, P(S)(OR5)NR5 -, P(S)(N(R5R5’)N(R5), Rs, R5O; E( and E2 are independently S, R5S, C(O)S, C(O)NH, C(O)O, C(O)R5S, C(=NH)NH, C(=NH)N(R5), C(=NH)S, -C=N-, C(=S)S, C(O)S, C(=S)NH, C(=S)N(R5), Ar-S, NC(O)CH2S, ArC(O)CH2S, S-S, 6 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 o wherein the chemical bond in the middle of two atoms means it can link either adjoining two atoms; Li, L2 and L’ are independently a linker, or a linker which has a functional group on the linker that enables reaction with a cell-binding agent (CBA), Q. Lj, L2 and L’ are independently preferred a releasable linker, which has the formula of: —Ww—(Aa)r—Tt—; or —Ww— (Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—; wherein: -W- is a Stretcher unit; w is 0 or 1; -Aa- is independently an Amino Acid unit; r is independently an integer ranging from 0 to 100; -T- is a Spacer unit, which can be a linear alkyl or branched alkyl, or polyethylene glycol spacer; and t is 0, or 1-100. The Stretcher unit W may independently contain a self-immolative spacer, peptidyl units, a hydrazone bond, a disulfide, an ester, or a thioether bond; w is 1 or 2 or 3; Preferably Li and L2 are independently selected from O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3 ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula (OCH2CH2)pOR3, or (OCH2CH-(CH3))pOR3, or NH(CH2CH2O)PR3, or NH(CH2CH(CH3)O)PR3> or N[(CH2CH2O)pR3]-[(CH2CH2O)p R3’], or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; Ci-Cs of alkyl; C2-Cs of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3- Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa)r, r =l-12(one to 12 amino acid units), which is composed from natural or unnatural amino acids, or the same or different sequences of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit; Q is a cell binding molecule, or a functional group that enables reaction with a cell-binding agent, or a functional group capable of reacting with a linker attached on a cell binding agent. The function group is chosen from a thiol, an amine, a hydrazine, an alkoxylamino, a disulfide substituent, a maleimido, a haloacetyl group, an A-hydroxy succinimide ester, a keton, an ester, an aldehyde, an alkynyl, an alkenyl, or protected thiol or disulfide group, such as SAc, SSRi or SSAr. Ar is aromatic group or hetero aromatic group. Q is preferably a cell-binding agent/ molecule, selected from the group consisting of an antibody, a single chain antibody, an 7 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 10 antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, a domain antibody fragment that binds to the target cell, an adnectin that mimics antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient¬ transport molecule (a transferrin), and a binding peptide, protein, or small molecule attached on albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid. In a second embodiment, the present invention discloses mono-linkage of conjugation of PBDs derivatives to a cell binding molecule, as shown in Formula (II), (III) and (IV), for targeted treatment of cell proliferation. wherein ==, , X, X’, Y, Y’, Z, Z’,1, F, m, m’, n, q, q’, Ri, Rf, R2, R2’, R3, R3’, R4, R4’, V, V*, U, U’ Li, Lz>. G, Q,Ei, and E2 are the same defined as in Formula (I). In a third embodiment, the present invention discloses a therapeutic composition comprising: (1) an effective amount of one or more of the pyrrolo[2,l-c][1,4]benzodiazepine derivatives linked to a cell binding agent as the conjugate structure of Formula (I), (II), (III) or 8 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV); and (2) a pharmaceutically acceptable carrier, diluent, or excipient, of Formula (I) ~ (IV) of the application, to kill target cells or tissues containing target cells. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the synthesis of cross-linked benzodiazepine dimers. Figure 2 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 3 shows the synthesis of cross-linked benzodiazepine dimers. Figure 4 shows the synthesis of cross-linked benzodiazepine dimers and a conjugate. Figure 5 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 6 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 7 shows the synthesis of benzodiazepine dimers. Figure 8 shows the synthesis of cross-linked benzodiazepine dimers and a conjugate. Figure 9 shows the synthesis of benzodiazepine dimers. Figure 10 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 11 shows the synthesis of benzodiazepine dimers. Figure 12 shows the synthesis of conjugates of benzodiazepine dimers and their linkers. Figure 13 shows the synthesis of the cross-linkers for conjugates of benzodiazepine dimers. Figure 14 shows the synthesis of the cross-linkers for conjugates of benzodiazepine dimers. Figure 15 shows the synthesis of the cross-linkers for conjugates of benzodiazepine dimers. Figure 16 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 17 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 18 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 19 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 20 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 21 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 22 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 23 shows the synthesis of cross-linked benzodiazepine dimers. Figure 24 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 25 shows the synthesis of cross-linked benzodiazepine dimers. Figure 26 shows the synthesis of cross-linked benzodiazepine dimers. Figure 27 shows the synthesis of cross-linked benzodiazepine dimers. Figure 28 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. 9 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Figure 29 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 30 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 31 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 32 shows the synthesis of conjugates of benzodiazepine dimers. Figure 33 shows the in vivo activity of conjugates of cross-linked benzodiazepine dimers. Figurre 34 shows the hematoxylin and eosin stainings of mouse liver tissues after dosing the conjugates CC-4, CC-29, T-DM1 and PBS buffer in ICR mice on day 5. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS: “Alkyl” refers to an aliphatic hydrocarbon group or univalent groups derived from alkane by removal of one or two hydrogen atoms from carbon atoms. It may be straight or branched having Ct-Cs (1 to 8 carbon atoms) in the chain. "Branched" means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, Lpropyl, n-butyl, /-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methyl-hexyl, 2,2- dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2- methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A Ci-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-C8 alkyl,-O-(Ci-C8 alkyl), -aryl, -C(O)R’, -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR’, - C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-C8 alkyl and aryl. “Halogen” refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom. “Heteroalkyl” refers to C2-C8 alkyl in which one to four carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. “Carbocycle” refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicycle [5,6] or [6,6] system. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, - cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, - 1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. A “C3-C8 carbocycle” refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated 10 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 nonaromatic carbocyclic ring. A Cs-Cg carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-Cs alkyl,-O-(Ci-Cg alkyl), -aryl, -C(O)R', - OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R’)2, -NHC(O)R', -SR', -S(O)R',-S(O)2R’, - OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-Cg alkyl and aryl. “Alkenyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, octenyl. “Alkynyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, npentynyl, hexylynyl, heptynyl, and octynyl. “Alkylene” refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3- propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like. “Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (-CH=CH-). “Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl. “Aryl” or Ar refers to an aromatic or hetero aromatic group, composed of one or several rings, comprising three to fourteen carbon atoms, preferentially six to ten carbon atoms. The term of “hetero aromatic group” refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N. The term aryl or Ar also refers to an aromatic group, wherein one or several H atoms are replaced independently by -R’, -halogen, -OR’, or -SR’, -NR’R”, -N=NR’, -N=R’, -NR’R”,- NO2, -S(O)R’, -S(O)2R’, -S(O)2OR’, -OS(O)2OR’, -PR’R”, -P(O)R’R”, -P(OR’)(OR”), - P(O)(OR’)(OR”) or -OP(O)(OR’)(OR”) wherein R’, R” are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts. 11 5 10 15 20 25 30 “Heterocycle” refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226. Preferred nonaromatic heterocyclic include epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused systems resulting from the condensation with a phenyl group. The term “heteroaryl” or aromatic heterocycles refers to a 3 to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi-, or multi-cyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl- 7V-oxide, as well as the fused systems resulting from the condensation with a phenyl group. “Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”, “heterocyclic” and the like refer also to the corresponding “alkylene”, “cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”, “heterocyclene” and the likes which are formed by the removal of two hydrogen atoms. “Arylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2- naphthylethan-l-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. “Hcteroarylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl radical. Examples of heteroarylalkyl groups are 2-benzimidazolylmethyl, 2-ftuylethyl. Examples of a “hydroxyl protecting group” includes, methoxymethyl ether, 2- methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, Z-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate. 12 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 “Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, a halide (e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. A preferred leaving group is selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions or for Mitsunobu reactions. The following abbreviations may be used herein and have the indicated definitions: Boc, tert-butoxy carbonyl; BroP, bromotrispyrrolidinophosphonium hexafluorophosphate; CDI, 1,1*- carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DEAD is diethylazodicarboxylate, DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl-aluminium hydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethyl phosphorocyanidate; DMA, N,N-dimethyl acetamide; DMAP, 4-(N, Ndimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DTPA is diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDC, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; EtOAc is ethyl acetate; Fmoc is N-(9-fluorenylmethoxycarbonyl); HATU, O-(7-azabenzotriazol-l-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS, N-Hydroxysuc-cinimide; MeCN is acetonitrile; MeOH is methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate-buffered saline (pH 7.0-7.5); Ph is phenyl; phe is L-phenylalanine; PyBrop is bromo-tris-pyrrolidinophosphonium hexafluorophosphate; PEG, polyethylene glycol; SEC, size-exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Vai, valine; TLC is thin layer chromatography; UV is ultraviolet. The “amino acid(s)” can be natural and/or unnatural amino acids, preferably alpha-amino acids. Natural amino acids are those encoded by the genetic code, which are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine. The unnatural amino acids are derived forms of proteinogenic amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gammaaminobutyric acid (the neurotransmitter), ornithine, citrulline, beta alanine (3-aminopropanoic acid), gamma-carboxyglutamate, selenocysteine (present in many noneukaryotes as well as most 13 5 10 15 20 25 30 35 eukaryotes, but not coded direcfly by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formyhnetiiionine (which is often the initial amino acid of proteins in bacteria, mitochondria, and chloroplasts), 5-hydroxytryptophan, L-dihydroxyphcnylalaninc, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimetics. Analogs are compounds having the same general H2N(R)CHCO2H structure of a natural amino acid, except that the R group is not one found among the natural amino acids. Examples of analogs include homoserine, norleucine, methionine-sulfoxide, and methionine methyl sulfonium. Preferably, an amino acid mimetic is a compound that has a structure different from the general chemical structure of an alpha-amino acid but functions in a manner similar to one. The term "unnatural amino acid" is intended to represent the "D" stereochemical form, the natural amino acids being of the "L" form. When 1~8 amino acids are used in this patent application, amino acid sequence is then preferably a cleavage recognition sequence for a protease. Many cleavage recognition sequences are known in the art. See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995). In particular, the sequence is selected from the group consisting of Val-Cit, Ala-Vai, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-LeuLys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Asp-Lys, Asp-Glu, Glu-Lys, Lys, Cit, Ser, and Glu. The “glycoside” is a molecule in which a sugar group is bonded through its anomeric carbon to another group via a glycosidic bond. Glycosides can be linked by an O- (an Oglycoside), N- (a glycosylamine), S-(a thioglycoside), or C- (a C-glycoside) glycosidic bond. Its core the empirical formula is Cm(H2O)„ (where m could be different from n, and m and n are < 36), Glycoside herein includes glucose (dextrose), fructose (levulose) allose, altrose, mannose, gulose, iodose, galactose, talose, galactosamine, glucosamine, sialic add, A-acetylglucosamine, sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrins, raffinose, Glucuronic acid (glucuronide), and stachyose. It can be in D form or L form, 5 atoms cyclic furanose forms, 6 atoms cyclic pyranose forms, or acyclic form, a-isomer (the -OH of the anomeric carbon below the plane of1he carbon atoms of Haworth projection), or a p-isomer (the -OH of the anomeric carbon above the plane of Haworth projection). It is used herein as a monosaccharide, disaccharide, polyols, or oligosaccharides containing 3-6 sugar units. The term "antibody," as used herein, refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that 14 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce auto¬ immune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. Antibodies useful in the invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab’) fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope¬ binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens. An “enantiomer”, also known as an “optical isomer”, is one of two stereoisomers that are mirror images of each other that are non-superposable (not identical), much as one’s left and right hands are the same except for being reversed along one axis (the hands cannot be made to appear identical simply by reorientation). A single chiral atom or similar structural feature in a compound causes that compound to have two possible structures which are non-superposable, each a mirror image of the other. The presence of multiple chiral features in a given compound increases the number of geometric forms possible, though there may be some perfect-mirrorimage pairs. Enantiopure compounds refer to samples having, within the limits of detection, molecules of only one chirality. When present in a symmetric environment, enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light (+/—) by equal amounts but in opposite directions (although the polarized light can be considered an asymmetric medium). They are sometimes called optical isomers for this reason. A mixture of equal parts of an optically active isomer and its enantiomer is termed racemic and has zero net rotation of plane-polarized light because the positive rotation of each (+) form is exactly counteracted by the negative rotation of a (—) one. Enantiomer members often have different chemical reactions with other enantiomer substances. Since many biological molecules are enantiomers, there is sometimes a marked difference in the effects of two enantiomers on biological organisms. In drugs, for example, often only one of a drug's enantiomers is responsible for the desired physiologic effects, while the other enantiomer is less active, inactive, or sometimes even productive of adverse effects. Owing to this discovery, drugs composed of only one enantiomer ("enantiopure") can be developed to enhance the pharmacological efficacy and sometimes eliminate some side effects. 15 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Isotopes are variants of a particular chemical element which differs in neutron number. All isotopes of a given element have the same number of protons in each atom. Each atomic number identifies a specific element, but not the isotope; an atom of a given element may have a wide range in its number of neutrons. The number of nucleons (both protons and neutrons) in the nucleus is the atom's mass number, and each isotope of a given element has a different mass number. For example, carbon-12, carbon-13 and carbon-14 are three isotopes of the element carbon with mass numbers 12, 13 and 14 respectively. The atomic number of carbon is 6, which means that every carbon atom has 6 protons, so that the neutron numbers of these isotopes are 6, 7 and 8 respectively. Hydrogen atom has three isotopes of protium ^H), deuterium (2H), and tritium (3H), which deuterium has twice the mass of protium and tritium has three times the mass of protium. Isotopic substitution can be used to determine the mechanism of a chemical reaction and via the kinetic isotope effect. Isotopic substitution can be used to study how the body affects a specific xenobiotic/chemical after administration through the mechanisms of absorption and distribution, as well as the metabolic changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), and the effects and routes of excretion of the metabolites of the drug. This study is called pharmacokinetics (PK). Isotopic substitution can be used to study of the biochemical and physiologic effects of drugs. The effects can include those manifested within animals (including humans), microorganisms, or combinations of organisms (for example, infection). This study is called pharmacodynamics (PD). The effects can include those manifested within animals (including humans), microorganisms, or combinations of organisms (for example, infection). Both together influence dosing, benefit, and adverse effects of the drug, isotopes can contain a stable (non-radioactive) or an unstable element. Isotopic substitution of a drug may have a different thrapeutical efficacy of the original drug. “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. “Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine. “Pharmaceutically acceptable excipient” includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media 16 5 10 15 20 25 30 35 and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositionsis contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations. As used herein, "pharmaceutical salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium. The pharmaceutical salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared via reaction the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, U^ed., Mack Publishing Company, Easton, PA, 1985, p. 1418. "Administering" or “administration" refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device. “Therapeutically effective amount” means an amount of a compound/ medicament according to the present invention effective in preventing or treating the herein referred pathological condition. The term “patient”, or “patient in need thereof’, is intended for an animal or a human being affected or likely to be affected with the herein referred pathological condition. Preferably, the patient is human. 17 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 In the context of cancer, the term "treating" includes any or all of: preventing growth of tumor cells or cancer cells, preventing replication of tumor cells or cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease. In the context of an autoimmune disease, the term "treating" includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells capable of producing an autoimmune antibody, lessening the autoimmuneantibody burden and ameliorating one or more symptoms of an autoimmune disease. In the context of an infectious disease, the term "treating" includes any or all of: preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease. Examples of a "mammal” or “animal" include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. The term “compound”, "cytotoxic agent”, "cytotoxic compound," "cytotoxic dimer" and "cytotoxic dimer compound" are used interchangeably. They are intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof. The term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs, and prodrug salts of a compound of all the formulae disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salt" "prodrug," "prodrug salt," "conjugates," "conjugates salt," "solvate," "hydrate," or "polymorph" in certain aspects of the invention described in this application shall not be interpreted as an intended omission of these forms in other aspects of the invention where the term "compound" is used without recitation of these other forms. The term "imine reactive reagent" refers to a reagent that is capable of reacting with an imine group. Examples of imine reactive reagent includes, but is not limited to, sulfites (H2SO3, H2SO2 or a salt of HSOf, SO? or HSÛ2’ formed with a cation), metabisulfite (H2S2O5 or a salt of S2O? formed with a cation), mono, di, tri, and tetra-thiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3 or a salt of PO3S3-, PO2S23-, POS33 or PS43’ formed with a cation), thio phosphate esters ((RsOjaPSfORs), R5SH, R5SOH, R5SO2H, R5SO3H), various amines (hydroxyl amine (NH2OH), hydrazine (NH2NH2), NH2OR5, R5NHR5-, NH2R5), NH2-CO-NH2, NH2-C(=S)- NH2), thiosulfate (H2S2O3 or a salt of §2O32' formed with a cation), dithionite (H2S2O4 or a salt of S2O42” formed with a cation), phosphorodithioate (P(=S)(OR5)(SH)(OH) or a salt thereof formed with a cation), hydroxamic acid (R5C(=O)NHOH or a salt formed with a cation), hydrazide 18 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (R5CONHNH2), formaldehyde sulfoxylate (HOCH2SO2H or a salt of HOCH2SO2 formed with a cation, such as HOCH2SO2'Na+), glycated nucleotide (such as GDP-mannose), fludarabine or a mixture thereof, wherein R5 and R5' are each independently a linear or branched alkyl having 1 to 8 carbon atoms and are substituted with at least one substituent selected from —N(Rs)(Rs>), - COjH. -SO3H, and -PO3H; R5 and Rs can be further optionally substituted with a substituent for an alkyl described herein; Preferably, the cation is a monovalent cation, such as Na+ or K+. Preferably, the imine reactive reagent is selected from sulfites, hydroxyl amine, urea and hydrazine. More preferably, the imine reactive reagent is NaHSOa or KHSO3. “Cell binding agents” or “Cell binding molecules” may be of any kind presently known, or that become known, and include peptides and non-peptides. Generally, these can be antibodies (especially monoclonal antibodies) or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, growth factors, nutrient-transport molecules (such as transferrin), or any other cell binding molecule or substance (such as vitamins). More specific examples of cell binding agents that can be used include: monoclonal antibodies; single chain antibodies; fragments of antibodies such as Fab, Fab’, F(ab’)2, Fv, {Parham, 131 J. Immunol. 2895-2902 (1983); Spring et al, 113 J. Immunol. 470-478 (1974); Nisonoff et al, 89 Arch. Biochem. Biophys. 230-244 (I960)}, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens; interferons; peptides; lymphokines such as IL-2, IL-3, IL-4, IL-6; hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such as androgens and estrogens; growth factors and colony-stimulating factors such as EGF, TGFa, insufin like growth factor (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF {Burgess, 5 Immunology Today 155-158 (1984)}; vitamins, such as folate and; transferrin {O’Keefe et al, 260 J. Biol. Chern. 932-937 (1985)}. Monoclonal antibody technology permits the production of extremely selective cell binding agents in the form of specific monoclonal antibodies. Particularly well known in the art are techniques for creating monoclonal antibodies produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins such as viral coat proteins. Selection of the appropriate cell binding agent is a matter of choice that depends upon the particular cell population that is to be targeted, but in general monoclonal antibodies are preferred if an appropriate one is available. CROSS-LINKED PBDs AND THEIR CONJUGATES. 19 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 The cross-linked PBD dimer derivatives that have conjugated or can be conjugated to a cell-binding molecule for targeted treatment of cell proliferation, have the structure of Formula (I) as shown below: or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers; wherein: represents an optional single bond or can be absent; ===== represents an optional single bond or a double bond; V and V’, the same or different, are independently selected from the group consisting of H, OH, -NHOH; ORs(an ether); OCORs (an ester); OCOORs(a carbonate); NR5R5’, NR5COR5’, or NRgNRs’NRs” (an amine); OCONR5R5’ (a carbamate); NRs(C=NH)NR5’R5’’ ( a guanidinum); NRsCONRs’Rs” (a urea); OCSNHR5 (a thiocarbamate); -SH (a thiol); -SR5 (a sulfide ); SOR5 a sulphoxide (a sulphoxide ); SOOR5 (a sulfone); SO3, HSO ? HSO2, or a salt of HSO3', SO32' or - HSO< (a sulphite); OSO3 (a bisulphite); NR5SOOR5’ (a sulfonamide); H2S20s or a salt of SiO?2’(a metabisulfite); PO3SH3, PO2S2H2, POS3H2, PS4H2or a salt of PO3S3; PO2S23', POS/h PS43' (mono- ,di-, tri-, and tetra-thiophosphate); (R5O)2POSR< (a thiophosphate ester); HS2O3 or a salt of S2O32~ (a thiosulfate); HS2O4 or a salt of S2Oa2" (a dithionite); P(=S)(ORs)(S)(OH) (a phosphorodithioate) or a salt thereof form with a cation; -NR5OR5’ (a hydroxylamine derivative); RsC(=O)NOH (a hydroxamic acid ) or a salt formed with a cation; HOCH2SO2', or its salts (a formaldehyde sulfoxylate ); NR5COR5 (an amide); O-glycoside; N3 (an azido); CN (a cyano); X (a halo); C(R5)(R5’)(R5”) (a trialkyl), OP(O)(OR5)(NHR5 ) or OP(O)(NHR5)(NHR5) (a phosphoramidate (phosphoramidic acid), or P(Rs)(R5>)(R5”) triarylphosphonium; Aa (an amino acid), or NRsCO(Aa)t (a peptide ), wherein Aa is an amino acid or a polypeptide containing between t =1~ 100 amino acid units; an aminoacid-derived group, such as an a-, p-, y-, or œ-aminoacid, or an unnatural aminoacid; where R5, R5’ and R5” are described below; 1, m, q, T, m’ and q’ are independently a number of 0, 1, 2, 3, 4, or 5; n is l~30; 20 5 10 15 20 25 21 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 X, X’, Y and Y’ the same or different, independently, represent N, O. S, an alkyl, such as CH2 or CHR5, an alkene, such as =CH- or =CRs-, an ether, such as -C(OR5)H-.; Z and Z’ the same or different, independently, represent N, CH, CR5, COH, CNH2,CNHRs, or COR5,or Z and Z’ link together with -COR5OC-; wherein R5 is independently selected from Ci~Cg alkyl and aryl; G is -CH2-, O, -N(R5)-, S, -P(O)(OR5)-, -P(O)(N R5R5 )-, Z z are defined above; wherein Z and Z’ U and U’ are independently C(O), C(O)O, C(O)NH, C(O)N(R5), C(=NH), C(=NH)O, C(=NH)NH. C(=NH)N(R5), -C=N-, C(=S), C(O)S, C(S)NH, C(S)N(R5), S(O), S(O)O, S(O)NH, S(O)(OR5), S(O)(N(R5)), S(O2), S(O2)O, P(O)(OR5), P(O)(OR5)O, P(O)(NH2), P(O)(NR5R5 ), P(O)(OR5)NH-, P(O)(OR5)NR5-, P(O) (N(R5R5 ) (N(R5), P(S)(OR5), P(S)(OR5)O, P(S)(NH2), P(S)(NR5R5), P(S)(OR5)NH-, P(SXOR5)NR5 -, P(S)(N(R5R5’)N(R5), Rs, R5O; El and E2 are independently S, R5S, C(O)S, C(O)NH, C(O)O, C(O)R5S, C(=NH)NH, C(=NH)N(R5), C(=NH)S, -C=N-, C(=S)S, C(O)S, C(=S)NH, C(=S)N(R5), Ar-S, NC(O)CH2S, adjoining two atoms; Li, and L2 are independently a linker, or a linker which has a functional group on the linker that enables reaction with a cell-binding agent (CBA), Q. Li, and L2 are independently preferred a releasable linker, which has the formula of: —Ww—(Aa)r—Tt—; or —Ww—(Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—; wherein: -W- is a Stretcher unit; w is 0 or 1; -Aa- is independently an Amino Acid unit; r is independently an integer ranging from 0 to 100; -T- is a Spacer unit, which can be a linear alkyl or branched alkyl, or polyethylene glycol spacer; and t is 0, or 1-100. The Stretcher unit W may independently contain a self-immolative spacer, peptidyl units, a hydrazone bond, a disulfide, an ester, or a thioether bond; w is 1 or 2 or 3; Preferably Li, and L2 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 are independently selected from O, NH, N, S. P, NNH, NHNH, N(R3), N(R3)N(R3 ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula (OCH2CH2)POR3, or (OCH2CH-(CH3))POR3, orNH(CH2CH2O)pR3, or NH(CH2CH(CH3)O)PR3,or N[(CH2CH2O)pR3]-[(CH2CH2O)p’R3 ], or (OCH2CH2)PCOOR3, or CH2CH2(OCH2CH2)PCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; Ci-Cg of alkyl; C2-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-Cs of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa)r, r =l-12(one to 12 amino acid units), which is composed from natural or unnatural amino acids, or the same or different sequences of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit; Ri, R2, R3, R4, Rf, R2'. R3’, and R4’ are the same or different and independently chosen from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, -(OCH2CH2)tRs ( a polyethylene glycol unit), halogen, NH(C=NH)NH2 (a guanidinium), -OR5, -NR5R5, -NO2, -NCO, -NR5COR;', -SR5, -SOR5(a sulfoxide), -SO2R5 (a sulfone), —SO3’M+ or -SO3H (a sulfonate), -OSO3’M+ or OSO3H (a sulfate), -SO2NRsR5’ (a sulfonamide), CN (a cyano), N3 (an azido), --COR5, —OCOR5, - OCONR5R5', CF3, OR5, Aryl, heterocycle, or P^jRsRs’Rs” and the linking group (L”) with the reactive group or a cell binding agent bonded thereto when Q, Q’ and T are absent; Rs, Rs’ and Rs” are independently selected from H, Ci~Cs of alkyl, alkenyl, alkinyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts; In addition, Ri and R2 join together, or Rf and R2’ join together form a =O (ketone), =S, =NR, -C(=O)R, or a double bond containing group =CRsRs’; and R| and R2join together, or Rf and R2’ join together, or R3 and R4 join together or R3’ and Rf join together form a C3-Ci2 aromatic, heterocyclic, or heteroaryl ring; Q is a cell binding molecule, or a functional group that enables reaction with a cell-binding agent, or a functional group capable of reacting with a linker attached on a cell binding agent. The function group is chosen from a thiol, an amine, a hydrazine, an alkoxylamino, a disulfide substituent, a maleimido, a haloacetyl group, an A-hydroxy succinimide ester, a keton, an ester, an aldehyde, an alkynyl, an alkenyl, or protected thiol or disulfide group, such as SAc, SSRi or SSAr. Ar is aromatic group or hetero aromatic group. Q is preferably a cell-binding agent/ molecule, selected from the group consisting of an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, a domain antibody 22 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 fragment that binds to the target cell, an adnectin that mimics antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient¬ transport molecule (a transferrin), and a binding peptide, protein, or small molecule attached on albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid. The term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzyme-labile bond. It is appreciated that such physiological conditions resulting in bond breaking do not necessarily include a biological or metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or disulfide bond exchange reaction with a intracellular thiol, such as the amillimolar range of abundant of glutathione inside the malignant cells. The Stretcher unit (—W—), when present, may link a targeted binding molecular unit (CBA) to an amino acid unit (—Aa—), or links T when an Aa is not present. The Stretcher unit W may independently contain a self-immolative spacer, peptidyl units, a hydrazone bond, disulfide or thiolether bonds. In this regard a binding molecular (CBA) has a functional group that can form a bond with a functional group of a Stretcher. Useful functional groups that can be present on a binding molecule, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (—SH), amino, hydroxyl, oxyamino, alkynyl, heteroaromatic, carbonyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Preferred functional groups are sulfhydryl, carboxy and amino. Sulfhydryl groups can be generated by reduction of an intramolecular disulfide bond of a Ligand (such as a protein or an antibody). Alternatively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a cell¬ binding molecule using 2-iminothiolane (Traut's reagent) or thiolactone or another sulfhydryl generating reagent, such as modifies the cell-binding molecule with a disulfide bond linker, or a thiol ester following by reduction or hydrolysis respectively. Preferably Li, and L2 are independently a chain of atoms selected from C, N, O, S, Si, and P, having 0-500 atoms. The atoms used in forming the Li, and L2 may be combined in all chemically relevant ways, preferably are Ci C20 alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination above thereof. More preferably Li, and L2are, the same or different, independently selected from O, NH, S, NHNH, N(R?), N(Rs)N(R3’), Ci-Cg alkyl, amide, amines, imines, hydrazines, hydrazones; C2-Cg heteroalkyl, alkylcycloalkyl, ethers, esters, hydrazones, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, 23 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 amino acids, peptides, acyloxylamines, hydroxamic acids, or heterocycloalkyl; Cg-Cg aryl, Ar¬ alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; polyethyleneoxy unit of formula (OCTI2CT^ or (OCH2CH(CH3))POR3; or NH(CH2CH2O)pR3, or NH(CH2CH(CH3)O)pR3, or N[(C1^ or (OCH2CH2)PCOOR3, or CH2CH2(OCH2CH2)PCOOR3, wherein p and p’ are independently an integer selected from 0 to about 5000, or combination thereof; wherein R3 and R? are independently H; Ci-Cg alkyl; C2-Cg heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-Cg aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcyclo-alkyl, alkylcarbonyl, or heteroaryl; or C2-C« esters, ether, or amide; or 1-8 amino acids; or polyethyleneoxy having formula (OCH2CH2)por (OCH2CH(CH3))P, wherein p is an integer from 0 to about 5000, or combination above thereof; Optionally Li and L2 may independenly be composed of one or more linker components of 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoate ("SPP"), 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("MCC"), (4-acetyl)amino-benzoate ("SIAB"), 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), or natural or unnatural peptides having 1-8 natural or unnatural amino acid unites. The natural aminoacid is preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, alanine; Li and L2 may also independenly contain a self-immolative or a non-self-immolative component, peptidic units, a hydrazone bond, a disulfide, an ester, an oxime, an amide, or a thioether bond. The self-immolative unit includes, but is not limited to, aromatic compounds that are electronically similar to the para-aminobenzylcarbamoyl (PAB) groups such as 2- aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronide, and ortho or para-aminobenzylacetals; Preferably, the self-immolative linker component has one of the following structures: 24 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 10 wherein the (*) atom is the point of attachment of additional spacer or releasable linker units, or the cytotoxic agent, and/or the binding molecule (CBA); X , Y , Z and Z are independently NH, O, or S; Z1 is H, OH, NHRi, ORi, SRi, COXiRi, wherein Xi and Ri are defined above; v is 0 or 1; U1 is independently H, OH, Ci~C6 alkyl, (OCH2CH2)n. F, Cl, Br, I, OR5, SRs, NR5R5’, N=NR5, N=R5,NR5R5’,NO2,SOR5R5’, SO2R5, SO3R5, OSO3R5, PR5R5’, POR5R5’, PO2R5R5’, OPO(OR5)(OR5’), or OCH2PO(OR5(OR5’), wherein R5 and R5’ are independently selected from H, C|~C8 alkyl; C2~C8 alkenyl, alkynyl, heteroalkyl, or amino acid; C3~C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or glycoside; or pharmaceutical cation salts; The non-self-immolative linker component is one of the following structures: (CH2)nCO(OCH2CH2)rOCH3 (CH2)nCON(CII2CH2O)rCOCII3 *(CH2CH2O)r*. 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein the (*) atom is the point of attachment of additional spacer or releasable linkers, the cytotoxic agents, and/or the binding molecules; X1, Y1, U1, R5, R5’ are defined as above; r is 0~100; m and n are 0~6 independently; 10 Further preferably, Li and L2 may independently be a releasable linker. The term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile or enzyme-labile bond. It is appreciated that such physiological conditions resulting in bond breaking do not necessarily include a biological or 15 metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or 26 5 10 15 20 25 30 35 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 disulfide bond exchange reaction with a intracellular thiol, such as a millimolar range of abundant of glutathione inside the malignant cells; Examples of the releasable linkers Li or L2 include, but not limited: -(CR5R6)m(Aa)r(CR7R8)n(OCH2CH2)r, -(CR5R6)m(CR7R8)n(Aa)r(OCH2CH2)r, -(Aa)r- (CR5R6)m(CR7R8)n(OCH2CH2)r, -(CR5R6)m(CR7R8)n(OCH2CH2)I(Aa)r, -(CRsRglm- (CR7=CR8)(CR9Rio)n(Aa) -(CR5R6)m(NRiiCO)(Aa)t(CR9Rio)n-(OCH2CH2)r-, - (CR5R6)m(Aa)t(NR11CO)(CR9R10)n(OCH2CH2)r-,-(CR5R6)m(OCO)(Aa)t(CR9Rio)n.(OCH2CH2)r-, -(CR5R6)m(OCNR7)(Aa)t(CR9Rio)n(OCH2CH2)r-, -(CRsRôMCOXAaMCRçRwMOCH^H^ (CR5R6)m(NRiiCO)(Aa)t(CR9Rio)n(OCH2CH2)r-, -(CR5R6)m.(OCO)(Aa)t(CR9Rio)n-(OCH2CH2)I-, -(CR5R6)n>(OCNR7)(Aa)t(CR9Ri0)n(OCH2CH2)r-, -(CR5R6)m(CO)(Aa)t(CR9Rio)n-(OCH2CH2)r-, - (CR5R6)m-phenyl-CO(Aa)t(CR7R8)n-, XCR5R6)m-furyl-CO(Aa)t(CR7R8)n-, -(CR5R6)moxazolyl-CO(Aa)t(CR7Rs)n-, -(CR5R6)m-thiazolyl-CO(Aa)t(CCR7R8)n-, -(CRsR/ûr thienyl-CCXCRzRsX-, -(CRsRâX-imidazolyl-CO-CCRvRsln-, -(CR5R6)t-morpholino-CO(Aa)t. (CR7Rs)n-, -(CR5R6)tpiperazino-CO(Aa)t.(CR7Rs)n-, -(CRsRôlt-N-methylpiperazin-COCAalt. (CR7R8)n-, -(CR5R)m-(Aa)tphenyl-, -(CR5R6)m-(Aa)tfuryl-, -(CR5R6)m-oxazolyl(Aa)t-, -(CR5R6)mthiazolyl(Aa)t-, -(CR5R6)m-thienyl-(Aa)t-, -(CR5R6)m-imidazolyl(Aa)t-, -(C RsRélmmorpholino-(Aa)t-, -(CR5R6)m-piperazino-(Aa)t-, -(CRsR«)m-N-methylpiperazino-(Aa)t-, -K(CR5R6)m(Aa)r(CR7R8)Q(OCH2CH2)r, -K(CR5R6)m(CR7R8)n(Aa)XOCH2CH2)t-, -K(Aa)r- (CR5R6)m(CR7R8)n(OCH2CH2)t-, -K(CR5R6)m(CR7R8)n(OCH2CH2)r(Aa)r, -K(CR5R6)m- (CR7=CR8)(CR9Rio)n(Aa)t(OCH2CH2)r-,-K(CR5R6)m(NRiiCO)(Aa)t(CR9Rio)n(OCH2CH2)r- , -K(CR5R6)m(Aa)t(NRnCO)(CR9Rio)n(OCH2CH2)r-, -K(CR5R6)m(OCO)(Aa)t(CR9Ri0)n- (OCH2CH2)r-,-K(CR5R6)In(OCNR7)(Aa)t(CR9Rio)n(OCH2CH2)r-, -K(CR5R6)m(CO)(Aa)t. (CR9R10)n(OCH2CH2)r-,-K(CR5R6)m(NR11CO)(Aa)l(CR9R1o)n(OCH2CH2)r-, -K(CR5R6)m- (OCO)(Aa)t(CR9R]0)n(OCH2CH2)r-, -K(CR5R6)m(OCNR7)(Aa)t(CR9Rio)n(OCH2CH^^ -K- (CR5R6)m(CO)(Aa)t(CR9Rio)n(OCH2CH2)r-, -K(CR5R6)m-phenyl-CO(Aa)t(CR7R8)n-, -K- (CR5R6)m-furyl-CO(Aa)t.(CR7Rs)IÎ-, -K(CR5R6)m-oxazolyl-CO(Aa)t(CR7R8)n-, -K(CR5R6)mthiazolyl-CO(Aa)t.(CR7R8)n-, -K(CR5R6)t-thienyl-CO(CR7R8)n-, -KfCRsR^timidazolyl-CO- (CR7R8)n-, -K(CR5R6)tmorpholino-CO(Aa)t(CR7R8)n-, -K(CR5Rô)tpiperazino-CO(Aa)t. (CR7R8)n-, -K(CR5R6)rN-methylpiperazinCO(Aa)t(CR7R8)n-, -K(CR5R)m(Aa)tphenyl, -K- (CR5R6)m-(Aa)tfiiryl-, -K(CR5R6)m-oxazolyl(Aa)t-, -K(CR5R6)m-thiazolyl(Aa)r, -K(CR5R6)mthienyl-(Aa)t-, -K(CRsR6)m-iniidazolyl(Aa)t-, -K(CRsR6)m-morpholino(Aa)t-, -KCCRsRfOmpiperazino-(Aa)tG, -K(CR5R6)mN-methylpiperazino(Aa)r; wherein m, Aa, m, and n are described above; t and r are 0- 100 independently; R3, R4, R5, Rô, R7, and R8 are independently chosen from H; halide; Ci~C8 alkyl; C2~C8 aryl, alkenyl, alkynyl, ether, ester, amine or amide, which optionally substituted by one or more halide, CN, NRiR2, CF3, ORi, Aryl, heterocycle, 27 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 10 15 S(O)Rb SO2Ri.-CO2H, -SO3H, -ORb -CO2Ri, -CONRb PO2R,R., PO3H or P(O)R!R2R3; K is NRb -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -ON-NH-, -C(=O)NH-NH-, O, S, Se, B, Het (heterocyclic or heteroaromatic ring having C3-Cg), or peptides containing 1- 20 of the same or different amino acids; Additionally U, U’, E, E’, Li and L2may independently contain one or more units of the following hydrophilic structures: X2, X3> X4, X5, orX6, are independently selected from NH; NHNH; N(R3); N(R3)N(R3>); O; S; CiC6 alkyl; C2-Cô heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-Cg aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or 1~8 28 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 amino acids; wherein R, and R3’ are independently H;Cj-Cx alkyl; C2-C8 hetero-alkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or C2-C8 esters, ether, or amide; or polyethyleneoxy unit having formula (OCH2CH2)por (OCH2CH(CH3))P, wherein p is an integer 5 from 0 to about 5000, or combination above thereof; More preferably, Li, or L2, are independently linear alkyl having from 1-6 carbon atoms, or polyethyleneoxy unit havingformula (OCHiCHalp, p = 1-5000,or a peptide containing1-4 units of aminoacids (L or D form), or combination above. In addition, U, U’, Li, L2, L’, Ei or E2 may independently be composed of one or more 10 following components as shown below: i-maleimidocaproyl (MC) maleimido propanoyl (MP), (k oxobutanoic K . acid, -.4 HO^ 0 A 15 phenylalanine (ala-phe), c V\ 5 s• 1 -j HO’yj ,. thio-maleido, thio-amino O thio-amino-oxobutenoic acid, h2 H 0 valine-citrulline (val-cit), «-y alanine- '— lysine-phenylalanine (lys-phe), O lysine-alanine (lys-ala), aminobenzyloxycarbonyl (PAB), O 0 4-thio-pentanoate (SPP), 29 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 5 “S O 4-(NO maleimidoethyl (ME), aryl-thiol (PySS), (4-acctyl)aminobcnzoatc (SIAB), aminobenzylthio, diaminobenzylthio, /-N C H alkoxy amino (AOA), dithio, O alkylsulfonyl, O OH OH phosphinic acid, NN,N’-dimethylphosphon-amidic acid, hydrazine, N‘ H 4-methyl-4-dithio-pentanoic (MPDP), OI O H ~ II Al sulfon-bisamide, O ii Phosphondiamide, V J. maleimidomethyl)cyclo-hexane-1-carboxylate (MCC), c SO3‘ alkylphosphonamide, I 8 1 I OH O 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), n O methylphosphonamidic acid, O H ' HN^ç O 4-thio-butyrate (SPDB), ~ ethyleneoxy (EO), ,N. :n—n: N,N’-dimethylphosphondiamide, triazole, alkylsulfonamide, O F ° , oxylbenzylthio, o^s —S—J dioxylbenzylthio, —S—J3 amino-oxylbenzylthio, 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 L- or D-, natural or unnatural peptides containing 1-20 amino acids; wherein a connecting bond in the middle of atoms means that it can connect either neighbor carbon atom bonds; wavery line 5 is the site wherein another bond can be connected to; Alternatively, U, U’, Ei, orE2, can be independently absent. The compounds of the general Formula (I) having geometrical and stereoisomers are also a part of the invention. A preferred stereoisomer of the Formula (I) is presented by the following Formula (la), 31 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein Zi is OH, NH2, ORi, NHRb NRiR2, SR|. NHR|COXjRb ORiCOXiRi.or »5 j iRi n I» 1 I» m, m’, n, q, q’, Rb RA R2, R2’, R3, RA R4, R4’, V, V’, U, U’ Lb L2. Ei, E2 and Q are the same as in Formula (I). In more preferred embodiment according to Formula (I), the conjugate of cross-linked PBD dimer derivatives of the invention have the Formula (1-01) ~ (1-18) below: 32 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 33 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (1-05), (1-06), (1-07), 34 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (1-13), 36 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (1-15), (1-16), 37 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 38 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein V, V’, n and q are defined the same above; mAb is a cell-binding molecule, preferably an antibody; r, r’ and r” are independently 0 - 200. In another embodiment, the present invention discloses mono-linkage of conjugation of 5 PBDs derivatives to a cell binding molecule, as shown in Formula (II), (III) and (IV), for targeted treatment of cell proliferation: 39 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein =, — , X, X’, Y, Y’, Z, Z’,1, 1’, m, m’, n, q, q’, Rj. R(\ R2. R2’, R3, R3\ R4, RA V, V’, U, U’ Li, L2, G, Q.Ei, and E2 are the same defined as in Formula (I). In preferred embodiment according to Formula (IT), (III) and (IV), the conjugates of PBD derivatives of the invention having the Formula (11-01) ~ (11-11), (in-01)~(III-06) and (IV-01)~(IV- 5 11) below: (11-01), (11-02), (H-03), 40 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (H-04), (11-05), (II-06), (11-07), 41 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 42 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 (UI-03). 43 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (III-04), (in-05), 44 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV-01), (IV-02), (IV-03), (IV-04), 45 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV-05), (IV-06), (TV-07), (IV-08), 46 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV-09), (IV-10), (IV-11), wherein =====, —, m, m’, n, q, and q’ are the same defined as in Formula (I); r, r’, and r’’ are independently 0- 200, m3 is 0 - 30. In another preferred embodiment, the conjugates of Formula (I), (II) (III), (IV) and (V) are 10 prepared from coupling of a cell-binding molecule with a PBD dimer derivative having the Formula (V) (VI), (VII), and (VIII) accordingly as shown below: 47 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein ==, — , X, X’, Y, Y’, Z, Z’, 1, 1’, m, m’, n, q, q’, Ri, Ri’, R2, R2’, R3, R3’, R4, R4’, V, V’, U, U’ Li, L2, Ei, and E2 are the same defined as in Formula (I); wherein E3 and E’3 are independently selected from: 48 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 acyl halide (acid halide). ethenesulfonyl; acryl (acryloyl); O 2-(dinitrophenoxy)acetyl; O2N- 2-(nitrophenoxy)acetyl; ^2N F 2-(fluorophenoxy)-acetyl; F o x2A2 2- (difluorophenoxy)-acetyl; 2-(((trifluoromethyl)-sulfonyl)oxy)acetyl; 2-(pentafluorophenoxy)acetyl; 49 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 P-lactam, or h2nhn- PCT/CN2018/094586hydrazide,s=c=n-£ isothiocyanate; wherein Xi’ O and X3’ are independently F, Cl, Br, I or Lv3; X2’ is O, NH, N(Ri), or CH2; R3 and R5 are independently H, Ri, aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -Ri, -halogen, -ORi, -SRi, -NR1R2, - NO2, -S(O)Ri, - S(O)2Ri, or -COORi; LV3 is a leaving group selected from methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, phenylthio, pyridinylthio, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluoro-phenoxy1, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazole-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxyl, 2-ethyl-5-phenylisoxazoliumyl, phenyloxadiazol-yl (ODA), oxadiazol-yl, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions, wherein R1 and R2 are defined above; More preferably, E3 and E’3 are independently selected from -SH, -S-SCH3, -S-SAc, -S-SPyridine. -S-S-Ar(-NO2), -S-cell binding agent, or any one of the following formulas: 50 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 wherein D is H, -NCF. SO3H, or F; wherein Ri, R2, R3, R4. r. m and n are defined above; w and w’ are independently 0, 1 or 2; wherein R5 and R5’ are independently selected from Ci~Cô alkyl, aryl, cyclic, cyclohetero, H, or M (wherein M isNa, K, Ca, ammonium or the other pharmaceutically acceptable salt); In certain embodiments, the PBD derivatives of Formula (V), (VI), (VII), and (VIII) are represented by the following Formulas (V-01)~(V-20), (VI-01) ~ (VI-05), (VH-01)~ (VII-06), (VIII-01) -(Vin-06) accordingly below: (V-03), 51 GA 03105541 2021-01-04 52 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 53 GA 03105541 2021-01-04 54 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 55 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 (VI-03), 56 GA 03105541 2021-01-04 57 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 58 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 59 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (VII-03), 60 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 (vm-02), (VIII-04), 61 5 10 15 20 (VIII-06), wherein U, U’, V, V’, n, n’, and L as defined above; Re and Re’ are independently selected from Ci~Ce alkyl, aryl, cyclic, cyclohetero, halogen, haloalkyl, alkoxy, haloalkoxy alkylamino, - NO2, -CN or H; Xi and Xf are independently H, F, Cl, Br, I, OTs (tosylate), OMs (mesylate), nitrophenol OAr(NO2), OAr(NO2)2dinitrophenol, OAr(F) monofluorophenol, OAr(F)5 pentafluorophenol, OAr(F)2 difluorophenol,N-hydroxysuccinimide (NHS), phenol, tetrafluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1- hydroxybenzotriazole, or 2-ethyl-5-phenylisoxazolium-3'-sulfonate. SYNTHESIS OF THE CROSS-LINKED PBD DIMER DERIVATIVES OF FORMULA (V), (VI), (VII) AND (VIII) AS CYTOTOXIC AGENTS. The compounds and process of the present invention can be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by application or adaptation of1he methods described in the examples, or variations thereon as appreciated by the skilled artisan. The appropriate modifications and substitutions will be readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. hi particular, such methods can be found in Richard C. Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, Two Volume Set, 2nd Edition, Wiley Publishers, 2010. Because the cytotoxic agents of the present invention may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless 62 Date Reçue/Date Received 2024-05-09 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers. The cytotoxic agents of the present invention may be prepared by a variety of synthetic routes. The reagents and starting materials are commercially available, or readily synthesized by well-known techniques by one of ordinary skill in the arts. All substituents, unless otherwise indicated, are as previously defined. In the synthetic reactions of the cytotoxic agents of the present invention, it may be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice, for examples see Peter G. M. Wuts, Theodora W. Greene in Greene's Protective Groups in Organic Synthesis, 4th Edition, John Wiley and Sons, 2006; Ian T. Harrison, Shuyen Harrison in Compendium of Organic Synthetic Methods, Vol 1,2 Vols. 1& 2 By Ian T. Harrison & Shuyen Harrison , Vols 3-5 by Louis S. Hegedus, Leroy Wade Vols 6-Vol 12 by Michael B. Smith, John Wiley and Sons, 2006-2012. Normally the synthetic reactions are carried out in suitable solvents, temperatures and time. A variety of solvents which have no adverse effect on the reaction or on the reagents involved can be used in a synthetic reaction of the cytotoxic agent. Examples of suitable solvents include: hydrocarbons, which may be aromatic, aliphatic or cycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene, toluene and xylene; hydrocarbons containing halogens, such as chloroform, dichloromethane, dichloroethane; amides, such as dimethylactamide or dimethylformamide; alcohols such as ethanol and methanol and ethers, such as diethyl ether and tetrahydrofuran. The reactions can take place over a wide range of temperatures, from -100 °C- 300°C. More preferably from 0°C to 150°C. The time required for the synthetic reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents and can be from 5 second to 4 weeks, more preferably from 10 min to 24 hours. In addition, the cytotoxic agents prepared may be isolated or purified from the reaction mixture by conventional means, such as evaporating or distilling off the solvent from the reaction mixture, or after distilling off the solvent from the reaction mixture, pouring the residue into water followed by extraction with a water-immiscible organic solvent and then distilling off the solvent from the extract. It may also involve various well known techniques, such as re-crystallization, re-precipitation or the various chromatography techniques, 63 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 notably column chromatography, preparative thin layer chromatography, or high performance liquid chromatography. Some of the synthetic reactions of the cytotoxic agents and their conjugates to a cell binding agent are further exampled but not restricted in the Figures 1~32 and in the examples of the following description. THE CONJUGATES OF CROSS-LINKED PBD DIMERS TO A CELL-BINDING MOLECULE. The present invention provides a conjugate molecule comprising at least one PBD derivative covalently linked to a cell-binding agent (Q) through a linking group of a conjugate linker. Preferably said conjugate comprises one to twenty molecules of cross-linked PBD dimer derivatives according to the invention covalently linked to a cell-binding agent through a linking group of a linker on a cross-linked PBD dimer derivative. As stated above, the conjugates of a cell surface binding molecule - cross-linked PBD dimer derivatives are illustrated in the Formula (I), (II), (III) and (IV) above. Drug loading may range from 1 to 30 drug moieties (D) per antibody and is preferred the average number of 2-8 drug moieties per antibody in a molecule of Formula (I) ~ (IV). The average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of the conjugates in terms of the drug loading may also be determined. In some instances, separation, purification, and characterization of homogeneous the conjugates where Drug loading is a certain value from the conjugates with the drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. The Cell binding agents (CBA) may be of any kind and include peptides and non-peptides. Generally, the cell binding agents include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies (polyconal and monoclonal antibodies); single chain antibodies; fragments of antibodies such as Fab, Fab', F(ab')2, Fv, [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR’s, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens; antibody mimetic, such as an affibody; domain antibodies (dAb); nanobodies; unibodies; DARPins; anticalins; versabodies; duocalins; lipocalins; vimers; interferons (such as type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, GM-CSF, interferon¬ gamma (IFN-y); hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such as androgens and estrogens, 64 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 melanocyte-stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factors (TGF), such as TGFa, TGFp, insulin and insulin like growth factors (IGF-!, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-158 (1984)]; vaccinia growth factors (VGF); fibroblast growth factors (FGFs); smaller molecular weight proteins, poly-peptide, peptides and peptide hormones, such as bombesin, gastrin, gastrin¬ releasing peptide; platelet-derived growth factors; interleukin and cytokines, such as interleukin- 2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors, granulocyte-macrophage colony¬ stimulating factor (GM-CSF); vitamins, such as folate; apoproteins and glycoproteins, such as transferrin {O’Keefe et al, 260 J. Biol. Chern. 932-937 (1985)}; sugar-binding proteins or lipoproteins, such as lectins; cell nutrient-transport molecules; and small molecular inhibitors, such as prostate-specific membrane antigen (PSMA) inhibitors and small molecular tyrosine kinase inhibitors (TKI), non-peptides or any other cell binding molecule or substance, such as bioactive polymers (Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); dendrimers (Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci. 2009, 106, 685- 90); nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chern. 2008, 19, 1309-12); liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9); viral capsides (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93). In general monoclonal antibodies are preferred as a cell-surface binding agent if an appropriate one is available. The linker used for the conjugation of this invention includes, but not limited to, a disulfide linker, a thioether linker, an amide bonded linker, a peptidase-labile linker, a photolabile linker, an acid-labile linkers (such as hydrazone liner), an esterase-labile linker, an oxidatively labile linker, a metabolically labile linker, a biochemically labile linker. Preferably, the linker is linked to the cell binding agent via a function reactive towards for instance thiol and amino functions of the cell binding agent coming from reduced disulfide bonds and lysine residues respectively. More particularly, said derivative is linked through the — CO— group to the amino function of the lysine residue of said cell binding agent, so as to form an amide bond. In addition, the linker may be composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine¬ citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af'), glycine-glycine, paminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), Nsuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("SMCC"), N-Succinimidyl (4- iodo-acetyl)aminobenzoate ("SIAB"), ethyleneoxy (—CH2CH2O—) as one or more repeating 65 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 units ("EO" or "PEO”). The linker may be a "cleavable linker," facilitating release of a drug in the cell. Additional linker components are known in the art and some are illustrated below: Oeo nu R| R, H O . ’ nhnh r7 Og-*. * c, 66 GA 03105541 2021-01-04 67 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Rs and R9 are independently selected from -Ci~Cs alkylene-, — Ci~C? carbocyclo-, -O-( Ci~Cs alkyl)-, -arylene-, — Ci~Cs alkylene-arylene-, -arylene, -Ci~Cs alkylene-, -Ci~Cg alkylene-( Ci~Cs carbocyclo)-, -(C3--C7 carbocyclo)- Ci~Cs alkylene-, -Cs-Cs heterocyclo-, -C|~Cs alkylene-( C ~Cs heterocyclo)-, -( C3~Cs heterocyclo)- C1-C9 alkylene-, -(CH2CH2O)k-, - (CH(CH3)CH2O)t-, and -( CH2CH2O)k-CH2-; k is an integer ranging from 1-30; X’”, Y’” and Z”’ are independently selected from NH, O or S; Q, Ri and R2are described above. In a preferred embodiment, conjugates of die invention are antibody/cytotoxic agent, antibody fragment/cytotoxic agent, diabody/cytotoxic agent, tri(a)body/cytotoxic agent, epidermal growth factor (EGF)/cytotoxic agent, prostate specific membrane antigen (PSMA) inhibitor/cytotoxic agent, melanocyte stimulating hormone (MSH)/cytotoxic agent, thyroid stimulating hormone (TSH)/cytotoxic agent, polyclonal antibody/cytotoxic agent, somatostatin/cytotoxic agent, folate/cytotoxic agent, matriptase inhibitor/cytotoxic agent, estrogen/cytotoxic agent, estrogen analogue/cytotoxic agent, designed ankyrin repeat proteins (DARPins)/cytotoxic agent, androgen/cytotoxic agent, and androgen analogue/cytotoxic agent. In a more preferred embodiment, cell binding molecule of the invention are monoclonal antibody. Examples of antibodies used for conjugation of cyotoxic agents in this prevention include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab (anti-TNF-a), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-a); Afutuzumab (anti-CD20), Alacizumab pcgol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab (antiCEA), Anatumomab ( anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (antiHLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin (CD62L), Atlizumab 68 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (a chain of IL-2 receptor), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCLll (eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, antiVEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE, anti-CD19). Brentuximab (cAClO, anti-CD30 TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab (Haris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pcgol (Cimzia anti-TNF-a), Cetuximab (Erbitux, IMCC225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzumab (anti-MUCl), Conatumumab (anti-TRAIL-R2), CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAbl7-lA, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CDIla), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (antiSLAMF7), Elsilimomab (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrin av03), Exbivirumab ( anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti¬ interferon receptor), Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFNy), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-p), Galiximab (antiCD80), Gantenerumab (anti- beta amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, antiGPNMB), Golimumab (Simponi, anti-TNF-a), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-a), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain of IL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti- CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti- IL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti- 69 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 CD40), Lumiliximab (anti- CD23 (IgE receptor), Mapatumumab (anti-TRAIL-Rl), Maslimomab (anti- T-cell receptor), Matuzumab (anti-EGFR). Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin ou), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-a), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R a), Omalizumab (Xolair, anti-IgE Fc region), Oportuzumab (antiEpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (antilipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti- Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUCl), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor). Rontalizumab (anti-IFN-a), Rovelizumab (LeukArrest, anti-CDll, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-a), Siltuximab (anti-IL-6), Siplizumab (antiCD2), (Smart) MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab (anti¬ sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte antigen), Tacatuzumab (anti-alphafetoprotein), Tadocizumab (anti-integrin anbPa), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAlL-R2). TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, (antiHER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus), Urtoxazumab (anti- Escherichia coli), Ustekinumab (Stelara, 70 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 anti-IL-12, IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrin cuP?), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3), Volociximab (anti-integrin aspi), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab (antiCD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst, for melanoma and solid tumors), J591 (anti-PSMA, Weill Cornell Medical School for prostate cancers), 225.28S [anti-HMW-MAA (High molecular weight-melanomaassociated antigen), Sorin Radiofarmaci S.R.L. (Milan, Italy) for melanoma], COL-1 (antiCEACAM3, CGM1, from Nat. Cancer Inst. USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, for prostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus), ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis toxic shock], MEDI-500 [T10B9, anti-CD3, TRap (T cell receptor alpha/beta), complex, from Medlmmune Inc for Graft-versus-host disease], RING SCAN [ antiTAG 72 (tumour associated glycoprotein 72), from Neoprobe Corp, for Breast, Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTDl (Tumorassociated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen; M4S; tumor antigen 17-1A; CD326, from NeoRx Corp, for Colon, Ovarian, Prostate cancers and NHL]; LymphoCide, Smart ID10, Oncolym, Allomune, anti-VEGF, CEAcide, IMC-1C11, and Cetuximab. Other antibodies as binding ligands include, but are not limited to, are antibodies against the following antigens: Aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin’s disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin’s lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma), CD66e (cancers), CD70 (metastatic renal cell carcinoma and 71 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas), CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast, colorectal and lung cancers), DLL4 (A-like-4), EGFR (Epidermal Growth Factor Receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, Heme-oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3 idiotype (cancers), Heat shock proteins (cancers), HER1 (lung, stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor (interleukin 2 receptor,T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman’s disease, IL6 dependent tumors), Integrins (avp3, a5pi, a6p4, alip3, a5p5, avp5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane-spanning 4- domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), Paratope of anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers), PSMA (prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associated glycoprotein 72, AML, gastric, colorectal, ovarian cancers), T cell transmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cell carcinoma), TRAIL-RI (Tumor necrosis apoprosis Inducing ligand Receptor 1,lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor associated antigens recognized by antibodies have been reviewed (Gerber, et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer Immunol Immunother. 54(3),187-207 (2005). Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76). Examples of these antigens that antibodies against are: Many other Cluster of Differentiations (CD2, CD2R, CD3, 72 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8, CD8a. CD8b, CD9, CD10, CDlla, CDllb, CDllc. CD12, CD12w, CD13, CD14, CD15, CD15s, CD15u. CD16, CD16a, CD16b, CD17, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55,CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71. CD72, CD73, CD74, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CDwll3, CD114, CD115, CD116, CD117, CD118, CD119, CDwll9, CD120a, CD120b, CD121a. CD12lb. CDwl21b, CD122, CD123, CDwl23, CD124, CD125, CDwl25, CD126, CD127, CD128, CDwl28, CD129, CD130, CD131, CDwl31, CD132, CD133, CD134, CD135, CD136, CDwl36, CD137, CDwl37, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CDwl45, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CDwl56c, CD157, CD158a, CD158b, CD159a, CD159b, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CDwl86, CD187, CD188, CD189, CD190, Cdl91, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CDwl98, CD199, CDwl99, CD200, CD200a, CD200b, CD201, CD202, CD202b, CD203, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210, CD212, CD213al, CD213a2, CDw217, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235ab, CD235b, CD236, CD236R, CD238, CD239, CD240, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265, CD266, CD267, CD268, CD269, CD271, CD273, CD274, CD275, CD276 (B7-H3), CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, 73 5 10 15 20 25 30 35 CDw338, CD339), Annexin Al, Nucleolin, Endoglin (CD105), ROBO4, Amino-peptidase N, like-3 (DLL3), E -like-4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 nonmutant, NYESO-1, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, Proteinase3 (PR1), bcrabl, Tyrosinase, Survivin, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgen receptor, Cyclin Bl, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothclin, PSCA, MAGE Al, sLe(a), CYP1B1, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-P, MAD-CT-2, Fos-related antigen 1. Production of antibodies used in the present invention involves in vivo or in vitro procedures or combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonal antibody is typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen (Kohler, G.; Milstein, C. (1975). Nature 256: 495-497). The detailed procedures are described in “Antibodies—A Laboratory Manual”, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988). Particularly monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins. Splénocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact specified receptors or inhibit receptor activity on target cells. A monoclonal antibody used in tire present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridomathat secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques, such as using protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusive chromatographies (particularly by affinity for the specific antigen after Protein A, and sizing column 74 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 chromatography); centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, 20% fetal calf serum and with an anti¬ foaming agent, such as polyoxyethylene-polyoxypropylene block copolymer. In addition, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with an oncovirus, such as Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also be produced via an anti-receptor peptide or peptides containing the carboxyl terminal as described well-known in the art. See Niman et al., Proc. Nad. Acad. Sei. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry 34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen for producing anti-receptor peptide monoclonal antibodies. There are also a number of other well-known techniques for making monoclonal antibodies as binding molecules in this invention. Particularly useful are methods of making fully human antibodies. One method is phage display technology which can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity enrichment. Phage display has been thoroughly described in the literature and the construction and screening of phage display libraries are well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13 (1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson et al., Nature 352:264-628 (1991); Huse et al., Science 246:1275-1281 (1989), Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001) (O'Brien et al., ed., Human Press, Totowa, N.J.), and in certain embodiments, in Lee et al. J. Mol. Biol. 340:1073-1093 (2004). Moncolonal antibodies derived by hybridoma technique from another species than human, such as mouse, can be humanized to avoid human anti-mouse antibodies when infused into humans. Among the more common methods of humanization of antibodies are complementarity¬ determining region grafting and resurfacing. These methods have been extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al, Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 1;13:1619-33 (2008); Lazar et al, Mol Immunol. 44(8):1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. USA.103(1O):3557-62 (2006). 75 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys, or other mammals, carrying large portions of the human immunoglobulin heavy and light chains, with an immunogen. Examples of such mice are: the Xenomouse. (Abgenix, Inc.), the HuMAbMouse (Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. No. 6,596,541 , 6,207,418, No. 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,545, Nos. 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In human therapy, murine variable regions and human constant regions can also be fused to construct called “chimeric antibodies” that are considerably less immunogenic in man than murine mAbs (Kipriyanov et al, Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002)). In addition, site-directed mutagenesis in the variable region of an antibody can result in an antibody with higher affinity and specificity for its antigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002)); Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchanging constant regions of a mAb can improve its ability to mediate effector functions of binding and cytotoxicity. Antibodies immunospecific for a malignant cell antigen can also be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a malignant cell antigen can be obtained commercially, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. DNA encoding hybridoma-derived monoclonal antibodies or phage display Fv clones of the antibody can be readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells (Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992)). Antibodies can also be produced by using an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and property assembled antibodies. Such modulation isaccomplished at least in part by simultaneously modulating translational strengths for the polypeptide components. After the fermentation which is known in the art, the produced antibody protein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. 76 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 The exemplary purification procedures: fractionation on immunoaffinity (such as Protein A columns) or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. Apart from an antibody, a peptide or protein that bind/block/target or in some other way interact with the epitopes or corresponding receptors on a targeted cell can be used as a binding molecule. These peptides or proteins could be any random peptide or proteins that have an affinity for the epitopes or corresponding receptors and they don’t necessarily have to be of the immunoglobulin family. These peptides can be isolated by similar techniques as for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49). The use of peptides from such random peptide libraries can be similar to antibodies and antibody fragments. The binding molecules of peptides or proteins may be conjugated on or linked to a large molecules or materials, such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, as long as such attachment permits the peptide or protein to retain its antigen binding specificity. The cell-binding molecule/ligands or cell receptor agonists can be Ig-based and non-Igbased protein scaffold molecules. The Ig-Based scaffolds can be selected, but not limited, from Nanobody (a derivative of VHH (camelid Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775—97); Domain antibodies (dAb, a derivative of VH or VL domain) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-90); Bispecific T cell Engager (BiTE, a bispecific diabody) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); Dual Affinity ReTargeting (DART, abispecific diabody) (Moore P. A. P, et al. 2011, Blood 117(17), 4542—51); Tetravalent tandem antibodies (TandAb, a dimerized bispecific diabody) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341). The Non-Ig scaffolds can be selected, but not limited, from Anticalin (a derivative of Lipocalins) (Skerra A. 2008, FEBS J., 275(11): 2677-83; Beste G, et al, 1999 Proc. Nat. Acad. USA. 96(5):1898-903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2): 337-50; Skerra, A. 2007, Curr Opin Biotechnol. 18(4): 295-304; Skerra, A. 2008, FEBS J. 275(11):2677-83); Adnectins (10th FN3 (Fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4):1141-51; Batori V, 2002, Protein Eng. 15(12): 1015-20; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2): 363—71; Hackel, B. J, 2010, Protein Eng. Des. Sei. 23(4): 211-19); Designed Ankyrin Repeat Proteins (DARPins) (a derivative of ankrin repeat (AR) proteins) (Boersma, Y.L, et al, 2011 Curr Opin Biotechnol. 22(6): 849-57), e.g. DARPin C9, DARPin Ec4 and DARPin E69_LZ3JE01 (Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 2674-83; Patricia M-K. M., et al, Clin Cancer Res. 2011; 17(1):100-10; Boersma Y. L, et al, 2011 J. Biol. Chern. 286(48), 41273—85); Avimers (a domain A/low-density lipoprotein (LDL) receptor) (Boersma Y. 77 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12):1556-61). Examples of the small molecule structures of the cell-binding molecules/ligands or cell receptor agonists of the patent application are the following: LB01 (Folate), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (Somatostatin), LB06 (Somatostatin), LB07 (Octreotide, a Somatostatin analog), LB08 (Lanreotide, a Somatostatin analog), LB09 (Vapreotide (Sanvar) , a Somatostatin analog), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (Gastrin releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (cobalamin, vitamin B12 analog), LB17 (cobalamin, vitamin B12 analog), LB18 (for avP3 integrin receptor, cyclic RGD pentapeptide), LB19 (hetero-bivalent peptide ligand for VEGF receptor), LB20 (Neuromedin B), LB21 (bombesin for a G-protein coupled receptor), LB22 (TLR2 for a Toll¬ like receptor,), LB23 (for an androgen receptor), LB24 (Cilengitide/cyclo(-RGDfV-) for an av intergrin receptor, LB23 (Fludrocortisone), LB25 (Rifabutin analog), LB26 (Rifabutin analog), LB27 (Rifabutin analog), LB28 (Fludrocortisone), LB29 (Dexamethasone), LB30 (fluticasone propionate), LB31 (Beclometasone dipropionate), LB32 (Triamcinolone acetonide), LB33 (Prednisone), LB34 (Prednisolone), LB35 (Methylprednisolone), LB36 (Betamethasone), LB37 (Irinotecan analog), LB38 (Crizotinib analog), LB39 (Bortezomib analog), LB40 (Carfilzomib analog), LB41 (Carfilzomib analog), LB42 (Leuprolide analog), LB43 (Triptorelin analog), LB44 (Clindamycin), LB45 (Liraglutide analog), LB46 (Semaglutide analog), LB47 (Retapamulin analog), LB48 (Indibulin analog), LB49 (Vinblastine analog), LB50 (Lixisenatide analog), LB51 (Osimertinib analog), LB52 (a neucleoside analog), LB53 (Erlotinib analog) and LB54 (Lapatinib analog) which are shown in the following structures: HOOC O O W^/^X4- A A/ HOOC N N COOH H H HOOC^N^ tOOH U n 0 °rOH LB01 (Folate conjugate), LB02 (PMSA ligand conjugate), LB03 (PMSA ligand conjugate), 78 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 LB08 (Lanreotide, a Somatostatin analog), 79 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 analog). LB12 (Gastrin releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), 80 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), 81 GA 03105541 2021-01-04 WO 2020/006722 LB18 (for avp3 intcgrin receptor, cyclic RGD pentapeptide), PCT/CN2018/094586 Ac LB19 (hetero-bivalent peptide ligand conjugate for VEGF receptor), G-N-L-W-A-T-G-H-F-M-NH2 LB20 (Neuromedin B), H CL Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-GIy-His-Leu-Met— N—£ LB21 (bombesin conjugate for a G-protein coupled receptor), 10 LB24 (Cilengitide/cyclo(-RGDfV-) conjugate for an av intergrin receptor) 82 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 5 83 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 84 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 LB38 (Crizotinib analog). LB39 (Bortezomib analog), wherein Y5, is N, CH, C(C1), C(CH3), or C(COORi); Ri is H, Ci-C6 Alkyl, C3-C8 Ar; 85 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 LB44 (Clindamycin), <5 -HN-H-A-Q-G-T-F-T-S-D H^j-K-A-A-Q-G-Q-L-Y-S-S-V Q-F-I-A-W-L-V-R-G-R-G-COOH LB45 (Liraglutide analog), -H-AIB-Q-G-T-F-T-S-D^ SjV>-a-a-q-g-Q-l-y-s-s-v Q-F-I-A-W-L-V-R-G-R-G-COOH LB46 (Semaglutide analog), 86 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 HOOC-H-G-E-G-T-F-T-S-D-L-S-K-Q^l g-g-n-k-l-w-e-i-f-l-r-v-a-e-e-f; ^-S-S-G-A-P-P-S-K-K-K-K-K-^ ^s/^N' H LB50 (Lixisenatide analog), wherein“^>-rux” is the site to link the side chain linker of the present patent; X4,and Yi are independently O, NH, NHNH, NR,, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(Ri), N(R|)C(O)N(R,). CH2, C(O)NHNHC(O) andC(O)NR,: X, is H, CH2, OH, O, C(O), C(O)NH, C(O)N(Ri), Ru NHRi, NR,, C(O)Ri or C(O)O; X5 is H, CH3, F, or Cl; Mi and M2 are independently H, Na, K, Ca, Mg, NH4, N(RiR2R3 R4); Ri. R2, R3and R4 are defined in Formula (I); 87 5 10 15 20 25 30 Any one of several different reactive groups on a cell binding agent, preferably on an antibody, can be a conjugation site, such as g-amino groups in lysine residues, pendant carbohydrate moietics, carboxylic acid groups, disulfide groups, and thiol groups. For reviews on antibody reactive groups suitable for conjugation, see, e.g., Hermanson, G.T. (2008). Bioconjugate Techniques, Academic Press; Garnett, Adv. Drug Delivery Rev. 53 (2001), 171- 216 and Dubowchik and Walker, Pharmacology & Therapeutics 83 (1999), 67-123. The cytotoxic agents, cross-linked PBD dimers of this invention can be directly conjugated (linked) to a cell binding agent, or via a bifunctional linker or a cross-linking agent to a cell binding agent The bifunctional linker possess two reactive groups; one of which is capable of reacting with a cell binding agent while the other one reacts with one or more molecules of cytotoxic agent of the invention. The bifunctional crosslinkers are well known in the art (see, for example, U.S.Pat. No. 5,208,020; Isalm and Dent in Bioconjugation chapter 5, p 218-363, Groves Dictionaries Inc. New York, 1999). Examples of bifunctional linker are: N-succinimidyl- 3-(2-pyridyldithio)-propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), Nsuccinimidyl-4-(2-pyridyl-dithio)pentanoate (SPP), N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB), 2-iminothiolane, N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), Nsuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SNPP), N-sulfosuccinimidyl-4-(5-nitro-2- pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)- pentanoate (SMNP), N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SSNPP), 4- succinimidyl-oxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-sulfosuccinimidyl- 4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SSMNP); N-succinimidyl-4-methyl-4-(2- pyridyldithio)pentanoate (SMPDP), N-succinimidyl-4-(5-N,N-dimethyl-carboxamido-2- pyridyldithio) butyrate (SCPB), N-sulfosuccinimidyl-4-(5-N,N-dimethyl-carboxamido-2- pyridyldithio) butyrate (SSCPB), N-succinimidyl-4,4-dimethyl-4-(2-pyridyl-dithio)pentanoate (SDMPDP), succinimidyl-4-(N-maleimidomcthyl)cyclohcxane-l-carboxylate (SMCQ, Nsuccinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), , bis-maleimidopolyethyleneglycol (BMPEG), BM(PEG)i~20,N-(P-maleimidopropyloxy)-succinimide ester (BMPS), iminothiolane (IT), dimethyl adipimidate HC1or derivatives of imidoesters, active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene), gamma-maleimidobutyric acid Nsuccinimidyl ester (GMBS), E-nialeimido-caproic acid N-hydroxysuccinimide ester (EMCS), 5- malcnnidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l- 88 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 carboxy-(6-amidocaproate) (a "long chain" analog of SMCC (LC-SMCC)). m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromo-acetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), Nsuccinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(beta-maleimidopropionamido)-hexanoate (SMPH), succinimidyl-(4-vinyl-sulfonyl)benzoate (SVSB), dithiobismaleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3- dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate (sulfo-SMCC), sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobcnzoyl-Nhydroxysulfosuccinimide ester (sulfo-MBS), N-(gamma-maleimidobutryloxy)- sulfosuccinimdeester (sulfo-GMBS), N-(epsilon-maleimidocaproyloxy)-sulfo-succimido ester (sulfo-EMCS), N-(kappa-maleimidoundecanoyloxy)-sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB ); or the commercially available linkers (such as from Thermo Scientific’s Pierce: Imidoester Crosslinkers: DMA (Dimethyl adipimidate-2 HCl), DMP (Dimethyl pimelimidate*2 HCl), DMS (Dimethyl Suberimidate«2 HCl), DTBP (Dimethyl 3,3'-dithiobispropionimidate«2 HC1); NHS-ester Crosslinkers-Amine Reactive: BS(PEG)s (Bis(succinimidyl) penta(ethylence glycol), BS(PEG)g(Bis(succinimidyl) nona(ethylence glycol), BS3 (Bis[sulfosuccinimidyl] suberate), BSOCOES (Bis[2- (succinimidooxycarbonyloxy)-cthyljsulfone), DSG (Disuccinimidyl glutarate), DSP (Dithiobis[succinimidyl propionate]), DSS (Disuccinimidyl suberate), DST (Disuccinimidyl tartarate), DTSSP (3,3'-Dithiobis[sulfo-succinimidylpropionate]), EGS (Ethylene glycol bis[succinimidylsuccinate]), Sulfo-EGS (Ethylene glycol bis[sulfo-succinimidylsuccinate]), TSAT (Tris-succinimidyl aminotriacetate), DFDNB (l,5-Difluoro-2,4-dinitrobenzene); Amineto-Sulfhydryl Crosslinkers: Sulfo-SIAB (Sulfosuccinimidyl (4-iodoacetyl)aminobenzoate), SIAB (Succinimidyl (4-iodoacetyl)aminobenzoate), SBAP (Succinimidyl 3-(bromoacetamido)- propionate), SIA (Succinimidyl iodoacetate), Sulfo-SMCC (Sulfosuccinimidyl-4-(Nmaleimidomethyl)-cyclohexane-l-carboxylate), SM(PEG)n (NHS-PEG-Maleimide Crosslinkers: Succinimidyl-([N-maleimidopropionamido])-#ethyleneglycol) ester, # =1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), LC-SMCC (Succinimidyl 4-(Nmaleimidomethyl) cyclohexane-l-carboxy-(6-amidocaproate)), Sulfo-EMCS (N-epsilonMaleimidocaproyl-oxysulfosuccinimide ester), EMCS (N-epsilon-Malemidocaproyloxysuccinimide ester), Sulfo-GMBS (N-gamma-Maleimidobutyryl-oxysulfosuccinimide ester), GMBS (N-gamma-Maleimidobutyryl-oxysuccinimide ester), Sulfo-KMUS (N-kappamaleimidoundecanoyl-oxysulfosuccinimide ester), Sulfo-MBS (m-maleimido-benzoyl-N- 89 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 hydroxysulfosuccinimidc ester), MBS (m-Malcimidobcnzoyl-N-hydroxysuccinimidc ester), Sulfo-SMPB ((Sulfo-succinimidyl 4-(p-maleiimidophenyl)butyrate), SMPB(Succinimidyl 4-(pmaleimido-phenyl)butyrate), AMAS N-(a-Maleimidoacetoxy) succinimide ester), BMPS (Nbeta-Maleimidopropyloxy-succinimide ester), SMPH (Succinimidyl 6-[(beta-maleimidopropionamido)hexanoate]), PEG12-SPDP (2-Pyridyldithiol-tetraoxaoctatriacontane-Nhydroxysuccinimide), PEG4-SPDP (2-Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide), Sulfo-LC-SPDP (Sulfosuccinimidyl 6-[3'-(2-pyridyldithio)propionamidoJhexanoate), LC-SPDP (Succinimidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate), SMPT (4- Succinimidyl-oxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene); Carboxyl-to-Amine Crosslinkers: DCC (Dicyclohexylcarbodiimide), EDC (l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide); Photoreactive Crosslinkers: ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide), NHS-Diazirine (SDA) Crosslinkers: SDA (NHS-Diazirine)(Succinimidyl 4,4'- azipentanoate), LC-SDA (NHS-LC-Diazirine) (Succinimidyl 6-(4,4'-azipentanamido)- hexanoate), SDAD (NHS-SS-Diazirine) (Succinimidyl 2-([4,4'-azipentanamido]ethyl)-l,3'- dithiopropionate), Sulfo-SDA (Sulfo-NHS-Diazirine) (Sulfosuccinimidyl 4,4’-azipentanoate), Sulfo-LC-SDA (Sulfo-NHS-LC-Diazirine) (Sulfosuccinimidyl 6-(4,4'-azipentanamido)- hexanoate), Sulfo-SDAD (Sulfo-NHS-SS-Diazirine)(Sulfosuccinimidyl 2-([4,4'- azipentanamido]ethyl)-l,3'-dithiopropionate), Sulfo-SANPAH (Sulfosuccinimidyl 6-(4'-azido-2'- nitrophenylamino)-hexanoate), SPB (Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate); Sulfhydrylto-Carbohydrate Crosslinkers: BMPH (N-beta-Maleimidopropionic acid hydrazide-TFA), EMCH (N-epsilon-Maleimidocaproic acid hydrazide-TFA), KMUH (N-kappa-malcimidoundecanoic acid hydrazide-TFA), MPBH (4-(4-N-Maleimidophenyl)butyric acid hydrazideHC1), PDPH (3-(2-Pyridyldithio)propionyl hydrazide); Sulfhydryl-to-Hydroxyl Crosslinkers: PMPI (p-Maleimidophenyl isocyanate); Sulfhydryl-to-Sulfhydryl Crosslinkers: BM(PEG)2 (1,8- Bismaleimido-diethyleneglycol), BM(PEG)3 (1,11-Bismaleimidotriethyleneglycol), BMB (1,4- Bismaleimidobutane), BMDB (l,4-Bismaleimidyl-2,3-dihydroxybutane), BMH (Bismaleimidohexane), BMOE (Bismaleimidoethane), DTME (Dithiobismaleimido-ethane), TMEA (Tris(2- maleimidoethyl)amine) and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The bis-maleimide or bis-2-pyridyldithiol reagents allow the attachment of the thiol group of a thiol-containing cell binding agent (such as antibody) to a thiol-containing drug moiety, label, or linker intermediate, in a sequential or concurrent fashion. Other functional groups besides maleimide and pyridyldithiol, which are reactive with a thiol group of a cell binding agent, drug moiety, label, or linker intermediate include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. 90 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 In additional embodiments, the linker may be composed of one or more linker components. The exemplary linker components are: 1. The self-immolative linker components: O wherein the (*) atom is the point of attachment of additional spacer or releasable linker units, or the cytotoxic agent, and/or the binding molecule (CBA); X1, Y1, Z2 and Z3 are independently NH, or O, or S; Z1 is H, or NH, or O or S independently, v is 0 or 1; Q1 is independently H, OH, Ci-Ce alkyl, (OCH2CH2)n F, Cl, Br, I, ORi, or SR!, NR^ N=NRX, N^NR^^ SORjR2, SO2Ri, SO3Ri, OSOd^, PRdC. POR1R2, PO2RiR2, OPO(OR,)(OR2). or OCH2PO(ORI(OR2) wherein Ri and R2 are described above, preferabably independently selected from H, Ci~Cg of alkyl; C2~Cg of alkenyl, alkynyl, heteroalkyl; Ca-Cg of aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl; or pharmaceutical cation salts 2. The examples of non-self-immolative linker components: (Œ2)nCO(OCH2CH2)rOCH3 (CH2)nCON(CH2CH2O)rCOŒ3 *(CH2CH2O)r* 9 9 91 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 92 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein the (*) atom is the point of attachment of additional spacer or releaseable linkers, the cytotoxic agents, and/or the binding molecules; X1, Y1. Q1, RI, R’, R” are defined above; r is 1-20, m and n are 1-6. 3. Exemplary linker components may include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("alaphe" or "af"), p-aminobenzyloxy-carbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("SMCC"), NSuccinimidyl (4-iodo-acetyl)amino-benzoate ("SIAB"), ethyleneoxy (—CH2CH2O—) as one or more repeating units ("EO" or "PEO"). Additional linker components are known in the art and some are described through this patent application. In additional embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (VC or val-cit), alanine-phenylalanine (af or alaphe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycineglycine (gly-gly-gly). Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. In the cell-binding agent - drug conjugates of the invention, cell-binding agent (Q) is conjugated to one or more drug moieties (Drug, or PBD derivatives), e.g. about 1 to about 20 drug moieties per cell-binding agent, through a bifunctional linker (L). The conjugate of Formula (I), (II), (III), and (IV) may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of a cell-binding agent with a bivalent linker reagent, to form Q-L, via a covalent bond, followed by reaction with a drug moiety Drug; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form Drug-L, via a covalent bond, followed by reaction with the nucleophilic group of a cell-binding agent. To synthesize the conjugates of Formula (I), (II), (III) or/and (IV) in general, a function group E3or/and E3’ on Formula (V), (VI), (VII) and (VIII) reacts one, two or more residues of a cell binding molecule at 0-60°C, pH 5-9.5 aqueous media with or without addition of 0-30% of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol, following by dialysis or chromatographic purification to form a conjugate compound of Formula (I), (II), (III) or/and 93 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV). Some of the residue (reacting group for conjugation) of the cell-binding molecule can be obtained through protein engineering. The thiol or amine groups on a cell-binding agents, such as an antibody, are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker reagents and drug-linker intermediates including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups; and (iv) disulfides, including pyridyl disulfides, via sulfide exchange. Nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents. Nucleophilic groups on antibodies or proteins can react to electrophilic groups on a function linker following by reaction with a cytotoxic agent, or directly react to a linkercytotoxic agent moiety to form covalent bond conjugate of a cell binding agent-cytotoxic agent. Nucleophilic groups on antibodies or proteins include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker-cytotoxic agent moieties including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges which may be made reactive by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethyl¬ phosphine (TCEP) (Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass), dithioerythritol (DTE), L-glutathione (GSH), 2-mercaptoethylamine (p-MEA), or/and beta mercaptoethanol (p-ME, 2-ME). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Alternatively, sulfhydryl groups can be introduced into antibodies through modification of lysine residues, e.g., by reacting lysine residues with 2-iminothiolane (Traut's reagent), resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing variant antibodies comprising one or more non-native cysteine amino acid residues). Thus free thiol on the cell binding agents can be conjugated to the thiol-reactive groups, such as, a maleimide, an iodoacetamide, a pyridyl disulfide, or other thiol-reactive groups on the cytotoxic agents, or linker-cytotoxic agent intermediates of the invention. Some 94 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 unconjugated free thiols on the antibodies can be reoxidized to reform interchain and intrachain disulfide bonds. Antibody-drug conjugates of the invention may also be produced by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one embodiment, an antibody is modified to introduce electrophilic moieties that are capable of reacting with nucleophilic substituents on the linker reagent or drug. In another embodiment, the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing Nterminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chern. 3:138-146; U.S. Pat. No. 5,362,852). Such an aldehyde can be reacted with a drug moiety or linker nucleophile. Examples of these kinds of two-step conjugations are depicted below: O _c SX E S-s r7-Drug X™ R'^R" R R H,N-CBA - CBA 1-20 O HjN-CBA I.N-R7 N^c]ja Drug-S^o g 7 r Drug—SH _ yE3 Drug “^E O Q O n Q O o Ar o H Drug Drug 95 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Br O s O E Drug-SH D™/ ¥^-»7 zE H2N-CBA s &Y\^R7 JJ% 'CBA5 o °2S^X^CBA « Br^ O o ° o ^«rug-s g o ° o ,,'^< E^ Drug ^/ E, j W Drug-SH H2N—CBA mug wherein E includes, but is not limited to, such as hydroxysuccinimidyl esters (NHS, SulfoNHS, etc), 4-nitrophcnyl esters, pentafluorophenyl esters, tetrafluorophenyl (includes sulfo¬ tetrafluorophenyl) esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. R’ and R” are independently H or CH3, orCaHs; J is F, Cl, Br, I, tosylate (TsO), mesylate (MsO), nitrophenol, dinitrophenol, or pentaflourophenol. It is to be understood that where more than one nucleophilic group on the cell binding agents, such as an antibody, reacts with a drug-linker intermediate or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of the cell binding agent-cytoxic agent conjugates with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography. In certain embodiments, a homogeneous conjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography. In the conjugation, The loading (drug/antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of lysine or cysteine residues is modified for control of the number and/or position of linker-drug attachments (such as thioMab or thioFab). The synthetic conjugate may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, and ion exchange or by dialysis. In some cases, a small molecule as a cell-binding agent (e.g. folic acid, melanocyte stimulating hormone, EGF etc) conjugated with a small molecular drugs can be purified by chromatography such as by HPLC, medium pressure column chromatography or ion exchange chromatography. 96 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 The aqueous solutions for the modification of cell-binding agents are buffered between pH 4 and 9, preferably between 6.0 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges. Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, hydroxypropyl-P-cyclodextrin, polyethylene glycols, sucrose and salts, for examples, NaCl and KC1. After the addition of the drug-linker of Formula (V), (VI), (VH) or Formula (VIII) into the solution containing the reduced cell-binding molecules, the reaction mixture is incubated at a temperature of from 4 °C to 55 °C, preferably at 15°C - ambient temperature. The progress of the reaction can be monitored by measuring the decrease in the absorption at a certain UV wavelength, such as at 252 nm, or increase in the absorption at a certain UV wavelength, such as 280 nm, or the other appropriate wavelength. After the reaction is complete, isolation of the modified cell-binding agent can be performed in a routine way, using for example a gel filtration chromatography, an ion exchange chromatography, an adsorptive chromatography or column chromatography over silica gel or alumina, crystallization, preparatory thin layer chromatography, ion exchange chromatography, or HPLC. The extent of modification can be assessed by measuring the absorbance of the nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxylamidopyridine dithione and dicarboxyl-amidopyridinc dithione group released via UV spectra. For the conjugation without a chromophore group, the modification or conjugation reaction can be monitored by LCMS, preferably by HPLC-MS/MS, UPLC-QTOF mass spectrometry, or Capilary electrophoresis -mass spectrometry (CE-MS). The side chain cross-linkers described herein have diverse functional groups that can react with any cell-binding molecules, particularly a modified cell¬ binding molecule that possess a suitable substituent. For examples, the modified cell-binding molecules bearing an amino or hydroxyl substituent can react with drugs bearing an N-hydroxysuccinimide (NHS) ester, the modified cell-binding molecules bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group. Additionally, the modified cell-binding molecules bearing a carbonyl (ketone or aldehyde) substituent either through protein engineering, enzymatical reaction or chemical modification can react with drugs bearing a hydrazide or an alkoxyamine. One skilled in the art can readily determine which modified drug-linker to be used based on the known reactivity of the available functional group on the modified cell-binding molecules. Other further examplary methods for preparing ADC are described in Figures 1-32 and examples in the description of the patent. 97 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 APPLICATION OF CONJUGATES OF CELL-BINDING AGENT WITH CROSS¬ LINKED PBD DIMERS. The cell-binding agent-cross-linked PBD dimer conjugate, preferably antibody- cross¬ linked PBD dimer conjugates (PBD dimer ADC) of the present invention may be used to treat various diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia and lymphoid malignancies. Others include neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic, including autoimmune, disorders. In specific embodiment, the conjugates of the invention are used in accordance with the compositions and methods of the invention for the treatment of cancers. The cancers include, but are not limited. Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependymoma, Medulloblastoma, Supratentorial Primitive Neuroectodermal and Pineal Tumors, Visual Pathway and Hypothalamic Glioma), Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell cancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Lymphoma (AIDS-Related, Central Nervous System, Cutaneous T-Cell, Hodgkin's Disease, Non-Hodgkin’s Disease, Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome, Myeloproliferative Disorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter (Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, Skin Cancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma, Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular 98 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Cancer, Wilms' Tumor In another specific embodiment, the compounds and the conjugates of the invention are used in accordance with the compositions and methods of the invention for the treatment or prevention of an autoimmune disease. The autoimmune diseases include, but are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease, Agammaglobulinemia, Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase syndrome. Arthritis, Atopic allergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger's disease, Bickerstaffs encephalitis, Blau syndrome, Bullous Pemphigoid, Castleman's disease, Chagas disease, Chronic Fatigue Immune Dysfunction Syndrome, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis, Chronic lyme disease, Chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Cranial arteritis, CREST syndrome, Crohns Disease (a type of idiopathic inflammatory bowel diseases), Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressier's syndrome, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto’s thyroiditis, Haemolytic anaemia, Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa, Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenic purpura), IgA nephropathy (Also Berger's disease), Inclusion body myositis, Inflammatory demyelinating polyneuopathy, Interstitial cystitis, Irritable Bowel Syndrome , Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eaton 99 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease (Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, Ménière's disease, Microscopic polyangiitis, MillerFisher syndrome, Mixed Connective Tissue Disease, Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, Multiple Myeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatrie Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Tumer syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome, Schnitzler syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome, Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathic inflammatory bowel diseases), Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome In another specific embodiment, a binding molecule used for the conjugate for the treatment or prevention of an autoimmune disease includes, but are not limited to, anti-elastin antibody; Abys against epithelial cells antibody; Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70; Anti-Jo; Anti-U.sub.IRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody, Anti-ganglioside antibody; AntiDesmogein 3 antibody; Anti-p62 antibody; Anti-spl00 antibody; Anti-Mitochondrial(M2) antibody; Rheumatoid factor antibody; Anti-MCV antibody; Anti-topoisomerase antibody; Anti¬ neutrophil cytoplasmic(cANCA) antibody. 100 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 In certain preferred embodiments, the binding molecule for the conjugate in the present invention, can bind to either a receptor or a receptor complex expressed on an activated lymphocyte which is associated with an autoimmune disease. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28,CD30, CD33, CD37, CD38, CD56, CD79, CD79b, CD90, CD152/CTLA-4, PD-1, or ICOS), a TNF receptor superfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-RI, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin (C-type, S-type, or Itype), or a complement control protein. In another specific embodiment, useful binding ligands that are immunospecific for a viral or a microbial antigen are humanized or human monoclonal antibodies. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein (e.g. HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuramimidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term "microbial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response. Examples of antibodies available 1for the viral or microbial infection include, but are not limited to, Palivizumab which is a humanized antirespiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542 which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir which is a human antibody for the treatment of hepatitis B virus; PROTVIR which is a humanized IgG.sub.l antibody for the treatment of cytomegalovirus; and anti-LPS antibodies. The binding molecules-cytotoxic agent conjugates of this invention can be used in the treatment of infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and 101 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever, Common cold (Acute viral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires’ disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, Measles, Melioidosis (Whitmore’s disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus 102 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 infection. Rickettsial infection, Rickettsialpox, Rift Valley fever, Rocky mountain spotted fever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber’s itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete’s foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia, West Nile Fever, White piedra (Tinea blanca), Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis. The binding molecules, more preferred antibodies described in this patent that are against pathogenic strains include, but are not limit, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitidcs, Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter genus, usually Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense; multiple parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4)- Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Human herpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, 103 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Fusobacterium genus, Clostridium pcrfringcns; other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza viruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus, Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous species of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii, Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformis and Spirillum minus, Respiratory syncytial virus, Rhinosporidium seeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea wemeckii, Trichophyton genus, Malassczia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris 104 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 trichiura, Mycobacterium tuberculosis, Francisclla tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus. Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms). Other antibodies as a binding ligand in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridac. Adenoviridae, Papovaviridae, Enteroviridae, Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi’s sarcomaassociated herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt's lymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cell cancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [such as Human Immunodeficiency Virus (AIDS)]; Central nervous system virus: [such as, JCV (Progressive multifocal leukoencephalopathy), MeV (Subacute sclerosing panencephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable) (Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviral meningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis, Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)]; Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis)); Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)]; Respiratory system/acute viral nasopharyngitis/viral pneumonia: [Epstein-Barr virus (EBV infection/Infectious mononucleosis), Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome) Orthomyxoviridae: Influenzavirus 105 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 A/B/C (Influcnza/Avian influenza), Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Human respiratory syncytial virus), hMPV]; Digestive system virus [MuV (Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus (Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis C virus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (Hepatitis G virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)]. FORMULATION AND APPLICATION OF THE CONJUGATES. The conjugates of the patent application are formulated to liquid, or suitable to be lyophilized and subsequently be reconstituted to a liquid formulation. A liquid formulation comprising 0.1 g/L ~300 g/L of concentration of a conjugate of the patent application as an active ingredient for delivery to a patient without high levels of antibody aggregation may include one or more polyols (e.g. sugars), a buffering agent with pH 4.5 to 7.5, a surfactant (e.g. polysorbate 20 or 80), an antioxidant (e.g. ascorbic acid and/or methionine), a tonicity agent (e.g. mannitol, sorbitol or NaCl), chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; a preservative (e.g. benzyl alcohol) and/or a free amino acid. In a preferred embodiment, the conjugate of the invention in vivo clinical use will be supplied as solutions or as a lyophilized solid (such as powder) that can be redissolved in sterile water for injection. The conjugate in a liquid formula or in the formulated lyophilized powder may take up 0.01%-99% by weight as major gradient in the formulation. The rest of the formulation is excipients which are comprised of one or more of the following compounds: 0.5%~25% of buffering reagents, 0%~20% of polyols, 0%~2.0% of surfactants, 0%~5% of preservatives, 0%-30% of Amino acids or bulky compounds, 0%~5% of Antioxidants, 0%~0.3% chelating agents. Suitable buffering agents for use in the formulations include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, tromethamine (tris(hydroxymethyl)- aminomethane) hydrochloride, or phosphate buffer. In addition, amino acid components can also be used as buffering agent. Such amino acid component includes without limitation arginine, glycine, glycylglycine, and histidine. The arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride¬ arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-argine succinate, etc. The formulations of 106 5 10 15 20 25 30 35 the buffers have a pH of 4.5 to pH 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salts in the buffer is from about 10 mM to about 500 mM.. A "polyol" that may optionally be included in the formulation is a substance with multiple hydroxyl groups. Polyols can be used as stabilizing excipients and/or isotonicity agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals ftom both physical and chemical degradation pathways. Preferentially excluded co-solvents increase the effective surface tension of solvent at the protein interface whereby the most energetically favorable structural conformations are those with the smallest surface areas. Polyols include sugars (reducing and nonreducing sugars), sugar alcohols and sugar acids. A "reducing sugar" is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a "nonreducing sugar" is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nonreducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol and glycerol. Sugar acids include L-gluconate and its metallic salts thereof. Preferably, a nonreducing sugar: sucrose or trehalose at a concentration of about from 0.01% to 15% is chosen in the formulation, wherein trehalose being preferred over sucrose, because of the solution stability of trehalose. A surfactant optionally in die formulations is selected from polysorbate (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and die like); poloxamer (e.g. poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407 or polyethylene-polypropylene glycol and the like); Triton™; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimcdiylaminc; sodium methyl cocoyl-, or disodium methyl olcyl-tauratc; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and coco ampho glycinate; and the MONAQUAT™ series (e.g. isostearyl ethylimidonium ethosulfate); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc.); etc. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters e.g. polysorbate 20, 40, 60 or 80 (Tween™ 20, 40, 60 or 80). The concentration of a surfactant is range from 0.0001% to about 1.0%. In certain embodiments, the surfactant concentration is from about 0.01% to about 0.1%. In one embodiment, the surfactant concentration is about 0.02%. 107 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 A "preservative" optionally in the formulations is a compound that essentially reduces bacterial action therein. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexaméthonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethyl-ammonium chlorides in which the alkyl groups are long-chain compounds), and benzéthonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The preservative is less than 5% in the formulation. Preferably 0.01% to 1%. In one embodiment, the preservative herein is benzyl alcohol. Suitable free amino acids optionally for use in the formulation, but are not limited to, are arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid. The inclusion of a basic amino acid is preferred i.e. arginine, lysine and/or histidine. If a composition includes histidine then this may act both as a buffering agent and a free amino acid, but when a histidine buffer is used it is typical to include a non-histidine free amino acid e.g. to include histidine buffer and lysine. An amino acid may be present in its Dand/or L-form, but the L-form is typical. The amino acid may be present as any suitable salt e.g. a hydrochloride salt, such as arginine-HCl. The concentration of an amino acid is range from 0.0001% to about 15.0%. Preferably 0.01% to 5%. The formulations can optionally comprise methionine or ascorbic acid as an antioxidant at a concentration of about from 0.01 mg/ml to 5 mg/ml; The formulations can optionally comprise chelating agent, e.g., EDTA, EGTA, etc., at a concentration of about from 0.01 mM to 2 mM. The final formulation may be adjusted to the preferred pH with an adjust agent (e.g. an acid, such as HCl, H2SO4, acetic acid, H3PO4, citric acid, etc., or a base, such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanol amine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc.) and the formulation should be controlled "isotonic" which is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. Other excipients which may be useful in either a liquid or lyophilized formulation of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluoronic F-127, cellulose, cyclodextrin, dextran (10, 40 and/or 70 kD), polydextrose, maltodextrin, ficoll, gelatin, hydroxypropylmeth, sodium phosphate, potassium phosphate, ZnCh, zinc, zinc oxide, sodium citrate, trisodium 108 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohols, or polyalcohols, hydrogenated forms of carbohydrate having a carbonyl group reduced to a primary or secondary hydroxyl group. Other contemplated excipients, which may be utilized in the aqueous pharmaceutical compositions of the patent application include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such sodium and the like. These and additional known pharmaceutical excipients and/or additives suitable for use in the formulations of the invention are known in the art, e.g., as listed in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005). In order to reduce patient pain during the injection of the formulation, a local analgesic agent can be used along with or prior to the injection of the formulation. Commonly used analgesics are: benzyl alcohol (0.01%-l%), Procaine hydrochloride (0.2%~2.0%), lidocaine hydrochloride (0.2%~2.0%), 2-tri-chloromethyl-2-propanol (0.3%~0.5%), tramadol, morphine, morphine sulfate, hydromorphone, oxycodone hydrochloride, dobutamine, gabapentin, cyclobenzaprine, trazodone, clonidine, codeine. The conjugates formulation of the patent application can be prepared as a pre-filled syringe solution, or a freezing-dry powder, or through high-efficient spray drying powder._A pharmaceutical container or a vessel is used to hold the pharmaceutical formulation of the conjugates. The vessel is a vial, bottle, pre-filled syringe, or pre-filled auto-injector syringe. In a further embodiment, the invention provides a method for preparing a formulation comprising the steps of: (a) lyophilizing the formulation comprising the conjugates, excipients, and a buffer system to a powder; and (b) reconstituting the lyophilized mixture of step (a) in a reconstitution medium such that the reconstituted formulation is stable. The formulation of step (a) may further comprise a stabilizer and one or more excipients selected from a group comprising bulking agent, salt, surfactant and preservative as hereinabove described. As reconstitution media several diluted organic acids or water, i.e. sterile water, bacteriostatic water for injection (BWFI) or may be used. The reconstitution medium may be selected from water, i.e. sterile water, bacteriostatic water for injection (BWFI) or the group consisting of acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solution of sodium 109 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 chloride, acidic solution of magnesium chloride and acidic solution of arginine, in an amount from about 10 to about 250 mM. A liquid pharmaceutical formulation of the conjugates of the patent application should exhibit a variety of pre-defined characteristics. One of the major concerns in liquid drug products is stability, as proteins/antibodies tend to form soluble and insoluble aggregates during manufacturing and storage. In addition, various chemical reactions can occur in solution (deamidation, oxidation, clipping, isomerization etc.) leading to an increase in degradation product levels and/or loss of bioactivity. Preferably, a conjugate in either liquid or loyphilizate formulation should exhibit a shelf life of more than 18 months at 25°C. More preferred a conjugate in either liquid or loyphilizate formulation should exhibit a shelf life of more than 24 months at 25°C. Most preferred liquid formulation should exhibit a shelf life of about 24 to 36 months at 2-8° C and the loyphilizate formulation should exhibit a shelf life of about preferably up to 60 months at 2-8° C. Both liquid and loyphilizate formulations should exhibit a shelf life for at least two years at -20° C, or -70° C. In certain embodiments, the formulation is stable following freezing (e. g., -20°C, or -70° C.) and thawing of the formulation, for example following 1, 2 or 3 cycles of freezing and thawing. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of drug/antibody(protein) ratio and aggregate formation (for example using UV, size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy¬ terminal sequence analysis; mass spectrometric analysis, or matrix-assisted laser desorption ionization/timc-of-flight mass spectrometry (MALDI/TOF MS), or HPLC-MS/MS; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS--C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomeriation), clipping/hydrolysis/ fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), Nterminal extension, C-terminal processing, glycosylation differences, etc. A stable conjugate should also "retains its biological activity" in a pharmaceutical formulation, if the biological activity of the conjugate at a given time, e. g. 12 month, within about 20%, preferably about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared as determined in an antigen binding assay, and/or in vitro, cytotoxic assay, for example. 110 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Examples of suitable protocols of conjugate administration are as follows. Conjugates are given daily, weekly, biweekly, triweekly, once every four weeks or monthly for 8~54 weeks as an i.v. bolus. Bolus doses are given in 50 to 1000 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can optionally be added. Dosages will be about 50 pg to 20 mg/kg of body weight per week, i.v. (range of 10 pg to 200 mg/kg per injection). 4~54 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by the skilled clinicians. Examples of in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen. Examples of ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells. For instance, clinical ex vivo treatment to remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplant in order to prevent graft-versus-host disease, can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the conjugate of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37 °C. The exact conditions of concentration and time of incubation (=dose) are readily determined by the skilled clinicians. After incubation the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment. Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite). Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any type including, for example, cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs; melanomas; autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft 111 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 versus host disease; viral infections, such as CMV infection, HIV infection, AIDS, etc.; bacterial infection; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by one skilled in the art. The identification of those subjects who are in need of treatment of herein-described diseases and conditions is well within the ability and knowledge of one skilled in the art. A veterinarian or a physician skilled in the art can readily identify, by the use of clinical tests, physical examination, medical/family history or biological and diagnostic tests, those subjects who are in need of such treatment. A therapeutically effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of subject; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristic of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. The amount of a conjugate which is required to achieve the desired biological effect, will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the diseased state of the patient, the route of administration, all factors which dictate the required dose amounts, delivery and regimen to be administered. In general terms, the conjugates of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 30% w/v conjugates for parenteral administration. Typical dose ranges are from 1 pg/kg to 0.1 g/kg of body weight daily; weekly, biweekly, triweekly, or monthly, a preferred dose range is from 0.01 mg/kg to 20 mg/kg of body weight weekly, biweekly, triweekly, or monthly, an equivalent dose in a human. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the route of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the conjugates by the chosen delivery route, and the speed (bolus or continuous infusion) and schedule of administrations (number of repetitions in a given period of time). The cell binding agent -cytotoxic agent conjugates of the present invention are also capable of being administered in unit dose forms, wherein the term “unit dose” means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, 112 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 remaining as a physically and chemically stable unit dose comprising either the active conjugate itself, or as a pharmaceutically acceptable composition, as described hereinafter. As such, typical total daily dose ranges are from 0.01 to 100 mg/kg of body weight. By way of general guidance, unit doses for humans range from 1 mg to 3000 mg per day, weekly, biweekly, or triweekly. Preferably the unit dose range is from 1 to 500 mg administered one to two times weekly, biweekly, or triweekly, and even more preferably from 10 mg to 500 mg, biweekly, or triweekly. Conjugates provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be prepared for use by I.V. or oral administration, particularly in the form of powders, tablets, simple capsules or soft gel capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, creams, lotions, gels or sprays, or via trans-dennal patches. The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington: The Science and Practice of Pharmacy, 21th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005. Preferred formulations include pharmaceutical compositions in which a compound of the present invention is formulated for oral or parenteral administration. For oral administration, tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings. In addition, the active compounds may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal. Preferred tablets contain lactose, cornstarch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, or talc in any combination. Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, buffered media, and saline. In particular, 113 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene¬ polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Alternative modes of administration include formulations for inhalation, which include such means as dry powder, aerosol, or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for buccal administration include, for example, lozenges or pastilles and may also include a flavored base, such as sucrose or acacia, and other excipients such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit-dose suppositories, with a solid based carrier, such as cocoa butter, and may include a salicylate. Formulations for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanolin, polyethylene glycols, alcohols, or their combinations. Formulations suitable for transdermal administration can be presented as discrete patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. In a specific embodiment, the cell binding agent -cytotoxic agent conjugates of this invention are administered concurrently with the other known or will be known therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other antibody-drug conjugates, resulting in a synergistic effect for effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease. In another specific embodiment, the synergistic drugs or radiation therapy are administered prior or subsequent to administration of a conjugate, in one aspect at least an hour, 12 hours, a day, a week, two weeks, three weeks, a month, in further aspects several months, prior or subsequent to administration of a conjugate of the invention. The synergistic agents are preferably selected from one or several of the following drugs: 1). Chemotherapeutic agents: a). Alkylating agents: such as [Nitrogen mustards: (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, trofosfamide); Nitrosoureas: (carmustine, lomustine); Alkylsulphonates: (busulfan, treosulfan); Triazenes: (dacarbazine); Platinum containing compounds: (carboplatin, cisplatin, oxaliplatin)]; b). Plant Alkaloids: such as [Vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine); Taxoids: (paclitaxel, docetaxol)]; c). DNA Topoisomerase Inhibitors: such as [EpipodophyIlins: (9- 114 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 aminocamptothccin, camptothecin, crisnatol, etoposide, etoposide phosphate, irinotecan, teniposide, topotecan,); Mitomycins: (mitomycin C)J; d). Anti-metabolites: such as {[Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate); IMP dehydrogenase Inhibitors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs: (5-Fluorouracil, doxifluridine, floxuridine, ratitrexed(Tomudex)); Cytosine analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, mercaptopurine, thioguanine)]}; e). Hormonal therapies: such as {Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide, flutamide)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrellin A); Cytokines: (Interferon-alpha, Interferon-gamma, tumor necrosis factor (TNFs), human proteins containing a TNF domain)]}; f). Kinase inhibitors, such as BIBW 2992 (antiEGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab, ispinesib; g). Others: such as gemcitabine, epoxomicins (e. g. carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins (such as l-methyl-4- phenylpyridinium ion), Cell cycle inhibitors (such as staurosporine), Actinomycins (such as Actinomycin D, dactinomycin), Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), Anthracyclines (such as daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDR inhibitors (such as verapamil), Ca2+ATPase inhibitors (such as thapsigargin), vismodegib, Histone deacetylase inhibitors (Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A) ; Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate. Disulfiram, Salinosporamide A. More detail lists of known and will be known anti-cancer drugs that can be used as a combination therapy (a synergistic effect) with the compounds and conjugates of the invention can be seen in National Cancer Institute (US) website (www.cancer.gov; www.cancer.gov/cancertopics/druginfo/alphalist), American Cancer Society (www.cancer.org/treatment/index) and Cancer Research UK (www.cancerrearchuk.org; (www.cancerresearchuk.org/cancer-help/about-cancer/treatment/cancer-drugs/) 115 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 2). An anti-autoimmune disease agent includes, but is not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus. 3). An anti-infectious disease agent includes, but is not limited to, a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol, florfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e). Cephems: carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefjpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f). Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g. tigecycline; g). p-Lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j). Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketoIide (telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin;1). Monobactams: aztreonam, tigemonam; m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cioxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam), mezlocillin, 116 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 meticillin, nafcillin. oxacillin, pcnamccillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin, colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q). Streptogramins: pristinamycin, quinupristin/dalfopristin); r). Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimcthoprim-sulfamcthoxazolc (co-trimoxazole); s). Steroid antibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g. tigecycline); u). Other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin; 4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturation inhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e). Nucleosides &_nucleotides: abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3’-fluoro¬ substituted 2’, 3’-dideoxynucleoside analogues (e.g. 3’-fluoro-2’,3’-dideoxythymidine (FLT) and 3’-fluoro-2’,3’-dideoxyguanosine (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. /M-thymidine and >8-1-2’-deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h). Other types of anti-virus drugs: 117 5 10 15 20 25 30 35 abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscamet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib. 5). Other immunolheraphy drugs: e.g. imiquimod, interferons (e.g. , granulocyte colony-stimulating factors, cytokines. Interleukins (IL-1 ~ IL-35), antibodies (e. g. trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab), Protein-bound drugs (e.g., Abraxanc), an antibody conjugated with drugs selected from calicheamicin derivative, of maytansine derivatives (DM1 and DM4), CC- 1065 and duocarmycin minor groove binders, potent Taxol™ derivatives, doxorubicin, auristatin antimitotic drugs (e. g. Trastuzumab-DMl, Inotuzumab ozogamicin, Brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab merlansine, AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, milatuzumab-doxorubicin, SGN-75 (anti-CD70), Anti-CD22-MCC-DMl, IMGN853, AntiCD22-MMAE, Anti-CD22-MMAF, Anti-CD22- calicheamicin. In a further embodiment,die synergistic agents are selected from one or several of the following drugs: Abatacept, Abiraterone acetate, Abraxane, Acetaminophen/hydrocodone, Acalabrutinib, aducanumab, Adalimumab, ADXS31-142, ADXS-HER2, afatinib dimaleate, aldesleukin, alectinib, alemtuzumab, Alitretinoin, ado-trastuzumab emtansine, Amphetamine/ dextroamphetamine, anastrozole, Aripiprazole, anthracyclines, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin, Avelumab, Axicabtagene ciloleucel, axitinib, belinostat, BCG Live, Bevacizumab, bexarotene, blinatumomab, Bortezomib, bosutinib, brentuximab vedotin, brigatinib, Budesonide, Budesonide/formoterol, Buprenorphine, Cabazitaxel, Cabozantinib, capmatinib, Capecitabine, carfilzomib, chimeric antigen receptor-engineered T (CAR-T) cells, Celecoxib, ceritinib. Cetuximab, Chidamide, Ciclosporin, Cinacalcet, crizotinib, Cobimetinib, Cosentyx, crizotinib, CTL019, Dabigatran, dabrafenib, dacarbazine, daclizumab, dacomotinib, daptomycin, Daratumumab, Darbepoetin alfa, Darunavir, dasatinib, denileukin diftitox, Denosumab, Depakote, Dexlansoprazole, Dcxmcthylphcnidatc, Dexamethasone, DigniCap Cooling System, Dinutuximab, Doxycycline, Duloxctinc, Duvelisib, durvalumab, elotuzumab, Emtricitabine/Rilpivirine/Tenofovir, disoproxil fumarate, Emtricitbine/tenofovir/efavirenz, Enoxaparin, ensartinib, Enzalutamide, Epoetin alfa, erlotinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, exemestane, everolimus, exenatide ER, Ezetimibe, Ezetimibe/simvastatin, Fenofibrate, Filgrastim, fmgolimod, Fluticasone propionate, Fluticasone/salmeterol, fulvestrant, gazyva, gefitinib, Glatiramer, Goserelin acetate, Icotinib, Imatinib, Ibritumomab tiuxetan, ibrutinib, idelalisib, ifosfamide, Infliximab, imiquimod, ImmuCyst, Immuno BCG, iniparib, Insulin aspart, Insulin detemir, Insulin glargine, Insulin 118 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 lispro, Interferon alfa, Interferon alfa-lb, Interferon alfa-2a, Interferon alfa-2b, Interferon beta, Interferon beta la, Interferon beta lb, Interferon gamma-la, lapatinib, Ipilimumab, Ipratropium bromide/salbutamol, Ixazomib, Kanuma, Lanreotide acetate, lenalidomide, lenaliomide, lenvatinib mesylate, letrozole, Levothyroxine, Levothyroxine, Lidocaine, Linezolid, Liraglutide, Lisdexamfetamine, LN-144, lorlatinib, Memantine, Methylphenidate, Metoprolol, Mekinist, mericitabine/Rilpivirine/ Tenofovir, Modafinil, Mometasone, Mycidac-C, Necitumumab, neratinib, Nilotinib, niraparib, Nivolumab, ofatumumab, obinutuzumab, olaparib, Olmesartan, Olmesartan/ hydrochlorothiazide, Omalizumab, Omega-3 fatty acid ethyl esters, Oncorine, Oseltamivir, Osimertinib, Oxycodone, palbociclib, Palivizumab, panitumumab, panobinostat, pazopanib, pembrolizumab, PD-1 antibody, PD-L1 antibody, Pemetrexed, pertuzumab, Pneumococcal conjugate vaccine, pomalidomide, Pregabalin, ProscaVax, Propranolol, Quetiapine, Rabeprazole, radium 223 chloride, Raloxifene, Raltegravir, ramucirumab, Ranibizumab, regorafenib, Rituximab, Rivaroxaban, romidepsin, Rosuvastatin, ruxolitinib phosphate, Salbutamol, savolitinib, semaglutide, Sevelamer, Sildenafil, siltuximab, Sipuleucel-T, Sitagliptin, Sitagliptin/metformin, Solifenacin, solanezumab, Sonidegib, Sorafenib, Sunitinib, tacrolimus, tacrimus, Tadalafil, tamoxifen, Tafinlar, Talimogene laherparepvec, talazoparib, Telaprevir, talazoparib, Temozolomide, temsirolimus, Tenofovir/emtricitabine, tenofovir disoproxil fumarate, Testosterone gel, Thalidomide, TICE BCG, Tiotropium bromide, Tisagenlecleucel, toremifene, trametinib, Trastuzumab, Trabectedin (ecteinascidin 743), trametinib, tremelimumab, Trifluridine/tipiracil, Tretinoin, Uro-BCG, Ustekinumab, Valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, ziv-aflibercept, Zostavax, and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients thereof, or a combination above thereof. The invention is further illustrated but not restricted by the description in the following examples. EXAMPLES The invention is further described in the following examples, which are not intended to limit the scope of the invention. Cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ), or The Shanghai Cell Culture Institute of Chinese Acadmy of Science, unless otherwise specified. Cell culture reagents were obtained from Invitrogen Corp., unless otherwise specified. All anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All other reagents and solvents were purchased as the highest grade available and 119 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 used without further purification. The preparative HPLC separations were performed with Varain PreStar HPLC. NMR spectra were recorded on Bruker 500 MHz Instrument. Chemical shifts (delta) are reported in parts per million (ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J) are reported in Hz. The mass spectral data were acquired on a Waters Xevo QTOF mass spectrum equipped with Waters Acquity UPLC separations module and Acquity TUV detector. Example 1. Synthesis of di-tert-butyl l,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-l,2- dicarboxylate. Boe Boc I I O To di-tert-butyl hydrazine-1,2-dicarboxylate (8.01 g, 34,4 mmol) in DMF (150 ml) was added NaH (60% in oil, 2.76 g, 68.8 mmol). After stirred at RT for 30 min, tert-butyl 2- bromoacetate (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, quenched with addition of methanol (3 ml), concentrated, diluted with EtOAc (100 ml) and water (100 ml), separated, and the aqueous layer was extracted with EtOAc (2 x 50 ml). The organic layers were combined, dried over MgSO4, filtered, evaporated, and purified by SiO2 column chromatography (EtOAc/Hexane 1:5 to 1:3) to afforded the title compound (12.98 g, 82% yield) as a colorless oil. MS ESI m/z calcd for C22H41N2O8 [M+H]+ 461.28, found 461.40. Example 2. Synthesis of 2,2’-(hydrazine-l,2-diyl)diacetic acid. Di-tert-butyl l,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) in 1,4-dioxane (40 ml) was added HC1 (12 M, 10 ml). The mixture was stirred for 30 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to afford the crude title product for the next step without further production (2.15 g, 103% yield, -93% pure). MS ESI m/z calcd for C4H9N2O4 [M+H]+ 149.05, found 149.40. Example 3. Synthesis of 2,2’-(l,2-bis((benzyloxy)carbonyl)hydrazine-l,2-diyl)diacetic acid. To a solution of 2,2'-(hydrazine-l,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in the mixture of THF (200 ml) and NaH2PO4 (0.1 M, 250 ml, pH 8.0) was added benzyl carbonochloridate (5.01 g, 29.47 mmol) in 4 portions in 2 h. The mixture was stirred for another 6 h, concentrated and purified on SiO2 column eluted with H2O/CH3CN (1:9) containing 1% formic acid to afford 120 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 the title compound (2.26 g, 73% yield, -95% pure). MS ESI m/z calcd for C20H2iN2O8 [M+H]+ 417.12, found 417.40. Example 4. Synthesis of dibenzyl l,2-bis(2-chloro-2-oxoethyl)hydrazine-l,2- dicarboxylate. 2,2’-(l,2-bis((benzyloxy)carbonyl)hydrazine-l,2-diyl)diacetic acid (350 mg, 0.841 mmol) in dichloroethane (30 ml) was added (COC1)2 (905 mg, 7.13 mmol), followed by addition of 0.030 ml of DMF. After stirred at RT for 2 h, the mixture was diluted with toluene, concentrated and co-evaporated with dichloroethane (2 x 20 ml) and toluene (2x15 ml) to dryness to afford the title crude product (which is not stable) for the next step without further purification (365 mg, 96% yield). MS ESI m/z calcd for C2oHl9Cl2N206 [M+H]+ 453.05, found 453.50. Example 5. Synthesis of di-tert-butyl 1,2-bis(2-(ter/-butoxy)-2-oxoethyl)hydrazine-l,2- dicarboxylate. To a suspension of NaH (0.259 g, 6.48mmol, 3.0 eq.) in anhydrous DMF (2 mL) at room temperature was added di-ter/-butyl hydrazine-1,2-dicarboxylate (0.50 g, 2.16 mmol, 1.0 eq. ) in anhydrous DMF (8 mL) in 10 minutes under nitrogen. The mixture was stirred at room temperature for 10 minutes and then cooled to 0°C. To which tert-butyl 2-bromoacetate(1.4 mL, 8.61mmol, 4.0 eq.) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred overnight. Saturated ammonium chloride solution (100 mL) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic solution was washed with water and brine, dried over anhydrous Na2SO4, concentrated and purified by SiO2 column chromatography (10:1 hexanes/ EtOAc) to give the title compound as a colourless oil (0.94 g, 99.6% yield). ESI MS m/z [M+Na]+ 483.4. Example 6. Synthesis of compound 2,2'-(hydrazine-1,2-diyl)diacetic acid. To a solution of di-te/7-butyl l,2-bis(2-(ter/-butoxy)-2-oxoethyl)hydrazine-1,2- dicarboxylate (0.94 g, 2.04 mmol) in DCM (4 mL) at 0 °C was added TFA (4 mL). The reaction was stirred for 30 minutes and then warmed to room temperature and stirred overnight. The mixture was concentrated, diluted with DCM, and concentrated. This operation was repeated for 121 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 three times to give a white solid. Trituration with DCM and a white solid was collected by filtration (0.232 g, 76.8% yield). ESI MS m/z [M+H]+ 149.2. Example 7. Synthesis of 2,2’-(l,2-bis(2-chloroacetyl)hydrazine - l,2-diyl)diacetic acid. To a solution of 2,2'-(hydrazine-l,2-diyl)diacetic acid (0.232 g, 1.57 mmol, 1.0 eq.) in anhydrous THF (10 mL) at 0 °C was added 2-chloroacetyl chloride (0.38 mL, 4.70 mmol, 3.0 eq.) in 10 minutes. The reaction was warmed to room temperature and stirred overnight and concentrated. The residue was co-evaporated with THF for three times to give a white solid (0.472 g, theoretical yield). ESI MS m/z [M+H]+ 301.1. Example 8. Synthesis of tert-butyl 2,8-dioxo-l,5-oxazocane-5-carboxylate. HOOC Boc2O/THI ^ HOOC^^ P2°5_ HOOC-XzNH E^O/NaOH* HOOC-X^X^ CmClT O To a solution of 3,3’-azanediyldipropanoic acid (10.00 g, 62.08 mmol) in 1.0 M NaOH (300 ml) at 4 °C was added di-tert-butyl dicarbonate (22.10 g, 101.3 mmol) in 200 ml THF in 1 h. After addition, the mixture was kept to stirring for 2 h at 4 °C. The mixture was carefully acidified to pH ~4 with 0.2 M H3PO4, concentrated in vacuo, extracted with CH2C12, dried over Na2SO4, evaporated and purified with flash SiO2 chromatography eluted with AcOH/MeOH/CHaCh (0.01:1:5) to afford 3,3'-((tert-butoxycarbonyl)azanediyl)dipropanoic acid (13.62 g, 84% yield). ESI MS m/z CnHi9NO6 [M+H] +, cacld. 262.27, found 262.40. To a solution of 3,3'-((tert-butoxycarbonyl)azanediyl)dipropanoic acid (8.0 g, 30.6 mmol) in CH2CI2 (500 ml) at 0 °C was added phosphorus pentoxide (8.70 g, 61.30 mmol). The mixture was stirred at 0 °C for 2 h and then r.t. for 1 h, filtered through short SiO2 column, and rinsed the column with EtOAc/CH2C12 (1:6). The filtrate was concentrated and triturated with EtOAc/hexane to afford the title compound (5.64 g, 74% yield). ESI MS m/z C11H17NO5 [M+H] +, cacld. 244.11, found 244.30. Example 9. Synthesis of tert-Butyl 3-((benzyloxy)amino)propanoate. O-benzylhydroxylamine hydrochloride salt (10.0 g, 62.7 mmol) in THF (100 ml) was added Et3N (15 ml) and tert-butyl acrylate (12.1 g, 94.5 mmol). The mixture was refluxed for overnight, concentrated and purified on SiO2 column eluted with EtOAc/Hexane (1:4) to afford 122 5 10 15 20 25 30 the title compound 3 (13.08 g, 83% yield). 1H NMR (CDCh) 7.49-7.25 (m, 5H), 4.75 (s, 2H), 3.20 (t, J=6.4Hz, 2H), 2.54 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI MS m/z+ CuHaiNNaOj (M+Na), cacld. 274.15, found 274.20. Example 10. Synthesis of tert-Butyl 3-(hydroxyamino)propanoate. ,OH tert-Butyl 3-((benzyloxy)ammo)propanoate (13.0 g, 51.76 mmol) in methanol (100 ml) was added Pd/C (0.85 g, 10%Pd, 50% wet) in a hydrogenation vessel. After the system was evacuated under vacuum and placed under 2 atm of hydrogen gas, the reaction mixture was stirred overnight at room temperature. The crude reaction was passed through a short pad of Celite™ rinsing with ethanol, concentrated and purified on SiO2 column eluted with MeOH/DCM (1:10-1:5) to afford the title compound (7.25 g, 87% yield). 1H NMR (CDCh) 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI MS m/z+ C7Hi5NNaO3 (M+Na), cacld. 184.10, found 184.30. Example 11. Synthesis of tert-Butyl 3-((tosyloxy)amino)propanoate. ,OH ,OTs “N' Tert-butyl 3-(hydroxyamino)propanoate (5.10 g, 31.65 mmol) in the mixture of DCM (50 ml) and pyridine (20 ml) was added tosylate chloride (12.05 g, 63.42) at 4°C. After addition, the mixture was stirred at room temperature overnight, concentrated and purified on SiOz column eluted with EtOAc/DCM (1:10-1:6) to afford the title compound (8.58 g, 86% yield).1HNMR (CDCh) 7.81 (s, 2H), 7.46 (s, 2H), 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4Hz, 2H), 2.41 (s, 3H), 1.49 (s, 9H); ESI MS m/z+ CiÆiNNaOsS (M+Na), cacld. 338.11, found 338.30. Example 12. Synthesis of di-tert-Butyl 3,3'-(hydrazine-l,2-diyl)dipropanoate. Tert-butyl 3-aminopropanoate (3.05 g, 21.01 mmol) in THF (80 ml) was added tert-Butyl 3-((tosyloxy)amino)propanoate (5.10 g, 16.18 mmol). The mixture was stirred at room temperature for 1 h and then 45 °C for 6 h. The mixture was concentrated and purified on SiO2 column eluted with CH3OH/DCM/Et3N (1:12:0.01-1:8:0.01) to afford the title compound (2.89 g, 62% yield).ESI MS m/z+ C14H28N2NaO4 (M+Na), cacld. 311.20, found 311.40. Example 13. Synthesis of di-tert-Butyl 3,3'-(l,2-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanoyl)hydrazine-1,2-diyl)dipropanoate. Date Reçue/Date Received 2022-07-07 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 3-Maleido-propanoic acid (1.00 g, 5.91 mmol) in DCM (50 ml) was added oxalyl dichloride (2.70 g. 21.25 mmol) and DMF (50 pL). The mixture was stirred at room temperature for 2 h, evaporated, and co-evaporated with DCM/toluene to obtain crude 3-maleido-propanoic acid chloride. To the compound di-tert-Butyl 3,3'-(hydrazine-l,2-diyl)dipropanoate (0.51 g, 1.76 mmol) in the mixture of DCM (35 ml) was added the crude 3-maleido-propanoic acid chloride. The mixture was stirred for overnight, evaporated, concentrated and purified on SiO2 column eluted with EtOAc/DCM (1:15-1:8) to afford the title compound (738 mg, 71% yield). ESI MS m/z+ C28H38N4NaOio (M+Na), cacld. 613.26, found 613.40. Example 14. Synthesis of 3,3'-(l,2-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanoyl)-hydrazine-1,2-diyl)dipropanoic acid. Compound 14 (700 mg, 1.18 mmol) in dioxane (4 ml) was added HCl (cone. 1 ml). The mixture was stirred for 30 min, diluted with EtOH (10 mL) and toluene (10 ml), evaporated and coevaporated with EtOH (10 ml) and toluene (10 ml) to afford the crude title product (560 mg) for next step without further purification. ESI MS m/z- C20H21N4O10 (M-H), cacld. 477.13, found 477.20. Example 15. Synthesis of Bis(2,5-dioxopyrrolidin-l-yl)-3,3'-(l,2-bis(3-(2,5-dioxo-2,5- dihydro-IH-pyrrol-l-yl)propanoyl)hydrazine-1,2-diyl)dipropanoate. To the crude compound 3,3'-(l,2-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)- hydrazine-l,2-diyl)dipropanoic acid (-560 mg, -1.17 mmol) in DMA (8 ml) was added NHS (400 mg, 3.47 mmol) and EDC (1.01 g, 5.26 mmol). The mixture was stirred for overnight, evaporated, concentrated and purified on SiO2 column eluted with EtOAc/DCM (1:12-1:7) to afford the title compound (520 mg, 65% yield in 2 steps). ESI MS m/z+ C^HisNeNaOu (M+Na), cacld. 695.17, found 695.40. 124 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 16. Synthesis of tert-Butyl 3-(2-(2-(2-hydroxyclhoxy)cthoxy)cthoxy)propanoatc. To 350 mL of anhydrous THF was added 80 mg (0.0025 mol) of sodium metal and triethylene glycol 150.1 g, 1.00 mol) with stirring. After the sodium had completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol) was added. The solution was stirred for 20 h at room temperature and neutralized with 8 mL of 1.0 M HC1. The solvent was removed in vacuo and the residue was suspended in brine (250 mL) and extracted with ethyl acetate (3 x 125 mL). The combined organic layers were washed with brine (100 mL) then water (100 mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried under vacuum to give 69.78 g (76% yields) of the title product. 'H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J=6.4 Hz), 3.59-3.72 (m. 14H). ESI MS m/z- C|3H25O6 (M-H), cacld. 277.17, found 277.20. Example 17. Synthesis of tert-Butyl 3-(2-(2-(2- (tosyloxy)ethoxy)ethoxy)ethoxy)propanoate. A solution of tert-Butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)cthoxy)propanoate (10.0 g, 35.95 mmol) in acetonitrile (50.0 mL) was treated with pyridine (20.0 mL). A solution of tosyl chloride (7.12 g, 37.3 mmol) in 50 mL acetonitrile was added dropwise via an addition funnel over 30 minutes. After 5 h TLC analysis revealed that the reaction was complete. The pyridine hydrochloride that had formed was filtered off and the solvent was removed. The residue was purified on silica gel by eluting from with 20% ethyl acetate in hexane to with neat ethyl acetate to give 11.2 g (76% yield) of the title compound. ’H NMR: 1.40 (s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4 Hz), 3.52-3.68 (m, 14H), 4.11 (t, 2H, J=4.8 Hz), 7.30 (d, 2H, J=8.0 Hz), 7.75 (d, 2H, J=8.0 Hz); ESI MS m/z+ C20H33O8S (M+H), cacld. 433.18, found 433.30. Example 18. Synthesis of tert-Butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate. NaN3 DMF To 50 mL of DMF was added tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)- propanoate (4.0 g, 9.25 mmol) and sodium azide (0.737 g, 11.3 mmol) with stirring. The reaction was heated to 80 °C. After 4 h TLC analysis revealed that the reaction was complete. The reaction was cooled to room temperature and quenched with water (25 mL). The aqueous layer was separated and extracted into ethyl acetate (3 x 35 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent removed in vacuo. The crude 125 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 azide product (2.24 g, 98% yield, about 93% pure by HPLC) was used for next step without further purification. ’H NMR (CDC13): 1.40 (s, 9H), 2.45 (t, 2H, J=6.4 Hz), 3.33 (t, 2H, J=5.2 Hz), 3.53-3.66 (m, 12H). ESI MS m/z+ C13H26N3O8 (M+H), cacld. 304.18, found 304.20. Example 19. Synthesis of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid. HC1 (1) Dioxane Tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate (2.20 g, 7.25 mmol) in 1.4- dioxane (40 ml) was added HC1(12 M, 10 ml). The mixture was stirred for 40 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to afford the crude title product for the next step without further production (1.88g, 105% yield, -92% pure by HPLC). MS ESI m/z calcd for C9Hi8N3O5 [M+H]+ 248.12, found 248.40. Example 20. Synthesis of 13-Amino-4,7,10-trioxadodecanoic acid tert-butyl ester, and 13- Amino-bis(4,7,10-trioxadodecanoic acid tert-Butyl Ester). The crude azide material 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (5.0 g, -14.84 mmol) was dissolved in ethanol (80 mL) and 300 mg of 10% Pd/C was added. The system was evacuated under vacuum and placed under 2 atm of hydrogen gas via hydrogenation reactor with vigorous stirring. The reaction was then stirred overnight at room temperature and TLC showed that the starting materials disappeared. The crude reaction was passed through a short pad of Celite rinsing with ethanol. The solvent was removed and the amine purified on silica gel using a mixture of methanol (from 5% to 15%) and 1% triethylamine in methylene chloride as the eluant to give 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (1.83 g, 44% yield, ESI MS m/z+ CuH^tNOs (M+H), cacld. 278.19, found 278.30) and 13-aminobis(4,7,10-trioxadodecanoic acid tert-butyl ester) (2.58 g, 32% yield, ESI MS m/z+ C26H52NO10 (M+H), cacld. 538.35, found 538.40). Example 21. Synthesis of 3-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)propanoic acid, HO salt. To 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (0.80 g, 2.89 mmol) in 30 mL of dioxane was 10 ml of HC1(36%) with stirring. After 0.5 h TLC analysis revealed that the reaction was complete, the reaction mixture was evaporated, and co-evaporated with EtOH and 126 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 EtOH/Toluene to form the title product in HC1 salt (>90% pure, 0.640 g, 86% yield) without further purification. ESI MS m/z+ C9H2oN05 (M+H), cacld. 222.12, found 222.20. Example 22. 13-Amino-bis(4,7,10-trioxadodecanoic acid, HC1 salt. To 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester) (1.00 g, 1.85 mmol) in 30 mL of dioxane was 10 ml of HO (36%) with stirring. After 0.5 h TLC analysis revealed that the reaction was complete, the reaction mixture was evaporated, and co-evaporated with EtOH and EtOH/Toluene to form the title product in HC1 salt (>90% pure, 0.71 g, 91% yield) without further purification. ESI MS m/z+ CisHsôNOio (M+H), cacld. 426.22, found 426.20. Example 23. Synthesis of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoatc. To a solution of 2,2’-(ethane-l,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight and then quenched by HC1 solution (20.0 mL, IN) at 0°C. THF was removed by rotary evaporation, brine (300 mL) was added and the resulting mixture was extracted with EtOAc (3 x 100 mL). The organic layers were washed with brine (3 x 300 mL), dried over anhydrous Na2SO4, filtered and concentrated to afford a colourless oil (30.20 g, 79.0% yield), which was used without further purification. MS ESI m/z calcd for C13H27O6 [M + H]+ 278.1729, found 278.1730. Example 24. Synthesis of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate. To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol, 2.0 eq.) in anhydrous DCM (220 mL) at 0 °C was added TEA (30.0 mL, 217.0 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight, and then washed with water (3 x 300 mL) and brine (300 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiO2 column chromatography (3:1 hexanes/ EtOAc) to give a colourless oil (39.4 g, 84.0% yield). MS ESI m/z calcd for C2oH3308S [M + H]+ 433.1818, found 433.2838. Example 25. Synthesis of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) propanoate. 127 5 10 15 20 25 30 ^3 Bu To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF(100 mL) was added NaN3(20.67 g, 316.6 mmol, 3.5 eq.). The mixture was stirred at room temperature overnight. Water (500 mL) was added and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with water (3 x 900 mL) and brine (900 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiO? column chromatography (5:1 hexanes/ EtOAc) to give a light yellow oil (23.8 g, 85.53% yield).MS ESI m/z calcd for Ci3H25O3N5Na |M + Na]+ 326.2, found 326.2. Example 26. Synthesis of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) propanoate. HjN Raney™-Ni (7.5 g, suspended in water) was washed with water (three times) and isopropyl alcohol (three times) and mixed with tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) propanoate (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under a H2 balloon at r.t. for 16 h and then filtered over a Celite pad, with washing of the pad with isopropyl alcohol. The filtrate was concentrated and purified by column chromatography (5-25% MeOH/DCM) to give a light yellow oil (2.60 g, 57% yield). MS ESI m/z calcd for CbH^NOs [M+H]+ 279.19; found 279.19. Example 27. Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and K2C03(83.00 g, 600 mmol, 3.0 eq.) in acetonitrile (350 mL) was added BnBr (57.0 mL, 480 mmol, 2.4 eq.). The mixture was refluxed overnight. Water (1 L) was added and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiÛ2 column chromatography (4:1 hexanes/ EtOAc) to give a colourless oil (50.97 g, 89.2% yield).MS ESI m/z calcd for Ci8H23NO2Na [M + Na]+ 309.1729, found 309.1967. Example 28. Synthesis of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate. Bn2Nxs^'°'^KO To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 eq.) , tert¬ butyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and M-BU4NI (6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL) was added sodium hydroxide solution (300 mL, 50%). The mixture was stirred overnight. The organic layer was separated and the water layer was extracted with EtOAc (3 x 100 mL). The organic layers were washed with water(3 x 300 mL) and brine (300 mL), dried 128 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 over anhydrous Na2SO4, filtered, concentrated and purified by SiOi column chromatography (7:1 hexanes/ EtOAc) to give a colourless oil (61.08 g, 89.4% yield). MS ESI m/z calcd for C25H36NO4 [M + H]+ 414.2566, found 414.2384. Example 29. Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate. H2N^ ° ^CO2'Bu To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THE (30 mL) and MeOH (60 mL) was added Pd/C (2.00 g, 10 wt%, 50% wet) in a hydrogenation bottle. The mixture was shaken at 1 atom pressure H2 overnight, filtered through Celite (filter aid), and the filtrate was concentrated to afford a colourless oil (10.58 g, 93.8% yield). MS ESI m/z calcd for C11H24NO4 [M + H]+ 234.1627, found 234.1810. Example 30. Synthesis of ter/-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate. To a solution of 2,2'-oxydiethanol (19.7 mL, 206.7 mmol, 3.0 eq.) in anhydrous THF (100 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then tert-butyl acrylate (10.0 mL, 68.9 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight, and brine (200 mL) was added and extracted with EtOAc (3 x 100 mL). The organic layers were washed with brine (3 x 300 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiÛ2 column chromatography (1:1 hexanes/ EtOAc) to give to a colourless oil (8.10 g, 49.4% yield). MS ESI m/z calcd for C11H23O5 [M +H]+ 235.1467, found 235.1667. Example 31. Synthesis of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate. To a solution of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (6.24 g, 26.63 mmol, 1.0 eq.) and TsCl (10.15 g, 53.27 mmol, 2.0 eq.) in anhydrous DCM(50 mL) at 0 °C was added pyridine (4.3 mL, 53.27 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight, and then washed with water (100 mL) and the water layer was extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiÛ2 column chromatography (5:1 hexanes/ EtOAc) to give a colourless oil (6.33 g, 61.3% yield). MS ESI m/z calcd for C18H27O7S [M + H]+ 389.1556, found 389.2809. Example 32. Synthesis of tert-butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate. N3 To a solution of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate (5.80 g, 14.93 mmol, 1.0 eq.) in anhydrous DMF (20 mL) was added NaNg (5.02 g, 77.22 mmol, 5.0 eq.). The 129 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 mixture was stirred at room temperature overnight. Water (120 mL) was added and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water (3 x 150 mL) and brine (150 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiO2 column chromatography (5:1 hexanes/ EtOAc) to give a colourless oil (3.73 g, 69.6% yield). MS ESI m/z calcd for CiiH22O3N4Na[M + H]+ 260.1532, found 260.2259. Example 33. Synthesis of /er/-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate. H2N' o^/CO/Bu /er/-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (0.18 g, 0.69 mmol) was dissolved in MeOH (3.0 mL, with 60 pL concentrated HC1) and hydrogenated with Pd/C (10 wt%, 20 mg) under a H2 balloon for 30 min. The catalyst was filtered through a Celite pad, with washing of the pad with MeOH. The filtrate was concentrated to give a colorless oil (0.15 g, 93% yield). MS ESI m/z calcd for CiiH24NO4 [M+H]+ 234.16; found 234.14. Example 34. Synthesis of 3-(2-(2-azidoethoxy)ethoxy)propanoic acid. n3x^°'^o /erZ-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (2.51 g, 9.68 mmol) dissolved in 1,4- dioxane (30 mL) was treated with 10 ml of HC1 (cone.) at r.t. The mixture was stirred for 35 min, diluted with EtOH (30 ml) and toluene (30 ml) and concentrated under vacuum. The crude mixture was purified on silica gel using a mixture of methanol (from 5% to 10%) and 1% formic acid in methylene chloride as the eluant to give title compound (1.63 g, 83% yield), ESI MS m/z C7H12N3O4 [M-Hf, cacld. 202.06, found 202.30. Example 35. Synthesis of 2,5-dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate. O To 3-(2-(2-azidoethoxy)ethoxy)propanoic acid (1.60 g, 7.87 mmol) in 30 mL of dichloromethane was added NHS (1.08 g, 9.39 mmol) and EDC (3.60 g, 18.75 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 10%) in methylene chloride as the eluant to give title compound (1.93 g, 82% yield). ESI MS m/z C11H17N4O6 [M+H]+, cacld.301.11, found 301.20. Example 36. Synthesis of 2,5-dioxopyrrolidin-l-yl 3-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)propanoate. 130 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (4.50 g, 18.21 mmol) in 80 mL of dichloromethane was added NHS (3.0 g, 26.08 mmol) and EDC (7.60 g, 39.58 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 10%) in methylene chloride as the eluant to give title compound (5.38 g, 86% yield). ESI MS m/z C13H20N4O7 [M+H]+, cacld.345.13, found 345.30. Example 37. Synthesis of (14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tertbutoxycarbonyl)-amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid To a solution of (S)-2-((S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)-4-(tertbutoxy)-4-oxobutanoic acid (2.81 g, 6.73 mmol) in the mixture of DMA (70 ml) and 0.1 M NaHiPO4 (50 ml, pH 7.5) was added 2,5-dioxopyrrolidin-l-yl 3-(2-(2-(2-azidoethoxy)ethoxy)- ethoxy)propanoate (3.50 g, 10.17). The mixture was stirred for 4 h, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 15%) in methylene chloride containing 0.5% acetic acid as the eluant to give title compound (3.35 g, 77% yield). ESI MS m/z C28H51N6O11 [M+H]+, cacld.647.35, found 647.80. Example 38. Synthesis of (14S,17S)-tert-butyl l-azido-14-(4-((tert-butoxycarbonyl)- amino)butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16- diazanonadecan-19-oate (14S,17S)-1-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)- amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid (3.30 g, 5.10 mmol) and (4-aminophenyl)methanol (0.75 g, 6.09) in DMA (25 ml) was added EDC (2.30 g, 11.97 mmol). The mixture was stirred for overnight, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 8%) in methylene chloride containing as the eluant to give title compound (3.18 g, 83% yield). ESI MS m/z C35H58N7On [M+HJ+, cacld.752.41, found 752.85. 131 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 39. Synthesis of (14S,17S)-tert-butyl l-amino-14-(4-((tertbutoxycarbonyl)amino)-butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazanonadecan-19-oate To a solution of (14S,17S)-tert-butyl l-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)- 17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19- oate (1.50 g, 1.99 mmol) in THF (35 mL) was added Pd/C (200 mg, 10% Pd, 50% wet) in a hydrogenation bottle. The mixture was shaken at 1 atom pressure H2 overnight, filtered through Celite (filter aid), and the filtrate was concentrated to afford the title compound (1.43 g, 99% yield) which was used immediately for the next step without further purification. ESI MS m/z CasHéoNsOn [M+H]+, cacld.726.42, found 726.70. Example 40. Synthesis of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6- diazapentadecan-1-oic acid To a solution of (S)-2-(2-amino-3-methylbutanamido)acetic acid (Val-Gly) (1.01 g, 5.80 mmol) in the mixture of DMA (50 ml) and 0.1 M NaH2PO4 (50 ml, pH 7.5) was added 2,5- dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (1.90 g, 6.33). The mixture was stirred for 4 h, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 15%) in methylene chloride containing 0.5% acetic acid as the eluant to give title compound (1.52 g, 73% yield). ESI MS m/z CuH^NsOe [M+H]+, cacld.360.18, found 360.40. Example 41. Synthesis of (S)-2,5-dioxopyrrolidin-l-yl 15-azido-5-isopropyl-4,7-dioxo- 10,13-dioxa-3,6-diazapentadecan-1-oate To a solution of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-loic acid (1.50 g, 4.17 mmol) in 40 mL of dichloromethane was added NHS (0.88 g, 7.65 mmol) and EDC (2.60 g, 13.54 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 20%) in methylene chloride as the eluant to give title compound (1.48 g, 78% yield). ESI MS m/z C18H29N6O8 [M+H]+, cacld.457.20, found 457.50. 132 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 42. Synthesis of 4-(((bcnzyloxy)carbonyl)amino)butanoic acid. CbzHNx^^CO2H A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H2O (40 mL) was cooled to 0 °C and treated with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reaction was allowed to warm to r.t. and stirred for 3 h. THF was removed under vacuum, the pH of the aqueous solution was adjusted to 1.5 by addition of 6 N HO. Extracted with ethyl acetate, and the organic layer was washed with brine, dried and concentrated to give the title compound (16.4 g, 92% yield). MS ESI m/z calcd for C12H16NO5 [M+H]+238.10, found 238.08. Example 43. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate. CbzH^J^x^^CO/Bu DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to a solution of 4- (((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and /-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring at r.t. overnight, the reaction was filtered and filtrate concentrated. The residue was dissolved in ethyl acetate and the washed with IN HC1, brine and dried over Na2SO4. Concentration and purification by column chromatography (10 to 50% EtOAc/hexanes) yielded the title compound (7.5 g, 37% yield). MS ESI m/z calcd for CiôHzaNCUNa [M+Na]+ 316.16, found 316.13. Example 44. Synthesis of ter/-butyl 4-aminobutanoate. IhN^^^CO/Bu ter/-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt%, 100 mg) then hydrogenated (1 atm) at room temperature for 3 h. The catalyst was filtered off and all volatiles were removed under vacuum to afford the title compound (272 mg, 90% yield). MS ESI m/z calcd for CgHigNCh [M+H]+ 160.13, found 160.13. Example 45. Synthesis of di-terz-butyl 3,3'-(benzylazanediyl)dipropanoate. O O 'BuO'X^z^Nx^zUkO,Bu Bn A mixture of phenylmethanamine (2.0 mL, 18.29 mmol, 1.0 eq) and terr-butyl acrylate (13.3 mL, 91.46 mmol, 5.0 eq) was refluxed at 80 °C overnight and then concentrated. The crude product was purified by SiO2column chromatography (20:1 hexanes/EtOAc) to give the title compound as colourless oil (5.10 g, 77% yield). ESI MS m/z: calcd for C21H34NO4[M+H]+ 133 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 364.2, found 364.2. *H NMR (400 MHz, CDC13) 5 7.38-7.21 (m, 5H), 3.58 (s, 2H), 2.76 (t, J = 7.0 Hz, 4H), 2.38 (t, J = 7.0 Hz, 4H), 1.43 (s, 17H). Example 46. Synthesis of di-Ze/7-butyl 3,3’-azanediyldipropanoate. O O /BuO'^z^N'^s*^KO/Bu H To a solution of di-ter/-butyl 3.3’-(benzylazanediyI)dipropanoate (1.37 g, 3.77 mmol, 1.0 equiv) in MeOH (10 mL) was added Pd/C (0.20 g, 10% Pd/C, 50% wet) in a hydrogenation bottle. The mixture was shaken overnight under H2 atmosphere and then filtered through a Celite pad. The filtrate was concentrated to give the title compound as colourless oil (1.22 g, 89% yield). ESI MS m/z: calcd for Ci4H28NO4 [M+H]+ 274.19, found 274.20. Example 47. Synthesis of Zerz-butyl 4-(2-(((benzyloxy)carbonyl)amino)propan amido)- butanoate. zBuO. NHCbz O 'N H To a solution of ter/-butyl 4-aminobutanoate (1.00 g, 6.28 mmol, 1.0 eq.) and Z-L-alaine (2.10 g, 9.42 mmol, 1.5 eq.) in anhydrous DCM (50 mL) at 0 °C were added HATU (3.10 g, 8.164 mmol, 1.3 eq.) and TEA (2.6 mL, 18.8 mmol, 3.0 eq.). The reaction was stirred at 0 °C for 10 min., then warmed to room temperature and stirred overnight. The mixture was diluted with DCM and washed with water and brine, dried over anhydrous Na2SO4, concentrated and purified by SiO2 column chromatography (10:3 petroleum ether/ethyl acetate) to give the title compound as a colorless oil (1.39 g, 61% yield). ESI MS m/z: calcd for Ci9H29N2O5Na [M+H]+ 387.2, found 387.2. Example 48. Synthesis of tert-butyl 4-(2-aminopropanamido)butanoate. To a solution of tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)propanamido) butanoate (1.39 g, 3.808 mmol, 1.0 eq.) in MeOH (12 mL) was added Pd/C (0.20 g, 10 wt%, 10% wet) in a hydrogenation bottle. The mixture was shaken for 2 h and then filtered through Celite (filter aid), concentrated to give the title compound as a light yellow oil (0.838 g, 95% yield). ESI MS m/z: calcd. for CiiH23N2O3[M+H]+ 231.16, found 231.15. Example 49. Synthesis of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid. O 134 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of terf-butyl 3-(2-(2-(dibcnzylamino)cthoxy)cthoxy)propanoatc (2.3g, 5.59 mmol, l.Oeq) in DCM (10 mL) at room temperature was added TFA (5 mL). After stirring for 90 min., the reaction mixture was diluted with anhydrous toluene and concentrated, this operation was repeated for three times to give the title compound as a light yellow oil (2.0 g, theoretical yield), which was directly used in the next step. ESI MS m/z calcd. for C21H28NO4 [M+H]+ 358.19, found358.19. Example 50. Synthesis of perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy) ethoxy)- propanoate. C6F5O^s^OxX^oxx^NBn2 To a solution of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid(2.00 g, 5.59 mmol, 1.0 eq.) in anhydrous DCM (30 mL) at 0 °C was added DIPEA until pH was neutral, and then PFP (1.54 g, 8.38 mmol, 1.5 eq.) and DIC (1.04 mL, 6.70 mmol, 1.2 eq.) were added. After 10 min. the reaction was warmed to room temperature and stirred overnight. The mixture was filtered, concentrated and purified by SiO2column chromatography (15:1 petroleum ether/ethyl acetate) to give the title compound as colourless oil (2.10 g, 72% yield). ESI MS m/z: calcd. for C27H27F5NO4 [M+H]+ 524.2, found 524.2. Example 51. Synthesis of /er/-butyl 2-benzyl-13-methyl-l1,14-dioxo-l-phenyl -5,8-dioxa- 2,12,15-triazanonadecan-19-oate. To a solution of tert-butyl 4-(2-aminopropanamido)butanoate (0.736 g, 3.2 mmol, 1.0 eq.) and perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy) ethoxy)propanoate (2.01 g, 3.84 mmol, 1.2 eq.) in anhydrous DMA (20 mL) at 0 °C was added DIPEA (1.7 mL, 9.6mmol, 3.0 eq.). After stirring at 0 °C for 10 min. the reaction was warmed to room temperature and stirred overnight. Water (100 mL) was added and the mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with water (3 x 200 mL) and brine (200 mL), dried over Na2SO4. filtered, concentrated and purified by SiO2 column chromatography (25:2 DCM/McOH) to give the title compound as a colourless oil (1.46 g, 80% yield). ESI MS m/z: calcd. for C32H48N3O6 [M+HJ+ 570.34, found570.33. Example 52. Synthesis of 2-benzyl-13-methyl-l1,14-dioxo-l-phenyl-5,8-dioxa -2,12,15- triazanonadecan-19-oic acid. 135 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of /erz-butyl 2-bcnzy1-13-methy1-11,14-dioxo-l-phenyl-5,8-dioxa-2.12,15- triazanonadecan-19-oate (0.057 g, 0.101 mmol, 1.0 eq) in DCM (3 mL ) at room temperature was added TFA (1 mL) and stirred for 40 min. The reaction was diluted with anhydrous toluene and then concentrated. This operation was repeated three times to give the title compound as a colourless oil (0.052 g, theoretical yield), which was used directly in the next step. ESI MS m/z: calcd for C28H4ON3O6[M+H]+ 514.28, found 514.28. Example 53. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid CbzHN^zx^COzH A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H2O (40 mL) was cooled to 0 °C and treated with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reaction was allowed to warm to r.t. and stirred for 3 h. THF was removed under vacuum, the pH of the aqueous solution was adjusted to 1.5 by addition of 6 N HC1. Extracted with ethyl acetate, and the organic layer was washed with brine, dried and concentrated to give the title compound (16.4 g, 92% yield). MS ESI m/z calcd for C12H16NO5 [M+H]+238.10, found 238.08. Example 54. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate. CbzHN^z'XzXCVBu DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g. 83 mmol) were added to a solution of 4- (((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring at r.t. overnight, the reaction was filtered and filtrate concentrated. The residue was dissolved in ethyl acetate and the washed with IN HO, brine and dried over Na2SCL. Concentration and purification by column chromatography (10 to 50% EtOAc/hexanes) yielded the title compound (7.5 g, 37% yield). MS ESI m/z calcd for Ci6H23NO4Na [M+Na]+316.16, found 316.13. Example 55. Synthesis of tert-butyl 4-aminobutanoate. H2N^x^CO2rBu /<?//-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt%, 100 mg) then hydrogenated (1 atm) at room temperature for 3 h. The catalyst was filtered off and all volatiles were removed under vacuum to afford the title compound (272 mg, 90% yield). MS ESI m/z calcd for CgHisNOi [M+H]+160.13, found 160.13. 136 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 56. Synthesis of tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetate. NHCbz HOBt/EDC DIPEA/DCM NHCbz 2-(((Benzyloxy)carbonyl)amino)propanoic acid (0.84g, 5mmol), tert-butyl 2-aminoacetate (0.66g, 5mmol), HOBt (0.68g, 5mmol), EDC (1.44g, 7.5mmol) were dissolved in DCM (20ml), followed by addition of DIPEA(1.7ml, lOmmol). The reaction mixture was stirred at RT overnight, washed with H2O (100ml), and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over MgSCh, filtered, evaporated under reduced pressure and the residue was purified on SiOz column to give the title product l(0.87g, 52%). ESI: m/z: calcd for C17H25N2O5 [M+H]+: 337.17, found 337.17. Example 57. Synthesis of 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetic acid. *^°Y^z\zNHCbZ TFA » HO\/X A^NHCbz * O H I DCM O H I Tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetate (0.25g, 0.74mmol) was dissolved in DCM (30ml), followed by addition of TFA (10ml). The mixture was stirred at RT overnight, concentrated to afford the title compound used for the next step without further purification. ESI: m/z: calcd for C13H17N2O5[M+H]+: 281.11, found 281.60. Example 58. Synthesis of di-tert-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa- 13,18- diazatriacont-15-yne-1,30-dioate. Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved in NMP (10 mL) and cooled to 0 °C, to which compound tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)- propanoate (2.06 g, 7.43 mmol, 2.4 eq.) was added, followed by DMTMM (2.39 g, 8.65 mmol, 2.8 eq.) in portions. The reaction was stirred at 0 °C for 6 h and then diluted with ethyl acetate and washed with water and brine. The organic solution was concentrated and triturated with a mixture solvent of ethyl acetate and petroleum ether. The solid was filtered off and the filtrate was concentrated and purified by column chromatography (80-90% EA/PE) to give a light yellow oil (2.26 g, >100% yield), which was used without further purification. MS ESI m/z [M+H]+ 633.30. Example 59. Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,l8-diaza triacont-15- yne-1,30-dioic acid. 137 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Compound di-ter/-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa- 13,18-diazatriacont-15- yne-1,30-dioate (2.26 g) was dissolved in dichloromethane (15 mL) and cooled to 0 °C then treated with TFA (15 mL). The reaction was warmed to r.t. and stirred for 45 min, and then the solvent and residual TFA was removed on rotovap. The crude product was purified by column chromatography (0-15% MeOH/DCM) to give a light yellow oil (1.39 g, 86% yield for two steps). MS ESI m/z [M+H]+ 521.24. Example 60. Synthesis of di-tert-butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo- 10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-l,42-dioate To a solution of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diaza triacont-15-yne-1,30- dioic acid (1.38 g, 2.65 mmol), tert-butyl 2-(2-aminopropanamido)propanoate (0.75 g, 3.47 mmol) in the mixture of DMA (40 ml) was added EDC (2.05 g, 10.67 mmol). The mixture was stirred for overnight, concentrated and purified on SiO^ column eluted with EtOAc/CH2C12 (1:5 to 1:1) to afford the title compound (2.01 g, 82% yield, ~95% pure by HPLC). MS ESI m/z calcd for C42H73N6O16 [M+H]+ 917.50, found 917.90. Example 61. Synthesis of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo- 10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-l,42-dioic acid Di-di-tert-butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33- hcxaoxa-3,6,19,24,37,40-hcxaazadotetracont-21-ync-l,42-dioatc (1.50 g, 1.63 mmol) was dissolved in the mixture of CH2CI2 (10 ml) and TFA (10 ml). The mixture was stirred for overnight, diluted with toluene (20 ml), concentrated to afford the title compound (1.33 g, 101% yield, ~92% pure by HPLC) which was used for the next step without further purification.. MS ESI m/z calcd for C34H56N6Oi6 [M+H]+ 805.37, found 805.85. 138 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 62. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 2,5,38,41-tetramethyl- 4,7,20.23,36,39-hcxaoxo-10,13.16,27,30.33-hcxaoxa-3,6,19,24,37,40-hexaazadotetracont-21- yne-l,42-dioate To a solution of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33- hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-1,42-dioic acid (1.30 g, 1.61 mmol) in the mixture of DMA (10 ml) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol). The mixture was stirred for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:4 to 2:1) to afford the title compound (1.33 g, 83% yield, -95% pure by HPLC). MS ESI m/z calcd for C42H63N8O20 [M+H]+ 999.40, found 999.95. Example 63. Synthesis of 2,3-bis(2-bromoacetamido)succinyl dichloride. 2,3-Diaminosuccinic acid (5.00 g, 33.77 mmol) in the mixture of THF/H2O/DIPEA (125 ml/125 ml/8 ml) was added 2-bromoacetyl bromide (25.0 g, 125.09 mmol). The mixture was stirred for overnight, evaporated and purified by SiO2 column chromatography (H2O/CH3CN 5:95) to afforded 2,3-bis(2-bromoacetamido)succinic acid (9.95 g, 76% yield) as light yellow oil. MS ESI m/z calcd for C8Hi1Br2N2O6 [M+H]+ 388.89, found 388.68. 2,3-bis(2-bromoacetamido)succinic acid (3.50 g, 9.02 mmol) in dichloromethane (80 ml) was added oxalyl dichloride (5.80 g, 46.05 mmol) and DMF (0.01 ml). The mixture was stirred for 2.5 h, diluted with toluene, concentrated and co-evaporated with dichloroethane (2 x 20 ml) and toluene (2x15 ml) to dryness to afford 2,3-bis(2-bromoacetamido)succinyl dichloride as crude product (which is not stable) for the next step without further purification (3.90 g, 102% yield). MS ESI m/z calcd for C8H9Br2Cl2N2O4 [M+H]+ 424.82, found 424.90. Example 64. Synthesis of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid. O O CbzHNNHCbz 139 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of 2,3-diaminosuccinic acid (4.05 g, 27.35 mmol) in the mixture of THF (250 ml) and NaHzPO4 (0.1 M, 250 ml, pH 8.0) was added benzyl carbonochloridate (15.0 g, 88.23 mmol) in 4 portions in 2 h. The mixture was stirred for another 6 h, concentrated and purified on SiOa column eluted with H2O/CH3CN (1:9) containing 1% formic acid to afford the title compound (8.65 g, 76% yield, -95% pure). MS ESI m/z calcd for C20H21N2O8 [M+H]+ 417.12, found 417.60. Example 65. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(((benzyloxy)carbonyl)- amino)succinate CbzHN NHCbz ° To a solution of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (4.25 g, 10.21 mmol) in the mixture of DMA (70 ml) was added NHS (3.60 g. 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred for overnight, concentrated and purified on S1O2 column eluted with EtOAc/CH2C12 (1:6) to afford the title compound (5.42 g, 87% yield, -95% pure). MS ESI m/z calcd for C28H27N4O12 [M+H]+ 611.15, found 611.60 Example 66. Synthesis of 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinic acid. 2,3-Diaminosuccinic acid (5.00 g, 33.77 mmol) in the mixture of THF/H2O/DIPEA (125 ml/125 ml/2 ml) was added maleic anhydride (6.68 g, 68.21 mmol). The mixture was stirred for overnight, evaporated to afforded 2,3-bis((Z)-3-carboxyacrylamido)succinic acid (11.05 g, 99% yield) as a white solid. MS ESI m/z calcd for C12H13N2O10 [M+H]+ 345.05, found 345.35. 2,3-bis((Z)-3-carboxyacrylamido)succinic acid (11.05 g, 33.43 mmol) in a mixture solution of HOAc (70 ml), DMF (10 ml) and toluene (50 ml) was added acetic anhydride (30 ml). The mixture was stirred for 2 h, reflux with Dean-Stark Trap at 100 °C for 6 h, concentrated, co¬ evaporated with EtOH (2 x 40 ml) and toluene (2 x 40 ml), and purified on SiO2 column eluted with H2O/CH3CN (1:10) to afford the title compound (7.90 g, 76% yield, -95% pure). MS ESI m/z calcd for C12H9N2O8 [M+H]+ 309.03, found 309.30. Example 67. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-lHpyrrol-1-yl)succinate 140 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinic acid (4.00 g, 12.98 mmol) in the mixture of DMF (70 ml) was added NHS (3.60 g, 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CIECb (1:6) to afford the title compound (5.73 g, 88% yield, ~96% pure by HPLC). MS ESI m/z calcd for C20H15N4O12 [M+H]+ 503.06, found 503.45. Example 68. Synthesis of (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tertbutoxycarbonyl)amino)butyl)-22,23-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3,42-bis((4- (hydroxymethyl)phenyl)carbamoyl)-5,8,21,24,37,40-hexaoxo-ll,14,17,28,31,34-hexaoxa- 4,7,20,25,38,41-hexaazatetratetracontane-l,44-dioate (14S,17S)-tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-((4- (hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate (1.43 g, 1.97 mmol) and 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinic acid (0.30 g, 0.97 mmol) in DMA (25 ml) was added EDC (1.30 g, 6.77 mmol). The mixture was stirred for overnight, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 8%) in methylene chloride containing as the eluant to give title compound (1.33 g, 80% yield). ESI MS m/z C82Hi23Ni2O28 [M+H]+, cacld.1722.85, found 1722.98.. Example 69. Synthesis of tert-butyl l-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa- 13,16-diazaoctadecan-18-oate To a solution of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (1.55 g, 6.27 mmol), tert-butyl 2-(2-aminopropanamido)propanoate (1.35 g, 6.27 mmol) in the mixture of DMA (60 ml) was added EDC (3.05 g, 15.88 mmol). The mixture was stirred for overnight, concentrated 141 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 and purified on SiCh column eluted with EiOAc/CILCb (1:3) to afford the title compound (2.42 g, 86% yield, -95% pure by HPLC). MS ESI m/z calcd for Ci9H36N5O7 [M+H]+ 446.25, found 446.60 Example 70. Synthesis of l-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecan-18-oic acid Dioxane Tert-butyl 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18- oate (2.20 g, 4.94 mmol) in 1,4-dioxane (40 ml) was added HC1 (12 M, 10 ml). The mixture was stirred for 40 ruin, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co¬ evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to afford the crude title product for the next step without further production (1.92g, 100% yield, -94% pure by HPLC). MS ESI m/z calcd for Ci5H28N5O7 [M+H]+ 390.19, found 390.45. Example 71. Synthesis of 21,22-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-2,5,38,41- tcjtrameth 1 ,2^),2^^3,.3Ci,Ï3 lic^xLao^xr? 10,1^3,1 ,21/,.30,^3^3 licjxiarixiha 13 ,1 ,2ï^l ,.3^7,^10 hexaazadotetracontane-1,42-dioic acid. 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid (1.90 g, 4.88 mmol) in DMA (40 ml) was added Pd/C (0.20 g, 50% wet). The system was evacuated under vacuum and placed under 2 atm of hydrogen gas via hydrogenation reactor with vigorous stirring. The reaction was then stirred for 6 h at room temperature and TLC showed that the starting materials disappeared. The crude reaction was passed through a short pad of Celite rinsing with ethanol. The solvent was concentrated under reduced pressure to afford the crude product, l-amino-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid in DMA which was used for the next step directly. ESI MS m/z+ C15H30N3O7 (M+H), cacld. 364.20, found 364.30. To the amino compound in DMA (-30 ml) was added 0.1 M NaH2PO4, pH 7.5 (20 ml), followed by addition of bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol- 1-yl)succinate (1.30 g, 2.59 mmol). The mixture was stirred overnight, concentrated and purified 142 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 on SiOi column eluted with 8% water on CH3CN to afford the title compound (1.97g, 81% yield). ESI MS m/z+ C42H63N802o (M+H), cacld. 999.41, found 999.95. Example 72. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 21,22-bis(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa- 3,6,19,24,37,40-hexaazadotetracontane-l,42-dioate To a solution of 21,22-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-2,5,38,41-tetramethyl- 4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3.6,19.24,37,40-hexaazadotetracontane- 1,42-dioic acid (1.50 g, 1.50 mmol) in the mixture of DMA (10 ml) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol). The mixture was stirred for overnight, concentrated and purified on SiCh column eluted with EtOAc/CH2C12 (1:4 to 2:1) to afford the title compound (1.50 g, 83% yield, -95% pure by HPLC). MS ESI m/z calcd for C50H69N10O24 [M+H]+ 1193.44, found 1193.95. To a solution of trans-4-hydroxy-L-proline methyl ester (18.0 g, 107.0 mmol) in the mixture of MeOH (150 ml) and sodium bicarbonate solution (2.0 M, 350 ml) was added Boc2O Example 73. Synthesis of (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloric. To a solution of trans-4-hydroxy-L-proline (15.0 g, 114.3 mmol) in dry methanol (250 mL) was added thionyl chloride (17 mL, 231 mmol) dropwise at 0 to 4 °C. The resulting mixture was stirred for at r.t. overnight, concentrated, crystallized with EtOH/hexane to provide the title compound (18.0 g, 87% yield). ESI MS m/z 168.2 ([M+Na]+). Example 74. Synthesis of (2S,4R)-l-ter/-butyl 2-methyl 4-hydroxypyrrolidine-l,2- dicarboxylate. z^jCO2Me ^CO^Me HO \—-NII ^Boc 143 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (30.0 g, 137.6 mmol) in three portions in 4 h. After stirring for an additional 4 h, the reaction was concentrated to ~35O ml and extracted with EtOAc (4 x 80 mL). The combined organic layers were washed with brine (100 mL), dried (MgSO4), filtered, concentrated and purified by SiOz column chromatography (1:1 hexanes/EtOAc) to give the title compound (22.54 g, 86% yield). ESI MS m/z 268.2 ([M+Na]+). Example 75. Synthesis of (S)-l-tert-butyl 2-methyl 4-oxopyrrolidine-l,2-dicarboxylate. /^v-COiMe The title compound prepared through Dess-Martin oxidation was described in: Franco Manfre et al. J. Org. Chern. 1992, 57, 2060-2065. Alternatively Swern oxidation procedure is as following: To a solution of (COC1)2 (13.0 ml, 74.38 mmol) in CH2CI2 (350 ml) cooled to -78 °C was added dry DMSO (26.0 mL). The solution was stirred at -78 °C for 15 min and then (2S,4R)-l-tert-butyl 2-methyl 4-hydroxypyrrolidine- 1,2-dicarboxyl ate (8.0 g, 32.63 mmol) in CH2O2 (100 ml) was added. After stirring at -78 °C for 2 h, triethylamine (50 ml, 180.3 mmol) was added dropwise, and the reaction solution was warmed to room temperature. The mixture was diluted with aq. NaH2PO4 solution (1.0 M, 400 ml) and phases separated. The aqueous layer was extracted with CH2Q2 (2 x 60 ml). The organic layers were combined, dried over MgSO4, filtered, concentrated and purified by SiO2 column chromatography (7:3 hexanes/EtOAc) to give the title compound (6.73 g, 85% yield). ESI MS m/z 266.2([M+Na]+). Example 76. Synthesis of (S)-l-tert-butyl 2-methyl 4-methylenepyrrolidine-l,2- dicarboxylate. kCO2Me To a suspension of methyltriphenylphosphonium bromide (19.62 g, 55.11 mmol) in THF (150 mL) at 0 °C was added potassium-t-butoxide (6.20 g, 55.30 mmol) in anhydrous THF (80 mL). After stirring at 0 °C for 2 h, the resulting yellow ylide was added to a solution of (S)-1- tert-butyl 2-methyl 4-oxopyrrolidine-l,2-dicarboxylate (6.70 g, 27.55 mmol) in THF (40 mL). After stirring at r.t. for 1 h, the reaction mixture was concentrated, diluted with EtOAc (200 mL), washed with H2O (150 mL), brine (150 mL), dried over MgSO4, concentrated and purified on SiOicolumn chromatography (9:1 hexanes/EtOAc) to yield the title compound (5.77 g, 87% yield). EI MS m/z 264 ([M+Na]+). Example 77. Synthesis of (S)-methyl 4-methylenepyrrolidine-2-carboxylate hydrochloride. NH'HCl ,CO2Me 144 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of (S)-l-terf-butyl 2-methyl 4-methylenepyrrolidine-l,2-dicarboxylate (5.70 g, 23.63 mmol) in EtOAc (40 ml) at 4 °C was added HCI (12 M, 10 ml). The mixture was stirred for 1 h, diluted with toluene (50 ml), concentrated, and crystallized with EtOH/hexane to yield the title compound as HCI salt (3.85 g, 92% yield). EI MS m/z 142.2 ([M+H]4). Example 78. Synthesis of (S)-tert-butyl 2-(hydroxymethyl)-4-methylenepyrrolidine-lcarboxylate. LiAlH4 THF To a solution of (S)-l-terf-butyl 2-methyl 4-methylencpyrrolidinc-l,2-dicarboxylate. (5.20 g, 21.56 mmol) in anhydrous THF (100 mL) at 0 °C was added LiAlH4 (15 ml, 2M in THF). After stirring at 0 °C for 4 h, the reaction was quenched by addition of methanol (5 ml) and water (20 ml). The reaction mixture was neutralized with 1 M HCI to pH 7, diluted with EtOAc (80 ml), filtered through Celite, separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over Na2SO4, concentrated and purified on SiO2column chromatography (1:5 EtOAc/DCM) to yield the title compound (3.77 g, 82% yield). EI MS m/z 236.40 ([M+Na]+). Example 79. Synthesis of (S)-(4-methylenepyrrolidin-2-yl)methanol, hydrochloride salt. =\^NHHC1 To a solution of (S)-tert-butyl 2-(hydroxymethyl)-4-methylenepyrrolidine-l-carboxylate (3.70 g, 17.36 mmol) in EtOAc (30 ml) at 4 °C was added HCI (12 M, 10 ml). The mixture was stirred for 1 h, diluted with toluene (50 ml), concentrated, and crystallized with EtOH/hexane to yield the title compound as HCI salt (2.43 g, 94% yield). EI MS m/z 115.1 ([M+H]+). Example 80. Synthesis of 4-(benzyloxy)-3-methoxybenzoic acid. BnO^x^. To a mixture of 4-hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) in ethanol (350 ml) and aq. NaOH solution (2.0 M, 350 ml) was added BnBr (140.0 g, 823.5 mmol). The mixture was stirred at 65 °C for 8 h, concentrated, co-evaporated with water (2 x 400 ml) and concentrated to -400 ml, acidified to pH 3.0 with 6 N HCI. The solid was collected by filtration, crystallized with EtOH, dried at 45 °C under vacuum to afford the title compound (63.6 g, 83% yield). ESI MS m/z 281.2 ([M+Na]+). Example 81. Synthesis of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid. 145 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 BnO.^z^NC^ MeCTx'^S:O2H To a solution of 4-(benzyloxy)-3-methoxybcnzoic acid (63.5 g, 246.0 mmol) in CH2C12 (400 ml) and HOAc (100 ml) was added HNO3 (fuming, 25.0 ml, 528.5 mmol). The mixture was stirred for 6 h, concentrated, crystallized with EtOH, dried at 40 °C under vacuum to afford the title compound (63.3 g, 85% yield). ESI MS m/z 326.1 ([M+Na]+). Example 82. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2- (hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone. BnO^NO, O A catalytic amount of DMF (30 pl) was added to a solution of 4-(benzyloxy)-5-mcthoxy-2- nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CH2CI2 (70 mL) and the resulting mixture was stirred at room temperature for 2 h. Excess CH2CI2 and oxalyl chloride was removed with rotavap. The acetyl chloride was re-suspended in fresh CH2CE (70 mL) and was added slowly to a pre-mixed solution of (S)-(4- methylenepyrrolidin-2-yl)methanol, hydrochloride salt (1.32 g, 8.91 mmol) and EtgN (6 mL) in CH2CI2 at 0 °C under N2 atmosphere. The reaction mixture was allowed to warm to r.t. and stirring was continued for 8 h. After removal of CH2C12 and EtaN, the residue was partitioned between H2O and EtOAc (70/70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried (MgSCU) and concentrated. Purification of the residue with flash chromatography (silica gel, 2:8 hexanes/EtOAc) yielded the title compound (2.80 g, 79% yield). EI MS m/z 421.2 ([M+Na]+). Example 83. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((tertbutyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-l-yl)methanone. (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidinl-yl)methanone (2.78 g, 8.52 mmol) in the mixture of DCM (10 ml) and pyridine (10 ml) was added tert-butylchlorodimethylsilane (2.50 g, 16.66 mmol). The mixture was stirred for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:6) to afford the title compound (3.62 g, 83% yield, -95% pure). MS ESI m/z calcd for C27H37N2O6S1 [M+H]+ 513.23, found 513.65. 146 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 84. Synthesis of (S)-(4-hydroxy-5-mcthoxy-2-nitrophcnyl)(2-(hydroxymcthyl)-4- methylenepyrrolidin-1-y1)methanone. BnO^- NO2 jj 1 As. HO^,NO2II J CH3SO3H DCM/PhSCH3 (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidinl-yl)methanone (2.80 g, 7.03 mmol) in the mixture of DCM (30 ml) and CH3SO3H (8 ml) was added PhSCH3 (2.00 g, 14.06 mmol). The mixture was stirred for 0.5 h, diluted with DCM (40 ml), neutralized with carefully addition of 0.1 M Na2CO3 solution. The mixture was separated and the aqueous solution was extracted with DCM (2 x 10 ml). The organic layers were combined, dried over Na2SO4, concentrated and purified on SiO2 column eluted with MeOH/CH2Cl2 (1:15 to 1:6) to afford the title compound (1.84 g, 85% yield, -95% pure). MS ESI m/z calcd for Ci4Hi7N2O6 [M+H]+ 309.10, found 309.30. Example 85. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,lphenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) (S)-(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-lyl)methanone (0.801 g, 2.60 mmol) in butanone (10 ml) was added Cs2CO3, ( 2.50 g, 7.67 mmol), followed by addition of 1,5-diiodopentane (415 mmol, 1.28 mmol). The mixture was stirred for 26 h, concentrated and purified on SiO2 column eluted with MeOH/CH2Cl2 (1:15 to 1:5) to afford the title compound (0.675 g, 77% yield, -95% pure). MS ESI m/z calcd for C33H4iN4Oi2 [M+H]+ 685.26, found 685.60. Example 86. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(2-amino-5-methoxy-4,lphenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2- (hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) (0.670 g, 0.98 mmol) in CH3OH (10 ml) was added Na2S2O4 (1.01 g, 5.80 mmol) in H2O (8 ml). The mixture was stirred at room temperature for 30 h. The reaction mixture was evaporated and co-evaporated with DMA (2 x 10 mL) and EtOH (2x10 ml)under high vacuum to dryness to afford the title compound (total 147 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 weight 1.63 g) containing inorganic salts which was used directly for the next step reaction (without further separation). EIMS m/z 647.32 (|M+NaJ+). Example 87. Synthesis of C-l (a PBD dimer analog having a bis-linker). (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tert-butoxycarbonyl)amino)butyl)-22,23- bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3,42-bis((4-(hydroxymethyl)phenyl)carbamoyl)- 5,8,21,24,37,40-hexaoxo-11,14,17,28,31,34-hexaoxa-4,7,20,25,38,41-hexaazatetratetracontane- 1,44-dioate (0.840 g, 0.488 mmol) in THF (8 mL) containing pyridine (0.100 ml, 1.24 mmol) at 0 °C was added dropwise of a solution of triphosgene (0.290 mg, 0.977 mmol) in THF (3.0 mL). The reaction mixture was stirred at 0 °C for 15 min then was used directly in the next step. (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2- (hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) containing inorganic salts (0.842 mg, -0.49 mmol) was suspended in EtOH (10 ml) at 0 °C was added the trichloride in THF prepared above. The mixture was stirred at 0 °C for 4 h, then warmed to RT for 1 h, concentrated, and purified by reverse phase HPLC (250 (L) mm x 10(d) mm, Cis column, 10-80% acetonitrile/water in 40 min, v =8 ml/min) to afford the title compound (561.1 mg, 48% yield in three steps). ESI MS m/z: calcd for C117H163N16O38 [M+H]+ 2400.12, found 2400.90. Example 88. Synthesis of C-2 (a PBD dimer analog having a bis-linker). Dess-Martin periodinane (138.0 mg, 0.329 mmol) was added to a solution of compound C- 148 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 1 (132.0 mg, 0.055 mmol) in DCM (5.0 mL) at 0 °C. The reaction mixture was warmed to RT and was stirred for 2 h. A saturated solution of NaHCOa/NazSOa (5.0 mL/5.0 mL) was then added and the mixture was extracted with DCM (3 x 25 mL). The combined organic layers were washed with NaHCO3/Na2SO3 (5.0 mL/5.0 mL), brine (10 mL), dried over Na2SO4, filtered, 5 concentrated and purified by reverse phase HPLC (250 (L) mm x 10(d) mm, Cis column, 10- 80% acetonitrile/water in 40 min, v =8 ml/min) to afford the title compound (103.1 mg, 78% yield) as a foam. ESI MS m/z: calcd for CmH^sNisOas [M+H]+ 2396.09, found 2396.65. Example 89. Synthesis of C-3 (a PBD dimer analog having a bis-linker). 10 C-2 compound (55.0 mg, 0.023 mmol) was dissolved in DCM (3 ml), followed by addition of TFA (3 ml) at 4 °C. The reaction mixture was then stirred at RT for 1 h, then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-3 (48.0 mg, 100% yield, 92% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure 15 product C-3 (42.1 mg, 88% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C99H)26N16Oa4 [M+H]+ 2083.86, found 2084.35. Example 90. Synthesis of C-4 (a PBD dimer analog having a bis-linker). 149 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 C-3 compound (35.0 mg, 0.017 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NaHaPOj (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,1l,14,17,20,23-octaoxahexacosan-26-oate (43.0 mg, 0.084 mmol) in 4 portions in 2 h. The reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-4 which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-4 (39.4 mg, 81% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C135H195N16O52 [M+H]+ 2872.30, found 2871.65. Example 91. Synthesis of C-5 (a PBD dimer analog having a bis-linker). To a solution of C-4 compound (35.0 mg, 0.012 mmol) and 2,5,8,11,14,17,20,23- octaoxapentacosan-25-amine (15.1 mg, 0.0394 mmol) in dry DMA (2 ml) was added EDC (30.0 mg, 0.156 mmol). The reaction mixture was stirred at RT for 14 h, concentrated, purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-5 (31.2 mg, 77% yield, 97% pure by HPLC) as a foam. ESI MS m/z: calcd for C161H249N18O62 [M+H]+ 3426.68, found 3427.21. Example 92. Synthesis of (S)-methyl l-(4-(bcnzyloxy)-5-methoxy-2-nitrobcnzoyl)-4- methylenepyrrolidine-2-carboxylate. A catalytic amount of DMF (30 pl) was added to a solution of 4-(benzyloxy)-5-methoxy-2- nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CH2CI2 (70 mL) and the resulting mixture was stirred at room temperature for 2 h. Excess CH2CI2 and oxalyl chloride was removed with rotavap. The acetyl chloride was re-suspended in fresh CH2O2 (70 mL) and was added slowly to a pre-mixed solution of (S)-methyl 4- 150 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 methylenepyrrolidine-2-carboxylate hydrochloride (1.58 g, 8.91 mmol) and Et2N (6 mL) in CH2C12 at 0 °C under N2 atmosphere. The reaction mixture was allowed to warm to r.t. and stirring was continued for 8 h. After removal of CH2C12 and Et2N, the residue was partitioned between H2O and EtOAc (70/70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried (MgSO4) and concentrated. Purification of the residue with flash chromatography (silica gel, 2:8 hcxanes/EtOAc) yielded the title compound (2.88 g, 76% yield). EI MS m/z 449.1 ([M+Na]+). Example 93. Synthesis of (S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4- mcthylencpyrro-lidinc-2-carbaldchyde. BnO^x^NO2 CHO To a vigorously stirred solution of (S)-methyl 1-(4-(bcnzyloxy)-5-mcthoxy-2-nitro benzoyl)-4-methylenepyrrolidine-2-carboxylate (2.80 g, 6.57 mmol) in anhydrous CH2C12(6O mL) was added DIBAL-H (IN in CH2C12, 10 mL) dropwise at -78 °C under N2 atmosphere. After the mixture was stirred for an additional 90 min, excess reagent was decomposed by addition of 2 ml of methanol, followed by 5% HCI (10 mL). The resulting mixture was allowed to warm to 0 °C. Layers were separated and the aqueous layer was further extracted with CH2C12 (3 x 50 mL). Combined organic layers were washed with brine, dried (MgSO4) and concentrated. Purification of the residue with flash chromatography (silica gel, 95:5 CHCL/MeOH) yielded the title compound (2.19 g, 84% yield). EIMS m/z 419.1 ([M+Na]+). Example 94. Synthesis of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-lHbenzo[e]-pyrrolo[1,2-a]azepin-5(11aH)-one. O A mixture of (S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4- methylenepyrro-lidine-2- carbaldehyde (2.18 g, 5.50 mmol) and Na2S2O4 (8.0 g, 45.97 mmol) in THF (60 ml) and H2O (40 ml) was stirred at room temperature for 20 h. Solvents were removed under high vacuum. The residue was re-suspended in MeOH (60 mL), and HCl (6M) was added dropwise until pH ~ 2 was reached. The resulting mixture was stirred at r.t. for 1 h. The reaction was worked-up by removing most of MeOH, then diluted with EtOAc (100 mL). The EtOAc solution was washed with sat. NaHCOa, brine, dried (MgSO4), and concentrated. Purification of the residue with flash chromatography (silica gel, 97:3 CHClj/McOH) yielded the title compound (1.52 g, 80%). EIMS m/z 372.1 ([M+Na]+). 151 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 95. Synthesis of (S)-8-hydroxy-7-mcthoxy-2-mcthylcnc-2,3 -dihydro-1Hbenzo[e]-pyrrolo[1.2-a]azcpin-5(11aH)-one. O To a solution of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3 -dihydro-lH-benzo[e]- pyrrolo[l,2-a]azepin-5(llaH)-one (1.50 g, 4.32 mmol) in 70 ml of CH2O2 was added 25 ml of CH3SO3H at 0 °C. The mixture was stirred at 0 °C for 10 min then r.t. for 2 h, diluted with CH2CI2, pH adjusted with cold 1.0 N NaHCO3 to 4 and filtered. The aqueous layer was extracted with CH2CI2 (3 x 60 ml). The organic layers were combined, dried over Na2SO4, filtered, evaporated and purified on SiO2 column chromatography (CH3OH/CH2CI2 1:15) to afford 811 mg (73% yield) of the title product. EIMS m/z 281.1 ([M+Na]+). Example 96. Synthesis of (llaS,lla'S)-8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylcne-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(l laH)-one). To a stirred suspended solution of Cs2CO3 (0.761 g, 2.33 mmol)in butanone (8 ml) were added (S)-8-hydroxy-7-methoxy-2-methylene-2,3 -dihydro-lH-benzo[e]-pyrrolo[l,2-a]azepin- 5(1laH)-onc (401 mg, 1.55 mmol) and 1,5-diiodopentane (240 mg, 0.740 mmol). The mixture was stirred at r.t. overnight, concentrated, and purified on SiÜ2 chromatography (EtOAc/CH2C12 1:10) to afford 337 mg (78% yield) of the title product. EIMS m/z 607.2 ([M+Na]^. Example 97. Synthesis of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,11a-hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(l laH)-one and (1laS,l la'S)- 8,8'-(pentane-1,5-diy1bis(oxy))bis(7-methoxy-2-methylene-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[l,2-a][1,4]diazepin-5(10H)-one) H 152 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of (llaS,lla'S)-8,8’-(pcntanc-l,5-diylbis(oxy))bis(7-mcthoxy-2-mcthylcnc- 2,3-dihydro- 1 H-benzo[c]pyrrolo[l,2-a]|T.4]diazepin-5(llaH)-one) (150 mg. 0.256 mmol) in anhydrous dichloromethane (1 mL) and absolute ethanol (1.5 mL) was added sodium borohydride in methoxyethyl ether (85pl, 0.5 M, 0.042mmol) at 0 °C. The ice bath was removed after 5 minutes and the mixture was stirred at room temperature for 3 hours, then cooled to 0 °C, quenched with saturated ammonium chloride, diluted with dichloromethane, and phases separated. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered through Celite and concentrated. The residue was purified by reverse phase HPLC (Cis column, acetonitrile/water). The corresponding fractions were extracted with dichloromethane and concentrated to afford the half reduced compound, (S)-7-mcthoxy-8-((5-(((S)-7-rncthoxy-2- methylene-5-oxo-2,3,5,10,ll,lla-hexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8- yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)- one (64.7 mg, 43%), MS m/z 609.2 ([M+Naf)- 625.3 ([M+K]+) and 627.2 ([M+Na+H2O]+); the fully reduced compound, (llaS,lla'S)-8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylene-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(10H)-one) (16.5 mg, 11%), MS m/z 611.2 ([M+Na]+), 627.2 ([M+K]+), 629.2 ([M+Na+H2O]+); and the unreacted starting material was also recovered (10.2 mg, 7%), MS m/z 607.2 ([M+Na]+), 625.2 ([M+Na+ELO]*). Example 98. Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy) propanoyl)-7- methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hcxahydro-1H-benzo[e]pyrrolo[l,2- a][l,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-1Hbenzo[e]pyrrolo[l,2-a][1,4]diazepin-5(11aH)-one. To the mixture of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(llaH)-one (60.0 mg, 0.102 mmol) and 2,5-dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (40.5 mg, 0.134 mmol) in dichloromethane (5 ml) was added EDC (100.5 mg, 0.520 mmol). The mixture was stirred at r.t. overnight, concentrated and purified on SiO2 column chromatography (EtOAc/CH2Cl2, 1:6) to afford 63.1 mg (81% yield) of the title product. ESI MS m/z C40H50N7O9 [M+H] +, cacld.772.36, found 772.30. 153 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 99. Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-aminoethoxy)ethoxy) propanoyl.)-7- methoxy-2-methylene-5-oxo-2,3,5,10,11.11a-hexahydro-1H-benzo[e]pyrrolo[l,2- a][l,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-lHbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one. To a solution of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy) propanoyl)-7-methoxy-2- methylcne-5-oxo-2.3,5,10,11,11a-hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2- a][l,4]diazepin-5(l laH)-one (60 mg, 0.078 mmol) in the mixture of THF (5 ml) and NaH2PO4 buffer solution (pH 7.5, 1.0 M, 0.7 ml) was added PPI13 (70 mg, 0.267 mmol). The mixture was stirred at r.t. overnight, concentrated and purified on Cis preparative HPLC, eluted with water/CH3CN (from 90% water to 35% water in 35 min) to afford 45.1 mg (79% yield) of the title product after drying under high vacuum. ESI MS m/z C40H52N5O9 [M+H]+, cacld.746.37, found 746.50. Example 100. Synthesis of (S)-N-(2-((S)-8-((5-(((llS,llaS)-10-((S)-15-azido-5-isopropyl- 4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-l-oyl)-ll-hydroxy-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-oxy)-7- methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin- 10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-3-methylbutanamide. H O To the mixture of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(llaH)-one (60.0 mg, 0.102 mmol) and (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oic acid (90.2 mg, 0.25 mmol) in DMA (8 ml) was added BrOP (240.2 mg, 0.618 mmol). The mixture was stirred at 60 °C overnight, concentrated and purified on SiO2 column chromatography (CH3OH/CH2C12, 1:10 to 1:5) to afford 97.1 mg (74% yield) of the title product. ESI MS m/z C61H87Ni4Oi7 [M+H] +, cacld.1287.63, found 1287.95. 154 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 101. Synthesis of (S)-N-(2-((S)-8-((5-(((l ISJlaSl-lO-tXSl-lS-amino-Sisopropy1-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oy1)-11-hydroxy-7-methoxy-2- methylene-5-oxo-2,3,5,10,l1,1la-hexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]- pyrrolo[l,2-a][l,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)- propanamido)-3-methylbutanamide (C-6). To a solution of (S)-N-(2-((S)-8-((5-(((llS,llaS)-10-((S)-15-azido-5-isopropyl-4,7-dioxo- 10,13-dioxa-3,6-diazapentadecan-1-oyl)-1l-hydroxy-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)pentyl)-oxy)-7- methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin- 10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-3-methylbutanamide (85 mg, 0.066 mmol) in the mixture of THF (5 ml) was added PPh3 (100 mg, 0.381 mmol). The mixture was stirred for 2 h, Na^PCU buffer solution (pH 7.5, 1.0 M, 0.7 ml) was then added and the mixture was stirred for 10 min. After confirmed by LC-MS to form (S)-N-(2-((S)-8-((5- (((HS,llaS)-10-((S)-15-amino-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-l-oyl)- 11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[l,2- a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[l,2-a][l,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)- propanamido)-3-methylbutanamide (ESI MS m/z C61H90N10O17 [M+Na]+, cacld.1257.66, found 1257.90), bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinate (33 mg, 0.066 mmol) was added. The mixture was continued to stir for 4 h, concentrated and purified on Cis preparative HPLC, eluted with water/CH3CN (from 90% water to 30% water in 35 min) to afford 40.1 mg (40% yield) of the title product C-5 after drying under high vacuum. ESI MS m/z C73H95N12O23 [M+H]+, cacld. 1507.66, found 1507.90. 155 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 102. Synthesis of 4,4'-(pentane-l,5-diylbis(oxy))bis(3-methoxybenzoic acid). MeO'^^COaH H2O/THF/NaOH, 65°C HO2C A solution of diiodopropane (19.0 g, 58.6 mmol) in THF (75 mL) was added dropwise over a period of 4 hours to a vigorously stirred solution of vanilic acid (20.0 g, 119 mmol) in THF (150 mL) and aqueous NaOH (340 mL) at 65°C in the absence of light (foil-wrapped flask). After heating at reflux for 48 hours in the dark, the solution was cooled and the THF removed by evaporation in vacuo. The residue was extracted with EA, The aqueous layer was separated and acidified to pH 2 with cone. HC1. The resultant precipitate collected by filtration, washed, dried and recrystallised from glacial acetic acid to afford the corresponding bis-carboxylic acid (14.0 g, 34.7 mmol). White solid, yield(60%).. Example 103. Synthesis of 4,4'-(pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid). HN03 HO2C';^^OMc HOAc IKLC^^OMe MeO''^'^CO2H To a suspention of 4,4’-(pentane-l,5-diylbis(oxy))bis(3-methoxybenzoic acid) (18.0 g, 66.8 mmol) in HOAc (80 mL, 1800 mmol) was added HNO3 (80 mL, 1778 mmol) dropwise at room temperature. After 2 h of stirring, the mixture was poured into 100 g ice and extrated with EA (2 x 200 mL). The organic layer was separated and washed with H2O(2 x 100 mL), then 4N NaOH (400 mL) was added. After extrated with EA (2 x 100 mL), the basic aqueous layer was separated and acidified to PH 2 with cone. HC1. The mixture was extrated with EA (2 x 250 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by flash chromatography (DCM/McOH = 4/1) to give 4,4'-(pentane-l,5- diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid) (6.1 g, 12.3 mmol) as a pale yellow solid in 18% yield. Ry 0.3 (DCM/MeOH = 3/1) Example 104. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,lphenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone). 156 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of 4,4'-(pcntanc-l,5-diylbis(oxy))bis(5-mcthoxy-2-nitrobcnzoic acid) (5.0 g, 10.0 mmol) and L-(+)-Prolinol (2.25 g, 22.3 mmol)in DMF (100 mL) was added TEA (4.0 g) at room temperature. After 10 min of stirring, HATU(10.77 g, 28.3 mmol) was added. The mixture was stirred at room temperature overnight. After completion of conversion, the mixture was diluted with H20(100 mL) and extrated with EA (2 x 100 mL) and DCM (2 x 50 mL), the combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by chromatography (DCM/MeOH = 15/1) to give (S)-((pentane-l,5-diylbis(oxy))bis(5- methoxy-2-nitro-4,l-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-l-yl)methanone) (6.0 g, 9.1 mmol) as a white foam in 91% yield. Example 105. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(2-amino-5-methoxy-4,lphenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-l-yl)methanone). Pd/C H2 MeOH To a solution of (S)-((pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,l-phenylene))- bis(((S)-2-(hydroxymethyl)pyrrolidin-l-yl)methanone) (6.0 g, 9.1 mmol) in MeOH (100 mL) was added 10% Pd/C (2.4 g), the mixture was stirred under hydrogen atmosphere at room temperature overnight. After 14 h of stirring, the Pd/C was removed by filtration and washed with MeOH. The filtrate was concentrated and the residue was purified by chromatography (DCM/MeOH = 10/1) to give (S)-((pentane-1.5-diylbis(oxy))bis(2-amino-5-methoxy-4,l-phenylene))bis(((S)-2- (hydroxymethyl)pyrrolidin-l-yl)methanone) (3.54 g, 5.9 mmol) as a white foam in 65% yield. Example 106. Synthesis of bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl) ((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2- (hydroxymethyl)pyrrolidine -l-carbonyl)-4-methoxy-5,l-phenylene))dicarbamate. triphosgene/DTEA/THF/0°C r.t. 157 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 O O To a solution of allyl ((S)-l-(((S)-l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2- yl)amino)-3-methyl-l-oxobutan-2-yl)carbamate (8.0 g, 21.3 mmol) in dry THF(300 mL) was added DIPEA (5.5 g, 40.3 mmol) and a solution of triphosgcnc(3.2 g, 10.8 mmol) in dry THF(50 5 mL) at 5°C. After 15 min of stirring, the solution was recooled to 5°C and a mixture of (S)- ((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis (((S)-2-(hydroxymethyl)- pyrrolidin-l-yl)methanone) (3.2 g, 5.3 mmol ) and DIPEA(2.75 g, 21.6 mmol) in dry THF (150 mL) was added. The resultant solution was allowed to warm to room temperature and stirred overnight. The THF removed by evaporation in vacuo. The residue was purified by 10 chromatography (DCM/MeOH = 20/1) to give bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido) propanamido)-benzyl)((S)-(pentane-l,5-diylbis(oxy))bis(2-((S)-2- (hydroxymethyl)pyrrolidine-l-carbonyl)-4-methoxy-5,1-phenylene))dicarbamate (7.0 g, 4.97 mmol) as a yellow foam in 94% yield. Example 107. Synthesis of (llS,llaS,irS,lla'S)-bis(4-((S)-2-((S)-2- 15 (((allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5- diylbis(oxy))bis(11-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate). o H Il 4 * o 1 HQ. 1 MeO'^^NH =^OH s/rN>0 O JLJ 0 o H * o H O r T n H I Y Dess-Martin periodinane DCM/r.t. /—\ L I (^NY^^OMe O O ' OA h = Oh q N A N O 1 rr "4 i r 158 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methyl butanamido) propanamido)benzyl)((S)-(pentane-l,5-diylbis(oxy))bis(2-((S)-2-(hydroxy-methyl)pyrrolidine -1- carbonyl)-4-methoxy-5,l-phenylene))dicarbamate (300 mg, 0.21 mmol) in dry DCM (15 mL) was added DMP (280 mg, 0.66 mmol) under nitrogen at room temperature. After completion of conversion, the reaction solution was added aqueous Na2SCh and followed by aqueous NaHCOa, the mixture was stirred for further 15 minutes and extracted with DCM (3 x 20 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by chromatography (DCM/MeOH = 20/1) to give (llS,llaS,H'S,lla'S)-bis(4-((S)-2- ((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)8,8'-(pentane-l,5- diy1bis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][l,4]diazepine-10(5H)-carboxylate) (270 mg, 0.19 mmol) as a off-white foam in 92% yield. Example 108. Synthesis of (llS,llaS,irS,lla'S)-bis(4-((S)-2-((S)-2- (((allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5- diylbis(oxy))bis(11-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate). To a solution of (llS,llaS,irS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl) amino)-3- methylbutanamido)propanamido)benzyl)8,8'-(pentane-l,5-diylbis(oxy))bis(l l-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate) (774 mg, 0.55 mmol) and pyrrolidine (196 mg, 2.76 mmol) in dry DCM (8 mL) was added Pd(ppha)4 (76 mg, 0.066 mmol). The reaction was flushed with argon and stirred for 2h at room temperature, after which the reaction was diluted with DCM and washed sequentially with 159 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 saturated aqueous NH4CI and brine. The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography (DCM/ MeOH = 6/1) to give (11S,1laS,ll'S,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy) carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)8,8’-(pentane-l,5-diylbis(oxy))bis(ll-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]-pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate) (420 mg, 0.34 mmol) as an off-white solid in 62% yield. Example 109. Synthesis of (S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanoic acid. ^^x^O^Cl H2N^CO2H K2CO3 N^CCfjH O + H2O/THF J Allyl chloroformate (24.8 g, 205 mmol) was added dropwise to a stirred solution of L-valine (20 g, 171 mmol) and K2CO3 (35.4 g, 257 mmol) in H2O (250 mL) and THE (250 mL). The reaction mixture was stirred at room temperature overnight, then the solvent was concentrated under reduced pressure and the remaining solution extracted with diethyl ether (100 mL). The aqueous portion was acidified to pH 2 with cone. HC1 and extracted with DCM (3 x 200 mL). The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated to afford the product (35 g, 174 mmol). White solid, yield(100% ).. Example 110. Synthesis of (S)-2,5-dioxopyrrolidin-l-yl 2-(((allyloxy)carbonyl)amino)-3- methylbutanoate. To a stirred solution of (S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanoic acid (35 g, 174 mmol) in dry DCM (500 mL) was added EDC (66.9 g, 348 mmol) and N-hydroxysuccinimide (30 g, 261 mmol) at room temperature. After 14 h of stirring, the reaction was diluted with DCM and washed with water and brine. The organic phase was dried over Na2SO4, filtered and concentrated to afforded the product (54.5 g) which was used in the next step without further purification.Yield: (100%) viscous colourless oil. Ry =0.5 (PE/EA = 2/1) Example 111. Synthesis of (S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-propanoic acid. To a solution of H-Ala-OH (15.7 g, 176 mmol) and NaHCO3 (15.5 g, 185 mmol) in THF (200 mL) and H2O (200 mL) was added a solution of (S)-2,5-dioxopyrrolidin-l-yl 2-(((allyloxy)- 160 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 carbonyl)amino)-3-methylbutanoate (50 g, 168 mmol) in THF (100 mL) at room temperature. After 72 hours of stirring, the THF was evaporated under reduced pressure. The residue was acidified to pH 3 with citric acid and extrated with EA (3 x 350 mL), the combined extracts was washed with brine, dried, filtered and concentrated to give a white solid. Trituration with diethyl ether (excess) afforded the pure product as a white powder(25.2 g, 93 mmol, 55%). Example 112. Synthesis of allyl ((S)-l-(((S)-l-((4-(hydroxymethyl)phenyl)amino)-loxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate. To a solution of (S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-propanoic acid (25.2 g, 92.6 mmol) and p-aminobenzyl alcohol (12.0 g, 97.6 mmol) in THF (300 mL) was added EEDQ (24.0 g, 97.2 mmol) at room temperature. After 18 hours of stirring, the solvent was evaporated under reduced pressure to give a pale brown solid. The solid was triturated with diethyl ether and filtered, washing with an excess of diethyl ether. This afforded the product as a white solid(40 g, 106 mmol, 100%). Example 113. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid. h2n^^co2h + Na2CO3H2O/THF Na2CO3 (41.1 g, 387 mmol) was added to a solution of 4-aminobutanoic acid (20 g, 193 mmol) in H2O (300 mL) at 5°C. After 10 min of stirring, a solution of CbzCl (33.2 mL, 232 mmol) in THF (100 mL) was added dropwise. The reaction was allowed to warm to room temperature and stirred overnight. After completion of conversion, the mixture was diluted with H2O (100 mL) and extrated with EA (2 x 100 mL). The aqueous layer was acidified to pH 2 with cone. HC1 and extracted with EA (3 x 100 mL). The combined organics were washed with brine, dried over NaiSO^ filtered and concentrated to give a white solid. Trituration with PE (excess) afforded the pure product as a white powder (31.6 g, 70%). Example 114. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate. M^O^N_^,CO2H g rn u + ,-BuOH DIC/4-DMAP dom - (BuOJV^.NHCbz O To a stirred solution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (5.9 g, 24.9 mmol) and tert-Butanol (14.7 g, 199 mmol) in dry DCM (250 mL) was added 4-DMAP (0.61 g, 5 161 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 mmol) and DIC (4.7 g, 37.3 mmol) at 0°C. After 16 h of stirring, the reaction was filtered and extracted with DCM (2 x 200 mL). The combined organic extract was washed with IN HC1 and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography (100% DCM) to give tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate (4.26 g, 14.5 mmol, 58%) viscous colourless oil. Example 115. Synthesis of tert-butyl 4-aminobutanoate. 9 Pd/C H2 ^A^^NHCbz 9 /-BuO MeOH* /-BuO < ^A^^NH2 2 To a solution of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate (1.69 g, 5.77 mmol) in MeOH (40 mL) was added 10% Pd/C (400 mg), the mixture was stirred under hydrogen atmosphere at room temperature overnight. After 14 h of stirring, the Pd/C was removed by filtration and washed with MeOH. The filtrate was concentrated to afford the product which was used in the next step without further purification(897 mg, 5.64 mmol), colorless liquid, yield (98%). Example 116. Synthesis of (2R,3S)-2,3-bis(benzylamino)succinic acid. 1IO2C. > HO2C ""'Br H2N HOoC vNHBnreflux ..chhdhEtOH HO2C^""NHBn To a solution of meso-2,3-dibromosuccinic acid (50 g, 181 mmol) in EtOH (400 mL) was added benzylamine (150 mL) dropwise. After completion of addition, the mixture was heated to 90°C and stirred overnight. The mixture was cooled to room temperature and diluted with IPO. 6N HC1 was added until pH 4 to give white precipitates. The precipitates were filtered, rinsed with H2O and dried to give (2R,3S)-2,3-bis(benzylamino)succinic acid(50 g, 152 mmol, 84%). Example 117. Synthesis of (2R,3S)-2,3-diaminosuccinic acid. HO2C rtNHBn Pd/C/H2 HO2C. „oNH2 1 * J HO2C ""NHBn AcOH HC1 HO2C "NH2 To a solution of (2R,3S)-2,3-bis(benzylamino)succinic acid (18 g, 55 mmol) in AcOH (100 mL) and HO (100 mL) was added 10% Pd/C (3 g), the mixture was stirred under hydrogen atmosphere at 50°C overnight. After 48 h of stirring, the Pd/C was removed by filtration and washed with H2O. The filtrate was concentrated and the residue was dissovled in IN NaOH (200 mL). AcOH was added until pH 5 to give white precipitates. The precipitates were filtered, rinsed with H2O and dried to give (2R,3S)-2,3-diaminosuccinic acid (8.7 g, 58.8 g, 100%). Example 118. Synthesis of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid. 162 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 HO2C oNH2 cbz0,4NNaOH HO2Œ.^NHCbz HO2C; '"NH2 THF HO2C^ 'NHCbz To a solution of (2R,3S)-2,3-diaminosuccinic acid (31.74 g, 214 mmol) in THF (220 mL) and 4N NaOH (214 mL) was added CbzCl (61 mL, 428 mmol) dropwise at 0°C. After completion of addition, the mixture was allowed to warm to room temperature and stirred for 2 h.The reaction was diluted with H?O (1600 mL) and extrated with EA (2 x 15600 mL). The aqueous layer was separated and acidified with conc.HCl until pH 2 was reached. The resultant solution was stirred for 1 h and standed at 5°C to give white precipitates. The precipitates were filtered, rinsed with H2O and dried to give 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (52.2 g, 125 mmol, 59%). Example 119. Synthesis of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4- diyl)dicarbamate. HO2(L ^NHCbz 1) Ac^o ]5() oc << NHCbz HO2C "'NHCbz 2) CHC13 ’""NHCbz The solution of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (5.0 g, 12 mmol) in Ac2O ( 37.5 mL) was refluxed for 20 min, cooled and concentrated to give resulting anhydride. The diastereomeric mixture was treat with CHCI3(37 mL), the insoluble mew-isomer was filtered and washed with PE to give crystals of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4- diyl)dicarbamate (2.0 g, 5 mmol, 42%) Example 120. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(((benzyloxy)carbonyl)- amino)succinyl)bis(azanediyl))dibutanoate. O NHCbz Z-BuO^ / O HN ? . NH2 AZ t-BuoVv + oZ HATU, DIPEA * A J3 UA '"NHCbz CbzHN*^ >-NH $ Ut-BU To a solution of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4-diyl)dicarbamate (2.03 g, 5.1 mmol) and tert-butyl 4-aminobutanoate (1.79 g, 11.3 mmol) in DMF (45 mL) was added DIPEA (1.98 g, 15.3 mmol) at 0°C. After 5 min of stirring, HATU(4.66 g, 12.3 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 2 h. After completion of conversion, the mixture was diluted with H2O (90 mL) and extrated with EA (2 x 200 mL) and DCM (2 x 90 mL), the combined organic extract was washed with brine and dried over Na2SO4. The majority of solvent was removed under reduced pressure and a white solid was precipitated, which was collected and dried to give di-tert-butyl 4,4’-(((2R,3S)-2,3-bis 163 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (((benzyloxy)carbonyl)amino)succinyl)bis(azanediyl))dibutanoate (2.8 g, 4.0 mmol) as a white solid in 80% yield. Example 121. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-diaminosuccinyl)bis- (azanediyl))dibutanoate. To a solution of 4,4'-(((2R,3S)-2,3-bis(((benzyloxy)carbonyl)amino)succinyl)bis- (azanediyl))dibutanoate (2.8 g, 4.0 mmol) in MeOH (100 mL) was added 10% Pd/C (1.1 g), the mixture was stirred under hydrogen atmosphere at room temperature overnight. After 18 h of stirring, the Pd/C was removed by filtration and washed with MeOH. The filtrate was concentrated to afford the product which was used in the next step without further purification(940 mg, 2.2 mmol), colorless liquid, yield(55%). Example 122. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)butanamido)succinyl)bis(azanediyl))dibutanoate. To a solution of di-tert-butyl 4,4’-(((2R,3S)-2,3-diaminosuccinyl)bis(azanediyl))- dibutanoate (940 mg, 2.19 mmol) and 4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)butanoic acid (840 mg, 4.59 mmol) in DMF (25 mL) was added DIPEA (1.13 g, 8.76 mmol) at 0°C. After 5 min of stirring, HATU (1.74 g, 4.58 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 1 h. After completion of conversion, the mixture was diluted with H2O (50 mL) and extracted with EA (2 x 100 mL) and DCM (2 x 50 mL), the combined organic extracts were washed with brine and dried over Na2SO4. The majority of solvent was removed under reduced pressure and a white solid was precipitated, which was collected and dried to give di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl)bis-(azanediyl))dibutanoate (1.36 g, 1.79 mmol) as a white solid in 82% yield. Example 123. Synthesis of 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl) bis(azanediyl))dibutanoic acid. 164 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl)bis(azanediyl))dibutanoate (1.36 g, 1.79 mmol) in DCM (15 mL) was 5 added TFA (30 mL) at room temperature 0°C. After 18 h of stirring, the reaction was concentrated and the residue was dissovled in dry toluene. The solvent was removed by evaporation in vacuo to give white precipitates which was used in the next step without further purification (1.3 mg, 2.0 mmol). yield(100%). Example 124. Synthesis of PBD prodcut C-7. 10 O O O O To a solution of (llS,llaS,l TS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl) amino)-3- methylbutanamido)propanamido)benzyl)8,8'-(pentane-l,5-diylbis(oxy))bis(ll-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- 15 carboxylate) (215 mg, 0.17 mmol) and 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-1Hpyrrol-l-yl)butanamido)succinyl)bis(azanediyl))dibutanoic acid(115 mg, 0.18 mmol) in DMF (18 mL) was added DIPEA (90 mg, 0.70 mmol) at 0°C. After 5 min of stirring, HATU(132 mg, 0.35 mmol) was added. The mixture was allowed to warm to room temperature and stirred overnight. After completion of conversion, the mixture was diluted with H2O (2 mL) and 165 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 extrated with EA (2 x 40 mL) and DCM (2 x 20 mL), the combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by pre-HPLC to give PBD product C-6 (10 mg) as a white powder. Example 125. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)butanamido)succinyl)bis(azanediyl))dibutanoate. To a solution of di-tert-butyl 4,4'-(((2R,3S)-2,3-diaminosuccinyl)bis(azanediyl))- dibutanoate (900 mg, 2.09 mmol) and 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoic acid (840 mg, 4.97 mmol) in DMF (25 mL) was added DIPEA (0.93 g, 7.21 mmol) at 0”C. After 5 min of stirring, EDC (1.74 g, 9.06 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 1 h. After completion of conversion, the mixture was diluted with H2O (50 mL) and extracted with EA (2 x 100 mL) and DCM (2 x 50 mL), the combined organic extracts were washed with brine and dried over Na2SO4. The majority of solvent was removed under reduced pressure and a white solid was precipitated, which was collected and dried to give di-tert-butyl 4,4’-(((2R,3S)-2,3-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)succinyl)bis-(azanediyl))dibutanoate (1.27 g, 1.79 mmol) as a white solid in 83% yield. ESI MS m/z+ C34H49N6O12, cacld. 733.33 (M+ H), found 733.55. Example 126. Synthesis of 4,4'-(((2R,3S)-2,3-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)succinyl)bis(azanediyl))dibutanoic acid. Di-tert-butyl 4,4’-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl)bis(azanediyl))dibutanoate (502.0 mg, 0.685 mmol) in 1,4-dioxane (8 ml) at 4°C was added cone. HC1 (3 ml). The mixture was then stirred at RT for 30 min, diluted with 1,4-dioxane (8 ml), concentrated, co-evaporated with dioxane/toluene (1:1, 2x10 ml) to dryness and crystallized with EtOH/Hexane to afford the title compound (289.0 g, 68% yield). ESI MS m/z-i- C26H33N6O12, cacld. 621.21 (M+ H), found 621.55. 166 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 127. Synthesis of allyl ((S)-3-methyl-l-(((S)-l-((4-((((4- nitrophenoxy)carbonyl)-oxy)methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-l-oxobutan-2- yl)carbamate. Allyl ((S)-l-(((S)-l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2-yl)amino)-3- methyl-l-oxobutan-2-yl)carbamate (2.21 g, 5.86 mmol) in the mixture of dry pyridine (5 ml) and CH2C12(2O ml) was added 4-nitrophenyl carbonochloridate (1.82 g, 9.05 mmol). The mixture was stirred at RT for 8 hour, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:12) to afford the title compound (2.63 g, 83% yield). MS ESI m/z calcd for C26H31N4O9 [M+H]+ 543.21, found 543.60 Example 128. Synthesis of (llaS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl) 8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylene-5-oxo-2,3,11,1la-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazcpine-10(5H)- carboxylate). (llaS,lla'S)-8,8'-(Pentane-l,5-diylbis(oxy))bis(7-methoxy-2-methylene-2,3,ll,llatetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(10H)-one) (288.2 mg, 0.490 mmol) in dry CH3CN (5 ml) was added allyl ((S)-3-methyl-l-(((S)-l-((4-((((4-nitrophenoxy)carbonyl)oxy)- methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-l-oxobutan-2-yl)carbamate (770.2 mg, 1.420 mmol) and DIPEA (2 ml). The mixture was stirred at 45 °C for 8 h, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:8) to afford the title compound (492.0 mg, 72% yield). MS ESI m/z calcd for C73H91N10O18 [M+H]+ 1395.64, found 1395.95. 167 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 129. Synthesis of (1 laS,l la,S)-bis(4-((S)-2-((S)-2-amino-3-mcthylbutanamido)- propanamido)benzyl) 8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2-methylene-5-oxo- 2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-carboxylate). To a solution of (llaS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2- methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate) (274.2 mg, 0.197 mmol) and pyrrolidine (49 mg, 6.90 mmol) in dry DCM (5 mL) was added Pd(pph3)4 (152.0 mg, 0.132 mmol). The reaction was flushed with argon and stirred for 2h at room temperature, after which the reaction was diluted with DCM and washed sequentially with saturated aqueous NH4CI and brine. The organic phase was dried over NanSO4, filtered and concentrated. The residue was purified by chromatography (DCM/MeOH/EtgN = 6/1/0.02) to give the title compound (166.7 mg, 69% yield) as an off-white solid. MS ESI m/z calcd for C65H83Nio014 [M+H]+ 1227.60, found 1227.93. (1laS,l la'S)-Bis(4-((S)-2-((S)-2-amino-3-methylbutanamido)-propanamido)benzyl) 8,8'- (pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-carboxylate) (151.1 mg, 0.123 mmol) and 4,4'- (((2R,3S)-2,3-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)succinyl)bis- (azanediyl))dibutanoic acid (77.1 mg, 0.124 mmol) in DMA (5 ml) was added EDC (95.2 mg, 0.496 mmol). The mixture was stirred at RT for 8 h, concentrated and purified on C-18 HPLC C18 3pm column (25 x 4 cm) using gradient elution with a mixture of (A) acetonitrile and (B) water /0.1% formic acid (gradient : 15% A : 85% B up to 25% A : 75% B over 5 minutes, 35% A : 65% B for 15 minutes, 60% A : 40% B down to 50% A : 50% B over 15 minute, 15% A : 168 5 10 15 20 25 169 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 85% B for 5 minutes) with a 8 mL/minutc flow rate. The fractions containing the title compound were pooled, evaporated and dried in a desiccator with P2O5 to afford the C-8 PBD compound (149.2 mg, 67% yield). MS ESI m/z calcd for C91H111N16O24 [M+H]+ 1811.79, found 1812.35. Example 131. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxylethyl)pyrrolidin-1-yl)methanone. 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (10.20 g, 33.65 mmol) and (S)-pyrrolidin-2- ylmethanol (3.85 g, 38.09 mmol) in dry DMF (150 ml) was added EDC (19.50 g, 101.56 mmol). The mixture was stirred at RT for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:4) to afford the title compound (11.56 g, 89% yield). MS ESI m/z calcd for C20H23N2O6 [M+H]+ 387.15, found 387.65. Example 132. Synthesis of (S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)pyrrolidine- 2-carbaldehyde. Dess-martin o o To a solution of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)- pyrrolidin-l-yl)methanone (3.80 g, 9.84 mmol) in dry DCM (15 mL) was added Dess-Martin periodinane (DMP) (5.80 g, 13.67 mmol) under nitrogen at room temperature. After completion of conversion, the reaction solution was added aqueous Na2SOa and followed by aqueous NaHCOa, the mixture was stirred for further 15 minutes and extracted with DCM (3 x 20 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by S1O2 chromatography (DCM/EtOAc = 4/1) to give the title compound (3.13 g, 83% yield) as an off-white foam. MS ESI m/z calcd for C20H21N2O6 [M+H]+ 385.13, found 385.60, 404.75 [M+H2O+H]+. Example 133. Synthesis of 8-hydroxy-7-methoxy-2,3,ll,lla-tetrahydro-lHbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(10H)-onc. o H rVYOH Q^^OMeM^OH QN^M-oMe To a solution of S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)pyrrolidine-2- carbaldehyde (3.00 g, 7.80 mmol) in methanol (75 mL) was Pd/C (10% Pd, 50% wet, 250 mg) in 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 a hydrogenation shaker. After air in the shaker was vacuumed out, hydrogen (5 Psi) was conducted in. The reaction vessel was shaked overnight and filtered through Celite. The filtrant was concentrated and purified by SiO2 chromatography (DCM/MeOH/Et3N = 4/1/0.05) to give the title compound (1.66 g, 86% yield) as a off-white foam. MS ESI m/z calcd for C13H17N2O3 [M+H]+ 249.12, found 249.50. Example 134. Synthesis of 4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4- ((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-hydroxy-7-methoxy-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine- 10(5H)-carboxylate. NHBoc O HN"7 H CO2‘Bu H To a solution of (14S,17S)-tert-butyl l-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)-17- ((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate (10.15 g, 13.50 mmol) in dry THF(300 mL) was added DIPEA (3.15 g, 24.41 mmol) and a solution of triphosgene (5.15 g, 17.36 mmol) in dry THF (50 mL) at 4-8 °C. After 15 min of stirring, the solution was recooled to 4-8 °C and then added dropwise to a solution of 8-hydroxy- 7-mcthoxy-2,3,l1,1 la-tctrahydro- lH-bcnzo[c]pyrrolo[ l,2-aj[ l,4Jdiazcpin-5(10H)-onc (2.92 g, 11.76 mmol ) in mixture of THF (100 mL) at 4-8 °C in 45 min. The resultant solution was allowed to warm to room temperature and stirred overnight. The mixture was diluted with tolune (50 ml), evaporated in vacuo and purified by SiO2 chromatography (DCM/MeOH = 15/1) to give the title compound (10.02 g, 82% yield) as a yellow foam. MS ESI m/z calcd for C50H74N9O15 [M+H]+ 1040.52, found 1040.90. Example 135. Synthesis of (S)-4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14- (4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecanamido)benzyl 8-(3-iodopropoxy)-7-methoxy-5-oxo-2,3,l1,1la-tetrahydro-lHbenzo[e]pyrrolo[1,2-aJ[1,4]diazepine-10(5H)-carboxylate. I O N' CO2tBu H To a solution of 4-((14S,17S)-l-azido-17-(2-(tcrt-butoxy)-2-oxoethyl)-14-(4-((tertbutoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8- 170 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[c]pyrrolo[1,2-a][1,4]diazepine- 10(5H)-carboxylate (2.02 g, 1.94 mmol) in butanone (50 ml) was added CS2CO3 (2.50 g, 7.67 mmol) and 1,3-diiodopropane (2.50 g, 8.45 mmol). The mixture was stirred at 45 °C under dark for 36 h, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:5) to afford the title compound (2.08 g, 90% yield). MS ESI m/z calcd for C52H77IN9O15 [M+H]+ 1194.45, found 1194.95. Example 136. Synthesis of (S)-2-((S)-l-azido-14-methyl-12-oxo-3,6,9-trioxa-13- azapentadecanamido)-N-(4-(hydroxymethyl)phenyl)propanamide. HO ,n3 v/-nh2 To a solution of (14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecan-18-oic acid (3.02 g, 7.75 mmol) and (4-aminophenyl)methanol (1.05 g, 8.53 mmol) in DMA was added EDC (4.90 g, 25.52 mmol). The mixture was stirred at RT for 14 h, concentrated and purified on S1O2 column eluted with EtOAc/CH2C12 (1:8 to 1:3) to afford the title compound (3.52 g, 92% yield). MS ESI m/z calcd for C22H35IN6O7 [M+H]+ 495.25, found 495.60. Example 137. Synthesis of (HR,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-11-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate. 1). Triphosgene/THF/DIPEA MeO Na2S2O4 O A mixture of (S)-(4-(bcnzyloxy)-5-mcthoxy-2-nitrophcnyl)(2-(hydroxymethyl)-4- methylene-pyrrolidin-l-yl)methanone (3.90 g, 9.80 mmol) and Na2S2O4 (6.0 g, 34.47 mmol) in THF (60 ml) and H2O (40 ml) was stirred at room temperature for 20 h, adjusted pH to 10 with Na2CO3, concentrated, purified on C-18 short column eluted with HaO/MeOH/EtgN (from 99.4/0.5/0.2 to 50/49.8/0.2). The fractions containing the reduced amino product were pooled, concentrated, diluted with THF (50 ml), then cooled to 4-8 °C. Separately to a solution of 2-(lazido-14-methyl-12-oxo-3,6,9-trioxa-13-azapentadecanamido)-N-(4-(hydroxy-methyl)phenyl)- 171 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 propanamidc (6.70 g, 13.56 mmol) in dry THF (150 mL) was added DIPEA (3.50 g. 27.12 mmol) and a solution of triphosgene (4.10 g, 13.80 mmol) in dry THF (20 mL) at 4-8 °C. After 15 min of stirring at 4-8 °C, the solution was added dropwise to the above amino solution at 4-8 °C for 45 min. The mixture was warmed to RT and continued to stir for 2 h, concentrated, extracted with CH2CI2 (3 x 30 ml), dried over Na2SO4, evaporated and and purified on SiCL column eluted with EtOAc/CHzCL (1:10 to 1:5) to afford the title compound (7.23 g, 83% yield in two steps). MS ESI m/z calcd for C45H57IN8O12 [M+H]+ 889.40, found 889.90. Example 138. Synthesis of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-ll-hydroxy-7-methoxy-2- methylcne-5-oxo-2.3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][1,4]diazepine-10(5H)- carboxylate. To a solution of (llR,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-1l-hydroxy-7-methoxy-2-methylene-5- oxo-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-cafboxylate (3.80 g, 4.27 mmol) in dry DCM (40 mL) was added Dess-Martin periodinane (DMP) (2.80 g, 6.60 mmol) under nitrogen at room temperature. After completion of conversion, the reaction solution was added aqueous Na2SO3 and followed by aqueous NaHCOg, the mixture was stirred for further 15 minutes and extracted with DCM (3 x 20 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by S1O2 chromatography (DCM/EtOAc — 5/1 to 2:1) to give the title compound (2.99 g, 79% yield) as an off-white foam. MS ESI m/z calcd for C44H55N8Oi2 [M+H]+ 886.39, found 886.80. Example 139. Synthesis of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecanamido)bcnzyl 8,ll-dihydroxy-7-methoxy-2-methylene-5-oxo- 2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-carboxylate. To a solution of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-l1-hydroxy-7-methoxy-2-methylene-5- 172 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 oxo-2,3,11,1la-tctrahydro-lH-bcnzo[c]pyrrolo[l,2-aJ[l,4]diazcpinc-10(5H)-carboxylatc (2.90 g, 3.27 mmol) in 40 ml of CH2O2 was added 15 ml of CH3SO3H at 0 °C. The mixture was stirred at 0 °C for 10 min then r.t. for 1 h, diluted with CH2CI2, pH adjusted with cold 1.0 N NaHCOa to 4 and filtered. The aqueous layer was extracted with CH2C12(3 x 60 ml). The organic layers were combined, dried over NajSO^ filtered, evaporated and purified on SiO2 column chromatography (CH3OH/CH2Q2 1:15 to 1:5 ) to afford 1.95 g (75% yield) of the title product. MS ESI m/z calcd for C37H48lN8Oi2 [M+HJ+ 797.34, found 797.90. Example 140. Synthesis of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-1-azido-17-(2- (tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa- 13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydrolH-benzo[e]pyrrolo[l,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-mcthoxy-2- methylene-5-oxo-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][1,4]diazepine-10(5H)- To a solution of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl 8,11-dihydroxy-7-methoxy-2-methylene-5-oxo- 2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (402 mg, 0.504 mmol) and (S)-4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tertbutoxycarbonyl)amino)-butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzy1 8- (3-iodopropoxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[l,2- a][l,4]diazepine-10(5H)-carboxylate (650 mg, 0.544 mmol) in butanone (50 ml) was added CS2CO3 (0.50 g, 1.53 mmol). The mixture was stirred at 45 °C under dark for 36 h, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:8 to 1:3) to afford the title compound (809 mg, 86% yield). MS ESI m/z calcd for C89Hi24Ni7O27 [M+H]+ 1862.89, found 1863.45. Example 141. Synthesis of (llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-amino- 17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,1la- 173 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate. (llS,llaS)-4-((14S,17S)-l-Azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2- oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecanamido)-benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,l1,1la-hexahydro-lHbenzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)propoxy)-ll-hydroxy-7-methoxy-2-methylene-5- oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (750 mg, 0.402 mmol) in THF (8 ml) was added Me3P (1.0 M in toluene, 2.0 ml, 2.0 mmol) at 0 - 4 °C under N2. After stirred for 5 min, the ice bath was removed and the reaction mixture was stirred at RT for 2 h. Then, water (1 ml) was added and the mixture was stirred for 10 min. The mixture was diluted with 1,4-dioxane (10 ml), concentrated and co-evaporated with dioxane/toluene to dryness to yield the crude amino product (725 mg, ~99% yield) which was used directly for next step without further purification. MS ESI m/z calcd for C89H128N13O27 [M+H]+ 1810.90, found 1811.50. Example 142. Synthesis of an asymmetrically cross-linked PBD dimer C-9. To the above crude amino compound ((llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl- 12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-lamino-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo- 174 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo- 2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[1,2-a][l,4]diazepin-8-yl)oxy)propoxy)-11- hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate) in dry DMA (8 ml) was added 4,4’-(((2R,3S)-2,3-bis(3- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)succinyl)bis(azanediyl))dibutanoic acid (248.0 mg, 0.400 mmol) and EDC (500.0 mg, 2.60 mmol). The mixture was astirred for 24 h, concentrated and purified on Cis preparative HPLC (17% C18, 250 mm x 50 mm), eluted with water/CH3CN (from 80% water to 30% water in 40 min, 9 ml/min) to afford 488.1 mg (51% yield) of the C-9 product after drying under high vacuum. ESI MS m/z C115H156N19O37 [M+H]+, cacld.2395.08, found 2395.90. Example 143. Synthesis of an unsymmetrically cross-linked PBD dimer C-10. C-9 compound (465.0 mg, 0.194 mmol) was dissolved in DCM (4 ml), followed by addition of TFA (2 ml) at 0-4 °C. The reaction mixture was then stirred at RT for 1 h, diluted with toluene (5 ml), then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-3 (48.0 mg, 100% yield, 92% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-10 (373.1 mg, 85% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C106H140N19O35 [M+H]+ 2238.97, found 2239.50. Example 144. Synthesis of an asymmetrically cross-linked PBD dimer C-ll. 175 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 C-10 compound (235.0 mg, 0.105 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NaH2PO4 (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (43.0 mg, 0.084 mmol) in 4 portions in 2 h. The 5 reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-ll which was further purified by reverse phase HPLC (250 (L) mm x 50(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-ll (215.5 mg, 78% yield, 95% pure ) as a foam. ESI MS m/z: calcd for C124H174N19O44 [M+H]+ 2633.20, found 2633.85. 10 Example 145. Synthesis of an asymmetrically cross-linked PBD dimer C-12. To a solution of C-ll compound (65.0 mg, 0.0246 mmol) and 2,5,8,11,14,17,20,23- octaoxapentacosan-25-amine (15.1 mg, 0.0394 mmol) in dry DMA (2 ml) was added EDC (30.0 mg, 0.156 mmol). The reaction mixture was stirred at RT for 15 h, concentrated, purified by 15 reverse phase HPLC (250 (L) mm x 30 (d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-12 (60.2 mg, 81% yield, 95% pure by HPLC) as a foam. ESI MS m/z: calcd for Ci4iH209N20O5i [M+H]+ 2998.43, found 2999.40. Example 146. Synthesis of an asymmetrically cross-linked PBD dimer C-13. 176 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To the crude amino compound ((llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-amino- 17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)propoxy)-ll-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)- carboxylate) (-120 mg, -0.0662 mmol) in dry THF (10 ml) was added 3,4-dibromofuran-2,5- dione (16.8 mg, 0,0661 mmol). The mixture was stirred at RT for 4 h, then EDC (50.2 mg, 0.261 mmol). The mixture was continuously stirred for 12 h, concentrated, purified by SiOi column, eluted with MeOH/CfECh (1:12 to 1:6) to afford the pure product C-13 (112.2 mg, 83% yield) as a foam. ESI MS m/z: calcd for €93^206^,3029 [M+H]+ 2046.7073, found 2046.8260. Example 147. Synthesis of an asymmetrically cross-linked PBD dimer C-14. C-13 compound (100.2 mg, 0.0489 mmol) was dissolved in DCM (4 ml), followed by addition of TFA (2 ml) at 0-4 °C. The reaction mixture was then stirred at RT for 1 h, diluted with toluene (5 ml), then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-14 (94.3 mg, 102% yield, 93% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-14 (76.6 mg, 83% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C84H109Br2Ni3O27 [M+H]+ 1890.5995, found 1890.6250, 1893.6565 [M+H+2]+. Example 148. Synthesis of an asymmetrically cross-linked PBD dimer C-15. 177 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 C-14 compound (55.0 mg, 0.0291 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NalEPOi (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,ll,14,17,20,23,26-nonaoxaoctacosan-28-oate (47.2 mg, 0.0875 mmol) in 4 portions in 2 h. The reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-15 which was further purified by reverse phase HPLC (250 (L) mm x 50(d) mm, Cis column, 20-60% acctonitrilc/water in 40 min, v =8 ml/min) to afford the pure product C-15 (215.5 mg, 78% yield, 95% pure ) as a foam. ESI MS m/z: calcd for Ci03H146Br2Ni3O37 [M+H]+ 2314.8309, found 2314.8575, 2316.8705 [M+H+2]+, 2318.1445 [M+H+4]+. Example 149. Synthesis of an asymmetrically cross-linked PBD dimer C-16. To the crude amino compound ((llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-amino- 17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)propoxy)-ll-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)- carboxylate) (~120 mg, -0.0662 mmol) in dry THF (10 ml) was added 3,4-furan-2,5-dione (6.5 mg, 0,0661 mmol). The mixture was stirred at RT for 4 h, then EDC (50.2 mg, 0.261 mmol). The mixture was continuously stirred for 12 h, concentrated, purified by SiO2column, eluted with MeOH/CH2C12 (1:12 to 1:6) to afford the pure product C-16 (107.3 mg, 86% yield) as a foam. ESIMS m/z: calcd for C93H128N13O29 [M+H]+ 1890.8941, found 1890.8990. 178 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 150. Synthesis of an asymmetrically cross-linked PBD dimer C-17. C-16 compound (85.5 mg, 0.0452 mmol) was dissolved in DCM (4 ml), followed by addition of TFA (2 ml) at 0-4 °C. The reaction mixture was then stirred at RT for 1 h, diluted with toluene (5 ml), then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-17 (81.3 mg, 104% yield, 92% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-17 (67.4 mg, 86% yield, 96% pure ) as a foam. ESI MS m/z: calcd for CsÆi12N13O27 [M+H]+ 1734.7785, found 1734.8285. Example 151. Synthesis of an asymmetrically cross-linked PBD dimer C-18. C-17 compound (53.0 mg, 0.0305 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NaH2PO4 (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,ll,14,17,20,23,26-nonaoxaoctacosan-28-oate (47.0 mg, 0.0874 mmol) in 4 portions in 2 h. The reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-18 which was further purified by reverse phase HPLC (250 (L) mm x 50(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-18 (53.25 mg, 83% yield, 95% pure ) as a foam. ESI MS m/z: calcd for C103H148N13O37 [M+H]+ 2159.0099, found 2159.0890. Example 152. Synthesis of an asymmetrically cross-linked PBD dimer C-19. 179 DEMANDE OU BREVET VOLUMINEUX LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND PLUS D’UN TOME. CECI EST LE TOME 1 DE 2 CONTENANT LES PAGES 1 À 179 NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE VOLUME THIS IS VOLUME 1 OF 2 CONTAINING PAGES 1 TO 179 NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME : NOTE POUR LE TOME / VOLUME NOTE: DEMANDE OU BREVET VOLUMINEUX LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND PLUS D’UN TOME. CECI EST LE TOME 1 DE 2 CONTENANT LES PAGES 1 À 179 NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE VOLUME THIS IS VOLUME 1 OF 2 CONTAINING PAGES 1 TO 179 NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME : NOTE POUR LE TOME / VOLUME NOTE: 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 CROSS-LINKED PYRROLOBENZODIAZEPINE DIMER (PBD) DERIVATIVE AND ITS CONJUGATES FIELD OF THE INVENTION The present invention relates to novel cross-linked cytotoxic agents, pyrrolobenzodiazepine dimer (PBD) derivatives, and their conjugates to a cell-binding molecule, a method for preparation of the conjugates and the therapeutic use of the conjugates. BACKGROUND OF THE INVENTION Antibody-drug conjugates (ADCs) have great promise as anti-cancer drugs, with four ADCs have been approved by FDA and over 100 others in clinical investigation by using several different classes of cytotoxic payloads such as maytansines (Zhao, Robert Y, et al 2011 J. Med. Chern. 54, 3606; Widdison, W.; et al 2006 J. Med. Chem., 49, 4392-4408); auristatins and its parent compound dolastatin (Doronina, S. O. et al, Nat. Biotechnol. 2003, 21 (7), 778-784; Maderna, A., 2014 J. Med. Chem., 57(24), 10527—10543); calicheamycins (Ricart, A. D. 2011 Clin Cancer Res 2011;17:6417-6427; Ricart A. D, et al Nat Clin Pract Oncol. 2007;4:245-255), duocarmycins and its analog CC-1065 (Elgersma, R. C. 2015 Mol. Pharmaceutics, 12 (6), 1813-1835; Zhao, Robert Y. et al, 2012 J. Med. Chem 55, 766-782), tubulysins (Li, J. Y. et al, 2016 Cancer Cell. 29(1):117-29; Tumey, L. N. et al 2016 ACS Med Chem Lett. 7(ll):977-982; Huang Y.Y., Zhao, Robert Y. et al, Med Chem. #44, 249th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO2014009774), Eribulins (US20170252458); camptothecin and its analog SN-38 (Cardillo. T. M, et al, 2015 Bioconjug Chem. 26(5):919-31; Cardillo. T. M, et al, 2011 Clin Cancer Res. 17(10): 3157-69; Doi T, et al, 2017 Lancet Oncol. 18(11):1512-1522; PCT/JP2013/006069; US20160333112); amanitin (Moldenhauer G, et al, J. Natl. Cancer Inst. 2012; 104(8):622-34; Zhao, Robert Y. et al, PCT/IB2016/052246); vinblastine (Laguzza, B. C. et al, 1989 J. Med. Chem., 32, 548-555; Starling, J. J., et al 1992 Bioconj. Chem., 3, 315-322); doxorubicin (Yang, H. M. and Reisfeld, R. A., 1988 Proc. Natl. Acad. Sci. USA, 85, 1189-93; Sapra, P., et al 2005 Clin Cancer Res 11, 5257-64; Geretti, E. et al, 2015 Mol Cancer Ther 14, 2060-71); Cryptophycin (Verma, V. A. et al, 2015 Bioorg. Med. Chem. Lett., 25 (4), 864-8); Eribulin (Furuuchi, K. et al, poster MORAb-202, 8th World ADC, Sept., 19, 2017; US2017252458); and pyrrolobenzodiazepine dimers (PBDs) (Mantaj, J. et al 2016 Angew. Chem. Int. Ed. 55, 2-29; D. Antonow and D. Thurston, Chem. Rev. 2011, 111, 2815-2864). In recent years, there has been increasing applications of the PBD class of cytotoxic payloads as ADC therapeutics (Kung Sutherland, M. S., et al 2013 Blood 122, 1455-63; Jeffrey, S. C., 2013, Bioconjug Chem. 1256-63); Saunders, L. R. et al 2015 Sci Transi Med., 7(302):302ral36; Flynn, M. J. et al 2016 Mol Cancer Ther.,15(11), 2709-272;. Zhao, Robert Y., WO2015028850; GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Donnell, A. F. et al 2017 Bioorg Med Chem Lett 27, 5267-5271) due to PBDs’ extream potency against most of the US National Cancer Institute’s (NCI’s) NCI 60-tumor-cell line panels along with unique mechanism of antitumor antibiotics activity (Mantaj, J. et al 2016 Angew. Chem. Int. Ed. 55, 2-29). 5 As shown in structures below, all PBDs are usually conjugated to an antibody with one conditionally stable linker, because it is normally believed that linkers of ADCs must be stable while the ADC is circulating in the blood to limit off-target toxicity, but allow for release of the drug once it is inside the target cancer cells (Alain Beck 2017 Nature Reviews Drug Discovery, doi:10.1038/nrd.2016.268). Unfortunately the most advanced in clinic evaluation of Vadastuxi- 10 mab talirine (CD33 antibody-PBD conjugate having a conditionally stable linker) (Stein, E. M., et al, 2018 Blood 131, 387-96) experienced a set-back because of liver toxicity that apparently killed four patients in the early stage trials (www.xconomy.com/seattle/2017/06/19). malennidocaproyl SGD-1910 । | SGD-la82 The SGD-1882 conjugate, studied by Seattle Genetics, O O | SG3199 SG3249 conjugate| | The SG3249 conjugate studied by ADC Therapeutics Ltd and by Medlmmune/AstraZeneca. The D-212 conjugate studied by Cellerant Therapeutics, 2 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Indolinobenzodiazepine dimer conjugate An IGN conjugate, studied by ImmunoGen, Inc., A Tomaymysin dimer (a PBD) conjugates for Sanofi, A PBD dimer conjugate, studied by Hangzhou DAC Biotech Co., Ltd, Linker-—mAb A PBD dimer conjugate, studied by Hangzhou DAC Biotech Co., Ltd, 10 The SG-3199 conjugate, studied by Stemcentrx/Abbvie and by ADC Therapeutics Ltd. 3 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 O O The SG-3199 conjugate, studied by Allozyne, Inc. The SG2057 conjugate, used by Gcncntcch Inc. O O A PBD conjugate via a traceless linker by Genentech Inc 10 Here we are disclosing the conjugation of PBD dimer derivatives through a dual cross linker that attached both N10 positions of the PBD dimer, thus DNA alkylation sites of the imine warheads are in the forms of prodrugs wherein the conjugate linkers have to be decomposed prior to converting back the potency. Indeed, the application of the dual cross-linking prodrug strategy of PBD conjugation demonstrated much wider thereapeutic windows than the single 15 linkage of PBD conjugates both in vitro and in vivo. It therefore may have much potentially better antitumor antibiotic activities for PBD conjugates in clinic applications. 4 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 SUMMARY OF THE INVENTION The first embodiment of this invention is to disclose a dual linkage of conjugation of PBDs via attachement of both N10 positions of pyrrolo[2,l-c][l,4]benzodiazepine derivatives to a cell binding molecule, as shown in Formula (I), for targeted treatment of cell proliferation. or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers; wherein: represents an optional single bond or can be absent; = represents an optional single bond or a double bond; V and V’, the same or different, are independently selected from the group consisting of H, OH, -NHOH; OR5(an ether); OCOR5 (an ester); OCOOR5(a carbonate); NR5R5’, NR5COR5’, or NRsNRs’NRs” (an amine); OCONR5R5’ (a carbamate); NR5(C=NH)NR5’R5’’ ( a guanidinum); NRsCONRs’Rs” (a urea); OCSNHR5 (a thiocarbamate); -SH (a thiol); -SR5 (a sulfide ); SOR5 a sulphoxide (a sulphoxide ); SOOR5 (a sulfone); SO3, HSO3, HSO2, or a salt of HSO3', SOs2- or - HSO2' (a sulphite); OSO3 (a bisulphite); NR5SOOR5’ (a sulfonamide); H2S2O5 or a salt of S2O52’(a metabisulfite); PO3SH3, PO2S2H2, POS3H2, PS4H2 or a salt of PO3S3’, PO2S23', POS3 ' , PS?'(mono- ,di-, tri-, and tetra-thiophosphatc); (RsO^OSRs’ (a thiophosphate ester); HS2O3 or a salt of S2O32'(a thiosulfate); HS2O4 or a salt of S2O42' (a dithionite); P(=S)(OR5)(S)(OH) (a phosphorodithioate) or a salt thereof form with a cation; -NR5OR5’ (a hydroxylamine derivative); RsC(=O)NOH (a hydroxamic acid ) or a salt formed with a cation; HOCH2SO2', or its salts (a formaldehyde sulfoxylate ); NR5COR5 (an amide); N3 (an azido); CN (a cyano); X (a halo); CtRsXRs’XRs”) (a trialkyl), OP(O)(OR5)(NHR5’) or OP(O)(NHR5)(NHR5) (a phosphoramidate (phosphoramidic acid), or PlRsXRyXRs”) triarylphosphonium; Aa (an amino acid), or NRsCO(Aa)t (a peptide ), wherein Aa is an amino acid or a polypeptide containing between t =1- 100 amino acid units; an aminoacidderived group, such as an a-, 0-, y-, or co-aminoacid, or an unnatural aminoacid; where R5, R5’ and Rs” are described below; 5 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 1, m, q, 1’, m’ and q’ are independently a number of 0, 1, 2. 3, 4, or 5; n is l~30; X, X’, Y and Y’ the same or different, independently, represent N, O, S, an alkyl, such as CH2 or CHR5, an alkene, such as =CH- or =CRs-, an ether, such as C(ORs )H-.; Z and Z’ the same or different, independently, represent N, CH, CR5, COH, CNHi,CNHR5 or COR5. or Z and Z’ link together with -COR5OC-. R5 is independently selected from Ci~C8 alkyl and aryl; Ri, R2, R3, R4, Ri’, R2’, R3’, and R4’ are the same or different and independently chosen from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, -(OCH2CH2)tR5 ( a polyethylene glycol unit), halogen, NH(C=NH)NH2 (a guanidinium), -OR5, -NR5R5', -NO2, -NCO, -NR5COR5', -SR5, -SOR5(a sulfoxide), -SO2Rs (a sulfone), —SOfM+ or -SO3H (a sulfonate), -OSO3'M+ or OSO3H (a sulfate), -SO2NR5R5’ (a sulfonamide), CN (a cyano), N3 (an azido), —COR5, —OCOR5, - OCONR5R5', CF3, OR5, Aryl, heterocycle, or P^jRsRs’Rs” and the linking group (L”) with the reactive group or a cell binding agent bonded thereto when Q, Q’ and T are absent; R5, R5’ and R5” are independently selected from H, Ci~Cg of alkyl, alkenyl, alkinyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts; In addition, Ri and R2 join together, or Rf and R2’ join together form a =O (ketone), =S, =NR, -C(=O)R, or a double bond containing group =CRsR5’; and Ri and R2join together, or Rf and R2’ join together, or R3 and R4 join together or R3’ and R4’ join together form a C3-C12 aromatic, heterocyclic, or heteroaryl ring; G is -CH2-, O, -N(Rs)-, S, -P(O)(OR5)-, -P(O)(N R5R5 )-, are defined above; ; wherein Z and Z’ U and U’ are independently C(O), C(O)O, C(O)NH, C(O)N(R5), C(=NH), C(=NH)O, C(=NH)NH, C(=NH)N(R5), -C=N-, C(=S), C(O)S, C(S)NH, C(S)N(R5), S(O), S(O)O, S(O)NH, S(O)(OR5), S(O)(N(R5)), S(O2), S(O2)O, P(O)(OR5), P(O)(OR5)O, P(O)(NH2), P(O)(NR5R5 ), P(O)(OR5)NH-, P(O)(OR5)NR5’-, P(O) (N(R5R5 ) (N(R5), P(S)(OR5), P(S)(OR5)O, P(S)(NH2), P(S)(NR5R5 ), P(S)(OR5)NH-, P(S)(OR5)NR5 -, P(S)(N(R5R5’)N(R5), Rs, R5O; E( and E2 are independently S, R5S, C(O)S, C(O)NH, C(O)O, C(O)R5S, C(=NH)NH, C(=NH)N(R5), C(=NH)S, -C=N-, C(=S)S, C(O)S, C(=S)NH, C(=S)N(R5), Ar-S, NC(O)CH2S, ArC(O)CH2S, S-S, 6 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 o wherein the chemical bond in the middle of two atoms means it can link either adjoining two atoms; Li, L2 and L’ are independently a linker, or a linker which has a functional group on the linker that enables reaction with a cell-binding agent (CBA), Q. Lj, L2 and L’ are independently preferred a releasable linker, which has the formula of: —Ww—(Aa)r—Tt—; or —Ww— (Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—; wherein: -W- is a Stretcher unit; w is 0 or 1; -Aa- is independently an Amino Acid unit; r is independently an integer ranging from 0 to 100; -T- is a Spacer unit, which can be a linear alkyl or branched alkyl, or polyethylene glycol spacer; and t is 0, or 1-100. The Stretcher unit W may independently contain a self-immolative spacer, peptidyl units, a hydrazone bond, a disulfide, an ester, or a thioether bond; w is 1 or 2 or 3; Preferably Li and L2 are independently selected from O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3 ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula (OCH2CH2)pOR3, or (OCH2CH-(CH3))pOR3, or NH(CH2CH2O)PR3, or NH(CH2CH(CH3)O)PR3> or N[(CH2CH2O)pR3]-[(CH2CH2O)p R3’], or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; Ci-Cs of alkyl; C2-Cs of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3- Cg of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa)r, r =l-12(one to 12 amino acid units), which is composed from natural or unnatural amino acids, or the same or different sequences of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit; Q is a cell binding molecule, or a functional group that enables reaction with a cell-binding agent, or a functional group capable of reacting with a linker attached on a cell binding agent. The function group is chosen from a thiol, an amine, a hydrazine, an alkoxylamino, a disulfide substituent, a maleimido, a haloacetyl group, an A-hydroxy succinimide ester, a keton, an ester, an aldehyde, an alkynyl, an alkenyl, or protected thiol or disulfide group, such as SAc, SSRi or SSAr. Ar is aromatic group or hetero aromatic group. Q is preferably a cell-binding agent/ molecule, selected from the group consisting of an antibody, a single chain antibody, an 7 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 10 antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, a domain antibody fragment that binds to the target cell, an adnectin that mimics antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient¬ transport molecule (a transferrin), and a binding peptide, protein, or small molecule attached on albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid. In a second embodiment, the present invention discloses mono-linkage of conjugation of PBDs derivatives to a cell binding molecule, as shown in Formula (II), (III) and (IV), for targeted treatment of cell proliferation. wherein ==, , X, X’, Y, Y’, Z, Z’,1, F, m, m’, n, q, q’, Ri, Rf, R2, R2’, R3, R3’, R4, R4’, V, V*, U, U’ Li, Lz>. G, Q,Ei, and E2 are the same defined as in Formula (I). In a third embodiment, the present invention discloses a therapeutic composition comprising: (1) an effective amount of one or more of the pyrrolo[2,l-c][1,4]benzodiazepine derivatives linked to a cell binding agent as the conjugate structure of Formula (I), (II), (III) or 8 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV); and (2) a pharmaceutically acceptable carrier, diluent, or excipient, of Formula (I) ~ (IV) of the application, to kill target cells or tissues containing target cells. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the synthesis of cross-linked benzodiazepine dimers. Figure 2 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 3 shows the synthesis of cross-linked benzodiazepine dimers. Figure 4 shows the synthesis of cross-linked benzodiazepine dimers and a conjugate. Figure 5 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 6 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 7 shows the synthesis of benzodiazepine dimers. Figure 8 shows the synthesis of cross-linked benzodiazepine dimers and a conjugate. Figure 9 shows the synthesis of benzodiazepine dimers. Figure 10 shows the synthesis of an antibody conjugate of cross-linked benzodiazepine dimers. Figure 11 shows the synthesis of benzodiazepine dimers. Figure 12 shows the synthesis of conjugates of benzodiazepine dimers and their linkers. Figure 13 shows the synthesis of the cross-linkers for conjugates of benzodiazepine dimers. Figure 14 shows the synthesis of the cross-linkers for conjugates of benzodiazepine dimers. Figure 15 shows the synthesis of the cross-linkers for conjugates of benzodiazepine dimers. Figure 16 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 17 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 18 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 19 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 20 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 21 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 22 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 23 shows the synthesis of cross-linked benzodiazepine dimers. Figure 24 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 25 shows the synthesis of cross-linked benzodiazepine dimers. Figure 26 shows the synthesis of cross-linked benzodiazepine dimers. Figure 27 shows the synthesis of cross-linked benzodiazepine dimers. Figure 28 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. 9 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Figure 29 shows the synthesis of conjugates of cross-linked benzodiazepine dimers. Figure 30 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 31 shows the synthesis of the intermediates for conjugates of benzodiazepine dimers. Figure 32 shows the synthesis of conjugates of benzodiazepine dimers. Figure 33 shows the in vivo activity of conjugates of cross-linked benzodiazepine dimers. Figurre 34 shows the hematoxylin and eosin stainings of mouse liver tissues after dosing the conjugates CC-4, CC-29, T-DM1 and PBS buffer in ICR mice on day 5. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS: “Alkyl” refers to an aliphatic hydrocarbon group or univalent groups derived from alkane by removal of one or two hydrogen atoms from carbon atoms. It may be straight or branched having Ct-Cs (1 to 8 carbon atoms) in the chain. "Branched" means that one or more lower C numbers of alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, Lpropyl, n-butyl, /-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methyl-hexyl, 2,2- dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2- methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. A Ci-C8 alkyl group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-C8 alkyl,-O-(Ci-C8 alkyl), -aryl, -C(O)R’, -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR’, - C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-C8 alkyl and aryl. “Halogen” refers to fluorine, chlorine, bromine or iodine atom; preferably fluorine and chlorine atom. “Heteroalkyl” refers to C2-C8 alkyl in which one to four carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. “Carbocycle” refers to a saturated or unsaturated ring having 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as a bicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicycle [5,6] or [6,6] system. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, - cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, - 1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. A “C3-C8 carbocycle” refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated 10 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 nonaromatic carbocyclic ring. A Cs-Cg carbocycle group can be unsubstituted or substituted with one or more groups including, but not limited to, -Ci-Cs alkyl,-O-(Ci-Cg alkyl), -aryl, -C(O)R', - OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R’)2, -NHC(O)R', -SR', -S(O)R',-S(O)2R’, - OH, -halogen, -N3, -NH2, -NH(R'), -N(R') 2 and -CN; where each R' is independently selected from -Ci-Cg alkyl and aryl. “Alkenyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, heptenyl, octenyl. “Alkynyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond which may be straight or branched having 2 to 8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, npentynyl, hexylynyl, heptynyl, and octynyl. “Alkylene” refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3- propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like. “Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (-CH=CH-). “Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl. “Aryl” or Ar refers to an aromatic or hetero aromatic group, composed of one or several rings, comprising three to fourteen carbon atoms, preferentially six to ten carbon atoms. The term of “hetero aromatic group” refers one or several carbon on aromatic group, preferentially one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferentially by O, S, and N. The term aryl or Ar also refers to an aromatic group, wherein one or several H atoms are replaced independently by -R’, -halogen, -OR’, or -SR’, -NR’R”, -N=NR’, -N=R’, -NR’R”,- NO2, -S(O)R’, -S(O)2R’, -S(O)2OR’, -OS(O)2OR’, -PR’R”, -P(O)R’R”, -P(OR’)(OR”), - P(O)(OR’)(OR”) or -OP(O)(OR’)(OR”) wherein R’, R” are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts. 11 5 10 15 20 25 30 “Heterocycle” refers to a ring system in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group of O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226. Preferred nonaromatic heterocyclic include epoxy, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, as well as the fused systems resulting from the condensation with a phenyl group. The term “heteroaryl” or aromatic heterocycles refers to a 3 to 14, preferably 5 to 10 membered aromatic hetero, mono-, bi-, or multi-cyclic ring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl- 7V-oxide, as well as the fused systems resulting from the condensation with a phenyl group. “Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”, “heterocyclic” and the like refer also to the corresponding “alkylene”, “cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”, “heterocyclene” and the likes which are formed by the removal of two hydrogen atoms. “Arylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2- naphthylethan-l-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. “Hcteroarylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced with a heteroaryl radical. Examples of heteroarylalkyl groups are 2-benzimidazolylmethyl, 2-ftuylethyl. Examples of a “hydroxyl protecting group” includes, methoxymethyl ether, 2- methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, Z-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate. 12 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 “Leaving group” refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art, and examples include, a halide (e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. A preferred leaving group is selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, anhydrides formed its self, or formed with the other anhydride, e.g. acetyl anhydride, formyl anhydride; or an intermediate molecule generated with a condensation reagent for peptide coupling reactions or for Mitsunobu reactions. The following abbreviations may be used herein and have the indicated definitions: Boc, tert-butoxy carbonyl; BroP, bromotrispyrrolidinophosphonium hexafluorophosphate; CDI, 1,1*- carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DEAD is diethylazodicarboxylate, DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl-aluminium hydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethyl phosphorocyanidate; DMA, N,N-dimethyl acetamide; DMAP, 4-(N, Ndimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DTPA is diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDC, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; EtOAc is ethyl acetate; Fmoc is N-(9-fluorenylmethoxycarbonyl); HATU, O-(7-azabenzotriazol-l-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS, N-Hydroxysuc-cinimide; MeCN is acetonitrile; MeOH is methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate-buffered saline (pH 7.0-7.5); Ph is phenyl; phe is L-phenylalanine; PyBrop is bromo-tris-pyrrolidinophosphonium hexafluorophosphate; PEG, polyethylene glycol; SEC, size-exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Vai, valine; TLC is thin layer chromatography; UV is ultraviolet. The “amino acid(s)” can be natural and/or unnatural amino acids, preferably alpha-amino acids. Natural amino acids are those encoded by the genetic code, which are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine. The unnatural amino acids are derived forms of proteinogenic amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gammaaminobutyric acid (the neurotransmitter), ornithine, citrulline, beta alanine (3-aminopropanoic acid), gamma-carboxyglutamate, selenocysteine (present in many noneukaryotes as well as most 13 5 10 15 20 25 30 35 eukaryotes, but not coded direcfly by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formyhnetiiionine (which is often the initial amino acid of proteins in bacteria, mitochondria, and chloroplasts), 5-hydroxytryptophan, L-dihydroxyphcnylalaninc, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimetics. Analogs are compounds having the same general H2N(R)CHCO2H structure of a natural amino acid, except that the R group is not one found among the natural amino acids. Examples of analogs include homoserine, norleucine, methionine-sulfoxide, and methionine methyl sulfonium. Preferably, an amino acid mimetic is a compound that has a structure different from the general chemical structure of an alpha-amino acid but functions in a manner similar to one. The term "unnatural amino acid" is intended to represent the "D" stereochemical form, the natural amino acids being of the "L" form. When 1~8 amino acids are used in this patent application, amino acid sequence is then preferably a cleavage recognition sequence for a protease. Many cleavage recognition sequences are known in the art. See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995). In particular, the sequence is selected from the group consisting of Val-Cit, Ala-Vai, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-LeuLys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Asp-Lys, Asp-Glu, Glu-Lys, Lys, Cit, Ser, and Glu. The “glycoside” is a molecule in which a sugar group is bonded through its anomeric carbon to another group via a glycosidic bond. Glycosides can be linked by an O- (an Oglycoside), N- (a glycosylamine), S-(a thioglycoside), or C- (a C-glycoside) glycosidic bond. Its core the empirical formula is Cm(H2O)„ (where m could be different from n, and m and n are < 36), Glycoside herein includes glucose (dextrose), fructose (levulose) allose, altrose, mannose, gulose, iodose, galactose, talose, galactosamine, glucosamine, sialic add, A-acetylglucosamine, sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrins, raffinose, Glucuronic acid (glucuronide), and stachyose. It can be in D form or L form, 5 atoms cyclic furanose forms, 6 atoms cyclic pyranose forms, or acyclic form, a-isomer (the -OH of the anomeric carbon below the plane of1he carbon atoms of Haworth projection), or a p-isomer (the -OH of the anomeric carbon above the plane of Haworth projection). It is used herein as a monosaccharide, disaccharide, polyols, or oligosaccharides containing 3-6 sugar units. The term "antibody," as used herein, refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that 14 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce auto¬ immune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. Antibodies useful in the invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F(ab’) fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope¬ binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens. An “enantiomer”, also known as an “optical isomer”, is one of two stereoisomers that are mirror images of each other that are non-superposable (not identical), much as one’s left and right hands are the same except for being reversed along one axis (the hands cannot be made to appear identical simply by reorientation). A single chiral atom or similar structural feature in a compound causes that compound to have two possible structures which are non-superposable, each a mirror image of the other. The presence of multiple chiral features in a given compound increases the number of geometric forms possible, though there may be some perfect-mirrorimage pairs. Enantiopure compounds refer to samples having, within the limits of detection, molecules of only one chirality. When present in a symmetric environment, enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light (+/—) by equal amounts but in opposite directions (although the polarized light can be considered an asymmetric medium). They are sometimes called optical isomers for this reason. A mixture of equal parts of an optically active isomer and its enantiomer is termed racemic and has zero net rotation of plane-polarized light because the positive rotation of each (+) form is exactly counteracted by the negative rotation of a (—) one. Enantiomer members often have different chemical reactions with other enantiomer substances. Since many biological molecules are enantiomers, there is sometimes a marked difference in the effects of two enantiomers on biological organisms. In drugs, for example, often only one of a drug's enantiomers is responsible for the desired physiologic effects, while the other enantiomer is less active, inactive, or sometimes even productive of adverse effects. Owing to this discovery, drugs composed of only one enantiomer ("enantiopure") can be developed to enhance the pharmacological efficacy and sometimes eliminate some side effects. 15 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Isotopes are variants of a particular chemical element which differs in neutron number. All isotopes of a given element have the same number of protons in each atom. Each atomic number identifies a specific element, but not the isotope; an atom of a given element may have a wide range in its number of neutrons. The number of nucleons (both protons and neutrons) in the nucleus is the atom's mass number, and each isotope of a given element has a different mass number. For example, carbon-12, carbon-13 and carbon-14 are three isotopes of the element carbon with mass numbers 12, 13 and 14 respectively. The atomic number of carbon is 6, which means that every carbon atom has 6 protons, so that the neutron numbers of these isotopes are 6, 7 and 8 respectively. Hydrogen atom has three isotopes of protium ^H), deuterium (2H), and tritium (3H), which deuterium has twice the mass of protium and tritium has three times the mass of protium. Isotopic substitution can be used to determine the mechanism of a chemical reaction and via the kinetic isotope effect. Isotopic substitution can be used to study how the body affects a specific xenobiotic/chemical after administration through the mechanisms of absorption and distribution, as well as the metabolic changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), and the effects and routes of excretion of the metabolites of the drug. This study is called pharmacokinetics (PK). Isotopic substitution can be used to study of the biochemical and physiologic effects of drugs. The effects can include those manifested within animals (including humans), microorganisms, or combinations of organisms (for example, infection). This study is called pharmacodynamics (PD). The effects can include those manifested within animals (including humans), microorganisms, or combinations of organisms (for example, infection). Both together influence dosing, benefit, and adverse effects of the drug, isotopes can contain a stable (non-radioactive) or an unstable element. Isotopic substitution of a drug may have a different thrapeutical efficacy of the original drug. “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. “Pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine. “Pharmaceutically acceptable excipient” includes any carriers, diluents, adjuvants, or vehicles, such as preserving or antioxidant agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media 16 5 10 15 20 25 30 35 and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositionsis contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations. As used herein, "pharmaceutical salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium. The pharmaceutical salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared via reaction the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, U^ed., Mack Publishing Company, Easton, PA, 1985, p. 1418. "Administering" or “administration" refers to any mode of transferring, delivering, introducing or transporting a pharmaceutical drug or other agent to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. Also contemplated by the present invention is utilization of a device or instrument in administering an agent. Such device may utilize active or passive transport and may be slow-release or fast-release delivery device. “Therapeutically effective amount” means an amount of a compound/ medicament according to the present invention effective in preventing or treating the herein referred pathological condition. The term “patient”, or “patient in need thereof’, is intended for an animal or a human being affected or likely to be affected with the herein referred pathological condition. Preferably, the patient is human. 17 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 In the context of cancer, the term "treating" includes any or all of: preventing growth of tumor cells or cancer cells, preventing replication of tumor cells or cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease. In the context of an autoimmune disease, the term "treating" includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells capable of producing an autoimmune antibody, lessening the autoimmuneantibody burden and ameliorating one or more symptoms of an autoimmune disease. In the context of an infectious disease, the term "treating" includes any or all of: preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease. Examples of a "mammal” or “animal" include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. The term “compound”, "cytotoxic agent”, "cytotoxic compound," "cytotoxic dimer" and "cytotoxic dimer compound" are used interchangeably. They are intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof. The term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs, and prodrug salts of a compound of all the formulae disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salt" "prodrug," "prodrug salt," "conjugates," "conjugates salt," "solvate," "hydrate," or "polymorph" in certain aspects of the invention described in this application shall not be interpreted as an intended omission of these forms in other aspects of the invention where the term "compound" is used without recitation of these other forms. The term "imine reactive reagent" refers to a reagent that is capable of reacting with an imine group. Examples of imine reactive reagent includes, but is not limited to, sulfites (H2SO3, H2SO2 or a salt of HSOf, SO? or HSÛ2’ formed with a cation), metabisulfite (H2S2O5 or a salt of S2O? formed with a cation), mono, di, tri, and tetra-thiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3 or a salt of PO3S3-, PO2S23-, POS33 or PS43’ formed with a cation), thio phosphate esters ((RsOjaPSfORs), R5SH, R5SOH, R5SO2H, R5SO3H), various amines (hydroxyl amine (NH2OH), hydrazine (NH2NH2), NH2OR5, R5NHR5-, NH2R5), NH2-CO-NH2, NH2-C(=S)- NH2), thiosulfate (H2S2O3 or a salt of §2O32' formed with a cation), dithionite (H2S2O4 or a salt of S2O42” formed with a cation), phosphorodithioate (P(=S)(OR5)(SH)(OH) or a salt thereof formed with a cation), hydroxamic acid (R5C(=O)NHOH or a salt formed with a cation), hydrazide 18 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (R5CONHNH2), formaldehyde sulfoxylate (HOCH2SO2H or a salt of HOCH2SO2 formed with a cation, such as HOCH2SO2'Na+), glycated nucleotide (such as GDP-mannose), fludarabine or a mixture thereof, wherein R5 and R5' are each independently a linear or branched alkyl having 1 to 8 carbon atoms and are substituted with at least one substituent selected from —N(Rs)(Rs>), - COjH. -SO3H, and -PO3H; R5 and Rs can be further optionally substituted with a substituent for an alkyl described herein; Preferably, the cation is a monovalent cation, such as Na+ or K+. Preferably, the imine reactive reagent is selected from sulfites, hydroxyl amine, urea and hydrazine. More preferably, the imine reactive reagent is NaHSOa or KHSO3. “Cell binding agents” or “Cell binding molecules” may be of any kind presently known, or that become known, and include peptides and non-peptides. Generally, these can be antibodies (especially monoclonal antibodies) or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, growth factors, nutrient-transport molecules (such as transferrin), or any other cell binding molecule or substance (such as vitamins). More specific examples of cell binding agents that can be used include: monoclonal antibodies; single chain antibodies; fragments of antibodies such as Fab, Fab’, F(ab’)2, Fv, {Parham, 131 J. Immunol. 2895-2902 (1983); Spring et al, 113 J. Immunol. 470-478 (1974); Nisonoff et al, 89 Arch. Biochem. Biophys. 230-244 (I960)}, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens; interferons; peptides; lymphokines such as IL-2, IL-3, IL-4, IL-6; hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such as androgens and estrogens; growth factors and colony-stimulating factors such as EGF, TGFa, insufin like growth factor (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF {Burgess, 5 Immunology Today 155-158 (1984)}; vitamins, such as folate and; transferrin {O’Keefe et al, 260 J. Biol. Chern. 932-937 (1985)}. Monoclonal antibody technology permits the production of extremely selective cell binding agents in the form of specific monoclonal antibodies. Particularly well known in the art are techniques for creating monoclonal antibodies produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins such as viral coat proteins. Selection of the appropriate cell binding agent is a matter of choice that depends upon the particular cell population that is to be targeted, but in general monoclonal antibodies are preferred if an appropriate one is available. CROSS-LINKED PBDs AND THEIR CONJUGATES. 19 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 The cross-linked PBD dimer derivatives that have conjugated or can be conjugated to a cell-binding molecule for targeted treatment of cell proliferation, have the structure of Formula (I) as shown below: or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers; wherein: represents an optional single bond or can be absent; ===== represents an optional single bond or a double bond; V and V’, the same or different, are independently selected from the group consisting of H, OH, -NHOH; ORs(an ether); OCORs (an ester); OCOORs(a carbonate); NR5R5’, NR5COR5’, or NRgNRs’NRs” (an amine); OCONR5R5’ (a carbamate); NRs(C=NH)NR5’R5’’ ( a guanidinum); NRsCONRs’Rs” (a urea); OCSNHR5 (a thiocarbamate); -SH (a thiol); -SR5 (a sulfide ); SOR5 a sulphoxide (a sulphoxide ); SOOR5 (a sulfone); SO3, HSO ? HSO2, or a salt of HSO3', SO32' or - HSO< (a sulphite); OSO3 (a bisulphite); NR5SOOR5’ (a sulfonamide); H2S20s or a salt of SiO?2’(a metabisulfite); PO3SH3, PO2S2H2, POS3H2, PS4H2or a salt of PO3S3; PO2S23', POS/h PS43' (mono- ,di-, tri-, and tetra-thiophosphate); (R5O)2POSR< (a thiophosphate ester); HS2O3 or a salt of S2O32~ (a thiosulfate); HS2O4 or a salt of S2Oa2" (a dithionite); P(=S)(ORs)(S)(OH) (a phosphorodithioate) or a salt thereof form with a cation; -NR5OR5’ (a hydroxylamine derivative); RsC(=O)NOH (a hydroxamic acid ) or a salt formed with a cation; HOCH2SO2', or its salts (a formaldehyde sulfoxylate ); NR5COR5 (an amide); O-glycoside; N3 (an azido); CN (a cyano); X (a halo); C(R5)(R5’)(R5”) (a trialkyl), OP(O)(OR5)(NHR5 ) or OP(O)(NHR5)(NHR5) (a phosphoramidate (phosphoramidic acid), or P(Rs)(R5>)(R5”) triarylphosphonium; Aa (an amino acid), or NRsCO(Aa)t (a peptide ), wherein Aa is an amino acid or a polypeptide containing between t =1~ 100 amino acid units; an aminoacid-derived group, such as an a-, p-, y-, or œ-aminoacid, or an unnatural aminoacid; where R5, R5’ and R5” are described below; 1, m, q, T, m’ and q’ are independently a number of 0, 1, 2, 3, 4, or 5; n is l~30; 20 5 10 15 20 25 21 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 X, X’, Y and Y’ the same or different, independently, represent N, O. S, an alkyl, such as CH2 or CHR5, an alkene, such as =CH- or =CRs-, an ether, such as -C(OR5)H-.; Z and Z’ the same or different, independently, represent N, CH, CR5, COH, CNH2,CNHRs, or COR5,or Z and Z’ link together with -COR5OC-; wherein R5 is independently selected from Ci~Cg alkyl and aryl; G is -CH2-, O, -N(R5)-, S, -P(O)(OR5)-, -P(O)(N R5R5 )-, Z z are defined above; wherein Z and Z’ U and U’ are independently C(O), C(O)O, C(O)NH, C(O)N(R5), C(=NH), C(=NH)O, C(=NH)NH. C(=NH)N(R5), -C=N-, C(=S), C(O)S, C(S)NH, C(S)N(R5), S(O), S(O)O, S(O)NH, S(O)(OR5), S(O)(N(R5)), S(O2), S(O2)O, P(O)(OR5), P(O)(OR5)O, P(O)(NH2), P(O)(NR5R5 ), P(O)(OR5)NH-, P(O)(OR5)NR5-, P(O) (N(R5R5 ) (N(R5), P(S)(OR5), P(S)(OR5)O, P(S)(NH2), P(S)(NR5R5), P(S)(OR5)NH-, P(SXOR5)NR5 -, P(S)(N(R5R5’)N(R5), Rs, R5O; El and E2 are independently S, R5S, C(O)S, C(O)NH, C(O)O, C(O)R5S, C(=NH)NH, C(=NH)N(R5), C(=NH)S, -C=N-, C(=S)S, C(O)S, C(=S)NH, C(=S)N(R5), Ar-S, NC(O)CH2S, adjoining two atoms; Li, and L2 are independently a linker, or a linker which has a functional group on the linker that enables reaction with a cell-binding agent (CBA), Q. Li, and L2 are independently preferred a releasable linker, which has the formula of: —Ww—(Aa)r—Tt—; or —Ww—(Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—; wherein: -W- is a Stretcher unit; w is 0 or 1; -Aa- is independently an Amino Acid unit; r is independently an integer ranging from 0 to 100; -T- is a Spacer unit, which can be a linear alkyl or branched alkyl, or polyethylene glycol spacer; and t is 0, or 1-100. The Stretcher unit W may independently contain a self-immolative spacer, peptidyl units, a hydrazone bond, a disulfide, an ester, or a thioether bond; w is 1 or 2 or 3; Preferably Li, and L2 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 are independently selected from O, NH, N, S. P, NNH, NHNH, N(R3), N(R3)N(R3 ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula (OCH2CH2)POR3, or (OCH2CH-(CH3))POR3, orNH(CH2CH2O)pR3, or NH(CH2CH(CH3)O)PR3,or N[(CH2CH2O)pR3]-[(CH2CH2O)p’R3 ], or (OCH2CH2)PCOOR3, or CH2CH2(OCH2CH2)PCOOR3, wherein p and p’ are independently an integer selected from 0 to about 1000, or combination thereof; Ci-Cg of alkyl; C2-Cg of heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-Cs of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa)r, r =l-12(one to 12 amino acid units), which is composed from natural or unnatural amino acids, or the same or different sequences of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit; Ri, R2, R3, R4, Rf, R2'. R3’, and R4’ are the same or different and independently chosen from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, -(OCH2CH2)tRs ( a polyethylene glycol unit), halogen, NH(C=NH)NH2 (a guanidinium), -OR5, -NR5R5, -NO2, -NCO, -NR5COR;', -SR5, -SOR5(a sulfoxide), -SO2R5 (a sulfone), —SO3’M+ or -SO3H (a sulfonate), -OSO3’M+ or OSO3H (a sulfate), -SO2NRsR5’ (a sulfonamide), CN (a cyano), N3 (an azido), --COR5, —OCOR5, - OCONR5R5', CF3, OR5, Aryl, heterocycle, or P^jRsRs’Rs” and the linking group (L”) with the reactive group or a cell binding agent bonded thereto when Q, Q’ and T are absent; Rs, Rs’ and Rs” are independently selected from H, Ci~Cs of alkyl, alkenyl, alkinyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutical salts; In addition, Ri and R2 join together, or Rf and R2’ join together form a =O (ketone), =S, =NR, -C(=O)R, or a double bond containing group =CRsRs’; and R| and R2join together, or Rf and R2’ join together, or R3 and R4 join together or R3’ and Rf join together form a C3-Ci2 aromatic, heterocyclic, or heteroaryl ring; Q is a cell binding molecule, or a functional group that enables reaction with a cell-binding agent, or a functional group capable of reacting with a linker attached on a cell binding agent. The function group is chosen from a thiol, an amine, a hydrazine, an alkoxylamino, a disulfide substituent, a maleimido, a haloacetyl group, an A-hydroxy succinimide ester, a keton, an ester, an aldehyde, an alkynyl, an alkenyl, or protected thiol or disulfide group, such as SAc, SSRi or SSAr. Ar is aromatic group or hetero aromatic group. Q is preferably a cell-binding agent/ molecule, selected from the group consisting of an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, a domain antibody 22 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 fragment that binds to the target cell, an adnectin that mimics antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient¬ transport molecule (a transferrin), and a binding peptide, protein, or small molecule attached on albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid. The term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzyme-labile bond. It is appreciated that such physiological conditions resulting in bond breaking do not necessarily include a biological or metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or disulfide bond exchange reaction with a intracellular thiol, such as the amillimolar range of abundant of glutathione inside the malignant cells. The Stretcher unit (—W—), when present, may link a targeted binding molecular unit (CBA) to an amino acid unit (—Aa—), or links T when an Aa is not present. The Stretcher unit W may independently contain a self-immolative spacer, peptidyl units, a hydrazone bond, disulfide or thiolether bonds. In this regard a binding molecular (CBA) has a functional group that can form a bond with a functional group of a Stretcher. Useful functional groups that can be present on a binding molecule, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (—SH), amino, hydroxyl, oxyamino, alkynyl, heteroaromatic, carbonyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl. Preferred functional groups are sulfhydryl, carboxy and amino. Sulfhydryl groups can be generated by reduction of an intramolecular disulfide bond of a Ligand (such as a protein or an antibody). Alternatively, sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of a cell¬ binding molecule using 2-iminothiolane (Traut's reagent) or thiolactone or another sulfhydryl generating reagent, such as modifies the cell-binding molecule with a disulfide bond linker, or a thiol ester following by reduction or hydrolysis respectively. Preferably Li, and L2 are independently a chain of atoms selected from C, N, O, S, Si, and P, having 0-500 atoms. The atoms used in forming the Li, and L2 may be combined in all chemically relevant ways, preferably are Ci C20 alkylene, alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination above thereof. More preferably Li, and L2are, the same or different, independently selected from O, NH, S, NHNH, N(R?), N(Rs)N(R3’), Ci-Cg alkyl, amide, amines, imines, hydrazines, hydrazones; C2-Cg heteroalkyl, alkylcycloalkyl, ethers, esters, hydrazones, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, 23 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 amino acids, peptides, acyloxylamines, hydroxamic acids, or heterocycloalkyl; Cg-Cg aryl, Ar¬ alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; polyethyleneoxy unit of formula (OCTI2CT^ or (OCH2CH(CH3))POR3; or NH(CH2CH2O)pR3, or NH(CH2CH(CH3)O)pR3, or N[(C1^ or (OCH2CH2)PCOOR3, or CH2CH2(OCH2CH2)PCOOR3, wherein p and p’ are independently an integer selected from 0 to about 5000, or combination thereof; wherein R3 and R? are independently H; Ci-Cg alkyl; C2-Cg heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-Cg aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcyclo-alkyl, alkylcarbonyl, or heteroaryl; or C2-C« esters, ether, or amide; or 1-8 amino acids; or polyethyleneoxy having formula (OCH2CH2)por (OCH2CH(CH3))P, wherein p is an integer from 0 to about 5000, or combination above thereof; Optionally Li and L2 may independenly be composed of one or more linker components of 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), 4-thiopentanoate ("SPP"), 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("MCC"), (4-acetyl)amino-benzoate ("SIAB"), 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), or natural or unnatural peptides having 1-8 natural or unnatural amino acid unites. The natural aminoacid is preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, alanine; Li and L2 may also independenly contain a self-immolative or a non-self-immolative component, peptidic units, a hydrazone bond, a disulfide, an ester, an oxime, an amide, or a thioether bond. The self-immolative unit includes, but is not limited to, aromatic compounds that are electronically similar to the para-aminobenzylcarbamoyl (PAB) groups such as 2- aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronide, and ortho or para-aminobenzylacetals; Preferably, the self-immolative linker component has one of the following structures: 24 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 10 wherein the (*) atom is the point of attachment of additional spacer or releasable linker units, or the cytotoxic agent, and/or the binding molecule (CBA); X , Y , Z and Z are independently NH, O, or S; Z1 is H, OH, NHRi, ORi, SRi, COXiRi, wherein Xi and Ri are defined above; v is 0 or 1; U1 is independently H, OH, Ci~C6 alkyl, (OCH2CH2)n. F, Cl, Br, I, OR5, SRs, NR5R5’, N=NR5, N=R5,NR5R5’,NO2,SOR5R5’, SO2R5, SO3R5, OSO3R5, PR5R5’, POR5R5’, PO2R5R5’, OPO(OR5)(OR5’), or OCH2PO(OR5(OR5’), wherein R5 and R5’ are independently selected from H, C|~C8 alkyl; C2~C8 alkenyl, alkynyl, heteroalkyl, or amino acid; C3~C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or glycoside; or pharmaceutical cation salts; The non-self-immolative linker component is one of the following structures: (CH2)nCO(OCH2CH2)rOCH3 (CH2)nCON(CII2CH2O)rCOCII3 *(CH2CH2O)r*. 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein the (*) atom is the point of attachment of additional spacer or releasable linkers, the cytotoxic agents, and/or the binding molecules; X1, Y1, U1, R5, R5’ are defined as above; r is 0~100; m and n are 0~6 independently; 10 Further preferably, Li and L2 may independently be a releasable linker. The term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile or enzyme-labile bond. It is appreciated that such physiological conditions resulting in bond breaking do not necessarily include a biological or 15 metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis or substitution reaction, for example, an endosome having a lower pH than cytosolic pH, and/or 26 5 10 15 20 25 30 35 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 disulfide bond exchange reaction with a intracellular thiol, such as a millimolar range of abundant of glutathione inside the malignant cells; Examples of the releasable linkers Li or L2 include, but not limited: -(CR5R6)m(Aa)r(CR7R8)n(OCH2CH2)r, -(CR5R6)m(CR7R8)n(Aa)r(OCH2CH2)r, -(Aa)r- (CR5R6)m(CR7R8)n(OCH2CH2)r, -(CR5R6)m(CR7R8)n(OCH2CH2)I(Aa)r, -(CRsRglm- (CR7=CR8)(CR9Rio)n(Aa) -(CR5R6)m(NRiiCO)(Aa)t(CR9Rio)n-(OCH2CH2)r-, - (CR5R6)m(Aa)t(NR11CO)(CR9R10)n(OCH2CH2)r-,-(CR5R6)m(OCO)(Aa)t(CR9Rio)n.(OCH2CH2)r-, -(CR5R6)m(OCNR7)(Aa)t(CR9Rio)n(OCH2CH2)r-, -(CRsRôMCOXAaMCRçRwMOCH^H^ (CR5R6)m(NRiiCO)(Aa)t(CR9Rio)n(OCH2CH2)r-, -(CR5R6)m.(OCO)(Aa)t(CR9Rio)n-(OCH2CH2)I-, -(CR5R6)n>(OCNR7)(Aa)t(CR9Ri0)n(OCH2CH2)r-, -(CR5R6)m(CO)(Aa)t(CR9Rio)n-(OCH2CH2)r-, - (CR5R6)m-phenyl-CO(Aa)t(CR7R8)n-, XCR5R6)m-furyl-CO(Aa)t(CR7R8)n-, -(CR5R6)moxazolyl-CO(Aa)t(CR7Rs)n-, -(CR5R6)m-thiazolyl-CO(Aa)t(CCR7R8)n-, -(CRsR/ûr thienyl-CCXCRzRsX-, -(CRsRâX-imidazolyl-CO-CCRvRsln-, -(CR5R6)t-morpholino-CO(Aa)t. (CR7Rs)n-, -(CR5R6)tpiperazino-CO(Aa)t.(CR7Rs)n-, -(CRsRôlt-N-methylpiperazin-COCAalt. (CR7R8)n-, -(CR5R)m-(Aa)tphenyl-, -(CR5R6)m-(Aa)tfuryl-, -(CR5R6)m-oxazolyl(Aa)t-, -(CR5R6)mthiazolyl(Aa)t-, -(CR5R6)m-thienyl-(Aa)t-, -(CR5R6)m-imidazolyl(Aa)t-, -(C RsRélmmorpholino-(Aa)t-, -(CR5R6)m-piperazino-(Aa)t-, -(CRsR«)m-N-methylpiperazino-(Aa)t-, -K(CR5R6)m(Aa)r(CR7R8)Q(OCH2CH2)r, -K(CR5R6)m(CR7R8)n(Aa)XOCH2CH2)t-, -K(Aa)r- (CR5R6)m(CR7R8)n(OCH2CH2)t-, -K(CR5R6)m(CR7R8)n(OCH2CH2)r(Aa)r, -K(CR5R6)m- (CR7=CR8)(CR9Rio)n(Aa)t(OCH2CH2)r-,-K(CR5R6)m(NRiiCO)(Aa)t(CR9Rio)n(OCH2CH2)r- , -K(CR5R6)m(Aa)t(NRnCO)(CR9Rio)n(OCH2CH2)r-, -K(CR5R6)m(OCO)(Aa)t(CR9Ri0)n- (OCH2CH2)r-,-K(CR5R6)In(OCNR7)(Aa)t(CR9Rio)n(OCH2CH2)r-, -K(CR5R6)m(CO)(Aa)t. (CR9R10)n(OCH2CH2)r-,-K(CR5R6)m(NR11CO)(Aa)l(CR9R1o)n(OCH2CH2)r-, -K(CR5R6)m- (OCO)(Aa)t(CR9R]0)n(OCH2CH2)r-, -K(CR5R6)m(OCNR7)(Aa)t(CR9Rio)n(OCH2CH^^ -K- (CR5R6)m(CO)(Aa)t(CR9Rio)n(OCH2CH2)r-, -K(CR5R6)m-phenyl-CO(Aa)t(CR7R8)n-, -K- (CR5R6)m-furyl-CO(Aa)t.(CR7Rs)IÎ-, -K(CR5R6)m-oxazolyl-CO(Aa)t(CR7R8)n-, -K(CR5R6)mthiazolyl-CO(Aa)t.(CR7R8)n-, -K(CR5R6)t-thienyl-CO(CR7R8)n-, -KfCRsR^timidazolyl-CO- (CR7R8)n-, -K(CR5R6)tmorpholino-CO(Aa)t(CR7R8)n-, -K(CR5Rô)tpiperazino-CO(Aa)t. (CR7R8)n-, -K(CR5R6)rN-methylpiperazinCO(Aa)t(CR7R8)n-, -K(CR5R)m(Aa)tphenyl, -K- (CR5R6)m-(Aa)tfiiryl-, -K(CR5R6)m-oxazolyl(Aa)t-, -K(CR5R6)m-thiazolyl(Aa)r, -K(CR5R6)mthienyl-(Aa)t-, -K(CRsR6)m-iniidazolyl(Aa)t-, -K(CRsR6)m-morpholino(Aa)t-, -KCCRsRfOmpiperazino-(Aa)tG, -K(CR5R6)mN-methylpiperazino(Aa)r; wherein m, Aa, m, and n are described above; t and r are 0- 100 independently; R3, R4, R5, Rô, R7, and R8 are independently chosen from H; halide; Ci~C8 alkyl; C2~C8 aryl, alkenyl, alkynyl, ether, ester, amine or amide, which optionally substituted by one or more halide, CN, NRiR2, CF3, ORi, Aryl, heterocycle, 27 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 10 15 S(O)Rb SO2Ri.-CO2H, -SO3H, -ORb -CO2Ri, -CONRb PO2R,R., PO3H or P(O)R!R2R3; K is NRb -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -ON-NH-, -C(=O)NH-NH-, O, S, Se, B, Het (heterocyclic or heteroaromatic ring having C3-Cg), or peptides containing 1- 20 of the same or different amino acids; Additionally U, U’, E, E’, Li and L2may independently contain one or more units of the following hydrophilic structures: X2, X3> X4, X5, orX6, are independently selected from NH; NHNH; N(R3); N(R3)N(R3>); O; S; CiC6 alkyl; C2-Cô heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-Cg aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or 1~8 28 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 amino acids; wherein R, and R3’ are independently H;Cj-Cx alkyl; C2-C8 hetero-alkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or C2-C8 esters, ether, or amide; or polyethyleneoxy unit having formula (OCH2CH2)por (OCH2CH(CH3))P, wherein p is an integer 5 from 0 to about 5000, or combination above thereof; More preferably, Li, or L2, are independently linear alkyl having from 1-6 carbon atoms, or polyethyleneoxy unit havingformula (OCHiCHalp, p = 1-5000,or a peptide containing1-4 units of aminoacids (L or D form), or combination above. In addition, U, U’, Li, L2, L’, Ei or E2 may independently be composed of one or more 10 following components as shown below: i-maleimidocaproyl (MC) maleimido propanoyl (MP), (k oxobutanoic K . acid, -.4 HO^ 0 A 15 phenylalanine (ala-phe), c V\ 5 s• 1 -j HO’yj ,. thio-maleido, thio-amino O thio-amino-oxobutenoic acid, h2 H 0 valine-citrulline (val-cit), «-y alanine- '— lysine-phenylalanine (lys-phe), O lysine-alanine (lys-ala), aminobenzyloxycarbonyl (PAB), O 0 4-thio-pentanoate (SPP), 29 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 5 “S O 4-(NO maleimidoethyl (ME), aryl-thiol (PySS), (4-acctyl)aminobcnzoatc (SIAB), aminobenzylthio, diaminobenzylthio, /-N C H alkoxy amino (AOA), dithio, O alkylsulfonyl, O OH OH phosphinic acid, NN,N’-dimethylphosphon-amidic acid, hydrazine, N‘ H 4-methyl-4-dithio-pentanoic (MPDP), OI O H ~ II Al sulfon-bisamide, O ii Phosphondiamide, V J. maleimidomethyl)cyclo-hexane-1-carboxylate (MCC), c SO3‘ alkylphosphonamide, I 8 1 I OH O 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), n O methylphosphonamidic acid, O H ' HN^ç O 4-thio-butyrate (SPDB), ~ ethyleneoxy (EO), ,N. :n—n: N,N’-dimethylphosphondiamide, triazole, alkylsulfonamide, O F ° , oxylbenzylthio, o^s —S—J dioxylbenzylthio, —S—J3 amino-oxylbenzylthio, 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 L- or D-, natural or unnatural peptides containing 1-20 amino acids; wherein a connecting bond in the middle of atoms means that it can connect either neighbor carbon atom bonds; wavery line 5 is the site wherein another bond can be connected to; Alternatively, U, U’, Ei, orE2, can be independently absent. The compounds of the general Formula (I) having geometrical and stereoisomers are also a part of the invention. A preferred stereoisomer of the Formula (I) is presented by the following Formula (la), 31 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein Zi is OH, NH2, ORi, NHRb NRiR2, SR|. NHR|COXjRb ORiCOXiRi.or »5 j iRi n I» 1 I» m, m’, n, q, q’, Rb RA R2, R2’, R3, RA R4, R4’, V, V’, U, U’ Lb L2. Ei, E2 and Q are the same as in Formula (I). In more preferred embodiment according to Formula (I), the conjugate of cross-linked PBD dimer derivatives of the invention have the Formula (1-01) ~ (1-18) below: 32 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 33 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (1-05), (1-06), (1-07), 34 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (1-13), 36 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (1-15), (1-16), 37 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 38 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein V, V’, n and q are defined the same above; mAb is a cell-binding molecule, preferably an antibody; r, r’ and r” are independently 0 - 200. In another embodiment, the present invention discloses mono-linkage of conjugation of 5 PBDs derivatives to a cell binding molecule, as shown in Formula (II), (III) and (IV), for targeted treatment of cell proliferation: 39 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein =, — , X, X’, Y, Y’, Z, Z’,1, 1’, m, m’, n, q, q’, Rj. R(\ R2. R2’, R3, R3\ R4, RA V, V’, U, U’ Li, L2, G, Q.Ei, and E2 are the same defined as in Formula (I). In preferred embodiment according to Formula (IT), (III) and (IV), the conjugates of PBD derivatives of the invention having the Formula (11-01) ~ (11-11), (in-01)~(III-06) and (IV-01)~(IV- 5 11) below: (11-01), (11-02), (H-03), 40 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (H-04), (11-05), (II-06), (11-07), 41 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 42 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 (UI-03). 43 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (III-04), (in-05), 44 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV-01), (IV-02), (IV-03), (IV-04), 45 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV-05), (IV-06), (TV-07), (IV-08), 46 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV-09), (IV-10), (IV-11), wherein =====, —, m, m’, n, q, and q’ are the same defined as in Formula (I); r, r’, and r’’ are independently 0- 200, m3 is 0 - 30. In another preferred embodiment, the conjugates of Formula (I), (II) (III), (IV) and (V) are 10 prepared from coupling of a cell-binding molecule with a PBD dimer derivative having the Formula (V) (VI), (VII), and (VIII) accordingly as shown below: 47 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein ==, — , X, X’, Y, Y’, Z, Z’, 1, 1’, m, m’, n, q, q’, Ri, Ri’, R2, R2’, R3, R3’, R4, R4’, V, V’, U, U’ Li, L2, Ei, and E2 are the same defined as in Formula (I); wherein E3 and E’3 are independently selected from: 48 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 acyl halide (acid halide). ethenesulfonyl; acryl (acryloyl); O 2-(dinitrophenoxy)acetyl; O2N- 2-(nitrophenoxy)acetyl; ^2N F 2-(fluorophenoxy)-acetyl; F o x2A2 2- (difluorophenoxy)-acetyl; 2-(((trifluoromethyl)-sulfonyl)oxy)acetyl; 2-(pentafluorophenoxy)acetyl; 49 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 P-lactam, or h2nhn- PCT/CN2018/094586hydrazide,s=c=n-£ isothiocyanate; wherein Xi’ O and X3’ are independently F, Cl, Br, I or Lv3; X2’ is O, NH, N(Ri), or CH2; R3 and R5 are independently H, Ri, aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by -Ri, -halogen, -ORi, -SRi, -NR1R2, - NO2, -S(O)Ri, - S(O)2Ri, or -COORi; LV3 is a leaving group selected from methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, phenylthio, pyridinylthio, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluoro-phenoxy1, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazole-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxyl, 2-ethyl-5-phenylisoxazoliumyl, phenyloxadiazol-yl (ODA), oxadiazol-yl, or an intermediate molecule generated with a condensation reagent for Mitsunobu reactions, wherein R1 and R2 are defined above; More preferably, E3 and E’3 are independently selected from -SH, -S-SCH3, -S-SAc, -S-SPyridine. -S-S-Ar(-NO2), -S-cell binding agent, or any one of the following formulas: 50 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 wherein D is H, -NCF. SO3H, or F; wherein Ri, R2, R3, R4. r. m and n are defined above; w and w’ are independently 0, 1 or 2; wherein R5 and R5’ are independently selected from Ci~Cô alkyl, aryl, cyclic, cyclohetero, H, or M (wherein M isNa, K, Ca, ammonium or the other pharmaceutically acceptable salt); In certain embodiments, the PBD derivatives of Formula (V), (VI), (VII), and (VIII) are represented by the following Formulas (V-01)~(V-20), (VI-01) ~ (VI-05), (VH-01)~ (VII-06), (VIII-01) -(Vin-06) accordingly below: (V-03), 51 GA 03105541 2021-01-04 52 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 53 GA 03105541 2021-01-04 54 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 55 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 (VI-03), 56 GA 03105541 2021-01-04 57 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 58 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 59 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (VII-03), 60 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 (vm-02), (VIII-04), 61 5 10 15 20 (VIII-06), wherein U, U’, V, V’, n, n’, and L as defined above; Re and Re’ are independently selected from Ci~Ce alkyl, aryl, cyclic, cyclohetero, halogen, haloalkyl, alkoxy, haloalkoxy alkylamino, - NO2, -CN or H; Xi and Xf are independently H, F, Cl, Br, I, OTs (tosylate), OMs (mesylate), nitrophenol OAr(NO2), OAr(NO2)2dinitrophenol, OAr(F) monofluorophenol, OAr(F)5 pentafluorophenol, OAr(F)2 difluorophenol,N-hydroxysuccinimide (NHS), phenol, tetrafluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1- hydroxybenzotriazole, or 2-ethyl-5-phenylisoxazolium-3'-sulfonate. SYNTHESIS OF THE CROSS-LINKED PBD DIMER DERIVATIVES OF FORMULA (V), (VI), (VII) AND (VIII) AS CYTOTOXIC AGENTS. The compounds and process of the present invention can be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by application or adaptation of1he methods described in the examples, or variations thereon as appreciated by the skilled artisan. The appropriate modifications and substitutions will be readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. hi particular, such methods can be found in Richard C. Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, Two Volume Set, 2nd Edition, Wiley Publishers, 2010. Because the cytotoxic agents of the present invention may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless 62 Date Reçue/Date Received 2024-05-09 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers. The cytotoxic agents of the present invention may be prepared by a variety of synthetic routes. The reagents and starting materials are commercially available, or readily synthesized by well-known techniques by one of ordinary skill in the arts. All substituents, unless otherwise indicated, are as previously defined. In the synthetic reactions of the cytotoxic agents of the present invention, it may be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups may be used in accordance with standard practice, for examples see Peter G. M. Wuts, Theodora W. Greene in Greene's Protective Groups in Organic Synthesis, 4th Edition, John Wiley and Sons, 2006; Ian T. Harrison, Shuyen Harrison in Compendium of Organic Synthetic Methods, Vol 1,2 Vols. 1& 2 By Ian T. Harrison & Shuyen Harrison , Vols 3-5 by Louis S. Hegedus, Leroy Wade Vols 6-Vol 12 by Michael B. Smith, John Wiley and Sons, 2006-2012. Normally the synthetic reactions are carried out in suitable solvents, temperatures and time. A variety of solvents which have no adverse effect on the reaction or on the reagents involved can be used in a synthetic reaction of the cytotoxic agent. Examples of suitable solvents include: hydrocarbons, which may be aromatic, aliphatic or cycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene, toluene and xylene; hydrocarbons containing halogens, such as chloroform, dichloromethane, dichloroethane; amides, such as dimethylactamide or dimethylformamide; alcohols such as ethanol and methanol and ethers, such as diethyl ether and tetrahydrofuran. The reactions can take place over a wide range of temperatures, from -100 °C- 300°C. More preferably from 0°C to 150°C. The time required for the synthetic reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents and can be from 5 second to 4 weeks, more preferably from 10 min to 24 hours. In addition, the cytotoxic agents prepared may be isolated or purified from the reaction mixture by conventional means, such as evaporating or distilling off the solvent from the reaction mixture, or after distilling off the solvent from the reaction mixture, pouring the residue into water followed by extraction with a water-immiscible organic solvent and then distilling off the solvent from the extract. It may also involve various well known techniques, such as re-crystallization, re-precipitation or the various chromatography techniques, 63 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 notably column chromatography, preparative thin layer chromatography, or high performance liquid chromatography. Some of the synthetic reactions of the cytotoxic agents and their conjugates to a cell binding agent are further exampled but not restricted in the Figures 1~32 and in the examples of the following description. THE CONJUGATES OF CROSS-LINKED PBD DIMERS TO A CELL-BINDING MOLECULE. The present invention provides a conjugate molecule comprising at least one PBD derivative covalently linked to a cell-binding agent (Q) through a linking group of a conjugate linker. Preferably said conjugate comprises one to twenty molecules of cross-linked PBD dimer derivatives according to the invention covalently linked to a cell-binding agent through a linking group of a linker on a cross-linked PBD dimer derivative. As stated above, the conjugates of a cell surface binding molecule - cross-linked PBD dimer derivatives are illustrated in the Formula (I), (II), (III) and (IV) above. Drug loading may range from 1 to 30 drug moieties (D) per antibody and is preferred the average number of 2-8 drug moieties per antibody in a molecule of Formula (I) ~ (IV). The average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of the conjugates in terms of the drug loading may also be determined. In some instances, separation, purification, and characterization of homogeneous the conjugates where Drug loading is a certain value from the conjugates with the drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. The Cell binding agents (CBA) may be of any kind and include peptides and non-peptides. Generally, the cell binding agents include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies (polyconal and monoclonal antibodies); single chain antibodies; fragments of antibodies such as Fab, Fab', F(ab')2, Fv, [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR’s, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens; antibody mimetic, such as an affibody; domain antibodies (dAb); nanobodies; unibodies; DARPins; anticalins; versabodies; duocalins; lipocalins; vimers; interferons (such as type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, GM-CSF, interferon¬ gamma (IFN-y); hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such as androgens and estrogens, 64 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 melanocyte-stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factors (TGF), such as TGFa, TGFp, insulin and insulin like growth factors (IGF-!, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-158 (1984)]; vaccinia growth factors (VGF); fibroblast growth factors (FGFs); smaller molecular weight proteins, poly-peptide, peptides and peptide hormones, such as bombesin, gastrin, gastrin¬ releasing peptide; platelet-derived growth factors; interleukin and cytokines, such as interleukin- 2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors, granulocyte-macrophage colony¬ stimulating factor (GM-CSF); vitamins, such as folate; apoproteins and glycoproteins, such as transferrin {O’Keefe et al, 260 J. Biol. Chern. 932-937 (1985)}; sugar-binding proteins or lipoproteins, such as lectins; cell nutrient-transport molecules; and small molecular inhibitors, such as prostate-specific membrane antigen (PSMA) inhibitors and small molecular tyrosine kinase inhibitors (TKI), non-peptides or any other cell binding molecule or substance, such as bioactive polymers (Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); dendrimers (Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci. 2009, 106, 685- 90); nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chern. 2008, 19, 1309-12); liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9); viral capsides (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93). In general monoclonal antibodies are preferred as a cell-surface binding agent if an appropriate one is available. The linker used for the conjugation of this invention includes, but not limited to, a disulfide linker, a thioether linker, an amide bonded linker, a peptidase-labile linker, a photolabile linker, an acid-labile linkers (such as hydrazone liner), an esterase-labile linker, an oxidatively labile linker, a metabolically labile linker, a biochemically labile linker. Preferably, the linker is linked to the cell binding agent via a function reactive towards for instance thiol and amino functions of the cell binding agent coming from reduced disulfide bonds and lysine residues respectively. More particularly, said derivative is linked through the — CO— group to the amino function of the lysine residue of said cell binding agent, so as to form an amide bond. In addition, the linker may be composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine¬ citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af'), glycine-glycine, paminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), Nsuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("SMCC"), N-Succinimidyl (4- iodo-acetyl)aminobenzoate ("SIAB"), ethyleneoxy (—CH2CH2O—) as one or more repeating 65 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 units ("EO" or "PEO”). The linker may be a "cleavable linker," facilitating release of a drug in the cell. Additional linker components are known in the art and some are illustrated below: Oeo nu R| R, H O . ’ nhnh r7 Og-*. * c, 66 GA 03105541 2021-01-04 67 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Rs and R9 are independently selected from -Ci~Cs alkylene-, — Ci~C? carbocyclo-, -O-( Ci~Cs alkyl)-, -arylene-, — Ci~Cs alkylene-arylene-, -arylene, -Ci~Cs alkylene-, -Ci~Cg alkylene-( Ci~Cs carbocyclo)-, -(C3--C7 carbocyclo)- Ci~Cs alkylene-, -Cs-Cs heterocyclo-, -C|~Cs alkylene-( C ~Cs heterocyclo)-, -( C3~Cs heterocyclo)- C1-C9 alkylene-, -(CH2CH2O)k-, - (CH(CH3)CH2O)t-, and -( CH2CH2O)k-CH2-; k is an integer ranging from 1-30; X’”, Y’” and Z”’ are independently selected from NH, O or S; Q, Ri and R2are described above. In a preferred embodiment, conjugates of die invention are antibody/cytotoxic agent, antibody fragment/cytotoxic agent, diabody/cytotoxic agent, tri(a)body/cytotoxic agent, epidermal growth factor (EGF)/cytotoxic agent, prostate specific membrane antigen (PSMA) inhibitor/cytotoxic agent, melanocyte stimulating hormone (MSH)/cytotoxic agent, thyroid stimulating hormone (TSH)/cytotoxic agent, polyclonal antibody/cytotoxic agent, somatostatin/cytotoxic agent, folate/cytotoxic agent, matriptase inhibitor/cytotoxic agent, estrogen/cytotoxic agent, estrogen analogue/cytotoxic agent, designed ankyrin repeat proteins (DARPins)/cytotoxic agent, androgen/cytotoxic agent, and androgen analogue/cytotoxic agent. In a more preferred embodiment, cell binding molecule of the invention are monoclonal antibody. Examples of antibodies used for conjugation of cyotoxic agents in this prevention include, but are not limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab (anti-TNF-a), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-a); Afutuzumab (anti-CD20), Alacizumab pcgol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab (antiCEA), Anatumomab ( anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (antiHLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin (CD62L), Atlizumab 68 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (a chain of IL-2 receptor), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCLll (eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, antiVEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE, anti-CD19). Brentuximab (cAClO, anti-CD30 TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab (Haris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pcgol (Cimzia anti-TNF-a), Cetuximab (Erbitux, IMCC225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzumab (anti-MUCl), Conatumumab (anti-TRAIL-R2), CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAbl7-lA, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CDIla), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (antiSLAMF7), Elsilimomab (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrin av03), Exbivirumab ( anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti¬ interferon receptor), Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFNy), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-p), Galiximab (antiCD80), Gantenerumab (anti- beta amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011, antiGPNMB), Golimumab (Simponi, anti-TNF-a), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-a), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain of IL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti- CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti- IL-13), Lemalesomab (anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti- 69 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 CD40), Lumiliximab (anti- CD23 (IgE receptor), Mapatumumab (anti-TRAIL-Rl), Maslimomab (anti- T-cell receptor), Matuzumab (anti-EGFR). Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin ou), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-a), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R a), Omalizumab (Xolair, anti-IgE Fc region), Oportuzumab (antiEpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (antilipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti- Pseudomonas aeruginosa), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUCl), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab (anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor). Rontalizumab (anti-IFN-a), Rovelizumab (LeukArrest, anti-CDll, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-a), Siltuximab (anti-IL-6), Siplizumab (antiCD2), (Smart) MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab (anti¬ sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte antigen), Tacatuzumab (anti-alphafetoprotein), Tadocizumab (anti-integrin anbPa), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factor A), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAlL-R2). TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, (antiHER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus), Urtoxazumab (anti- Escherichia coli), Ustekinumab (Stelara, 70 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 anti-IL-12, IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrin cuP?), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3), Volociximab (anti-integrin aspi), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab (antiCD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron Regulatory Protein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst, for melanoma and solid tumors), J591 (anti-PSMA, Weill Cornell Medical School for prostate cancers), 225.28S [anti-HMW-MAA (High molecular weight-melanomaassociated antigen), Sorin Radiofarmaci S.R.L. (Milan, Italy) for melanoma], COL-1 (antiCEACAM3, CGM1, from Nat. Cancer Inst. USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, for prostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus), ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for Sepsis toxic shock], MEDI-500 [T10B9, anti-CD3, TRap (T cell receptor alpha/beta), complex, from Medlmmune Inc for Graft-versus-host disease], RING SCAN [ antiTAG 72 (tumour associated glycoprotein 72), from Neoprobe Corp, for Breast, Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTDl (Tumorassociated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen; M4S; tumor antigen 17-1A; CD326, from NeoRx Corp, for Colon, Ovarian, Prostate cancers and NHL]; LymphoCide, Smart ID10, Oncolym, Allomune, anti-VEGF, CEAcide, IMC-1C11, and Cetuximab. Other antibodies as binding ligands include, but are not limited to, are antibodies against the following antigens: Aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin’s disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin’s lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma), CD66e (cancers), CD70 (metastatic renal cell carcinoma and 71 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancers), mucin (carcinomas), CD221 (solid tumors), CD227 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast, colorectal and lung cancers), DLL4 (A-like-4), EGFR (Epidermal Growth Factor Receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, Heme-oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor Receptor 2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (a cell surface antigen glyvolipid, melanoma), GD3 idiotype (cancers), Heat shock proteins (cancers), HER1 (lung, stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor (interleukin 2 receptor,T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman’s disease, IL6 dependent tumors), Integrins (avp3, a5pi, a6p4, alip3, a5p5, avp5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (Ovarian cancers), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane-spanning 4- domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), Paratope of anti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers), PSMA (prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associated glycoprotein 72, AML, gastric, colorectal, ovarian cancers), T cell transmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cell carcinoma), TRAIL-RI (Tumor necrosis apoprosis Inducing ligand Receptor 1,lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor associated antigens recognized by antibodies have been reviewed (Gerber, et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer Immunol Immunother. 54(3),187-207 (2005). Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76). Examples of these antigens that antibodies against are: Many other Cluster of Differentiations (CD2, CD2R, CD3, 72 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8, CD8a. CD8b, CD9, CD10, CDlla, CDllb, CDllc. CD12, CD12w, CD13, CD14, CD15, CD15s, CD15u. CD16, CD16a, CD16b, CD17, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55,CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71. CD72, CD73, CD74, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CDwll3, CD114, CD115, CD116, CD117, CD118, CD119, CDwll9, CD120a, CD120b, CD121a. CD12lb. CDwl21b, CD122, CD123, CDwl23, CD124, CD125, CDwl25, CD126, CD127, CD128, CDwl28, CD129, CD130, CD131, CDwl31, CD132, CD133, CD134, CD135, CD136, CDwl36, CD137, CDwl37, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CDwl45, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CDwl56c, CD157, CD158a, CD158b, CD159a, CD159b, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CDwl86, CD187, CD188, CD189, CD190, Cdl91, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CDwl98, CD199, CDwl99, CD200, CD200a, CD200b, CD201, CD202, CD202b, CD203, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210, CD212, CD213al, CD213a2, CDw217, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235ab, CD235b, CD236, CD236R, CD238, CD239, CD240, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265, CD266, CD267, CD268, CD269, CD271, CD273, CD274, CD275, CD276 (B7-H3), CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, 73 5 10 15 20 25 30 35 CDw338, CD339), Annexin Al, Nucleolin, Endoglin (CD105), ROBO4, Amino-peptidase N, like-3 (DLL3), E -like-4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 nonmutant, NYESO-1, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, Proteinase3 (PR1), bcrabl, Tyrosinase, Survivin, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgen receptor, Cyclin Bl, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothclin, PSCA, MAGE Al, sLe(a), CYP1B1, PLACl, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-P, MAD-CT-2, Fos-related antigen 1. Production of antibodies used in the present invention involves in vivo or in vitro procedures or combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well-known in the art, such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonal antibody is typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen (Kohler, G.; Milstein, C. (1975). Nature 256: 495-497). The detailed procedures are described in “Antibodies—A Laboratory Manual”, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988). Particularly monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whole virus, attenuated whole virus, and viral proteins. Splénocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact specified receptors or inhibit receptor activity on target cells. A monoclonal antibody used in tire present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridomathat secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques, such as using protein-A affinity chromatography; anion, cation, hydrophobic, or size exclusive chromatographies (particularly by affinity for the specific antigen after Protein A, and sizing column 74 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 chromatography); centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, 20% fetal calf serum and with an anti¬ foaming agent, such as polyoxyethylene-polyoxypropylene block copolymer. In addition, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with an oncovirus, such as Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also be produced via an anti-receptor peptide or peptides containing the carboxyl terminal as described well-known in the art. See Niman et al., Proc. Nad. Acad. Sei. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry 34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen for producing anti-receptor peptide monoclonal antibodies. There are also a number of other well-known techniques for making monoclonal antibodies as binding molecules in this invention. Particularly useful are methods of making fully human antibodies. One method is phage display technology which can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity enrichment. Phage display has been thoroughly described in the literature and the construction and screening of phage display libraries are well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13 (1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson et al., Nature 352:264-628 (1991); Huse et al., Science 246:1275-1281 (1989), Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001) (O'Brien et al., ed., Human Press, Totowa, N.J.), and in certain embodiments, in Lee et al. J. Mol. Biol. 340:1073-1093 (2004). Moncolonal antibodies derived by hybridoma technique from another species than human, such as mouse, can be humanized to avoid human anti-mouse antibodies when infused into humans. Among the more common methods of humanization of antibodies are complementarity¬ determining region grafting and resurfacing. These methods have been extensively described, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al, Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 1;13:1619-33 (2008); Lazar et al, Mol Immunol. 44(8):1986-98 (2007); Li et al, Proc. Natl. Acad. Sci. USA.103(1O):3557-62 (2006). 75 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys, or other mammals, carrying large portions of the human immunoglobulin heavy and light chains, with an immunogen. Examples of such mice are: the Xenomouse. (Abgenix, Inc.), the HuMAbMouse (Medarex/BMS), the VelociMouse (Regeneron), see also U.S. Pat. No. 6,596,541 , 6,207,418, No. 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,545, Nos. 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In human therapy, murine variable regions and human constant regions can also be fused to construct called “chimeric antibodies” that are considerably less immunogenic in man than murine mAbs (Kipriyanov et al, Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002)). In addition, site-directed mutagenesis in the variable region of an antibody can result in an antibody with higher affinity and specificity for its antigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002)); Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchanging constant regions of a mAb can improve its ability to mediate effector functions of binding and cytotoxicity. Antibodies immunospecific for a malignant cell antigen can also be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a malignant cell antigen can be obtained commercially, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. DNA encoding hybridoma-derived monoclonal antibodies or phage display Fv clones of the antibody can be readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells (Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992)). Antibodies can also be produced by using an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and property assembled antibodies. Such modulation isaccomplished at least in part by simultaneously modulating translational strengths for the polypeptide components. After the fermentation which is known in the art, the produced antibody protein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. 76 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 The exemplary purification procedures: fractionation on immunoaffinity (such as Protein A columns) or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. Apart from an antibody, a peptide or protein that bind/block/target or in some other way interact with the epitopes or corresponding receptors on a targeted cell can be used as a binding molecule. These peptides or proteins could be any random peptide or proteins that have an affinity for the epitopes or corresponding receptors and they don’t necessarily have to be of the immunoglobulin family. These peptides can be isolated by similar techniques as for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49). The use of peptides from such random peptide libraries can be similar to antibodies and antibody fragments. The binding molecules of peptides or proteins may be conjugated on or linked to a large molecules or materials, such as, but is not limited, an albumin, a polymer, a liposome, a nano particle, as long as such attachment permits the peptide or protein to retain its antigen binding specificity. The cell-binding molecule/ligands or cell receptor agonists can be Ig-based and non-Igbased protein scaffold molecules. The Ig-Based scaffolds can be selected, but not limited, from Nanobody (a derivative of VHH (camelid Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775—97); Domain antibodies (dAb, a derivative of VH or VL domain) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-90); Bispecific T cell Engager (BiTE, a bispecific diabody) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); Dual Affinity ReTargeting (DART, abispecific diabody) (Moore P. A. P, et al. 2011, Blood 117(17), 4542—51); Tetravalent tandem antibodies (TandAb, a dimerized bispecific diabody) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341). The Non-Ig scaffolds can be selected, but not limited, from Anticalin (a derivative of Lipocalins) (Skerra A. 2008, FEBS J., 275(11): 2677-83; Beste G, et al, 1999 Proc. Nat. Acad. USA. 96(5):1898-903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2): 337-50; Skerra, A. 2007, Curr Opin Biotechnol. 18(4): 295-304; Skerra, A. 2008, FEBS J. 275(11):2677-83); Adnectins (10th FN3 (Fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4):1141-51; Batori V, 2002, Protein Eng. 15(12): 1015-20; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2): 363—71; Hackel, B. J, 2010, Protein Eng. Des. Sei. 23(4): 211-19); Designed Ankyrin Repeat Proteins (DARPins) (a derivative of ankrin repeat (AR) proteins) (Boersma, Y.L, et al, 2011 Curr Opin Biotechnol. 22(6): 849-57), e.g. DARPin C9, DARPin Ec4 and DARPin E69_LZ3JE01 (Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 2674-83; Patricia M-K. M., et al, Clin Cancer Res. 2011; 17(1):100-10; Boersma Y. L, et al, 2011 J. Biol. Chern. 286(48), 41273—85); Avimers (a domain A/low-density lipoprotein (LDL) receptor) (Boersma Y. 77 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12):1556-61). Examples of the small molecule structures of the cell-binding molecules/ligands or cell receptor agonists of the patent application are the following: LB01 (Folate), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (Somatostatin), LB06 (Somatostatin), LB07 (Octreotide, a Somatostatin analog), LB08 (Lanreotide, a Somatostatin analog), LB09 (Vapreotide (Sanvar) , a Somatostatin analog), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (Gastrin releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (cobalamin, vitamin B12 analog), LB17 (cobalamin, vitamin B12 analog), LB18 (for avP3 integrin receptor, cyclic RGD pentapeptide), LB19 (hetero-bivalent peptide ligand for VEGF receptor), LB20 (Neuromedin B), LB21 (bombesin for a G-protein coupled receptor), LB22 (TLR2 for a Toll¬ like receptor,), LB23 (for an androgen receptor), LB24 (Cilengitide/cyclo(-RGDfV-) for an av intergrin receptor, LB23 (Fludrocortisone), LB25 (Rifabutin analog), LB26 (Rifabutin analog), LB27 (Rifabutin analog), LB28 (Fludrocortisone), LB29 (Dexamethasone), LB30 (fluticasone propionate), LB31 (Beclometasone dipropionate), LB32 (Triamcinolone acetonide), LB33 (Prednisone), LB34 (Prednisolone), LB35 (Methylprednisolone), LB36 (Betamethasone), LB37 (Irinotecan analog), LB38 (Crizotinib analog), LB39 (Bortezomib analog), LB40 (Carfilzomib analog), LB41 (Carfilzomib analog), LB42 (Leuprolide analog), LB43 (Triptorelin analog), LB44 (Clindamycin), LB45 (Liraglutide analog), LB46 (Semaglutide analog), LB47 (Retapamulin analog), LB48 (Indibulin analog), LB49 (Vinblastine analog), LB50 (Lixisenatide analog), LB51 (Osimertinib analog), LB52 (a neucleoside analog), LB53 (Erlotinib analog) and LB54 (Lapatinib analog) which are shown in the following structures: HOOC O O W^/^X4- A A/ HOOC N N COOH H H HOOC^N^ tOOH U n 0 °rOH LB01 (Folate conjugate), LB02 (PMSA ligand conjugate), LB03 (PMSA ligand conjugate), 78 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 LB08 (Lanreotide, a Somatostatin analog), 79 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 analog). LB12 (Gastrin releasing peptide receptor (GRPr), MBA), LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), 80 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), 81 GA 03105541 2021-01-04 WO 2020/006722 LB18 (for avp3 intcgrin receptor, cyclic RGD pentapeptide), PCT/CN2018/094586 Ac LB19 (hetero-bivalent peptide ligand conjugate for VEGF receptor), G-N-L-W-A-T-G-H-F-M-NH2 LB20 (Neuromedin B), H CL Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-GIy-His-Leu-Met— N—£ LB21 (bombesin conjugate for a G-protein coupled receptor), 10 LB24 (Cilengitide/cyclo(-RGDfV-) conjugate for an av intergrin receptor) 82 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 5 83 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 84 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 LB38 (Crizotinib analog). LB39 (Bortezomib analog), wherein Y5, is N, CH, C(C1), C(CH3), or C(COORi); Ri is H, Ci-C6 Alkyl, C3-C8 Ar; 85 WO 2020/006722 CA 03105541 2021-01-04 PCT/CN2018/094586 LB44 (Clindamycin), <5 -HN-H-A-Q-G-T-F-T-S-D H^j-K-A-A-Q-G-Q-L-Y-S-S-V Q-F-I-A-W-L-V-R-G-R-G-COOH LB45 (Liraglutide analog), -H-AIB-Q-G-T-F-T-S-D^ SjV>-a-a-q-g-Q-l-y-s-s-v Q-F-I-A-W-L-V-R-G-R-G-COOH LB46 (Semaglutide analog), 86 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 HOOC-H-G-E-G-T-F-T-S-D-L-S-K-Q^l g-g-n-k-l-w-e-i-f-l-r-v-a-e-e-f; ^-S-S-G-A-P-P-S-K-K-K-K-K-^ ^s/^N' H LB50 (Lixisenatide analog), wherein“^>-rux” is the site to link the side chain linker of the present patent; X4,and Yi are independently O, NH, NHNH, NR,, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(Ri), N(R|)C(O)N(R,). CH2, C(O)NHNHC(O) andC(O)NR,: X, is H, CH2, OH, O, C(O), C(O)NH, C(O)N(Ri), Ru NHRi, NR,, C(O)Ri or C(O)O; X5 is H, CH3, F, or Cl; Mi and M2 are independently H, Na, K, Ca, Mg, NH4, N(RiR2R3 R4); Ri. R2, R3and R4 are defined in Formula (I); 87 5 10 15 20 25 30 Any one of several different reactive groups on a cell binding agent, preferably on an antibody, can be a conjugation site, such as g-amino groups in lysine residues, pendant carbohydrate moietics, carboxylic acid groups, disulfide groups, and thiol groups. For reviews on antibody reactive groups suitable for conjugation, see, e.g., Hermanson, G.T. (2008). Bioconjugate Techniques, Academic Press; Garnett, Adv. Drug Delivery Rev. 53 (2001), 171- 216 and Dubowchik and Walker, Pharmacology & Therapeutics 83 (1999), 67-123. The cytotoxic agents, cross-linked PBD dimers of this invention can be directly conjugated (linked) to a cell binding agent, or via a bifunctional linker or a cross-linking agent to a cell binding agent The bifunctional linker possess two reactive groups; one of which is capable of reacting with a cell binding agent while the other one reacts with one or more molecules of cytotoxic agent of the invention. The bifunctional crosslinkers are well known in the art (see, for example, U.S.Pat. No. 5,208,020; Isalm and Dent in Bioconjugation chapter 5, p 218-363, Groves Dictionaries Inc. New York, 1999). Examples of bifunctional linker are: N-succinimidyl- 3-(2-pyridyldithio)-propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), Nsuccinimidyl-4-(2-pyridyl-dithio)pentanoate (SPP), N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB), 2-iminothiolane, N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), Nsuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SNPP), N-sulfosuccinimidyl-4-(5-nitro-2- pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)- pentanoate (SMNP), N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SSNPP), 4- succinimidyl-oxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-sulfosuccinimidyl- 4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SSMNP); N-succinimidyl-4-methyl-4-(2- pyridyldithio)pentanoate (SMPDP), N-succinimidyl-4-(5-N,N-dimethyl-carboxamido-2- pyridyldithio) butyrate (SCPB), N-sulfosuccinimidyl-4-(5-N,N-dimethyl-carboxamido-2- pyridyldithio) butyrate (SSCPB), N-succinimidyl-4,4-dimethyl-4-(2-pyridyl-dithio)pentanoate (SDMPDP), succinimidyl-4-(N-maleimidomcthyl)cyclohcxane-l-carboxylate (SMCQ, Nsuccinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), , bis-maleimidopolyethyleneglycol (BMPEG), BM(PEG)i~20,N-(P-maleimidopropyloxy)-succinimide ester (BMPS), iminothiolane (IT), dimethyl adipimidate HC1or derivatives of imidoesters, active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene), gamma-maleimidobutyric acid Nsuccinimidyl ester (GMBS), E-nialeimido-caproic acid N-hydroxysuccinimide ester (EMCS), 5- malcnnidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l- 88 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 carboxy-(6-amidocaproate) (a "long chain" analog of SMCC (LC-SMCC)). m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromo-acetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), Nsuccinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(beta-maleimidopropionamido)-hexanoate (SMPH), succinimidyl-(4-vinyl-sulfonyl)benzoate (SVSB), dithiobismaleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3- dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate (sulfo-SMCC), sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobcnzoyl-Nhydroxysulfosuccinimide ester (sulfo-MBS), N-(gamma-maleimidobutryloxy)- sulfosuccinimdeester (sulfo-GMBS), N-(epsilon-maleimidocaproyloxy)-sulfo-succimido ester (sulfo-EMCS), N-(kappa-maleimidoundecanoyloxy)-sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB ); or the commercially available linkers (such as from Thermo Scientific’s Pierce: Imidoester Crosslinkers: DMA (Dimethyl adipimidate-2 HCl), DMP (Dimethyl pimelimidate*2 HCl), DMS (Dimethyl Suberimidate«2 HCl), DTBP (Dimethyl 3,3'-dithiobispropionimidate«2 HC1); NHS-ester Crosslinkers-Amine Reactive: BS(PEG)s (Bis(succinimidyl) penta(ethylence glycol), BS(PEG)g(Bis(succinimidyl) nona(ethylence glycol), BS3 (Bis[sulfosuccinimidyl] suberate), BSOCOES (Bis[2- (succinimidooxycarbonyloxy)-cthyljsulfone), DSG (Disuccinimidyl glutarate), DSP (Dithiobis[succinimidyl propionate]), DSS (Disuccinimidyl suberate), DST (Disuccinimidyl tartarate), DTSSP (3,3'-Dithiobis[sulfo-succinimidylpropionate]), EGS (Ethylene glycol bis[succinimidylsuccinate]), Sulfo-EGS (Ethylene glycol bis[sulfo-succinimidylsuccinate]), TSAT (Tris-succinimidyl aminotriacetate), DFDNB (l,5-Difluoro-2,4-dinitrobenzene); Amineto-Sulfhydryl Crosslinkers: Sulfo-SIAB (Sulfosuccinimidyl (4-iodoacetyl)aminobenzoate), SIAB (Succinimidyl (4-iodoacetyl)aminobenzoate), SBAP (Succinimidyl 3-(bromoacetamido)- propionate), SIA (Succinimidyl iodoacetate), Sulfo-SMCC (Sulfosuccinimidyl-4-(Nmaleimidomethyl)-cyclohexane-l-carboxylate), SM(PEG)n (NHS-PEG-Maleimide Crosslinkers: Succinimidyl-([N-maleimidopropionamido])-#ethyleneglycol) ester, # =1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), LC-SMCC (Succinimidyl 4-(Nmaleimidomethyl) cyclohexane-l-carboxy-(6-amidocaproate)), Sulfo-EMCS (N-epsilonMaleimidocaproyl-oxysulfosuccinimide ester), EMCS (N-epsilon-Malemidocaproyloxysuccinimide ester), Sulfo-GMBS (N-gamma-Maleimidobutyryl-oxysulfosuccinimide ester), GMBS (N-gamma-Maleimidobutyryl-oxysuccinimide ester), Sulfo-KMUS (N-kappamaleimidoundecanoyl-oxysulfosuccinimide ester), Sulfo-MBS (m-maleimido-benzoyl-N- 89 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 hydroxysulfosuccinimidc ester), MBS (m-Malcimidobcnzoyl-N-hydroxysuccinimidc ester), Sulfo-SMPB ((Sulfo-succinimidyl 4-(p-maleiimidophenyl)butyrate), SMPB(Succinimidyl 4-(pmaleimido-phenyl)butyrate), AMAS N-(a-Maleimidoacetoxy) succinimide ester), BMPS (Nbeta-Maleimidopropyloxy-succinimide ester), SMPH (Succinimidyl 6-[(beta-maleimidopropionamido)hexanoate]), PEG12-SPDP (2-Pyridyldithiol-tetraoxaoctatriacontane-Nhydroxysuccinimide), PEG4-SPDP (2-Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide), Sulfo-LC-SPDP (Sulfosuccinimidyl 6-[3'-(2-pyridyldithio)propionamidoJhexanoate), LC-SPDP (Succinimidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate), SMPT (4- Succinimidyl-oxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene); Carboxyl-to-Amine Crosslinkers: DCC (Dicyclohexylcarbodiimide), EDC (l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide); Photoreactive Crosslinkers: ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide), NHS-Diazirine (SDA) Crosslinkers: SDA (NHS-Diazirine)(Succinimidyl 4,4'- azipentanoate), LC-SDA (NHS-LC-Diazirine) (Succinimidyl 6-(4,4'-azipentanamido)- hexanoate), SDAD (NHS-SS-Diazirine) (Succinimidyl 2-([4,4'-azipentanamido]ethyl)-l,3'- dithiopropionate), Sulfo-SDA (Sulfo-NHS-Diazirine) (Sulfosuccinimidyl 4,4’-azipentanoate), Sulfo-LC-SDA (Sulfo-NHS-LC-Diazirine) (Sulfosuccinimidyl 6-(4,4'-azipentanamido)- hexanoate), Sulfo-SDAD (Sulfo-NHS-SS-Diazirine)(Sulfosuccinimidyl 2-([4,4'- azipentanamido]ethyl)-l,3'-dithiopropionate), Sulfo-SANPAH (Sulfosuccinimidyl 6-(4'-azido-2'- nitrophenylamino)-hexanoate), SPB (Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate); Sulfhydrylto-Carbohydrate Crosslinkers: BMPH (N-beta-Maleimidopropionic acid hydrazide-TFA), EMCH (N-epsilon-Maleimidocaproic acid hydrazide-TFA), KMUH (N-kappa-malcimidoundecanoic acid hydrazide-TFA), MPBH (4-(4-N-Maleimidophenyl)butyric acid hydrazideHC1), PDPH (3-(2-Pyridyldithio)propionyl hydrazide); Sulfhydryl-to-Hydroxyl Crosslinkers: PMPI (p-Maleimidophenyl isocyanate); Sulfhydryl-to-Sulfhydryl Crosslinkers: BM(PEG)2 (1,8- Bismaleimido-diethyleneglycol), BM(PEG)3 (1,11-Bismaleimidotriethyleneglycol), BMB (1,4- Bismaleimidobutane), BMDB (l,4-Bismaleimidyl-2,3-dihydroxybutane), BMH (Bismaleimidohexane), BMOE (Bismaleimidoethane), DTME (Dithiobismaleimido-ethane), TMEA (Tris(2- maleimidoethyl)amine) and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The bis-maleimide or bis-2-pyridyldithiol reagents allow the attachment of the thiol group of a thiol-containing cell binding agent (such as antibody) to a thiol-containing drug moiety, label, or linker intermediate, in a sequential or concurrent fashion. Other functional groups besides maleimide and pyridyldithiol, which are reactive with a thiol group of a cell binding agent, drug moiety, label, or linker intermediate include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. 90 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 In additional embodiments, the linker may be composed of one or more linker components. The exemplary linker components are: 1. The self-immolative linker components: O wherein the (*) atom is the point of attachment of additional spacer or releasable linker units, or the cytotoxic agent, and/or the binding molecule (CBA); X1, Y1, Z2 and Z3 are independently NH, or O, or S; Z1 is H, or NH, or O or S independently, v is 0 or 1; Q1 is independently H, OH, Ci-Ce alkyl, (OCH2CH2)n F, Cl, Br, I, ORi, or SR!, NR^ N=NRX, N^NR^^ SORjR2, SO2Ri, SO3Ri, OSOd^, PRdC. POR1R2, PO2RiR2, OPO(OR,)(OR2). or OCH2PO(ORI(OR2) wherein Ri and R2 are described above, preferabably independently selected from H, Ci~Cg of alkyl; C2~Cg of alkenyl, alkynyl, heteroalkyl; Ca-Cg of aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl; or pharmaceutical cation salts 2. The examples of non-self-immolative linker components: (Œ2)nCO(OCH2CH2)rOCH3 (CH2)nCON(CH2CH2O)rCOŒ3 *(CH2CH2O)r* 9 9 91 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 5 92 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 wherein the (*) atom is the point of attachment of additional spacer or releaseable linkers, the cytotoxic agents, and/or the binding molecules; X1, Y1. Q1, RI, R’, R” are defined above; r is 1-20, m and n are 1-6. 3. Exemplary linker components may include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("alaphe" or "af"), p-aminobenzyloxy-carbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("SMCC"), NSuccinimidyl (4-iodo-acetyl)amino-benzoate ("SIAB"), ethyleneoxy (—CH2CH2O—) as one or more repeating units ("EO" or "PEO"). Additional linker components are known in the art and some are described through this patent application. In additional embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (VC or val-cit), alanine-phenylalanine (af or alaphe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycineglycine (gly-gly-gly). Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. In the cell-binding agent - drug conjugates of the invention, cell-binding agent (Q) is conjugated to one or more drug moieties (Drug, or PBD derivatives), e.g. about 1 to about 20 drug moieties per cell-binding agent, through a bifunctional linker (L). The conjugate of Formula (I), (II), (III), and (IV) may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of a cell-binding agent with a bivalent linker reagent, to form Q-L, via a covalent bond, followed by reaction with a drug moiety Drug; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form Drug-L, via a covalent bond, followed by reaction with the nucleophilic group of a cell-binding agent. To synthesize the conjugates of Formula (I), (II), (III) or/and (IV) in general, a function group E3or/and E3’ on Formula (V), (VI), (VII) and (VIII) reacts one, two or more residues of a cell binding molecule at 0-60°C, pH 5-9.5 aqueous media with or without addition of 0-30% of water mixable (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylene diol, following by dialysis or chromatographic purification to form a conjugate compound of Formula (I), (II), (III) or/and 93 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (IV). Some of the residue (reacting group for conjugation) of the cell-binding molecule can be obtained through protein engineering. The thiol or amine groups on a cell-binding agents, such as an antibody, are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker reagents and drug-linker intermediates including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups; and (iv) disulfides, including pyridyl disulfides, via sulfide exchange. Nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents. Nucleophilic groups on antibodies or proteins can react to electrophilic groups on a function linker following by reaction with a cytotoxic agent, or directly react to a linkercytotoxic agent moiety to form covalent bond conjugate of a cell binding agent-cytotoxic agent. Nucleophilic groups on antibodies or proteins include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker-cytotoxic agent moieties including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges which may be made reactive by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethyl¬ phosphine (TCEP) (Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass), dithioerythritol (DTE), L-glutathione (GSH), 2-mercaptoethylamine (p-MEA), or/and beta mercaptoethanol (p-ME, 2-ME). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Alternatively, sulfhydryl groups can be introduced into antibodies through modification of lysine residues, e.g., by reacting lysine residues with 2-iminothiolane (Traut's reagent), resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into an antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing variant antibodies comprising one or more non-native cysteine amino acid residues). Thus free thiol on the cell binding agents can be conjugated to the thiol-reactive groups, such as, a maleimide, an iodoacetamide, a pyridyl disulfide, or other thiol-reactive groups on the cytotoxic agents, or linker-cytotoxic agent intermediates of the invention. Some 94 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 unconjugated free thiols on the antibodies can be reoxidized to reform interchain and intrachain disulfide bonds. Antibody-drug conjugates of the invention may also be produced by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one embodiment, an antibody is modified to introduce electrophilic moieties that are capable of reacting with nucleophilic substituents on the linker reagent or drug. In another embodiment, the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing Nterminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chern. 3:138-146; U.S. Pat. No. 5,362,852). Such an aldehyde can be reacted with a drug moiety or linker nucleophile. Examples of these kinds of two-step conjugations are depicted below: O _c SX E S-s r7-Drug X™ R'^R" R R H,N-CBA - CBA 1-20 O HjN-CBA I.N-R7 N^c]ja Drug-S^o g 7 r Drug—SH _ yE3 Drug “^E O Q O n Q O o Ar o H Drug Drug 95 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Br O s O E Drug-SH D™/ ¥^-»7 zE H2N-CBA s &Y\^R7 JJ% 'CBA5 o °2S^X^CBA « Br^ O o ° o ^«rug-s g o ° o ,,'^< E^ Drug ^/ E, j W Drug-SH H2N—CBA mug wherein E includes, but is not limited to, such as hydroxysuccinimidyl esters (NHS, SulfoNHS, etc), 4-nitrophcnyl esters, pentafluorophenyl esters, tetrafluorophenyl (includes sulfo¬ tetrafluorophenyl) esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. R’ and R” are independently H or CH3, orCaHs; J is F, Cl, Br, I, tosylate (TsO), mesylate (MsO), nitrophenol, dinitrophenol, or pentaflourophenol. It is to be understood that where more than one nucleophilic group on the cell binding agents, such as an antibody, reacts with a drug-linker intermediate or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of the cell binding agent-cytoxic agent conjugates with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography. In certain embodiments, a homogeneous conjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography. In the conjugation, The loading (drug/antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of lysine or cysteine residues is modified for control of the number and/or position of linker-drug attachments (such as thioMab or thioFab). The synthetic conjugate may be purified by standard biochemical means, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, and ion exchange or by dialysis. In some cases, a small molecule as a cell-binding agent (e.g. folic acid, melanocyte stimulating hormone, EGF etc) conjugated with a small molecular drugs can be purified by chromatography such as by HPLC, medium pressure column chromatography or ion exchange chromatography. 96 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 The aqueous solutions for the modification of cell-binding agents are buffered between pH 4 and 9, preferably between 6.0 and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH ranges. Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES, and MOPS buffers, which can contain additional components, such as cyclodextrins, hydroxypropyl-P-cyclodextrin, polyethylene glycols, sucrose and salts, for examples, NaCl and KC1. After the addition of the drug-linker of Formula (V), (VI), (VH) or Formula (VIII) into the solution containing the reduced cell-binding molecules, the reaction mixture is incubated at a temperature of from 4 °C to 55 °C, preferably at 15°C - ambient temperature. The progress of the reaction can be monitored by measuring the decrease in the absorption at a certain UV wavelength, such as at 252 nm, or increase in the absorption at a certain UV wavelength, such as 280 nm, or the other appropriate wavelength. After the reaction is complete, isolation of the modified cell-binding agent can be performed in a routine way, using for example a gel filtration chromatography, an ion exchange chromatography, an adsorptive chromatography or column chromatography over silica gel or alumina, crystallization, preparatory thin layer chromatography, ion exchange chromatography, or HPLC. The extent of modification can be assessed by measuring the absorbance of the nitropyridine thione, dinitropyridine dithione, pyridine thione, carboxylamidopyridine dithione and dicarboxyl-amidopyridinc dithione group released via UV spectra. For the conjugation without a chromophore group, the modification or conjugation reaction can be monitored by LCMS, preferably by HPLC-MS/MS, UPLC-QTOF mass spectrometry, or Capilary electrophoresis -mass spectrometry (CE-MS). The side chain cross-linkers described herein have diverse functional groups that can react with any cell-binding molecules, particularly a modified cell¬ binding molecule that possess a suitable substituent. For examples, the modified cell-binding molecules bearing an amino or hydroxyl substituent can react with drugs bearing an N-hydroxysuccinimide (NHS) ester, the modified cell-binding molecules bearing a thiol substituent can react with drugs bearing a maleimido or haloacetyl group. Additionally, the modified cell-binding molecules bearing a carbonyl (ketone or aldehyde) substituent either through protein engineering, enzymatical reaction or chemical modification can react with drugs bearing a hydrazide or an alkoxyamine. One skilled in the art can readily determine which modified drug-linker to be used based on the known reactivity of the available functional group on the modified cell-binding molecules. Other further examplary methods for preparing ADC are described in Figures 1-32 and examples in the description of the patent. 97 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 APPLICATION OF CONJUGATES OF CELL-BINDING AGENT WITH CROSS¬ LINKED PBD DIMERS. The cell-binding agent-cross-linked PBD dimer conjugate, preferably antibody- cross¬ linked PBD dimer conjugates (PBD dimer ADC) of the present invention may be used to treat various diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia and lymphoid malignancies. Others include neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic, including autoimmune, disorders. In specific embodiment, the conjugates of the invention are used in accordance with the compositions and methods of the invention for the treatment of cancers. The cancers include, but are not limited. Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependymoma, Medulloblastoma, Supratentorial Primitive Neuroectodermal and Pineal Tumors, Visual Pathway and Hypothalamic Glioma), Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor, Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell cancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid, Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell, Lymphoma (AIDS-Related, Central Nervous System, Cutaneous T-Cell, Hodgkin's Disease, Non-Hodgkin’s Disease, Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome, Myeloproliferative Disorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine, Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter (Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, Skin Cancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma, Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular 98 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Cancer, Wilms' Tumor In another specific embodiment, the compounds and the conjugates of the invention are used in accordance with the compositions and methods of the invention for the treatment or prevention of an autoimmune disease. The autoimmune diseases include, but are not limited, Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease, Agammaglobulinemia, Alopecia areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase syndrome. Arthritis, Atopic allergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger's disease, Bickerstaffs encephalitis, Blau syndrome, Bullous Pemphigoid, Castleman's disease, Chagas disease, Chronic Fatigue Immune Dysfunction Syndrome, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis, Chronic lyme disease, Chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Cranial arteritis, CREST syndrome, Crohns Disease (a type of idiopathic inflammatory bowel diseases), Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressier's syndrome, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto’s thyroiditis, Haemolytic anaemia, Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa, Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenic purpura), IgA nephropathy (Also Berger's disease), Inclusion body myositis, Inflammatory demyelinating polyneuopathy, Interstitial cystitis, Irritable Bowel Syndrome , Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease, Lambert-Eaton 99 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease (Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, Ménière's disease, Microscopic polyangiitis, MillerFisher syndrome, Mixed Connective Tissue Disease, Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, Multiple Myeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatrie Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Tumer syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome, Schnitzler syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome, Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathic inflammatory bowel diseases), Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome In another specific embodiment, a binding molecule used for the conjugate for the treatment or prevention of an autoimmune disease includes, but are not limited to, anti-elastin antibody; Abys against epithelial cells antibody; Anti-Basement Membrane Collagen Type IV Protein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody, T-cells antibody; Thyroglobulin Antibody, Anti SCL-70; Anti-Jo; Anti-U.sub.IRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody, Anti-ganglioside antibody; AntiDesmogein 3 antibody; Anti-p62 antibody; Anti-spl00 antibody; Anti-Mitochondrial(M2) antibody; Rheumatoid factor antibody; Anti-MCV antibody; Anti-topoisomerase antibody; Anti¬ neutrophil cytoplasmic(cANCA) antibody. 100 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 In certain preferred embodiments, the binding molecule for the conjugate in the present invention, can bind to either a receptor or a receptor complex expressed on an activated lymphocyte which is associated with an autoimmune disease. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28,CD30, CD33, CD37, CD38, CD56, CD79, CD79b, CD90, CD152/CTLA-4, PD-1, or ICOS), a TNF receptor superfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-RI, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin (C-type, S-type, or Itype), or a complement control protein. In another specific embodiment, useful binding ligands that are immunospecific for a viral or a microbial antigen are humanized or human monoclonal antibodies. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein (e.g. HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuramimidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term "microbial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response. Examples of antibodies available 1for the viral or microbial infection include, but are not limited to, Palivizumab which is a humanized antirespiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542 which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir which is a human antibody for the treatment of hepatitis B virus; PROTVIR which is a humanized IgG.sub.l antibody for the treatment of cytomegalovirus; and anti-LPS antibodies. The binding molecules-cytotoxic agent conjugates of this invention can be used in the treatment of infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and 101 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever, Common cold (Acute viral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremic syndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires’ disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever, Measles, Melioidosis (Whitmore’s disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis, Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus 102 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 infection. Rickettsial infection, Rickettsialpox, Rift Valley fever, Rocky mountain spotted fever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber’s itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea pedis (Athlete’s foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viral pneumonia, West Nile Fever, White piedra (Tinea blanca), Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis. The binding molecules, more preferred antibodies described in this patent that are against pathogenic strains include, but are not limit, Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (Human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitidcs, Machupo virus, Borrelia genus, Clostridium botulinum, Sabia, Brucella genus, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter genus, usually Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense; multiple parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4)- Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcus genus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Human herpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, 103 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Fusobacterium genus, Clostridium pcrfringcns; other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza viruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus, Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous species of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii, Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformis and Spirillum minus, Respiratory syncytial virus, Rhinosporidium seeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea wemeckii, Trichophyton genus, Malassczia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris 104 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 trichiura, Mycobacterium tuberculosis, Francisclla tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus. Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms). Other antibodies as a binding ligand in this invention for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridac. Adenoviridae, Papovaviridae, Enteroviridae, Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervical cancer, Anal cancer), Kaposi’s sarcomaassociated herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt's lymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cell cancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [such as Human Immunodeficiency Virus (AIDS)]; Central nervous system virus: [such as, JCV (Progressive multifocal leukoencephalopathy), MeV (Subacute sclerosing panencephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable) (Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviral meningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis, Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)]; Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis)); Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)]; Respiratory system/acute viral nasopharyngitis/viral pneumonia: [Epstein-Barr virus (EBV infection/Infectious mononucleosis), Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome) Orthomyxoviridae: Influenzavirus 105 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 A/B/C (Influcnza/Avian influenza), Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Human respiratory syncytial virus), hMPV]; Digestive system virus [MuV (Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus (Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis C virus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (Hepatitis G virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)]. FORMULATION AND APPLICATION OF THE CONJUGATES. The conjugates of the patent application are formulated to liquid, or suitable to be lyophilized and subsequently be reconstituted to a liquid formulation. A liquid formulation comprising 0.1 g/L ~300 g/L of concentration of a conjugate of the patent application as an active ingredient for delivery to a patient without high levels of antibody aggregation may include one or more polyols (e.g. sugars), a buffering agent with pH 4.5 to 7.5, a surfactant (e.g. polysorbate 20 or 80), an antioxidant (e.g. ascorbic acid and/or methionine), a tonicity agent (e.g. mannitol, sorbitol or NaCl), chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; a preservative (e.g. benzyl alcohol) and/or a free amino acid. In a preferred embodiment, the conjugate of the invention in vivo clinical use will be supplied as solutions or as a lyophilized solid (such as powder) that can be redissolved in sterile water for injection. The conjugate in a liquid formula or in the formulated lyophilized powder may take up 0.01%-99% by weight as major gradient in the formulation. The rest of the formulation is excipients which are comprised of one or more of the following compounds: 0.5%~25% of buffering reagents, 0%~20% of polyols, 0%~2.0% of surfactants, 0%~5% of preservatives, 0%-30% of Amino acids or bulky compounds, 0%~5% of Antioxidants, 0%~0.3% chelating agents. Suitable buffering agents for use in the formulations include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, tromethamine (tris(hydroxymethyl)- aminomethane) hydrochloride, or phosphate buffer. In addition, amino acid components can also be used as buffering agent. Such amino acid component includes without limitation arginine, glycine, glycylglycine, and histidine. The arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride¬ arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-argine succinate, etc. The formulations of 106 5 10 15 20 25 30 35 the buffers have a pH of 4.5 to pH 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salts in the buffer is from about 10 mM to about 500 mM.. A "polyol" that may optionally be included in the formulation is a substance with multiple hydroxyl groups. Polyols can be used as stabilizing excipients and/or isotonicity agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals ftom both physical and chemical degradation pathways. Preferentially excluded co-solvents increase the effective surface tension of solvent at the protein interface whereby the most energetically favorable structural conformations are those with the smallest surface areas. Polyols include sugars (reducing and nonreducing sugars), sugar alcohols and sugar acids. A "reducing sugar" is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a "nonreducing sugar" is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nonreducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol and glycerol. Sugar acids include L-gluconate and its metallic salts thereof. Preferably, a nonreducing sugar: sucrose or trehalose at a concentration of about from 0.01% to 15% is chosen in the formulation, wherein trehalose being preferred over sucrose, because of the solution stability of trehalose. A surfactant optionally in die formulations is selected from polysorbate (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and die like); poloxamer (e.g. poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407 or polyethylene-polypropylene glycol and the like); Triton™; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimcdiylaminc; sodium methyl cocoyl-, or disodium methyl olcyl-tauratc; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and coco ampho glycinate; and the MONAQUAT™ series (e.g. isostearyl ethylimidonium ethosulfate); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc.); etc. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters e.g. polysorbate 20, 40, 60 or 80 (Tween™ 20, 40, 60 or 80). The concentration of a surfactant is range from 0.0001% to about 1.0%. In certain embodiments, the surfactant concentration is from about 0.01% to about 0.1%. In one embodiment, the surfactant concentration is about 0.02%. 107 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 A "preservative" optionally in the formulations is a compound that essentially reduces bacterial action therein. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexaméthonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethyl-ammonium chlorides in which the alkyl groups are long-chain compounds), and benzéthonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The preservative is less than 5% in the formulation. Preferably 0.01% to 1%. In one embodiment, the preservative herein is benzyl alcohol. Suitable free amino acids optionally for use in the formulation, but are not limited to, are arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid. The inclusion of a basic amino acid is preferred i.e. arginine, lysine and/or histidine. If a composition includes histidine then this may act both as a buffering agent and a free amino acid, but when a histidine buffer is used it is typical to include a non-histidine free amino acid e.g. to include histidine buffer and lysine. An amino acid may be present in its Dand/or L-form, but the L-form is typical. The amino acid may be present as any suitable salt e.g. a hydrochloride salt, such as arginine-HCl. The concentration of an amino acid is range from 0.0001% to about 15.0%. Preferably 0.01% to 5%. The formulations can optionally comprise methionine or ascorbic acid as an antioxidant at a concentration of about from 0.01 mg/ml to 5 mg/ml; The formulations can optionally comprise chelating agent, e.g., EDTA, EGTA, etc., at a concentration of about from 0.01 mM to 2 mM. The final formulation may be adjusted to the preferred pH with an adjust agent (e.g. an acid, such as HCl, H2SO4, acetic acid, H3PO4, citric acid, etc., or a base, such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanol amine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc.) and the formulation should be controlled "isotonic" which is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. Other excipients which may be useful in either a liquid or lyophilized formulation of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluoronic F-127, cellulose, cyclodextrin, dextran (10, 40 and/or 70 kD), polydextrose, maltodextrin, ficoll, gelatin, hydroxypropylmeth, sodium phosphate, potassium phosphate, ZnCh, zinc, zinc oxide, sodium citrate, trisodium 108 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohols, or polyalcohols, hydrogenated forms of carbohydrate having a carbonyl group reduced to a primary or secondary hydroxyl group. Other contemplated excipients, which may be utilized in the aqueous pharmaceutical compositions of the patent application include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such sodium and the like. These and additional known pharmaceutical excipients and/or additives suitable for use in the formulations of the invention are known in the art, e.g., as listed in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005). In order to reduce patient pain during the injection of the formulation, a local analgesic agent can be used along with or prior to the injection of the formulation. Commonly used analgesics are: benzyl alcohol (0.01%-l%), Procaine hydrochloride (0.2%~2.0%), lidocaine hydrochloride (0.2%~2.0%), 2-tri-chloromethyl-2-propanol (0.3%~0.5%), tramadol, morphine, morphine sulfate, hydromorphone, oxycodone hydrochloride, dobutamine, gabapentin, cyclobenzaprine, trazodone, clonidine, codeine. The conjugates formulation of the patent application can be prepared as a pre-filled syringe solution, or a freezing-dry powder, or through high-efficient spray drying powder._A pharmaceutical container or a vessel is used to hold the pharmaceutical formulation of the conjugates. The vessel is a vial, bottle, pre-filled syringe, or pre-filled auto-injector syringe. In a further embodiment, the invention provides a method for preparing a formulation comprising the steps of: (a) lyophilizing the formulation comprising the conjugates, excipients, and a buffer system to a powder; and (b) reconstituting the lyophilized mixture of step (a) in a reconstitution medium such that the reconstituted formulation is stable. The formulation of step (a) may further comprise a stabilizer and one or more excipients selected from a group comprising bulking agent, salt, surfactant and preservative as hereinabove described. As reconstitution media several diluted organic acids or water, i.e. sterile water, bacteriostatic water for injection (BWFI) or may be used. The reconstitution medium may be selected from water, i.e. sterile water, bacteriostatic water for injection (BWFI) or the group consisting of acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solution of sodium 109 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 chloride, acidic solution of magnesium chloride and acidic solution of arginine, in an amount from about 10 to about 250 mM. A liquid pharmaceutical formulation of the conjugates of the patent application should exhibit a variety of pre-defined characteristics. One of the major concerns in liquid drug products is stability, as proteins/antibodies tend to form soluble and insoluble aggregates during manufacturing and storage. In addition, various chemical reactions can occur in solution (deamidation, oxidation, clipping, isomerization etc.) leading to an increase in degradation product levels and/or loss of bioactivity. Preferably, a conjugate in either liquid or loyphilizate formulation should exhibit a shelf life of more than 18 months at 25°C. More preferred a conjugate in either liquid or loyphilizate formulation should exhibit a shelf life of more than 24 months at 25°C. Most preferred liquid formulation should exhibit a shelf life of about 24 to 36 months at 2-8° C and the loyphilizate formulation should exhibit a shelf life of about preferably up to 60 months at 2-8° C. Both liquid and loyphilizate formulations should exhibit a shelf life for at least two years at -20° C, or -70° C. In certain embodiments, the formulation is stable following freezing (e. g., -20°C, or -70° C.) and thawing of the formulation, for example following 1, 2 or 3 cycles of freezing and thawing. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of drug/antibody(protein) ratio and aggregate formation (for example using UV, size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy¬ terminal sequence analysis; mass spectrometric analysis, or matrix-assisted laser desorption ionization/timc-of-flight mass spectrometry (MALDI/TOF MS), or HPLC-MS/MS; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS--C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomeriation), clipping/hydrolysis/ fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), Nterminal extension, C-terminal processing, glycosylation differences, etc. A stable conjugate should also "retains its biological activity" in a pharmaceutical formulation, if the biological activity of the conjugate at a given time, e. g. 12 month, within about 20%, preferably about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared as determined in an antigen binding assay, and/or in vitro, cytotoxic assay, for example. 110 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Examples of suitable protocols of conjugate administration are as follows. Conjugates are given daily, weekly, biweekly, triweekly, once every four weeks or monthly for 8~54 weeks as an i.v. bolus. Bolus doses are given in 50 to 1000 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mL of a concentrated solution of human serum albumin, 100 mg/mL) can optionally be added. Dosages will be about 50 pg to 20 mg/kg of body weight per week, i.v. (range of 10 pg to 200 mg/kg per injection). 4~54 weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc., can be determined by the skilled clinicians. Examples of in vitro uses include treatments of cell cultures in order to kill all cells except for desired variants that do not express the target antigen; or to kill variants that express undesired antigen. Examples of ex vivo uses include treatments of hematopoietic stem cells (HSC) prior to the performance of the transplantation (HSCT) into the same patient in order to kill diseased or malignant cells. For instance, clinical ex vivo treatment to remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplant in order to prevent graft-versus-host disease, can be carried out as follows. Bone marrow is harvested from the patient or other individual and then incubated in medium containing serum to which is added the conjugate of the invention, concentrations range from about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37 °C. The exact conditions of concentration and time of incubation (=dose) are readily determined by the skilled clinicians. After incubation the bone marrow cells are washed with medium containing serum and returned to the patient by i.v. infusion according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment. Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any types of cancer, autoimmune diseases, graft rejections, and infections (viral, bacterial or parasite). Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of killing selected cell populations include malignancy of any type including, for example, cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs; melanomas; autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft 111 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 versus host disease; viral infections, such as CMV infection, HIV infection, AIDS, etc.; bacterial infection; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by one skilled in the art. The identification of those subjects who are in need of treatment of herein-described diseases and conditions is well within the ability and knowledge of one skilled in the art. A veterinarian or a physician skilled in the art can readily identify, by the use of clinical tests, physical examination, medical/family history or biological and diagnostic tests, those subjects who are in need of such treatment. A therapeutically effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of subject; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristic of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. The amount of a conjugate which is required to achieve the desired biological effect, will vary depending upon a number of factors, including the chemical characteristics, the potency, and the bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the diseased state of the patient, the route of administration, all factors which dictate the required dose amounts, delivery and regimen to be administered. In general terms, the conjugates of this invention may be provided in an aqueous physiological buffer solution containing 0.1 to 30% w/v conjugates for parenteral administration. Typical dose ranges are from 1 pg/kg to 0.1 g/kg of body weight daily; weekly, biweekly, triweekly, or monthly, a preferred dose range is from 0.01 mg/kg to 20 mg/kg of body weight weekly, biweekly, triweekly, or monthly, an equivalent dose in a human. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the route of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the conjugates by the chosen delivery route, and the speed (bolus or continuous infusion) and schedule of administrations (number of repetitions in a given period of time). The cell binding agent -cytotoxic agent conjugates of the present invention are also capable of being administered in unit dose forms, wherein the term “unit dose” means a single dose which is capable of being administered to a patient, and which can be readily handled and packaged, 112 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 remaining as a physically and chemically stable unit dose comprising either the active conjugate itself, or as a pharmaceutically acceptable composition, as described hereinafter. As such, typical total daily dose ranges are from 0.01 to 100 mg/kg of body weight. By way of general guidance, unit doses for humans range from 1 mg to 3000 mg per day, weekly, biweekly, or triweekly. Preferably the unit dose range is from 1 to 500 mg administered one to two times weekly, biweekly, or triweekly, and even more preferably from 10 mg to 500 mg, biweekly, or triweekly. Conjugates provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be prepared for use by I.V. or oral administration, particularly in the form of powders, tablets, simple capsules or soft gel capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically in ointments, creams, lotions, gels or sprays, or via trans-dennal patches. The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington: The Science and Practice of Pharmacy, 21th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005. Preferred formulations include pharmaceutical compositions in which a compound of the present invention is formulated for oral or parenteral administration. For oral administration, tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings. In addition, the active compounds may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal. Preferred tablets contain lactose, cornstarch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, or talc in any combination. Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, buffered media, and saline. In particular, 113 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene¬ polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Alternative modes of administration include formulations for inhalation, which include such means as dry powder, aerosol, or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for buccal administration include, for example, lozenges or pastilles and may also include a flavored base, such as sucrose or acacia, and other excipients such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit-dose suppositories, with a solid based carrier, such as cocoa butter, and may include a salicylate. Formulations for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanolin, polyethylene glycols, alcohols, or their combinations. Formulations suitable for transdermal administration can be presented as discrete patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. In a specific embodiment, the cell binding agent -cytotoxic agent conjugates of this invention are administered concurrently with the other known or will be known therapeutic agents such as the chemotherapeutic agent, the radiation therapy, immunotherapy agents, autoimmune disorder agents, anti-infectious agents or the other antibody-drug conjugates, resulting in a synergistic effect for effective treatment or prevention of a cancer, or an autoimmune disease, or an infectious disease. In another specific embodiment, the synergistic drugs or radiation therapy are administered prior or subsequent to administration of a conjugate, in one aspect at least an hour, 12 hours, a day, a week, two weeks, three weeks, a month, in further aspects several months, prior or subsequent to administration of a conjugate of the invention. The synergistic agents are preferably selected from one or several of the following drugs: 1). Chemotherapeutic agents: a). Alkylating agents: such as [Nitrogen mustards: (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, trofosfamide); Nitrosoureas: (carmustine, lomustine); Alkylsulphonates: (busulfan, treosulfan); Triazenes: (dacarbazine); Platinum containing compounds: (carboplatin, cisplatin, oxaliplatin)]; b). Plant Alkaloids: such as [Vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine); Taxoids: (paclitaxel, docetaxol)]; c). DNA Topoisomerase Inhibitors: such as [EpipodophyIlins: (9- 114 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 aminocamptothccin, camptothecin, crisnatol, etoposide, etoposide phosphate, irinotecan, teniposide, topotecan,); Mitomycins: (mitomycin C)J; d). Anti-metabolites: such as {[Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate); IMP dehydrogenase Inhibitors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs: (5-Fluorouracil, doxifluridine, floxuridine, ratitrexed(Tomudex)); Cytosine analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, mercaptopurine, thioguanine)]}; e). Hormonal therapies: such as {Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate); Anti-androgens: (bicalutamide, flutamide)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrellin A); Cytokines: (Interferon-alpha, Interferon-gamma, tumor necrosis factor (TNFs), human proteins containing a TNF domain)]}; f). Kinase inhibitors, such as BIBW 2992 (antiEGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab, ispinesib; g). Others: such as gemcitabine, epoxomicins (e. g. carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins (such as l-methyl-4- phenylpyridinium ion), Cell cycle inhibitors (such as staurosporine), Actinomycins (such as Actinomycin D, dactinomycin), Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), Anthracyclines (such as daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDR inhibitors (such as verapamil), Ca2+ATPase inhibitors (such as thapsigargin), vismodegib, Histone deacetylase inhibitors (Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A) ; Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate. Disulfiram, Salinosporamide A. More detail lists of known and will be known anti-cancer drugs that can be used as a combination therapy (a synergistic effect) with the compounds and conjugates of the invention can be seen in National Cancer Institute (US) website (www.cancer.gov; www.cancer.gov/cancertopics/druginfo/alphalist), American Cancer Society (www.cancer.org/treatment/index) and Cancer Research UK (www.cancerrearchuk.org; (www.cancerresearchuk.org/cancer-help/about-cancer/treatment/cancer-drugs/) 115 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 2). An anti-autoimmune disease agent includes, but is not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g. amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus. 3). An anti-infectious disease agent includes, but is not limited to, a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol, florfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e). Cephems: carbacephem (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefjpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f). Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g. tigecycline; g). p-Lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j). Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k). Macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketoIide (telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin;1). Monobactams: aztreonam, tigemonam; m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (carindacillin), cioxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam), mezlocillin, 116 5 10 15 20 25 30 35 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 meticillin, nafcillin. oxacillin, pcnamccillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin, colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q). Streptogramins: pristinamycin, quinupristin/dalfopristin); r). Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimcthoprim-sulfamcthoxazolc (co-trimoxazole); s). Steroid antibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g. tigecycline); u). Other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin; 4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturation inhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e). Nucleosides &_nucleotides: abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddl), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3’-fluoro¬ substituted 2’, 3’-dideoxynucleoside analogues (e.g. 3’-fluoro-2’,3’-dideoxythymidine (FLT) and 3’-fluoro-2’,3’-dideoxyguanosine (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. /M-thymidine and >8-1-2’-deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h). Other types of anti-virus drugs: 117 5 10 15 20 25 30 35 abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscamet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, seliciclib. 5). Other immunolheraphy drugs: e.g. imiquimod, interferons (e.g. , granulocyte colony-stimulating factors, cytokines. Interleukins (IL-1 ~ IL-35), antibodies (e. g. trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab), Protein-bound drugs (e.g., Abraxanc), an antibody conjugated with drugs selected from calicheamicin derivative, of maytansine derivatives (DM1 and DM4), CC- 1065 and duocarmycin minor groove binders, potent Taxol™ derivatives, doxorubicin, auristatin antimitotic drugs (e. g. Trastuzumab-DMl, Inotuzumab ozogamicin, Brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab merlansine, AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, milatuzumab-doxorubicin, SGN-75 (anti-CD70), Anti-CD22-MCC-DMl, IMGN853, AntiCD22-MMAE, Anti-CD22-MMAF, Anti-CD22- calicheamicin. In a further embodiment,die synergistic agents are selected from one or several of the following drugs: Abatacept, Abiraterone acetate, Abraxane, Acetaminophen/hydrocodone, Acalabrutinib, aducanumab, Adalimumab, ADXS31-142, ADXS-HER2, afatinib dimaleate, aldesleukin, alectinib, alemtuzumab, Alitretinoin, ado-trastuzumab emtansine, Amphetamine/ dextroamphetamine, anastrozole, Aripiprazole, anthracyclines, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin, Avelumab, Axicabtagene ciloleucel, axitinib, belinostat, BCG Live, Bevacizumab, bexarotene, blinatumomab, Bortezomib, bosutinib, brentuximab vedotin, brigatinib, Budesonide, Budesonide/formoterol, Buprenorphine, Cabazitaxel, Cabozantinib, capmatinib, Capecitabine, carfilzomib, chimeric antigen receptor-engineered T (CAR-T) cells, Celecoxib, ceritinib. Cetuximab, Chidamide, Ciclosporin, Cinacalcet, crizotinib, Cobimetinib, Cosentyx, crizotinib, CTL019, Dabigatran, dabrafenib, dacarbazine, daclizumab, dacomotinib, daptomycin, Daratumumab, Darbepoetin alfa, Darunavir, dasatinib, denileukin diftitox, Denosumab, Depakote, Dexlansoprazole, Dcxmcthylphcnidatc, Dexamethasone, DigniCap Cooling System, Dinutuximab, Doxycycline, Duloxctinc, Duvelisib, durvalumab, elotuzumab, Emtricitabine/Rilpivirine/Tenofovir, disoproxil fumarate, Emtricitbine/tenofovir/efavirenz, Enoxaparin, ensartinib, Enzalutamide, Epoetin alfa, erlotinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, exemestane, everolimus, exenatide ER, Ezetimibe, Ezetimibe/simvastatin, Fenofibrate, Filgrastim, fmgolimod, Fluticasone propionate, Fluticasone/salmeterol, fulvestrant, gazyva, gefitinib, Glatiramer, Goserelin acetate, Icotinib, Imatinib, Ibritumomab tiuxetan, ibrutinib, idelalisib, ifosfamide, Infliximab, imiquimod, ImmuCyst, Immuno BCG, iniparib, Insulin aspart, Insulin detemir, Insulin glargine, Insulin 118 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 lispro, Interferon alfa, Interferon alfa-lb, Interferon alfa-2a, Interferon alfa-2b, Interferon beta, Interferon beta la, Interferon beta lb, Interferon gamma-la, lapatinib, Ipilimumab, Ipratropium bromide/salbutamol, Ixazomib, Kanuma, Lanreotide acetate, lenalidomide, lenaliomide, lenvatinib mesylate, letrozole, Levothyroxine, Levothyroxine, Lidocaine, Linezolid, Liraglutide, Lisdexamfetamine, LN-144, lorlatinib, Memantine, Methylphenidate, Metoprolol, Mekinist, mericitabine/Rilpivirine/ Tenofovir, Modafinil, Mometasone, Mycidac-C, Necitumumab, neratinib, Nilotinib, niraparib, Nivolumab, ofatumumab, obinutuzumab, olaparib, Olmesartan, Olmesartan/ hydrochlorothiazide, Omalizumab, Omega-3 fatty acid ethyl esters, Oncorine, Oseltamivir, Osimertinib, Oxycodone, palbociclib, Palivizumab, panitumumab, panobinostat, pazopanib, pembrolizumab, PD-1 antibody, PD-L1 antibody, Pemetrexed, pertuzumab, Pneumococcal conjugate vaccine, pomalidomide, Pregabalin, ProscaVax, Propranolol, Quetiapine, Rabeprazole, radium 223 chloride, Raloxifene, Raltegravir, ramucirumab, Ranibizumab, regorafenib, Rituximab, Rivaroxaban, romidepsin, Rosuvastatin, ruxolitinib phosphate, Salbutamol, savolitinib, semaglutide, Sevelamer, Sildenafil, siltuximab, Sipuleucel-T, Sitagliptin, Sitagliptin/metformin, Solifenacin, solanezumab, Sonidegib, Sorafenib, Sunitinib, tacrolimus, tacrimus, Tadalafil, tamoxifen, Tafinlar, Talimogene laherparepvec, talazoparib, Telaprevir, talazoparib, Temozolomide, temsirolimus, Tenofovir/emtricitabine, tenofovir disoproxil fumarate, Testosterone gel, Thalidomide, TICE BCG, Tiotropium bromide, Tisagenlecleucel, toremifene, trametinib, Trastuzumab, Trabectedin (ecteinascidin 743), trametinib, tremelimumab, Trifluridine/tipiracil, Tretinoin, Uro-BCG, Ustekinumab, Valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, ziv-aflibercept, Zostavax, and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents, or excipients thereof, or a combination above thereof. The invention is further illustrated but not restricted by the description in the following examples. EXAMPLES The invention is further described in the following examples, which are not intended to limit the scope of the invention. Cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ), or The Shanghai Cell Culture Institute of Chinese Acadmy of Science, unless otherwise specified. Cell culture reagents were obtained from Invitrogen Corp., unless otherwise specified. All anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All other reagents and solvents were purchased as the highest grade available and 119 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 used without further purification. The preparative HPLC separations were performed with Varain PreStar HPLC. NMR spectra were recorded on Bruker 500 MHz Instrument. Chemical shifts (delta) are reported in parts per million (ppm) referenced to tetramethylsilane at 0.00 and coupling constants (J) are reported in Hz. The mass spectral data were acquired on a Waters Xevo QTOF mass spectrum equipped with Waters Acquity UPLC separations module and Acquity TUV detector. Example 1. Synthesis of di-tert-butyl l,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-l,2- dicarboxylate. Boe Boc I I O To di-tert-butyl hydrazine-1,2-dicarboxylate (8.01 g, 34,4 mmol) in DMF (150 ml) was added NaH (60% in oil, 2.76 g, 68.8 mmol). After stirred at RT for 30 min, tert-butyl 2- bromoacetate (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, quenched with addition of methanol (3 ml), concentrated, diluted with EtOAc (100 ml) and water (100 ml), separated, and the aqueous layer was extracted with EtOAc (2 x 50 ml). The organic layers were combined, dried over MgSO4, filtered, evaporated, and purified by SiO2 column chromatography (EtOAc/Hexane 1:5 to 1:3) to afforded the title compound (12.98 g, 82% yield) as a colorless oil. MS ESI m/z calcd for C22H41N2O8 [M+H]+ 461.28, found 461.40. Example 2. Synthesis of 2,2’-(hydrazine-l,2-diyl)diacetic acid. Di-tert-butyl l,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) in 1,4-dioxane (40 ml) was added HC1 (12 M, 10 ml). The mixture was stirred for 30 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to afford the crude title product for the next step without further production (2.15 g, 103% yield, -93% pure). MS ESI m/z calcd for C4H9N2O4 [M+H]+ 149.05, found 149.40. Example 3. Synthesis of 2,2’-(l,2-bis((benzyloxy)carbonyl)hydrazine-l,2-diyl)diacetic acid. To a solution of 2,2'-(hydrazine-l,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in the mixture of THF (200 ml) and NaH2PO4 (0.1 M, 250 ml, pH 8.0) was added benzyl carbonochloridate (5.01 g, 29.47 mmol) in 4 portions in 2 h. The mixture was stirred for another 6 h, concentrated and purified on SiO2 column eluted with H2O/CH3CN (1:9) containing 1% formic acid to afford 120 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 the title compound (2.26 g, 73% yield, -95% pure). MS ESI m/z calcd for C20H2iN2O8 [M+H]+ 417.12, found 417.40. Example 4. Synthesis of dibenzyl l,2-bis(2-chloro-2-oxoethyl)hydrazine-l,2- dicarboxylate. 2,2’-(l,2-bis((benzyloxy)carbonyl)hydrazine-l,2-diyl)diacetic acid (350 mg, 0.841 mmol) in dichloroethane (30 ml) was added (COC1)2 (905 mg, 7.13 mmol), followed by addition of 0.030 ml of DMF. After stirred at RT for 2 h, the mixture was diluted with toluene, concentrated and co-evaporated with dichloroethane (2 x 20 ml) and toluene (2x15 ml) to dryness to afford the title crude product (which is not stable) for the next step without further purification (365 mg, 96% yield). MS ESI m/z calcd for C2oHl9Cl2N206 [M+H]+ 453.05, found 453.50. Example 5. Synthesis of di-tert-butyl 1,2-bis(2-(ter/-butoxy)-2-oxoethyl)hydrazine-l,2- dicarboxylate. To a suspension of NaH (0.259 g, 6.48mmol, 3.0 eq.) in anhydrous DMF (2 mL) at room temperature was added di-ter/-butyl hydrazine-1,2-dicarboxylate (0.50 g, 2.16 mmol, 1.0 eq. ) in anhydrous DMF (8 mL) in 10 minutes under nitrogen. The mixture was stirred at room temperature for 10 minutes and then cooled to 0°C. To which tert-butyl 2-bromoacetate(1.4 mL, 8.61mmol, 4.0 eq.) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred overnight. Saturated ammonium chloride solution (100 mL) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic solution was washed with water and brine, dried over anhydrous Na2SO4, concentrated and purified by SiO2 column chromatography (10:1 hexanes/ EtOAc) to give the title compound as a colourless oil (0.94 g, 99.6% yield). ESI MS m/z [M+Na]+ 483.4. Example 6. Synthesis of compound 2,2'-(hydrazine-1,2-diyl)diacetic acid. To a solution of di-te/7-butyl l,2-bis(2-(ter/-butoxy)-2-oxoethyl)hydrazine-1,2- dicarboxylate (0.94 g, 2.04 mmol) in DCM (4 mL) at 0 °C was added TFA (4 mL). The reaction was stirred for 30 minutes and then warmed to room temperature and stirred overnight. The mixture was concentrated, diluted with DCM, and concentrated. This operation was repeated for 121 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 three times to give a white solid. Trituration with DCM and a white solid was collected by filtration (0.232 g, 76.8% yield). ESI MS m/z [M+H]+ 149.2. Example 7. Synthesis of 2,2’-(l,2-bis(2-chloroacetyl)hydrazine - l,2-diyl)diacetic acid. To a solution of 2,2'-(hydrazine-l,2-diyl)diacetic acid (0.232 g, 1.57 mmol, 1.0 eq.) in anhydrous THF (10 mL) at 0 °C was added 2-chloroacetyl chloride (0.38 mL, 4.70 mmol, 3.0 eq.) in 10 minutes. The reaction was warmed to room temperature and stirred overnight and concentrated. The residue was co-evaporated with THF for three times to give a white solid (0.472 g, theoretical yield). ESI MS m/z [M+H]+ 301.1. Example 8. Synthesis of tert-butyl 2,8-dioxo-l,5-oxazocane-5-carboxylate. HOOC Boc2O/THI ^ HOOC^^ P2°5_ HOOC-XzNH E^O/NaOH* HOOC-X^X^ CmClT O To a solution of 3,3’-azanediyldipropanoic acid (10.00 g, 62.08 mmol) in 1.0 M NaOH (300 ml) at 4 °C was added di-tert-butyl dicarbonate (22.10 g, 101.3 mmol) in 200 ml THF in 1 h. After addition, the mixture was kept to stirring for 2 h at 4 °C. The mixture was carefully acidified to pH ~4 with 0.2 M H3PO4, concentrated in vacuo, extracted with CH2C12, dried over Na2SO4, evaporated and purified with flash SiO2 chromatography eluted with AcOH/MeOH/CHaCh (0.01:1:5) to afford 3,3'-((tert-butoxycarbonyl)azanediyl)dipropanoic acid (13.62 g, 84% yield). ESI MS m/z CnHi9NO6 [M+H] +, cacld. 262.27, found 262.40. To a solution of 3,3'-((tert-butoxycarbonyl)azanediyl)dipropanoic acid (8.0 g, 30.6 mmol) in CH2CI2 (500 ml) at 0 °C was added phosphorus pentoxide (8.70 g, 61.30 mmol). The mixture was stirred at 0 °C for 2 h and then r.t. for 1 h, filtered through short SiO2 column, and rinsed the column with EtOAc/CH2C12 (1:6). The filtrate was concentrated and triturated with EtOAc/hexane to afford the title compound (5.64 g, 74% yield). ESI MS m/z C11H17NO5 [M+H] +, cacld. 244.11, found 244.30. Example 9. Synthesis of tert-Butyl 3-((benzyloxy)amino)propanoate. O-benzylhydroxylamine hydrochloride salt (10.0 g, 62.7 mmol) in THF (100 ml) was added Et3N (15 ml) and tert-butyl acrylate (12.1 g, 94.5 mmol). The mixture was refluxed for overnight, concentrated and purified on SiO2 column eluted with EtOAc/Hexane (1:4) to afford 122 5 10 15 20 25 30 the title compound 3 (13.08 g, 83% yield). 1H NMR (CDCh) 7.49-7.25 (m, 5H), 4.75 (s, 2H), 3.20 (t, J=6.4Hz, 2H), 2.54 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI MS m/z+ CuHaiNNaOj (M+Na), cacld. 274.15, found 274.20. Example 10. Synthesis of tert-Butyl 3-(hydroxyamino)propanoate. ,OH tert-Butyl 3-((benzyloxy)ammo)propanoate (13.0 g, 51.76 mmol) in methanol (100 ml) was added Pd/C (0.85 g, 10%Pd, 50% wet) in a hydrogenation vessel. After the system was evacuated under vacuum and placed under 2 atm of hydrogen gas, the reaction mixture was stirred overnight at room temperature. The crude reaction was passed through a short pad of Celite™ rinsing with ethanol, concentrated and purified on SiO2 column eluted with MeOH/DCM (1:10-1:5) to afford the title compound (7.25 g, 87% yield). 1H NMR (CDCh) 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI MS m/z+ C7Hi5NNaO3 (M+Na), cacld. 184.10, found 184.30. Example 11. Synthesis of tert-Butyl 3-((tosyloxy)amino)propanoate. ,OH ,OTs “N' Tert-butyl 3-(hydroxyamino)propanoate (5.10 g, 31.65 mmol) in the mixture of DCM (50 ml) and pyridine (20 ml) was added tosylate chloride (12.05 g, 63.42) at 4°C. After addition, the mixture was stirred at room temperature overnight, concentrated and purified on SiOz column eluted with EtOAc/DCM (1:10-1:6) to afford the title compound (8.58 g, 86% yield).1HNMR (CDCh) 7.81 (s, 2H), 7.46 (s, 2H), 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4Hz, 2H), 2.41 (s, 3H), 1.49 (s, 9H); ESI MS m/z+ CiÆiNNaOsS (M+Na), cacld. 338.11, found 338.30. Example 12. Synthesis of di-tert-Butyl 3,3'-(hydrazine-l,2-diyl)dipropanoate. Tert-butyl 3-aminopropanoate (3.05 g, 21.01 mmol) in THF (80 ml) was added tert-Butyl 3-((tosyloxy)amino)propanoate (5.10 g, 16.18 mmol). The mixture was stirred at room temperature for 1 h and then 45 °C for 6 h. The mixture was concentrated and purified on SiO2 column eluted with CH3OH/DCM/Et3N (1:12:0.01-1:8:0.01) to afford the title compound (2.89 g, 62% yield).ESI MS m/z+ C14H28N2NaO4 (M+Na), cacld. 311.20, found 311.40. Example 13. Synthesis of di-tert-Butyl 3,3'-(l,2-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanoyl)hydrazine-1,2-diyl)dipropanoate. Date Reçue/Date Received 2022-07-07 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 3-Maleido-propanoic acid (1.00 g, 5.91 mmol) in DCM (50 ml) was added oxalyl dichloride (2.70 g. 21.25 mmol) and DMF (50 pL). The mixture was stirred at room temperature for 2 h, evaporated, and co-evaporated with DCM/toluene to obtain crude 3-maleido-propanoic acid chloride. To the compound di-tert-Butyl 3,3'-(hydrazine-l,2-diyl)dipropanoate (0.51 g, 1.76 mmol) in the mixture of DCM (35 ml) was added the crude 3-maleido-propanoic acid chloride. The mixture was stirred for overnight, evaporated, concentrated and purified on SiO2 column eluted with EtOAc/DCM (1:15-1:8) to afford the title compound (738 mg, 71% yield). ESI MS m/z+ C28H38N4NaOio (M+Na), cacld. 613.26, found 613.40. Example 14. Synthesis of 3,3'-(l,2-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanoyl)-hydrazine-1,2-diyl)dipropanoic acid. Compound 14 (700 mg, 1.18 mmol) in dioxane (4 ml) was added HCl (cone. 1 ml). The mixture was stirred for 30 min, diluted with EtOH (10 mL) and toluene (10 ml), evaporated and coevaporated with EtOH (10 ml) and toluene (10 ml) to afford the crude title product (560 mg) for next step without further purification. ESI MS m/z- C20H21N4O10 (M-H), cacld. 477.13, found 477.20. Example 15. Synthesis of Bis(2,5-dioxopyrrolidin-l-yl)-3,3'-(l,2-bis(3-(2,5-dioxo-2,5- dihydro-IH-pyrrol-l-yl)propanoyl)hydrazine-1,2-diyl)dipropanoate. To the crude compound 3,3'-(l,2-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoyl)- hydrazine-l,2-diyl)dipropanoic acid (-560 mg, -1.17 mmol) in DMA (8 ml) was added NHS (400 mg, 3.47 mmol) and EDC (1.01 g, 5.26 mmol). The mixture was stirred for overnight, evaporated, concentrated and purified on SiO2 column eluted with EtOAc/DCM (1:12-1:7) to afford the title compound (520 mg, 65% yield in 2 steps). ESI MS m/z+ C^HisNeNaOu (M+Na), cacld. 695.17, found 695.40. 124 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 16. Synthesis of tert-Butyl 3-(2-(2-(2-hydroxyclhoxy)cthoxy)cthoxy)propanoatc. To 350 mL of anhydrous THF was added 80 mg (0.0025 mol) of sodium metal and triethylene glycol 150.1 g, 1.00 mol) with stirring. After the sodium had completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol) was added. The solution was stirred for 20 h at room temperature and neutralized with 8 mL of 1.0 M HC1. The solvent was removed in vacuo and the residue was suspended in brine (250 mL) and extracted with ethyl acetate (3 x 125 mL). The combined organic layers were washed with brine (100 mL) then water (100 mL), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was dried under vacuum to give 69.78 g (76% yields) of the title product. 'H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J=6.4 Hz), 3.59-3.72 (m. 14H). ESI MS m/z- C|3H25O6 (M-H), cacld. 277.17, found 277.20. Example 17. Synthesis of tert-Butyl 3-(2-(2-(2- (tosyloxy)ethoxy)ethoxy)ethoxy)propanoate. A solution of tert-Butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)cthoxy)propanoate (10.0 g, 35.95 mmol) in acetonitrile (50.0 mL) was treated with pyridine (20.0 mL). A solution of tosyl chloride (7.12 g, 37.3 mmol) in 50 mL acetonitrile was added dropwise via an addition funnel over 30 minutes. After 5 h TLC analysis revealed that the reaction was complete. The pyridine hydrochloride that had formed was filtered off and the solvent was removed. The residue was purified on silica gel by eluting from with 20% ethyl acetate in hexane to with neat ethyl acetate to give 11.2 g (76% yield) of the title compound. ’H NMR: 1.40 (s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4 Hz), 3.52-3.68 (m, 14H), 4.11 (t, 2H, J=4.8 Hz), 7.30 (d, 2H, J=8.0 Hz), 7.75 (d, 2H, J=8.0 Hz); ESI MS m/z+ C20H33O8S (M+H), cacld. 433.18, found 433.30. Example 18. Synthesis of tert-Butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate. NaN3 DMF To 50 mL of DMF was added tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)- propanoate (4.0 g, 9.25 mmol) and sodium azide (0.737 g, 11.3 mmol) with stirring. The reaction was heated to 80 °C. After 4 h TLC analysis revealed that the reaction was complete. The reaction was cooled to room temperature and quenched with water (25 mL). The aqueous layer was separated and extracted into ethyl acetate (3 x 35 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent removed in vacuo. The crude 125 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 azide product (2.24 g, 98% yield, about 93% pure by HPLC) was used for next step without further purification. ’H NMR (CDC13): 1.40 (s, 9H), 2.45 (t, 2H, J=6.4 Hz), 3.33 (t, 2H, J=5.2 Hz), 3.53-3.66 (m, 12H). ESI MS m/z+ C13H26N3O8 (M+H), cacld. 304.18, found 304.20. Example 19. Synthesis of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid. HC1 (1) Dioxane Tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate (2.20 g, 7.25 mmol) in 1.4- dioxane (40 ml) was added HC1(12 M, 10 ml). The mixture was stirred for 40 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to afford the crude title product for the next step without further production (1.88g, 105% yield, -92% pure by HPLC). MS ESI m/z calcd for C9Hi8N3O5 [M+H]+ 248.12, found 248.40. Example 20. Synthesis of 13-Amino-4,7,10-trioxadodecanoic acid tert-butyl ester, and 13- Amino-bis(4,7,10-trioxadodecanoic acid tert-Butyl Ester). The crude azide material 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (5.0 g, -14.84 mmol) was dissolved in ethanol (80 mL) and 300 mg of 10% Pd/C was added. The system was evacuated under vacuum and placed under 2 atm of hydrogen gas via hydrogenation reactor with vigorous stirring. The reaction was then stirred overnight at room temperature and TLC showed that the starting materials disappeared. The crude reaction was passed through a short pad of Celite rinsing with ethanol. The solvent was removed and the amine purified on silica gel using a mixture of methanol (from 5% to 15%) and 1% triethylamine in methylene chloride as the eluant to give 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (1.83 g, 44% yield, ESI MS m/z+ CuH^tNOs (M+H), cacld. 278.19, found 278.30) and 13-aminobis(4,7,10-trioxadodecanoic acid tert-butyl ester) (2.58 g, 32% yield, ESI MS m/z+ C26H52NO10 (M+H), cacld. 538.35, found 538.40). Example 21. Synthesis of 3-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)propanoic acid, HO salt. To 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (0.80 g, 2.89 mmol) in 30 mL of dioxane was 10 ml of HC1(36%) with stirring. After 0.5 h TLC analysis revealed that the reaction was complete, the reaction mixture was evaporated, and co-evaporated with EtOH and 126 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 EtOH/Toluene to form the title product in HC1 salt (>90% pure, 0.640 g, 86% yield) without further purification. ESI MS m/z+ C9H2oN05 (M+H), cacld. 222.12, found 222.20. Example 22. 13-Amino-bis(4,7,10-trioxadodecanoic acid, HC1 salt. To 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester) (1.00 g, 1.85 mmol) in 30 mL of dioxane was 10 ml of HO (36%) with stirring. After 0.5 h TLC analysis revealed that the reaction was complete, the reaction mixture was evaporated, and co-evaporated with EtOH and EtOH/Toluene to form the title product in HC1 salt (>90% pure, 0.71 g, 91% yield) without further purification. ESI MS m/z+ CisHsôNOio (M+H), cacld. 426.22, found 426.20. Example 23. Synthesis of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoatc. To a solution of 2,2’-(ethane-l,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight and then quenched by HC1 solution (20.0 mL, IN) at 0°C. THF was removed by rotary evaporation, brine (300 mL) was added and the resulting mixture was extracted with EtOAc (3 x 100 mL). The organic layers were washed with brine (3 x 300 mL), dried over anhydrous Na2SO4, filtered and concentrated to afford a colourless oil (30.20 g, 79.0% yield), which was used without further purification. MS ESI m/z calcd for C13H27O6 [M + H]+ 278.1729, found 278.1730. Example 24. Synthesis of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate. To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol, 2.0 eq.) in anhydrous DCM (220 mL) at 0 °C was added TEA (30.0 mL, 217.0 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight, and then washed with water (3 x 300 mL) and brine (300 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiO2 column chromatography (3:1 hexanes/ EtOAc) to give a colourless oil (39.4 g, 84.0% yield). MS ESI m/z calcd for C2oH3308S [M + H]+ 433.1818, found 433.2838. Example 25. Synthesis of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) propanoate. 127 5 10 15 20 25 30 ^3 Bu To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF(100 mL) was added NaN3(20.67 g, 316.6 mmol, 3.5 eq.). The mixture was stirred at room temperature overnight. Water (500 mL) was added and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with water (3 x 900 mL) and brine (900 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiO? column chromatography (5:1 hexanes/ EtOAc) to give a light yellow oil (23.8 g, 85.53% yield).MS ESI m/z calcd for Ci3H25O3N5Na |M + Na]+ 326.2, found 326.2. Example 26. Synthesis of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) propanoate. HjN Raney™-Ni (7.5 g, suspended in water) was washed with water (three times) and isopropyl alcohol (three times) and mixed with tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) propanoate (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under a H2 balloon at r.t. for 16 h and then filtered over a Celite pad, with washing of the pad with isopropyl alcohol. The filtrate was concentrated and purified by column chromatography (5-25% MeOH/DCM) to give a light yellow oil (2.60 g, 57% yield). MS ESI m/z calcd for CbH^NOs [M+H]+ 279.19; found 279.19. Example 27. Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and K2C03(83.00 g, 600 mmol, 3.0 eq.) in acetonitrile (350 mL) was added BnBr (57.0 mL, 480 mmol, 2.4 eq.). The mixture was refluxed overnight. Water (1 L) was added and extracted with EtOAc (3 x 300 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiÛ2 column chromatography (4:1 hexanes/ EtOAc) to give a colourless oil (50.97 g, 89.2% yield).MS ESI m/z calcd for Ci8H23NO2Na [M + Na]+ 309.1729, found 309.1967. Example 28. Synthesis of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate. Bn2Nxs^'°'^KO To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 eq.) , tert¬ butyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and M-BU4NI (6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL) was added sodium hydroxide solution (300 mL, 50%). The mixture was stirred overnight. The organic layer was separated and the water layer was extracted with EtOAc (3 x 100 mL). The organic layers were washed with water(3 x 300 mL) and brine (300 mL), dried 128 Date Reçue/Date Received 2022-07-07 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 over anhydrous Na2SO4, filtered, concentrated and purified by SiOi column chromatography (7:1 hexanes/ EtOAc) to give a colourless oil (61.08 g, 89.4% yield). MS ESI m/z calcd for C25H36NO4 [M + H]+ 414.2566, found 414.2384. Example 29. Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate. H2N^ ° ^CO2'Bu To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THE (30 mL) and MeOH (60 mL) was added Pd/C (2.00 g, 10 wt%, 50% wet) in a hydrogenation bottle. The mixture was shaken at 1 atom pressure H2 overnight, filtered through Celite (filter aid), and the filtrate was concentrated to afford a colourless oil (10.58 g, 93.8% yield). MS ESI m/z calcd for C11H24NO4 [M + H]+ 234.1627, found 234.1810. Example 30. Synthesis of ter/-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate. To a solution of 2,2'-oxydiethanol (19.7 mL, 206.7 mmol, 3.0 eq.) in anhydrous THF (100 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then tert-butyl acrylate (10.0 mL, 68.9 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight, and brine (200 mL) was added and extracted with EtOAc (3 x 100 mL). The organic layers were washed with brine (3 x 300 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiÛ2 column chromatography (1:1 hexanes/ EtOAc) to give to a colourless oil (8.10 g, 49.4% yield). MS ESI m/z calcd for C11H23O5 [M +H]+ 235.1467, found 235.1667. Example 31. Synthesis of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate. To a solution of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (6.24 g, 26.63 mmol, 1.0 eq.) and TsCl (10.15 g, 53.27 mmol, 2.0 eq.) in anhydrous DCM(50 mL) at 0 °C was added pyridine (4.3 mL, 53.27 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight, and then washed with water (100 mL) and the water layer was extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiÛ2 column chromatography (5:1 hexanes/ EtOAc) to give a colourless oil (6.33 g, 61.3% yield). MS ESI m/z calcd for C18H27O7S [M + H]+ 389.1556, found 389.2809. Example 32. Synthesis of tert-butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate. N3 To a solution of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate (5.80 g, 14.93 mmol, 1.0 eq.) in anhydrous DMF (20 mL) was added NaNg (5.02 g, 77.22 mmol, 5.0 eq.). The 129 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 mixture was stirred at room temperature overnight. Water (120 mL) was added and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water (3 x 150 mL) and brine (150 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by SiO2 column chromatography (5:1 hexanes/ EtOAc) to give a colourless oil (3.73 g, 69.6% yield). MS ESI m/z calcd for CiiH22O3N4Na[M + H]+ 260.1532, found 260.2259. Example 33. Synthesis of /er/-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate. H2N' o^/CO/Bu /er/-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (0.18 g, 0.69 mmol) was dissolved in MeOH (3.0 mL, with 60 pL concentrated HC1) and hydrogenated with Pd/C (10 wt%, 20 mg) under a H2 balloon for 30 min. The catalyst was filtered through a Celite pad, with washing of the pad with MeOH. The filtrate was concentrated to give a colorless oil (0.15 g, 93% yield). MS ESI m/z calcd for CiiH24NO4 [M+H]+ 234.16; found 234.14. Example 34. Synthesis of 3-(2-(2-azidoethoxy)ethoxy)propanoic acid. n3x^°'^o /erZ-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (2.51 g, 9.68 mmol) dissolved in 1,4- dioxane (30 mL) was treated with 10 ml of HC1 (cone.) at r.t. The mixture was stirred for 35 min, diluted with EtOH (30 ml) and toluene (30 ml) and concentrated under vacuum. The crude mixture was purified on silica gel using a mixture of methanol (from 5% to 10%) and 1% formic acid in methylene chloride as the eluant to give title compound (1.63 g, 83% yield), ESI MS m/z C7H12N3O4 [M-Hf, cacld. 202.06, found 202.30. Example 35. Synthesis of 2,5-dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate. O To 3-(2-(2-azidoethoxy)ethoxy)propanoic acid (1.60 g, 7.87 mmol) in 30 mL of dichloromethane was added NHS (1.08 g, 9.39 mmol) and EDC (3.60 g, 18.75 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 10%) in methylene chloride as the eluant to give title compound (1.93 g, 82% yield). ESI MS m/z C11H17N4O6 [M+H]+, cacld.301.11, found 301.20. Example 36. Synthesis of 2,5-dioxopyrrolidin-l-yl 3-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)propanoate. 130 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (4.50 g, 18.21 mmol) in 80 mL of dichloromethane was added NHS (3.0 g, 26.08 mmol) and EDC (7.60 g, 39.58 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 10%) in methylene chloride as the eluant to give title compound (5.38 g, 86% yield). ESI MS m/z C13H20N4O7 [M+H]+, cacld.345.13, found 345.30. Example 37. Synthesis of (14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tertbutoxycarbonyl)-amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid To a solution of (S)-2-((S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)-4-(tertbutoxy)-4-oxobutanoic acid (2.81 g, 6.73 mmol) in the mixture of DMA (70 ml) and 0.1 M NaHiPO4 (50 ml, pH 7.5) was added 2,5-dioxopyrrolidin-l-yl 3-(2-(2-(2-azidoethoxy)ethoxy)- ethoxy)propanoate (3.50 g, 10.17). The mixture was stirred for 4 h, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 15%) in methylene chloride containing 0.5% acetic acid as the eluant to give title compound (3.35 g, 77% yield). ESI MS m/z C28H51N6O11 [M+H]+, cacld.647.35, found 647.80. Example 38. Synthesis of (14S,17S)-tert-butyl l-azido-14-(4-((tert-butoxycarbonyl)- amino)butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16- diazanonadecan-19-oate (14S,17S)-1-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)- amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid (3.30 g, 5.10 mmol) and (4-aminophenyl)methanol (0.75 g, 6.09) in DMA (25 ml) was added EDC (2.30 g, 11.97 mmol). The mixture was stirred for overnight, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 8%) in methylene chloride containing as the eluant to give title compound (3.18 g, 83% yield). ESI MS m/z C35H58N7On [M+HJ+, cacld.752.41, found 752.85. 131 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 39. Synthesis of (14S,17S)-tert-butyl l-amino-14-(4-((tertbutoxycarbonyl)amino)-butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazanonadecan-19-oate To a solution of (14S,17S)-tert-butyl l-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)- 17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19- oate (1.50 g, 1.99 mmol) in THF (35 mL) was added Pd/C (200 mg, 10% Pd, 50% wet) in a hydrogenation bottle. The mixture was shaken at 1 atom pressure H2 overnight, filtered through Celite (filter aid), and the filtrate was concentrated to afford the title compound (1.43 g, 99% yield) which was used immediately for the next step without further purification. ESI MS m/z CasHéoNsOn [M+H]+, cacld.726.42, found 726.70. Example 40. Synthesis of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6- diazapentadecan-1-oic acid To a solution of (S)-2-(2-amino-3-methylbutanamido)acetic acid (Val-Gly) (1.01 g, 5.80 mmol) in the mixture of DMA (50 ml) and 0.1 M NaH2PO4 (50 ml, pH 7.5) was added 2,5- dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (1.90 g, 6.33). The mixture was stirred for 4 h, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 15%) in methylene chloride containing 0.5% acetic acid as the eluant to give title compound (1.52 g, 73% yield). ESI MS m/z CuH^NsOe [M+H]+, cacld.360.18, found 360.40. Example 41. Synthesis of (S)-2,5-dioxopyrrolidin-l-yl 15-azido-5-isopropyl-4,7-dioxo- 10,13-dioxa-3,6-diazapentadecan-1-oate To a solution of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-loic acid (1.50 g, 4.17 mmol) in 40 mL of dichloromethane was added NHS (0.88 g, 7.65 mmol) and EDC (2.60 g, 13.54 mmol) with stirring. After 8 h TLC analysis revealed that the reaction was complete, the reaction mixture was concentrated and purified on silica gel using a mixture of ethyl acetate (from 5% to 20%) in methylene chloride as the eluant to give title compound (1.48 g, 78% yield). ESI MS m/z C18H29N6O8 [M+H]+, cacld.457.20, found 457.50. 132 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 42. Synthesis of 4-(((bcnzyloxy)carbonyl)amino)butanoic acid. CbzHNx^^CO2H A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H2O (40 mL) was cooled to 0 °C and treated with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reaction was allowed to warm to r.t. and stirred for 3 h. THF was removed under vacuum, the pH of the aqueous solution was adjusted to 1.5 by addition of 6 N HO. Extracted with ethyl acetate, and the organic layer was washed with brine, dried and concentrated to give the title compound (16.4 g, 92% yield). MS ESI m/z calcd for C12H16NO5 [M+H]+238.10, found 238.08. Example 43. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate. CbzH^J^x^^CO/Bu DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to a solution of 4- (((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and /-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring at r.t. overnight, the reaction was filtered and filtrate concentrated. The residue was dissolved in ethyl acetate and the washed with IN HC1, brine and dried over Na2SO4. Concentration and purification by column chromatography (10 to 50% EtOAc/hexanes) yielded the title compound (7.5 g, 37% yield). MS ESI m/z calcd for CiôHzaNCUNa [M+Na]+ 316.16, found 316.13. Example 44. Synthesis of ter/-butyl 4-aminobutanoate. IhN^^^CO/Bu ter/-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt%, 100 mg) then hydrogenated (1 atm) at room temperature for 3 h. The catalyst was filtered off and all volatiles were removed under vacuum to afford the title compound (272 mg, 90% yield). MS ESI m/z calcd for CgHigNCh [M+H]+ 160.13, found 160.13. Example 45. Synthesis of di-terz-butyl 3,3'-(benzylazanediyl)dipropanoate. O O 'BuO'X^z^Nx^zUkO,Bu Bn A mixture of phenylmethanamine (2.0 mL, 18.29 mmol, 1.0 eq) and terr-butyl acrylate (13.3 mL, 91.46 mmol, 5.0 eq) was refluxed at 80 °C overnight and then concentrated. The crude product was purified by SiO2column chromatography (20:1 hexanes/EtOAc) to give the title compound as colourless oil (5.10 g, 77% yield). ESI MS m/z: calcd for C21H34NO4[M+H]+ 133 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 364.2, found 364.2. *H NMR (400 MHz, CDC13) 5 7.38-7.21 (m, 5H), 3.58 (s, 2H), 2.76 (t, J = 7.0 Hz, 4H), 2.38 (t, J = 7.0 Hz, 4H), 1.43 (s, 17H). Example 46. Synthesis of di-Ze/7-butyl 3,3’-azanediyldipropanoate. O O /BuO'^z^N'^s*^KO/Bu H To a solution of di-ter/-butyl 3.3’-(benzylazanediyI)dipropanoate (1.37 g, 3.77 mmol, 1.0 equiv) in MeOH (10 mL) was added Pd/C (0.20 g, 10% Pd/C, 50% wet) in a hydrogenation bottle. The mixture was shaken overnight under H2 atmosphere and then filtered through a Celite pad. The filtrate was concentrated to give the title compound as colourless oil (1.22 g, 89% yield). ESI MS m/z: calcd for Ci4H28NO4 [M+H]+ 274.19, found 274.20. Example 47. Synthesis of Zerz-butyl 4-(2-(((benzyloxy)carbonyl)amino)propan amido)- butanoate. zBuO. NHCbz O 'N H To a solution of ter/-butyl 4-aminobutanoate (1.00 g, 6.28 mmol, 1.0 eq.) and Z-L-alaine (2.10 g, 9.42 mmol, 1.5 eq.) in anhydrous DCM (50 mL) at 0 °C were added HATU (3.10 g, 8.164 mmol, 1.3 eq.) and TEA (2.6 mL, 18.8 mmol, 3.0 eq.). The reaction was stirred at 0 °C for 10 min., then warmed to room temperature and stirred overnight. The mixture was diluted with DCM and washed with water and brine, dried over anhydrous Na2SO4, concentrated and purified by SiO2 column chromatography (10:3 petroleum ether/ethyl acetate) to give the title compound as a colorless oil (1.39 g, 61% yield). ESI MS m/z: calcd for Ci9H29N2O5Na [M+H]+ 387.2, found 387.2. Example 48. Synthesis of tert-butyl 4-(2-aminopropanamido)butanoate. To a solution of tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)propanamido) butanoate (1.39 g, 3.808 mmol, 1.0 eq.) in MeOH (12 mL) was added Pd/C (0.20 g, 10 wt%, 10% wet) in a hydrogenation bottle. The mixture was shaken for 2 h and then filtered through Celite (filter aid), concentrated to give the title compound as a light yellow oil (0.838 g, 95% yield). ESI MS m/z: calcd. for CiiH23N2O3[M+H]+ 231.16, found 231.15. Example 49. Synthesis of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid. O 134 5 10 15 20 25 30 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of terf-butyl 3-(2-(2-(dibcnzylamino)cthoxy)cthoxy)propanoatc (2.3g, 5.59 mmol, l.Oeq) in DCM (10 mL) at room temperature was added TFA (5 mL). After stirring for 90 min., the reaction mixture was diluted with anhydrous toluene and concentrated, this operation was repeated for three times to give the title compound as a light yellow oil (2.0 g, theoretical yield), which was directly used in the next step. ESI MS m/z calcd. for C21H28NO4 [M+H]+ 358.19, found358.19. Example 50. Synthesis of perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy) ethoxy)- propanoate. C6F5O^s^OxX^oxx^NBn2 To a solution of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid(2.00 g, 5.59 mmol, 1.0 eq.) in anhydrous DCM (30 mL) at 0 °C was added DIPEA until pH was neutral, and then PFP (1.54 g, 8.38 mmol, 1.5 eq.) and DIC (1.04 mL, 6.70 mmol, 1.2 eq.) were added. After 10 min. the reaction was warmed to room temperature and stirred overnight. The mixture was filtered, concentrated and purified by SiO2column chromatography (15:1 petroleum ether/ethyl acetate) to give the title compound as colourless oil (2.10 g, 72% yield). ESI MS m/z: calcd. for C27H27F5NO4 [M+H]+ 524.2, found 524.2. Example 51. Synthesis of /er/-butyl 2-benzyl-13-methyl-l1,14-dioxo-l-phenyl -5,8-dioxa- 2,12,15-triazanonadecan-19-oate. To a solution of tert-butyl 4-(2-aminopropanamido)butanoate (0.736 g, 3.2 mmol, 1.0 eq.) and perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy) ethoxy)propanoate (2.01 g, 3.84 mmol, 1.2 eq.) in anhydrous DMA (20 mL) at 0 °C was added DIPEA (1.7 mL, 9.6mmol, 3.0 eq.). After stirring at 0 °C for 10 min. the reaction was warmed to room temperature and stirred overnight. Water (100 mL) was added and the mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with water (3 x 200 mL) and brine (200 mL), dried over Na2SO4. filtered, concentrated and purified by SiO2 column chromatography (25:2 DCM/McOH) to give the title compound as a colourless oil (1.46 g, 80% yield). ESI MS m/z: calcd. for C32H48N3O6 [M+HJ+ 570.34, found570.33. Example 52. Synthesis of 2-benzyl-13-methyl-l1,14-dioxo-l-phenyl-5,8-dioxa -2,12,15- triazanonadecan-19-oic acid. 135 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of /erz-butyl 2-bcnzy1-13-methy1-11,14-dioxo-l-phenyl-5,8-dioxa-2.12,15- triazanonadecan-19-oate (0.057 g, 0.101 mmol, 1.0 eq) in DCM (3 mL ) at room temperature was added TFA (1 mL) and stirred for 40 min. The reaction was diluted with anhydrous toluene and then concentrated. This operation was repeated three times to give the title compound as a colourless oil (0.052 g, theoretical yield), which was used directly in the next step. ESI MS m/z: calcd for C28H4ON3O6[M+H]+ 514.28, found 514.28. Example 53. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid CbzHN^zx^COzH A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H2O (40 mL) was cooled to 0 °C and treated with a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reaction was allowed to warm to r.t. and stirred for 3 h. THF was removed under vacuum, the pH of the aqueous solution was adjusted to 1.5 by addition of 6 N HC1. Extracted with ethyl acetate, and the organic layer was washed with brine, dried and concentrated to give the title compound (16.4 g, 92% yield). MS ESI m/z calcd for C12H16NO5 [M+H]+238.10, found 238.08. Example 54. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate. CbzHN^z'XzXCVBu DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g. 83 mmol) were added to a solution of 4- (((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring at r.t. overnight, the reaction was filtered and filtrate concentrated. The residue was dissolved in ethyl acetate and the washed with IN HO, brine and dried over Na2SCL. Concentration and purification by column chromatography (10 to 50% EtOAc/hexanes) yielded the title compound (7.5 g, 37% yield). MS ESI m/z calcd for Ci6H23NO4Na [M+Na]+316.16, found 316.13. Example 55. Synthesis of tert-butyl 4-aminobutanoate. H2N^x^CO2rBu /<?//-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt%, 100 mg) then hydrogenated (1 atm) at room temperature for 3 h. The catalyst was filtered off and all volatiles were removed under vacuum to afford the title compound (272 mg, 90% yield). MS ESI m/z calcd for CgHisNOi [M+H]+160.13, found 160.13. 136 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 56. Synthesis of tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetate. NHCbz HOBt/EDC DIPEA/DCM NHCbz 2-(((Benzyloxy)carbonyl)amino)propanoic acid (0.84g, 5mmol), tert-butyl 2-aminoacetate (0.66g, 5mmol), HOBt (0.68g, 5mmol), EDC (1.44g, 7.5mmol) were dissolved in DCM (20ml), followed by addition of DIPEA(1.7ml, lOmmol). The reaction mixture was stirred at RT overnight, washed with H2O (100ml), and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over MgSCh, filtered, evaporated under reduced pressure and the residue was purified on SiOz column to give the title product l(0.87g, 52%). ESI: m/z: calcd for C17H25N2O5 [M+H]+: 337.17, found 337.17. Example 57. Synthesis of 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetic acid. *^°Y^z\zNHCbZ TFA » HO\/X A^NHCbz * O H I DCM O H I Tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetate (0.25g, 0.74mmol) was dissolved in DCM (30ml), followed by addition of TFA (10ml). The mixture was stirred at RT overnight, concentrated to afford the title compound used for the next step without further purification. ESI: m/z: calcd for C13H17N2O5[M+H]+: 281.11, found 281.60. Example 58. Synthesis of di-tert-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa- 13,18- diazatriacont-15-yne-1,30-dioate. Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved in NMP (10 mL) and cooled to 0 °C, to which compound tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)- propanoate (2.06 g, 7.43 mmol, 2.4 eq.) was added, followed by DMTMM (2.39 g, 8.65 mmol, 2.8 eq.) in portions. The reaction was stirred at 0 °C for 6 h and then diluted with ethyl acetate and washed with water and brine. The organic solution was concentrated and triturated with a mixture solvent of ethyl acetate and petroleum ether. The solid was filtered off and the filtrate was concentrated and purified by column chromatography (80-90% EA/PE) to give a light yellow oil (2.26 g, >100% yield), which was used without further purification. MS ESI m/z [M+H]+ 633.30. Example 59. Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,l8-diaza triacont-15- yne-1,30-dioic acid. 137 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Compound di-ter/-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa- 13,18-diazatriacont-15- yne-1,30-dioate (2.26 g) was dissolved in dichloromethane (15 mL) and cooled to 0 °C then treated with TFA (15 mL). The reaction was warmed to r.t. and stirred for 45 min, and then the solvent and residual TFA was removed on rotovap. The crude product was purified by column chromatography (0-15% MeOH/DCM) to give a light yellow oil (1.39 g, 86% yield for two steps). MS ESI m/z [M+H]+ 521.24. Example 60. Synthesis of di-tert-butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo- 10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-l,42-dioate To a solution of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diaza triacont-15-yne-1,30- dioic acid (1.38 g, 2.65 mmol), tert-butyl 2-(2-aminopropanamido)propanoate (0.75 g, 3.47 mmol) in the mixture of DMA (40 ml) was added EDC (2.05 g, 10.67 mmol). The mixture was stirred for overnight, concentrated and purified on SiO^ column eluted with EtOAc/CH2C12 (1:5 to 1:1) to afford the title compound (2.01 g, 82% yield, ~95% pure by HPLC). MS ESI m/z calcd for C42H73N6O16 [M+H]+ 917.50, found 917.90. Example 61. Synthesis of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo- 10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-l,42-dioic acid Di-di-tert-butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33- hcxaoxa-3,6,19,24,37,40-hcxaazadotetracont-21-ync-l,42-dioatc (1.50 g, 1.63 mmol) was dissolved in the mixture of CH2CI2 (10 ml) and TFA (10 ml). The mixture was stirred for overnight, diluted with toluene (20 ml), concentrated to afford the title compound (1.33 g, 101% yield, ~92% pure by HPLC) which was used for the next step without further purification.. MS ESI m/z calcd for C34H56N6Oi6 [M+H]+ 805.37, found 805.85. 138 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 62. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 2,5,38,41-tetramethyl- 4,7,20.23,36,39-hcxaoxo-10,13.16,27,30.33-hcxaoxa-3,6,19,24,37,40-hexaazadotetracont-21- yne-l,42-dioate To a solution of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33- hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-yne-1,42-dioic acid (1.30 g, 1.61 mmol) in the mixture of DMA (10 ml) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol). The mixture was stirred for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:4 to 2:1) to afford the title compound (1.33 g, 83% yield, -95% pure by HPLC). MS ESI m/z calcd for C42H63N8O20 [M+H]+ 999.40, found 999.95. Example 63. Synthesis of 2,3-bis(2-bromoacetamido)succinyl dichloride. 2,3-Diaminosuccinic acid (5.00 g, 33.77 mmol) in the mixture of THF/H2O/DIPEA (125 ml/125 ml/8 ml) was added 2-bromoacetyl bromide (25.0 g, 125.09 mmol). The mixture was stirred for overnight, evaporated and purified by SiO2 column chromatography (H2O/CH3CN 5:95) to afforded 2,3-bis(2-bromoacetamido)succinic acid (9.95 g, 76% yield) as light yellow oil. MS ESI m/z calcd for C8Hi1Br2N2O6 [M+H]+ 388.89, found 388.68. 2,3-bis(2-bromoacetamido)succinic acid (3.50 g, 9.02 mmol) in dichloromethane (80 ml) was added oxalyl dichloride (5.80 g, 46.05 mmol) and DMF (0.01 ml). The mixture was stirred for 2.5 h, diluted with toluene, concentrated and co-evaporated with dichloroethane (2 x 20 ml) and toluene (2x15 ml) to dryness to afford 2,3-bis(2-bromoacetamido)succinyl dichloride as crude product (which is not stable) for the next step without further purification (3.90 g, 102% yield). MS ESI m/z calcd for C8H9Br2Cl2N2O4 [M+H]+ 424.82, found 424.90. Example 64. Synthesis of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid. O O CbzHNNHCbz 139 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of 2,3-diaminosuccinic acid (4.05 g, 27.35 mmol) in the mixture of THF (250 ml) and NaHzPO4 (0.1 M, 250 ml, pH 8.0) was added benzyl carbonochloridate (15.0 g, 88.23 mmol) in 4 portions in 2 h. The mixture was stirred for another 6 h, concentrated and purified on SiOa column eluted with H2O/CH3CN (1:9) containing 1% formic acid to afford the title compound (8.65 g, 76% yield, -95% pure). MS ESI m/z calcd for C20H21N2O8 [M+H]+ 417.12, found 417.60. Example 65. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(((benzyloxy)carbonyl)- amino)succinate CbzHN NHCbz ° To a solution of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (4.25 g, 10.21 mmol) in the mixture of DMA (70 ml) was added NHS (3.60 g. 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred for overnight, concentrated and purified on S1O2 column eluted with EtOAc/CH2C12 (1:6) to afford the title compound (5.42 g, 87% yield, -95% pure). MS ESI m/z calcd for C28H27N4O12 [M+H]+ 611.15, found 611.60 Example 66. Synthesis of 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinic acid. 2,3-Diaminosuccinic acid (5.00 g, 33.77 mmol) in the mixture of THF/H2O/DIPEA (125 ml/125 ml/2 ml) was added maleic anhydride (6.68 g, 68.21 mmol). The mixture was stirred for overnight, evaporated to afforded 2,3-bis((Z)-3-carboxyacrylamido)succinic acid (11.05 g, 99% yield) as a white solid. MS ESI m/z calcd for C12H13N2O10 [M+H]+ 345.05, found 345.35. 2,3-bis((Z)-3-carboxyacrylamido)succinic acid (11.05 g, 33.43 mmol) in a mixture solution of HOAc (70 ml), DMF (10 ml) and toluene (50 ml) was added acetic anhydride (30 ml). The mixture was stirred for 2 h, reflux with Dean-Stark Trap at 100 °C for 6 h, concentrated, co¬ evaporated with EtOH (2 x 40 ml) and toluene (2 x 40 ml), and purified on SiO2 column eluted with H2O/CH3CN (1:10) to afford the title compound (7.90 g, 76% yield, -95% pure). MS ESI m/z calcd for C12H9N2O8 [M+H]+ 309.03, found 309.30. Example 67. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-lHpyrrol-1-yl)succinate 140 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinic acid (4.00 g, 12.98 mmol) in the mixture of DMF (70 ml) was added NHS (3.60 g, 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CIECb (1:6) to afford the title compound (5.73 g, 88% yield, ~96% pure by HPLC). MS ESI m/z calcd for C20H15N4O12 [M+H]+ 503.06, found 503.45. Example 68. Synthesis of (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tertbutoxycarbonyl)amino)butyl)-22,23-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3,42-bis((4- (hydroxymethyl)phenyl)carbamoyl)-5,8,21,24,37,40-hexaoxo-ll,14,17,28,31,34-hexaoxa- 4,7,20,25,38,41-hexaazatetratetracontane-l,44-dioate (14S,17S)-tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-((4- (hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate (1.43 g, 1.97 mmol) and 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinic acid (0.30 g, 0.97 mmol) in DMA (25 ml) was added EDC (1.30 g, 6.77 mmol). The mixture was stirred for overnight, evaporated in vacuo, purified on silica gel using a mixture of methanol (from 5% to 8%) in methylene chloride containing as the eluant to give title compound (1.33 g, 80% yield). ESI MS m/z C82Hi23Ni2O28 [M+H]+, cacld.1722.85, found 1722.98.. Example 69. Synthesis of tert-butyl l-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa- 13,16-diazaoctadecan-18-oate To a solution of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (1.55 g, 6.27 mmol), tert-butyl 2-(2-aminopropanamido)propanoate (1.35 g, 6.27 mmol) in the mixture of DMA (60 ml) was added EDC (3.05 g, 15.88 mmol). The mixture was stirred for overnight, concentrated 141 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 and purified on SiCh column eluted with EiOAc/CILCb (1:3) to afford the title compound (2.42 g, 86% yield, -95% pure by HPLC). MS ESI m/z calcd for Ci9H36N5O7 [M+H]+ 446.25, found 446.60 Example 70. Synthesis of l-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecan-18-oic acid Dioxane Tert-butyl 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18- oate (2.20 g, 4.94 mmol) in 1,4-dioxane (40 ml) was added HC1 (12 M, 10 ml). The mixture was stirred for 40 ruin, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co¬ evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to afford the crude title product for the next step without further production (1.92g, 100% yield, -94% pure by HPLC). MS ESI m/z calcd for Ci5H28N5O7 [M+H]+ 390.19, found 390.45. Example 71. Synthesis of 21,22-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-2,5,38,41- tcjtrameth 1 ,2^),2^^3,.3Ci,Ï3 lic^xLao^xr? 10,1^3,1 ,21/,.30,^3^3 licjxiarixiha 13 ,1 ,2ï^l ,.3^7,^10 hexaazadotetracontane-1,42-dioic acid. 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid (1.90 g, 4.88 mmol) in DMA (40 ml) was added Pd/C (0.20 g, 50% wet). The system was evacuated under vacuum and placed under 2 atm of hydrogen gas via hydrogenation reactor with vigorous stirring. The reaction was then stirred for 6 h at room temperature and TLC showed that the starting materials disappeared. The crude reaction was passed through a short pad of Celite rinsing with ethanol. The solvent was concentrated under reduced pressure to afford the crude product, l-amino-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid in DMA which was used for the next step directly. ESI MS m/z+ C15H30N3O7 (M+H), cacld. 364.20, found 364.30. To the amino compound in DMA (-30 ml) was added 0.1 M NaH2PO4, pH 7.5 (20 ml), followed by addition of bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol- 1-yl)succinate (1.30 g, 2.59 mmol). The mixture was stirred overnight, concentrated and purified 142 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 on SiOi column eluted with 8% water on CH3CN to afford the title compound (1.97g, 81% yield). ESI MS m/z+ C42H63N802o (M+H), cacld. 999.41, found 999.95. Example 72. Synthesis of bis(2,5-dioxopyrrolidin-l-yl) 21,22-bis(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa- 3,6,19,24,37,40-hexaazadotetracontane-l,42-dioate To a solution of 21,22-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-2,5,38,41-tetramethyl- 4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3.6,19.24,37,40-hexaazadotetracontane- 1,42-dioic acid (1.50 g, 1.50 mmol) in the mixture of DMA (10 ml) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol). The mixture was stirred for overnight, concentrated and purified on SiCh column eluted with EtOAc/CH2C12 (1:4 to 2:1) to afford the title compound (1.50 g, 83% yield, -95% pure by HPLC). MS ESI m/z calcd for C50H69N10O24 [M+H]+ 1193.44, found 1193.95. To a solution of trans-4-hydroxy-L-proline methyl ester (18.0 g, 107.0 mmol) in the mixture of MeOH (150 ml) and sodium bicarbonate solution (2.0 M, 350 ml) was added Boc2O Example 73. Synthesis of (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloric. To a solution of trans-4-hydroxy-L-proline (15.0 g, 114.3 mmol) in dry methanol (250 mL) was added thionyl chloride (17 mL, 231 mmol) dropwise at 0 to 4 °C. The resulting mixture was stirred for at r.t. overnight, concentrated, crystallized with EtOH/hexane to provide the title compound (18.0 g, 87% yield). ESI MS m/z 168.2 ([M+Na]+). Example 74. Synthesis of (2S,4R)-l-ter/-butyl 2-methyl 4-hydroxypyrrolidine-l,2- dicarboxylate. z^jCO2Me ^CO^Me HO \—-NII ^Boc 143 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (30.0 g, 137.6 mmol) in three portions in 4 h. After stirring for an additional 4 h, the reaction was concentrated to ~35O ml and extracted with EtOAc (4 x 80 mL). The combined organic layers were washed with brine (100 mL), dried (MgSO4), filtered, concentrated and purified by SiOz column chromatography (1:1 hexanes/EtOAc) to give the title compound (22.54 g, 86% yield). ESI MS m/z 268.2 ([M+Na]+). Example 75. Synthesis of (S)-l-tert-butyl 2-methyl 4-oxopyrrolidine-l,2-dicarboxylate. /^v-COiMe The title compound prepared through Dess-Martin oxidation was described in: Franco Manfre et al. J. Org. Chern. 1992, 57, 2060-2065. Alternatively Swern oxidation procedure is as following: To a solution of (COC1)2 (13.0 ml, 74.38 mmol) in CH2CI2 (350 ml) cooled to -78 °C was added dry DMSO (26.0 mL). The solution was stirred at -78 °C for 15 min and then (2S,4R)-l-tert-butyl 2-methyl 4-hydroxypyrrolidine- 1,2-dicarboxyl ate (8.0 g, 32.63 mmol) in CH2O2 (100 ml) was added. After stirring at -78 °C for 2 h, triethylamine (50 ml, 180.3 mmol) was added dropwise, and the reaction solution was warmed to room temperature. The mixture was diluted with aq. NaH2PO4 solution (1.0 M, 400 ml) and phases separated. The aqueous layer was extracted with CH2Q2 (2 x 60 ml). The organic layers were combined, dried over MgSO4, filtered, concentrated and purified by SiO2 column chromatography (7:3 hexanes/EtOAc) to give the title compound (6.73 g, 85% yield). ESI MS m/z 266.2([M+Na]+). Example 76. Synthesis of (S)-l-tert-butyl 2-methyl 4-methylenepyrrolidine-l,2- dicarboxylate. kCO2Me To a suspension of methyltriphenylphosphonium bromide (19.62 g, 55.11 mmol) in THF (150 mL) at 0 °C was added potassium-t-butoxide (6.20 g, 55.30 mmol) in anhydrous THF (80 mL). After stirring at 0 °C for 2 h, the resulting yellow ylide was added to a solution of (S)-1- tert-butyl 2-methyl 4-oxopyrrolidine-l,2-dicarboxylate (6.70 g, 27.55 mmol) in THF (40 mL). After stirring at r.t. for 1 h, the reaction mixture was concentrated, diluted with EtOAc (200 mL), washed with H2O (150 mL), brine (150 mL), dried over MgSO4, concentrated and purified on SiOicolumn chromatography (9:1 hexanes/EtOAc) to yield the title compound (5.77 g, 87% yield). EI MS m/z 264 ([M+Na]+). Example 77. Synthesis of (S)-methyl 4-methylenepyrrolidine-2-carboxylate hydrochloride. NH'HCl ,CO2Me 144 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of (S)-l-terf-butyl 2-methyl 4-methylenepyrrolidine-l,2-dicarboxylate (5.70 g, 23.63 mmol) in EtOAc (40 ml) at 4 °C was added HCI (12 M, 10 ml). The mixture was stirred for 1 h, diluted with toluene (50 ml), concentrated, and crystallized with EtOH/hexane to yield the title compound as HCI salt (3.85 g, 92% yield). EI MS m/z 142.2 ([M+H]4). Example 78. Synthesis of (S)-tert-butyl 2-(hydroxymethyl)-4-methylenepyrrolidine-lcarboxylate. LiAlH4 THF To a solution of (S)-l-terf-butyl 2-methyl 4-methylencpyrrolidinc-l,2-dicarboxylate. (5.20 g, 21.56 mmol) in anhydrous THF (100 mL) at 0 °C was added LiAlH4 (15 ml, 2M in THF). After stirring at 0 °C for 4 h, the reaction was quenched by addition of methanol (5 ml) and water (20 ml). The reaction mixture was neutralized with 1 M HCI to pH 7, diluted with EtOAc (80 ml), filtered through Celite, separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over Na2SO4, concentrated and purified on SiO2column chromatography (1:5 EtOAc/DCM) to yield the title compound (3.77 g, 82% yield). EI MS m/z 236.40 ([M+Na]+). Example 79. Synthesis of (S)-(4-methylenepyrrolidin-2-yl)methanol, hydrochloride salt. =\^NHHC1 To a solution of (S)-tert-butyl 2-(hydroxymethyl)-4-methylenepyrrolidine-l-carboxylate (3.70 g, 17.36 mmol) in EtOAc (30 ml) at 4 °C was added HCI (12 M, 10 ml). The mixture was stirred for 1 h, diluted with toluene (50 ml), concentrated, and crystallized with EtOH/hexane to yield the title compound as HCI salt (2.43 g, 94% yield). EI MS m/z 115.1 ([M+H]+). Example 80. Synthesis of 4-(benzyloxy)-3-methoxybenzoic acid. BnO^x^. To a mixture of 4-hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) in ethanol (350 ml) and aq. NaOH solution (2.0 M, 350 ml) was added BnBr (140.0 g, 823.5 mmol). The mixture was stirred at 65 °C for 8 h, concentrated, co-evaporated with water (2 x 400 ml) and concentrated to -400 ml, acidified to pH 3.0 with 6 N HCI. The solid was collected by filtration, crystallized with EtOH, dried at 45 °C under vacuum to afford the title compound (63.6 g, 83% yield). ESI MS m/z 281.2 ([M+Na]+). Example 81. Synthesis of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid. 145 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 BnO.^z^NC^ MeCTx'^S:O2H To a solution of 4-(benzyloxy)-3-methoxybcnzoic acid (63.5 g, 246.0 mmol) in CH2C12 (400 ml) and HOAc (100 ml) was added HNO3 (fuming, 25.0 ml, 528.5 mmol). The mixture was stirred for 6 h, concentrated, crystallized with EtOH, dried at 40 °C under vacuum to afford the title compound (63.3 g, 85% yield). ESI MS m/z 326.1 ([M+Na]+). Example 82. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2- (hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone. BnO^NO, O A catalytic amount of DMF (30 pl) was added to a solution of 4-(benzyloxy)-5-mcthoxy-2- nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CH2CI2 (70 mL) and the resulting mixture was stirred at room temperature for 2 h. Excess CH2CI2 and oxalyl chloride was removed with rotavap. The acetyl chloride was re-suspended in fresh CH2CE (70 mL) and was added slowly to a pre-mixed solution of (S)-(4- methylenepyrrolidin-2-yl)methanol, hydrochloride salt (1.32 g, 8.91 mmol) and EtgN (6 mL) in CH2CI2 at 0 °C under N2 atmosphere. The reaction mixture was allowed to warm to r.t. and stirring was continued for 8 h. After removal of CH2C12 and EtaN, the residue was partitioned between H2O and EtOAc (70/70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried (MgSCU) and concentrated. Purification of the residue with flash chromatography (silica gel, 2:8 hexanes/EtOAc) yielded the title compound (2.80 g, 79% yield). EI MS m/z 421.2 ([M+Na]+). Example 83. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((tertbutyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-l-yl)methanone. (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidinl-yl)methanone (2.78 g, 8.52 mmol) in the mixture of DCM (10 ml) and pyridine (10 ml) was added tert-butylchlorodimethylsilane (2.50 g, 16.66 mmol). The mixture was stirred for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:6) to afford the title compound (3.62 g, 83% yield, -95% pure). MS ESI m/z calcd for C27H37N2O6S1 [M+H]+ 513.23, found 513.65. 146 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 84. Synthesis of (S)-(4-hydroxy-5-mcthoxy-2-nitrophcnyl)(2-(hydroxymcthyl)-4- methylenepyrrolidin-1-y1)methanone. BnO^- NO2 jj 1 As. HO^,NO2II J CH3SO3H DCM/PhSCH3 (S)-(4-(Benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidinl-yl)methanone (2.80 g, 7.03 mmol) in the mixture of DCM (30 ml) and CH3SO3H (8 ml) was added PhSCH3 (2.00 g, 14.06 mmol). The mixture was stirred for 0.5 h, diluted with DCM (40 ml), neutralized with carefully addition of 0.1 M Na2CO3 solution. The mixture was separated and the aqueous solution was extracted with DCM (2 x 10 ml). The organic layers were combined, dried over Na2SO4, concentrated and purified on SiO2 column eluted with MeOH/CH2Cl2 (1:15 to 1:6) to afford the title compound (1.84 g, 85% yield, -95% pure). MS ESI m/z calcd for Ci4Hi7N2O6 [M+H]+ 309.10, found 309.30. Example 85. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,lphenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) (S)-(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-lyl)methanone (0.801 g, 2.60 mmol) in butanone (10 ml) was added Cs2CO3, ( 2.50 g, 7.67 mmol), followed by addition of 1,5-diiodopentane (415 mmol, 1.28 mmol). The mixture was stirred for 26 h, concentrated and purified on SiO2 column eluted with MeOH/CH2Cl2 (1:15 to 1:5) to afford the title compound (0.675 g, 77% yield, -95% pure). MS ESI m/z calcd for C33H4iN4Oi2 [M+H]+ 685.26, found 685.60. Example 86. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(2-amino-5-methoxy-4,lphenylene))bis(((S)-2-(hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2- (hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) (0.670 g, 0.98 mmol) in CH3OH (10 ml) was added Na2S2O4 (1.01 g, 5.80 mmol) in H2O (8 ml). The mixture was stirred at room temperature for 30 h. The reaction mixture was evaporated and co-evaporated with DMA (2 x 10 mL) and EtOH (2x10 ml)under high vacuum to dryness to afford the title compound (total 147 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 weight 1.63 g) containing inorganic salts which was used directly for the next step reaction (without further separation). EIMS m/z 647.32 (|M+NaJ+). Example 87. Synthesis of C-l (a PBD dimer analog having a bis-linker). (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tert-butoxycarbonyl)amino)butyl)-22,23- bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3,42-bis((4-(hydroxymethyl)phenyl)carbamoyl)- 5,8,21,24,37,40-hexaoxo-11,14,17,28,31,34-hexaoxa-4,7,20,25,38,41-hexaazatetratetracontane- 1,44-dioate (0.840 g, 0.488 mmol) in THF (8 mL) containing pyridine (0.100 ml, 1.24 mmol) at 0 °C was added dropwise of a solution of triphosgene (0.290 mg, 0.977 mmol) in THF (3.0 mL). The reaction mixture was stirred at 0 °C for 15 min then was used directly in the next step. (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2- (hydroxymethyl)-4-methylenepyrrolidin-l-yl)methanone) containing inorganic salts (0.842 mg, -0.49 mmol) was suspended in EtOH (10 ml) at 0 °C was added the trichloride in THF prepared above. The mixture was stirred at 0 °C for 4 h, then warmed to RT for 1 h, concentrated, and purified by reverse phase HPLC (250 (L) mm x 10(d) mm, Cis column, 10-80% acetonitrile/water in 40 min, v =8 ml/min) to afford the title compound (561.1 mg, 48% yield in three steps). ESI MS m/z: calcd for C117H163N16O38 [M+H]+ 2400.12, found 2400.90. Example 88. Synthesis of C-2 (a PBD dimer analog having a bis-linker). Dess-Martin periodinane (138.0 mg, 0.329 mmol) was added to a solution of compound C- 148 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 1 (132.0 mg, 0.055 mmol) in DCM (5.0 mL) at 0 °C. The reaction mixture was warmed to RT and was stirred for 2 h. A saturated solution of NaHCOa/NazSOa (5.0 mL/5.0 mL) was then added and the mixture was extracted with DCM (3 x 25 mL). The combined organic layers were washed with NaHCO3/Na2SO3 (5.0 mL/5.0 mL), brine (10 mL), dried over Na2SO4, filtered, 5 concentrated and purified by reverse phase HPLC (250 (L) mm x 10(d) mm, Cis column, 10- 80% acetonitrile/water in 40 min, v =8 ml/min) to afford the title compound (103.1 mg, 78% yield) as a foam. ESI MS m/z: calcd for CmH^sNisOas [M+H]+ 2396.09, found 2396.65. Example 89. Synthesis of C-3 (a PBD dimer analog having a bis-linker). 10 C-2 compound (55.0 mg, 0.023 mmol) was dissolved in DCM (3 ml), followed by addition of TFA (3 ml) at 4 °C. The reaction mixture was then stirred at RT for 1 h, then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-3 (48.0 mg, 100% yield, 92% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure 15 product C-3 (42.1 mg, 88% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C99H)26N16Oa4 [M+H]+ 2083.86, found 2084.35. Example 90. Synthesis of C-4 (a PBD dimer analog having a bis-linker). 149 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 C-3 compound (35.0 mg, 0.017 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NaHaPOj (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,1l,14,17,20,23-octaoxahexacosan-26-oate (43.0 mg, 0.084 mmol) in 4 portions in 2 h. The reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-4 which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-4 (39.4 mg, 81% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C135H195N16O52 [M+H]+ 2872.30, found 2871.65. Example 91. Synthesis of C-5 (a PBD dimer analog having a bis-linker). To a solution of C-4 compound (35.0 mg, 0.012 mmol) and 2,5,8,11,14,17,20,23- octaoxapentacosan-25-amine (15.1 mg, 0.0394 mmol) in dry DMA (2 ml) was added EDC (30.0 mg, 0.156 mmol). The reaction mixture was stirred at RT for 14 h, concentrated, purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-5 (31.2 mg, 77% yield, 97% pure by HPLC) as a foam. ESI MS m/z: calcd for C161H249N18O62 [M+H]+ 3426.68, found 3427.21. Example 92. Synthesis of (S)-methyl l-(4-(bcnzyloxy)-5-methoxy-2-nitrobcnzoyl)-4- methylenepyrrolidine-2-carboxylate. A catalytic amount of DMF (30 pl) was added to a solution of 4-(benzyloxy)-5-methoxy-2- nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 mL, 22.50 mmol) in anhydrous CH2CI2 (70 mL) and the resulting mixture was stirred at room temperature for 2 h. Excess CH2CI2 and oxalyl chloride was removed with rotavap. The acetyl chloride was re-suspended in fresh CH2O2 (70 mL) and was added slowly to a pre-mixed solution of (S)-methyl 4- 150 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 methylenepyrrolidine-2-carboxylate hydrochloride (1.58 g, 8.91 mmol) and Et2N (6 mL) in CH2C12 at 0 °C under N2 atmosphere. The reaction mixture was allowed to warm to r.t. and stirring was continued for 8 h. After removal of CH2C12 and Et2N, the residue was partitioned between H2O and EtOAc (70/70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried (MgSO4) and concentrated. Purification of the residue with flash chromatography (silica gel, 2:8 hcxanes/EtOAc) yielded the title compound (2.88 g, 76% yield). EI MS m/z 449.1 ([M+Na]+). Example 93. Synthesis of (S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4- mcthylencpyrro-lidinc-2-carbaldchyde. BnO^x^NO2 CHO To a vigorously stirred solution of (S)-methyl 1-(4-(bcnzyloxy)-5-mcthoxy-2-nitro benzoyl)-4-methylenepyrrolidine-2-carboxylate (2.80 g, 6.57 mmol) in anhydrous CH2C12(6O mL) was added DIBAL-H (IN in CH2C12, 10 mL) dropwise at -78 °C under N2 atmosphere. After the mixture was stirred for an additional 90 min, excess reagent was decomposed by addition of 2 ml of methanol, followed by 5% HCI (10 mL). The resulting mixture was allowed to warm to 0 °C. Layers were separated and the aqueous layer was further extracted with CH2C12 (3 x 50 mL). Combined organic layers were washed with brine, dried (MgSO4) and concentrated. Purification of the residue with flash chromatography (silica gel, 95:5 CHCL/MeOH) yielded the title compound (2.19 g, 84% yield). EIMS m/z 419.1 ([M+Na]+). Example 94. Synthesis of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-lHbenzo[e]-pyrrolo[1,2-a]azepin-5(11aH)-one. O A mixture of (S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4- methylenepyrro-lidine-2- carbaldehyde (2.18 g, 5.50 mmol) and Na2S2O4 (8.0 g, 45.97 mmol) in THF (60 ml) and H2O (40 ml) was stirred at room temperature for 20 h. Solvents were removed under high vacuum. The residue was re-suspended in MeOH (60 mL), and HCl (6M) was added dropwise until pH ~ 2 was reached. The resulting mixture was stirred at r.t. for 1 h. The reaction was worked-up by removing most of MeOH, then diluted with EtOAc (100 mL). The EtOAc solution was washed with sat. NaHCOa, brine, dried (MgSO4), and concentrated. Purification of the residue with flash chromatography (silica gel, 97:3 CHClj/McOH) yielded the title compound (1.52 g, 80%). EIMS m/z 372.1 ([M+Na]+). 151 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 95. Synthesis of (S)-8-hydroxy-7-mcthoxy-2-mcthylcnc-2,3 -dihydro-1Hbenzo[e]-pyrrolo[1.2-a]azcpin-5(11aH)-one. O To a solution of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3 -dihydro-lH-benzo[e]- pyrrolo[l,2-a]azepin-5(llaH)-one (1.50 g, 4.32 mmol) in 70 ml of CH2O2 was added 25 ml of CH3SO3H at 0 °C. The mixture was stirred at 0 °C for 10 min then r.t. for 2 h, diluted with CH2CI2, pH adjusted with cold 1.0 N NaHCO3 to 4 and filtered. The aqueous layer was extracted with CH2CI2 (3 x 60 ml). The organic layers were combined, dried over Na2SO4, filtered, evaporated and purified on SiO2 column chromatography (CH3OH/CH2CI2 1:15) to afford 811 mg (73% yield) of the title product. EIMS m/z 281.1 ([M+Na]+). Example 96. Synthesis of (llaS,lla'S)-8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylcne-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(l laH)-one). To a stirred suspended solution of Cs2CO3 (0.761 g, 2.33 mmol)in butanone (8 ml) were added (S)-8-hydroxy-7-methoxy-2-methylene-2,3 -dihydro-lH-benzo[e]-pyrrolo[l,2-a]azepin- 5(1laH)-onc (401 mg, 1.55 mmol) and 1,5-diiodopentane (240 mg, 0.740 mmol). The mixture was stirred at r.t. overnight, concentrated, and purified on SiÜ2 chromatography (EtOAc/CH2C12 1:10) to afford 337 mg (78% yield) of the title product. EIMS m/z 607.2 ([M+Na]^. Example 97. Synthesis of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,11a-hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(l laH)-one and (1laS,l la'S)- 8,8'-(pentane-1,5-diy1bis(oxy))bis(7-methoxy-2-methylene-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[l,2-a][1,4]diazepin-5(10H)-one) H 152 5 10 15 20 25 30 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of (llaS,lla'S)-8,8’-(pcntanc-l,5-diylbis(oxy))bis(7-mcthoxy-2-mcthylcnc- 2,3-dihydro- 1 H-benzo[c]pyrrolo[l,2-a]|T.4]diazepin-5(llaH)-one) (150 mg. 0.256 mmol) in anhydrous dichloromethane (1 mL) and absolute ethanol (1.5 mL) was added sodium borohydride in methoxyethyl ether (85pl, 0.5 M, 0.042mmol) at 0 °C. The ice bath was removed after 5 minutes and the mixture was stirred at room temperature for 3 hours, then cooled to 0 °C, quenched with saturated ammonium chloride, diluted with dichloromethane, and phases separated. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered through Celite and concentrated. The residue was purified by reverse phase HPLC (Cis column, acetonitrile/water). The corresponding fractions were extracted with dichloromethane and concentrated to afford the half reduced compound, (S)-7-mcthoxy-8-((5-(((S)-7-rncthoxy-2- methylene-5-oxo-2,3,5,10,ll,lla-hexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8- yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)- one (64.7 mg, 43%), MS m/z 609.2 ([M+Naf)- 625.3 ([M+K]+) and 627.2 ([M+Na+H2O]+); the fully reduced compound, (llaS,lla'S)-8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylene-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(10H)-one) (16.5 mg, 11%), MS m/z 611.2 ([M+Na]+), 627.2 ([M+K]+), 629.2 ([M+Na+H2O]+); and the unreacted starting material was also recovered (10.2 mg, 7%), MS m/z 607.2 ([M+Na]+), 625.2 ([M+Na+ELO]*). Example 98. Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy) propanoyl)-7- methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hcxahydro-1H-benzo[e]pyrrolo[l,2- a][l,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-1Hbenzo[e]pyrrolo[l,2-a][1,4]diazepin-5(11aH)-one. To the mixture of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(llaH)-one (60.0 mg, 0.102 mmol) and 2,5-dioxopyrrolidin-l-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (40.5 mg, 0.134 mmol) in dichloromethane (5 ml) was added EDC (100.5 mg, 0.520 mmol). The mixture was stirred at r.t. overnight, concentrated and purified on SiO2 column chromatography (EtOAc/CH2Cl2, 1:6) to afford 63.1 mg (81% yield) of the title product. ESI MS m/z C40H50N7O9 [M+H] +, cacld.772.36, found 772.30. 153 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 99. Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-aminoethoxy)ethoxy) propanoyl.)-7- methoxy-2-methylene-5-oxo-2,3,5,10,11.11a-hexahydro-1H-benzo[e]pyrrolo[l,2- a][l,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-lHbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one. To a solution of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy) propanoyl)-7-methoxy-2- methylcne-5-oxo-2.3,5,10,11,11a-hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2- a][l,4]diazepin-5(l laH)-one (60 mg, 0.078 mmol) in the mixture of THF (5 ml) and NaH2PO4 buffer solution (pH 7.5, 1.0 M, 0.7 ml) was added PPI13 (70 mg, 0.267 mmol). The mixture was stirred at r.t. overnight, concentrated and purified on Cis preparative HPLC, eluted with water/CH3CN (from 90% water to 35% water in 35 min) to afford 45.1 mg (79% yield) of the title product after drying under high vacuum. ESI MS m/z C40H52N5O9 [M+H]+, cacld.746.37, found 746.50. Example 100. Synthesis of (S)-N-(2-((S)-8-((5-(((llS,llaS)-10-((S)-15-azido-5-isopropyl- 4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-l-oyl)-ll-hydroxy-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-oxy)-7- methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin- 10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-3-methylbutanamide. H O To the mixture of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2- methylene-2,3-dihydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(llaH)-one (60.0 mg, 0.102 mmol) and (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oic acid (90.2 mg, 0.25 mmol) in DMA (8 ml) was added BrOP (240.2 mg, 0.618 mmol). The mixture was stirred at 60 °C overnight, concentrated and purified on SiO2 column chromatography (CH3OH/CH2C12, 1:10 to 1:5) to afford 97.1 mg (74% yield) of the title product. ESI MS m/z C61H87Ni4Oi7 [M+H] +, cacld.1287.63, found 1287.95. 154 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 101. Synthesis of (S)-N-(2-((S)-8-((5-(((l ISJlaSl-lO-tXSl-lS-amino-Sisopropy1-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oy1)-11-hydroxy-7-methoxy-2- methylene-5-oxo-2,3,5,10,l1,1la-hexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]- pyrrolo[l,2-a][l,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)- propanamido)-3-methylbutanamide (C-6). To a solution of (S)-N-(2-((S)-8-((5-(((llS,llaS)-10-((S)-15-azido-5-isopropyl-4,7-dioxo- 10,13-dioxa-3,6-diazapentadecan-1-oyl)-1l-hydroxy-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)pentyl)-oxy)-7- methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin- 10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-3-methylbutanamide (85 mg, 0.066 mmol) in the mixture of THF (5 ml) was added PPh3 (100 mg, 0.381 mmol). The mixture was stirred for 2 h, Na^PCU buffer solution (pH 7.5, 1.0 M, 0.7 ml) was then added and the mixture was stirred for 10 min. After confirmed by LC-MS to form (S)-N-(2-((S)-8-((5- (((HS,llaS)-10-((S)-15-amino-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-l-oyl)- 11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[l,2- a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[l,2-a][l,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)- propanamido)-3-methylbutanamide (ESI MS m/z C61H90N10O17 [M+Na]+, cacld.1257.66, found 1257.90), bis(2,5-dioxopyrrolidin-l-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)succinate (33 mg, 0.066 mmol) was added. The mixture was continued to stir for 4 h, concentrated and purified on Cis preparative HPLC, eluted with water/CH3CN (from 90% water to 30% water in 35 min) to afford 40.1 mg (40% yield) of the title product C-5 after drying under high vacuum. ESI MS m/z C73H95N12O23 [M+H]+, cacld. 1507.66, found 1507.90. 155 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 102. Synthesis of 4,4'-(pentane-l,5-diylbis(oxy))bis(3-methoxybenzoic acid). MeO'^^COaH H2O/THF/NaOH, 65°C HO2C A solution of diiodopropane (19.0 g, 58.6 mmol) in THF (75 mL) was added dropwise over a period of 4 hours to a vigorously stirred solution of vanilic acid (20.0 g, 119 mmol) in THF (150 mL) and aqueous NaOH (340 mL) at 65°C in the absence of light (foil-wrapped flask). After heating at reflux for 48 hours in the dark, the solution was cooled and the THF removed by evaporation in vacuo. The residue was extracted with EA, The aqueous layer was separated and acidified to pH 2 with cone. HC1. The resultant precipitate collected by filtration, washed, dried and recrystallised from glacial acetic acid to afford the corresponding bis-carboxylic acid (14.0 g, 34.7 mmol). White solid, yield(60%).. Example 103. Synthesis of 4,4'-(pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid). HN03 HO2C';^^OMc HOAc IKLC^^OMe MeO''^'^CO2H To a suspention of 4,4’-(pentane-l,5-diylbis(oxy))bis(3-methoxybenzoic acid) (18.0 g, 66.8 mmol) in HOAc (80 mL, 1800 mmol) was added HNO3 (80 mL, 1778 mmol) dropwise at room temperature. After 2 h of stirring, the mixture was poured into 100 g ice and extrated with EA (2 x 200 mL). The organic layer was separated and washed with H2O(2 x 100 mL), then 4N NaOH (400 mL) was added. After extrated with EA (2 x 100 mL), the basic aqueous layer was separated and acidified to PH 2 with cone. HC1. The mixture was extrated with EA (2 x 250 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by flash chromatography (DCM/McOH = 4/1) to give 4,4'-(pentane-l,5- diylbis(oxy))bis(5-methoxy-2-nitrobenzoic acid) (6.1 g, 12.3 mmol) as a pale yellow solid in 18% yield. Ry 0.3 (DCM/MeOH = 3/1) Example 104. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,lphenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone). 156 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of 4,4'-(pcntanc-l,5-diylbis(oxy))bis(5-mcthoxy-2-nitrobcnzoic acid) (5.0 g, 10.0 mmol) and L-(+)-Prolinol (2.25 g, 22.3 mmol)in DMF (100 mL) was added TEA (4.0 g) at room temperature. After 10 min of stirring, HATU(10.77 g, 28.3 mmol) was added. The mixture was stirred at room temperature overnight. After completion of conversion, the mixture was diluted with H20(100 mL) and extrated with EA (2 x 100 mL) and DCM (2 x 50 mL), the combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by chromatography (DCM/MeOH = 15/1) to give (S)-((pentane-l,5-diylbis(oxy))bis(5- methoxy-2-nitro-4,l-phenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-l-yl)methanone) (6.0 g, 9.1 mmol) as a white foam in 91% yield. Example 105. Synthesis of (S)-((pentane-l,5-diylbis(oxy))bis(2-amino-5-methoxy-4,lphenylene))bis(((S)-2-(hydroxymethyl)pyrrolidin-l-yl)methanone). Pd/C H2 MeOH To a solution of (S)-((pentane-l,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,l-phenylene))- bis(((S)-2-(hydroxymethyl)pyrrolidin-l-yl)methanone) (6.0 g, 9.1 mmol) in MeOH (100 mL) was added 10% Pd/C (2.4 g), the mixture was stirred under hydrogen atmosphere at room temperature overnight. After 14 h of stirring, the Pd/C was removed by filtration and washed with MeOH. The filtrate was concentrated and the residue was purified by chromatography (DCM/MeOH = 10/1) to give (S)-((pentane-1.5-diylbis(oxy))bis(2-amino-5-methoxy-4,l-phenylene))bis(((S)-2- (hydroxymethyl)pyrrolidin-l-yl)methanone) (3.54 g, 5.9 mmol) as a white foam in 65% yield. Example 106. Synthesis of bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl) ((S)-(pentane-1,5-diylbis(oxy))bis(2-((S)-2- (hydroxymethyl)pyrrolidine -l-carbonyl)-4-methoxy-5,l-phenylene))dicarbamate. triphosgene/DTEA/THF/0°C r.t. 157 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 O O To a solution of allyl ((S)-l-(((S)-l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2- yl)amino)-3-methyl-l-oxobutan-2-yl)carbamate (8.0 g, 21.3 mmol) in dry THF(300 mL) was added DIPEA (5.5 g, 40.3 mmol) and a solution of triphosgcnc(3.2 g, 10.8 mmol) in dry THF(50 5 mL) at 5°C. After 15 min of stirring, the solution was recooled to 5°C and a mixture of (S)- ((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis (((S)-2-(hydroxymethyl)- pyrrolidin-l-yl)methanone) (3.2 g, 5.3 mmol ) and DIPEA(2.75 g, 21.6 mmol) in dry THF (150 mL) was added. The resultant solution was allowed to warm to room temperature and stirred overnight. The THF removed by evaporation in vacuo. The residue was purified by 10 chromatography (DCM/MeOH = 20/1) to give bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido) propanamido)-benzyl)((S)-(pentane-l,5-diylbis(oxy))bis(2-((S)-2- (hydroxymethyl)pyrrolidine-l-carbonyl)-4-methoxy-5,1-phenylene))dicarbamate (7.0 g, 4.97 mmol) as a yellow foam in 94% yield. Example 107. Synthesis of (llS,llaS,irS,lla'S)-bis(4-((S)-2-((S)-2- 15 (((allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5- diylbis(oxy))bis(11-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate). o H Il 4 * o 1 HQ. 1 MeO'^^NH =^OH s/rN>0 O JLJ 0 o H * o H O r T n H I Y Dess-Martin periodinane DCM/r.t. /—\ L I (^NY^^OMe O O ' OA h = Oh q N A N O 1 rr "4 i r 158 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methyl butanamido) propanamido)benzyl)((S)-(pentane-l,5-diylbis(oxy))bis(2-((S)-2-(hydroxy-methyl)pyrrolidine -1- carbonyl)-4-methoxy-5,l-phenylene))dicarbamate (300 mg, 0.21 mmol) in dry DCM (15 mL) was added DMP (280 mg, 0.66 mmol) under nitrogen at room temperature. After completion of conversion, the reaction solution was added aqueous Na2SCh and followed by aqueous NaHCOa, the mixture was stirred for further 15 minutes and extracted with DCM (3 x 20 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by chromatography (DCM/MeOH = 20/1) to give (llS,llaS,H'S,lla'S)-bis(4-((S)-2- ((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)8,8'-(pentane-l,5- diy1bis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][l,4]diazepine-10(5H)-carboxylate) (270 mg, 0.19 mmol) as a off-white foam in 92% yield. Example 108. Synthesis of (llS,llaS,irS,lla'S)-bis(4-((S)-2-((S)-2- (((allyloxy)carbonyl)-amino)-3-methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5- diylbis(oxy))bis(11-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate). To a solution of (llS,llaS,irS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl) amino)-3- methylbutanamido)propanamido)benzyl)8,8'-(pentane-l,5-diylbis(oxy))bis(l l-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate) (774 mg, 0.55 mmol) and pyrrolidine (196 mg, 2.76 mmol) in dry DCM (8 mL) was added Pd(ppha)4 (76 mg, 0.066 mmol). The reaction was flushed with argon and stirred for 2h at room temperature, after which the reaction was diluted with DCM and washed sequentially with 159 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 saturated aqueous NH4CI and brine. The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography (DCM/ MeOH = 6/1) to give (11S,1laS,ll'S,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy) carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)8,8’-(pentane-l,5-diylbis(oxy))bis(ll-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]-pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate) (420 mg, 0.34 mmol) as an off-white solid in 62% yield. Example 109. Synthesis of (S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanoic acid. ^^x^O^Cl H2N^CO2H K2CO3 N^CCfjH O + H2O/THF J Allyl chloroformate (24.8 g, 205 mmol) was added dropwise to a stirred solution of L-valine (20 g, 171 mmol) and K2CO3 (35.4 g, 257 mmol) in H2O (250 mL) and THE (250 mL). The reaction mixture was stirred at room temperature overnight, then the solvent was concentrated under reduced pressure and the remaining solution extracted with diethyl ether (100 mL). The aqueous portion was acidified to pH 2 with cone. HC1 and extracted with DCM (3 x 200 mL). The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated to afford the product (35 g, 174 mmol). White solid, yield(100% ).. Example 110. Synthesis of (S)-2,5-dioxopyrrolidin-l-yl 2-(((allyloxy)carbonyl)amino)-3- methylbutanoate. To a stirred solution of (S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanoic acid (35 g, 174 mmol) in dry DCM (500 mL) was added EDC (66.9 g, 348 mmol) and N-hydroxysuccinimide (30 g, 261 mmol) at room temperature. After 14 h of stirring, the reaction was diluted with DCM and washed with water and brine. The organic phase was dried over Na2SO4, filtered and concentrated to afforded the product (54.5 g) which was used in the next step without further purification.Yield: (100%) viscous colourless oil. Ry =0.5 (PE/EA = 2/1) Example 111. Synthesis of (S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)-propanoic acid. To a solution of H-Ala-OH (15.7 g, 176 mmol) and NaHCO3 (15.5 g, 185 mmol) in THF (200 mL) and H2O (200 mL) was added a solution of (S)-2,5-dioxopyrrolidin-l-yl 2-(((allyloxy)- 160 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 carbonyl)amino)-3-methylbutanoate (50 g, 168 mmol) in THF (100 mL) at room temperature. After 72 hours of stirring, the THF was evaporated under reduced pressure. The residue was acidified to pH 3 with citric acid and extrated with EA (3 x 350 mL), the combined extracts was washed with brine, dried, filtered and concentrated to give a white solid. Trituration with diethyl ether (excess) afforded the pure product as a white powder(25.2 g, 93 mmol, 55%). Example 112. Synthesis of allyl ((S)-l-(((S)-l-((4-(hydroxymethyl)phenyl)amino)-loxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate. To a solution of (S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)-propanoic acid (25.2 g, 92.6 mmol) and p-aminobenzyl alcohol (12.0 g, 97.6 mmol) in THF (300 mL) was added EEDQ (24.0 g, 97.2 mmol) at room temperature. After 18 hours of stirring, the solvent was evaporated under reduced pressure to give a pale brown solid. The solid was triturated with diethyl ether and filtered, washing with an excess of diethyl ether. This afforded the product as a white solid(40 g, 106 mmol, 100%). Example 113. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid. h2n^^co2h + Na2CO3H2O/THF Na2CO3 (41.1 g, 387 mmol) was added to a solution of 4-aminobutanoic acid (20 g, 193 mmol) in H2O (300 mL) at 5°C. After 10 min of stirring, a solution of CbzCl (33.2 mL, 232 mmol) in THF (100 mL) was added dropwise. The reaction was allowed to warm to room temperature and stirred overnight. After completion of conversion, the mixture was diluted with H2O (100 mL) and extrated with EA (2 x 100 mL). The aqueous layer was acidified to pH 2 with cone. HC1 and extracted with EA (3 x 100 mL). The combined organics were washed with brine, dried over NaiSO^ filtered and concentrated to give a white solid. Trituration with PE (excess) afforded the pure product as a white powder (31.6 g, 70%). Example 114. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate. M^O^N_^,CO2H g rn u + ,-BuOH DIC/4-DMAP dom - (BuOJV^.NHCbz O To a stirred solution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (5.9 g, 24.9 mmol) and tert-Butanol (14.7 g, 199 mmol) in dry DCM (250 mL) was added 4-DMAP (0.61 g, 5 161 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 mmol) and DIC (4.7 g, 37.3 mmol) at 0°C. After 16 h of stirring, the reaction was filtered and extracted with DCM (2 x 200 mL). The combined organic extract was washed with IN HC1 and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography (100% DCM) to give tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate (4.26 g, 14.5 mmol, 58%) viscous colourless oil. Example 115. Synthesis of tert-butyl 4-aminobutanoate. 9 Pd/C H2 ^A^^NHCbz 9 /-BuO MeOH* /-BuO < ^A^^NH2 2 To a solution of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate (1.69 g, 5.77 mmol) in MeOH (40 mL) was added 10% Pd/C (400 mg), the mixture was stirred under hydrogen atmosphere at room temperature overnight. After 14 h of stirring, the Pd/C was removed by filtration and washed with MeOH. The filtrate was concentrated to afford the product which was used in the next step without further purification(897 mg, 5.64 mmol), colorless liquid, yield (98%). Example 116. Synthesis of (2R,3S)-2,3-bis(benzylamino)succinic acid. 1IO2C. > HO2C ""'Br H2N HOoC vNHBnreflux ..chhdhEtOH HO2C^""NHBn To a solution of meso-2,3-dibromosuccinic acid (50 g, 181 mmol) in EtOH (400 mL) was added benzylamine (150 mL) dropwise. After completion of addition, the mixture was heated to 90°C and stirred overnight. The mixture was cooled to room temperature and diluted with IPO. 6N HC1 was added until pH 4 to give white precipitates. The precipitates were filtered, rinsed with H2O and dried to give (2R,3S)-2,3-bis(benzylamino)succinic acid(50 g, 152 mmol, 84%). Example 117. Synthesis of (2R,3S)-2,3-diaminosuccinic acid. HO2C rtNHBn Pd/C/H2 HO2C. „oNH2 1 * J HO2C ""NHBn AcOH HC1 HO2C "NH2 To a solution of (2R,3S)-2,3-bis(benzylamino)succinic acid (18 g, 55 mmol) in AcOH (100 mL) and HO (100 mL) was added 10% Pd/C (3 g), the mixture was stirred under hydrogen atmosphere at 50°C overnight. After 48 h of stirring, the Pd/C was removed by filtration and washed with H2O. The filtrate was concentrated and the residue was dissovled in IN NaOH (200 mL). AcOH was added until pH 5 to give white precipitates. The precipitates were filtered, rinsed with H2O and dried to give (2R,3S)-2,3-diaminosuccinic acid (8.7 g, 58.8 g, 100%). Example 118. Synthesis of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid. 162 5 10 15 20 25 CA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 HO2C oNH2 cbz0,4NNaOH HO2Œ.^NHCbz HO2C; '"NH2 THF HO2C^ 'NHCbz To a solution of (2R,3S)-2,3-diaminosuccinic acid (31.74 g, 214 mmol) in THF (220 mL) and 4N NaOH (214 mL) was added CbzCl (61 mL, 428 mmol) dropwise at 0°C. After completion of addition, the mixture was allowed to warm to room temperature and stirred for 2 h.The reaction was diluted with H?O (1600 mL) and extrated with EA (2 x 15600 mL). The aqueous layer was separated and acidified with conc.HCl until pH 2 was reached. The resultant solution was stirred for 1 h and standed at 5°C to give white precipitates. The precipitates were filtered, rinsed with H2O and dried to give 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (52.2 g, 125 mmol, 59%). Example 119. Synthesis of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4- diyl)dicarbamate. HO2(L ^NHCbz 1) Ac^o ]5() oc << NHCbz HO2C "'NHCbz 2) CHC13 ’""NHCbz The solution of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (5.0 g, 12 mmol) in Ac2O ( 37.5 mL) was refluxed for 20 min, cooled and concentrated to give resulting anhydride. The diastereomeric mixture was treat with CHCI3(37 mL), the insoluble mew-isomer was filtered and washed with PE to give crystals of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4- diyl)dicarbamate (2.0 g, 5 mmol, 42%) Example 120. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(((benzyloxy)carbonyl)- amino)succinyl)bis(azanediyl))dibutanoate. O NHCbz Z-BuO^ / O HN ? . NH2 AZ t-BuoVv + oZ HATU, DIPEA * A J3 UA '"NHCbz CbzHN*^ >-NH $ Ut-BU To a solution of dibenzyl ((3R,4S)-2,5-dioxotetrahydrofuran-3,4-diyl)dicarbamate (2.03 g, 5.1 mmol) and tert-butyl 4-aminobutanoate (1.79 g, 11.3 mmol) in DMF (45 mL) was added DIPEA (1.98 g, 15.3 mmol) at 0°C. After 5 min of stirring, HATU(4.66 g, 12.3 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 2 h. After completion of conversion, the mixture was diluted with H2O (90 mL) and extrated with EA (2 x 200 mL) and DCM (2 x 90 mL), the combined organic extract was washed with brine and dried over Na2SO4. The majority of solvent was removed under reduced pressure and a white solid was precipitated, which was collected and dried to give di-tert-butyl 4,4’-(((2R,3S)-2,3-bis 163 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 (((benzyloxy)carbonyl)amino)succinyl)bis(azanediyl))dibutanoate (2.8 g, 4.0 mmol) as a white solid in 80% yield. Example 121. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-diaminosuccinyl)bis- (azanediyl))dibutanoate. To a solution of 4,4'-(((2R,3S)-2,3-bis(((benzyloxy)carbonyl)amino)succinyl)bis- (azanediyl))dibutanoate (2.8 g, 4.0 mmol) in MeOH (100 mL) was added 10% Pd/C (1.1 g), the mixture was stirred under hydrogen atmosphere at room temperature overnight. After 18 h of stirring, the Pd/C was removed by filtration and washed with MeOH. The filtrate was concentrated to afford the product which was used in the next step without further purification(940 mg, 2.2 mmol), colorless liquid, yield(55%). Example 122. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)butanamido)succinyl)bis(azanediyl))dibutanoate. To a solution of di-tert-butyl 4,4’-(((2R,3S)-2,3-diaminosuccinyl)bis(azanediyl))- dibutanoate (940 mg, 2.19 mmol) and 4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)butanoic acid (840 mg, 4.59 mmol) in DMF (25 mL) was added DIPEA (1.13 g, 8.76 mmol) at 0°C. After 5 min of stirring, HATU (1.74 g, 4.58 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 1 h. After completion of conversion, the mixture was diluted with H2O (50 mL) and extracted with EA (2 x 100 mL) and DCM (2 x 50 mL), the combined organic extracts were washed with brine and dried over Na2SO4. The majority of solvent was removed under reduced pressure and a white solid was precipitated, which was collected and dried to give di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl)bis-(azanediyl))dibutanoate (1.36 g, 1.79 mmol) as a white solid in 82% yield. Example 123. Synthesis of 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl) bis(azanediyl))dibutanoic acid. 164 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To a solution of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl)bis(azanediyl))dibutanoate (1.36 g, 1.79 mmol) in DCM (15 mL) was 5 added TFA (30 mL) at room temperature 0°C. After 18 h of stirring, the reaction was concentrated and the residue was dissovled in dry toluene. The solvent was removed by evaporation in vacuo to give white precipitates which was used in the next step without further purification (1.3 mg, 2.0 mmol). yield(100%). Example 124. Synthesis of PBD prodcut C-7. 10 O O O O To a solution of (llS,llaS,l TS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl) amino)-3- methylbutanamido)propanamido)benzyl)8,8'-(pentane-l,5-diylbis(oxy))bis(ll-hydroxy-7- methoxy-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- 15 carboxylate) (215 mg, 0.17 mmol) and 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-1Hpyrrol-l-yl)butanamido)succinyl)bis(azanediyl))dibutanoic acid(115 mg, 0.18 mmol) in DMF (18 mL) was added DIPEA (90 mg, 0.70 mmol) at 0°C. After 5 min of stirring, HATU(132 mg, 0.35 mmol) was added. The mixture was allowed to warm to room temperature and stirred overnight. After completion of conversion, the mixture was diluted with H2O (2 mL) and 165 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 extrated with EA (2 x 40 mL) and DCM (2 x 20 mL), the combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by pre-HPLC to give PBD product C-6 (10 mg) as a white powder. Example 125. Synthesis of di-tert-butyl 4,4'-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)butanamido)succinyl)bis(azanediyl))dibutanoate. To a solution of di-tert-butyl 4,4'-(((2R,3S)-2,3-diaminosuccinyl)bis(azanediyl))- dibutanoate (900 mg, 2.09 mmol) and 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoic acid (840 mg, 4.97 mmol) in DMF (25 mL) was added DIPEA (0.93 g, 7.21 mmol) at 0”C. After 5 min of stirring, EDC (1.74 g, 9.06 mmol) was added. The mixture was allowed to warm to room temperature and stirred for 1 h. After completion of conversion, the mixture was diluted with H2O (50 mL) and extracted with EA (2 x 100 mL) and DCM (2 x 50 mL), the combined organic extracts were washed with brine and dried over Na2SO4. The majority of solvent was removed under reduced pressure and a white solid was precipitated, which was collected and dried to give di-tert-butyl 4,4’-(((2R,3S)-2,3-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)succinyl)bis-(azanediyl))dibutanoate (1.27 g, 1.79 mmol) as a white solid in 83% yield. ESI MS m/z+ C34H49N6O12, cacld. 733.33 (M+ H), found 733.55. Example 126. Synthesis of 4,4'-(((2R,3S)-2,3-bis(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)succinyl)bis(azanediyl))dibutanoic acid. Di-tert-butyl 4,4’-(((2R,3S)-2,3-bis(4-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)butanamido)succinyl)bis(azanediyl))dibutanoate (502.0 mg, 0.685 mmol) in 1,4-dioxane (8 ml) at 4°C was added cone. HC1 (3 ml). The mixture was then stirred at RT for 30 min, diluted with 1,4-dioxane (8 ml), concentrated, co-evaporated with dioxane/toluene (1:1, 2x10 ml) to dryness and crystallized with EtOH/Hexane to afford the title compound (289.0 g, 68% yield). ESI MS m/z-i- C26H33N6O12, cacld. 621.21 (M+ H), found 621.55. 166 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 127. Synthesis of allyl ((S)-3-methyl-l-(((S)-l-((4-((((4- nitrophenoxy)carbonyl)-oxy)methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-l-oxobutan-2- yl)carbamate. Allyl ((S)-l-(((S)-l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2-yl)amino)-3- methyl-l-oxobutan-2-yl)carbamate (2.21 g, 5.86 mmol) in the mixture of dry pyridine (5 ml) and CH2C12(2O ml) was added 4-nitrophenyl carbonochloridate (1.82 g, 9.05 mmol). The mixture was stirred at RT for 8 hour, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:12) to afford the title compound (2.63 g, 83% yield). MS ESI m/z calcd for C26H31N4O9 [M+H]+ 543.21, found 543.60 Example 128. Synthesis of (llaS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl) 8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2- methylene-5-oxo-2,3,11,1la-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazcpine-10(5H)- carboxylate). (llaS,lla'S)-8,8'-(Pentane-l,5-diylbis(oxy))bis(7-methoxy-2-methylene-2,3,ll,llatetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-5(10H)-one) (288.2 mg, 0.490 mmol) in dry CH3CN (5 ml) was added allyl ((S)-3-methyl-l-(((S)-l-((4-((((4-nitrophenoxy)carbonyl)oxy)- methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-l-oxobutan-2-yl)carbamate (770.2 mg, 1.420 mmol) and DIPEA (2 ml). The mixture was stirred at 45 °C for 8 h, concentrated and purified on SiO2 column eluted with EtOAc/CH2Cl2 (1:8) to afford the title compound (492.0 mg, 72% yield). MS ESI m/z calcd for C73H91N10O18 [M+H]+ 1395.64, found 1395.95. 167 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 129. Synthesis of (1 laS,l la,S)-bis(4-((S)-2-((S)-2-amino-3-mcthylbutanamido)- propanamido)benzyl) 8,8'-(pentane-l,5-diylbis(oxy))bis(7-methoxy-2-methylene-5-oxo- 2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-carboxylate). To a solution of (llaS,lla'S)-bis(4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2- methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate) (274.2 mg, 0.197 mmol) and pyrrolidine (49 mg, 6.90 mmol) in dry DCM (5 mL) was added Pd(pph3)4 (152.0 mg, 0.132 mmol). The reaction was flushed with argon and stirred for 2h at room temperature, after which the reaction was diluted with DCM and washed sequentially with saturated aqueous NH4CI and brine. The organic phase was dried over NanSO4, filtered and concentrated. The residue was purified by chromatography (DCM/MeOH/EtgN = 6/1/0.02) to give the title compound (166.7 mg, 69% yield) as an off-white solid. MS ESI m/z calcd for C65H83Nio014 [M+H]+ 1227.60, found 1227.93. (1laS,l la'S)-Bis(4-((S)-2-((S)-2-amino-3-methylbutanamido)-propanamido)benzyl) 8,8'- (pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1Hbenzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-carboxylate) (151.1 mg, 0.123 mmol) and 4,4'- (((2R,3S)-2,3-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)succinyl)bis- (azanediyl))dibutanoic acid (77.1 mg, 0.124 mmol) in DMA (5 ml) was added EDC (95.2 mg, 0.496 mmol). The mixture was stirred at RT for 8 h, concentrated and purified on C-18 HPLC C18 3pm column (25 x 4 cm) using gradient elution with a mixture of (A) acetonitrile and (B) water /0.1% formic acid (gradient : 15% A : 85% B up to 25% A : 75% B over 5 minutes, 35% A : 65% B for 15 minutes, 60% A : 40% B down to 50% A : 50% B over 15 minute, 15% A : 168 5 10 15 20 25 169 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 85% B for 5 minutes) with a 8 mL/minutc flow rate. The fractions containing the title compound were pooled, evaporated and dried in a desiccator with P2O5 to afford the C-8 PBD compound (149.2 mg, 67% yield). MS ESI m/z calcd for C91H111N16O24 [M+H]+ 1811.79, found 1812.35. Example 131. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxylethyl)pyrrolidin-1-yl)methanone. 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (10.20 g, 33.65 mmol) and (S)-pyrrolidin-2- ylmethanol (3.85 g, 38.09 mmol) in dry DMF (150 ml) was added EDC (19.50 g, 101.56 mmol). The mixture was stirred at RT for overnight, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:4) to afford the title compound (11.56 g, 89% yield). MS ESI m/z calcd for C20H23N2O6 [M+H]+ 387.15, found 387.65. Example 132. Synthesis of (S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)pyrrolidine- 2-carbaldehyde. Dess-martin o o To a solution of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)- pyrrolidin-l-yl)methanone (3.80 g, 9.84 mmol) in dry DCM (15 mL) was added Dess-Martin periodinane (DMP) (5.80 g, 13.67 mmol) under nitrogen at room temperature. After completion of conversion, the reaction solution was added aqueous Na2SOa and followed by aqueous NaHCOa, the mixture was stirred for further 15 minutes and extracted with DCM (3 x 20 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by S1O2 chromatography (DCM/EtOAc = 4/1) to give the title compound (3.13 g, 83% yield) as an off-white foam. MS ESI m/z calcd for C20H21N2O6 [M+H]+ 385.13, found 385.60, 404.75 [M+H2O+H]+. Example 133. Synthesis of 8-hydroxy-7-methoxy-2,3,ll,lla-tetrahydro-lHbenzo[e]pyrrolo[1,2-a][1,4]diazepin-5(10H)-onc. o H rVYOH Q^^OMeM^OH QN^M-oMe To a solution of S)-l-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)pyrrolidine-2- carbaldehyde (3.00 g, 7.80 mmol) in methanol (75 mL) was Pd/C (10% Pd, 50% wet, 250 mg) in 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 a hydrogenation shaker. After air in the shaker was vacuumed out, hydrogen (5 Psi) was conducted in. The reaction vessel was shaked overnight and filtered through Celite. The filtrant was concentrated and purified by SiO2 chromatography (DCM/MeOH/Et3N = 4/1/0.05) to give the title compound (1.66 g, 86% yield) as a off-white foam. MS ESI m/z calcd for C13H17N2O3 [M+H]+ 249.12, found 249.50. Example 134. Synthesis of 4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4- ((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-hydroxy-7-methoxy-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine- 10(5H)-carboxylate. NHBoc O HN"7 H CO2‘Bu H To a solution of (14S,17S)-tert-butyl l-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)-17- ((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazanonadecan-19-oate (10.15 g, 13.50 mmol) in dry THF(300 mL) was added DIPEA (3.15 g, 24.41 mmol) and a solution of triphosgene (5.15 g, 17.36 mmol) in dry THF (50 mL) at 4-8 °C. After 15 min of stirring, the solution was recooled to 4-8 °C and then added dropwise to a solution of 8-hydroxy- 7-mcthoxy-2,3,l1,1 la-tctrahydro- lH-bcnzo[c]pyrrolo[ l,2-aj[ l,4Jdiazcpin-5(10H)-onc (2.92 g, 11.76 mmol ) in mixture of THF (100 mL) at 4-8 °C in 45 min. The resultant solution was allowed to warm to room temperature and stirred overnight. The mixture was diluted with tolune (50 ml), evaporated in vacuo and purified by SiO2 chromatography (DCM/MeOH = 15/1) to give the title compound (10.02 g, 82% yield) as a yellow foam. MS ESI m/z calcd for C50H74N9O15 [M+H]+ 1040.52, found 1040.90. Example 135. Synthesis of (S)-4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14- (4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecanamido)benzyl 8-(3-iodopropoxy)-7-methoxy-5-oxo-2,3,l1,1la-tetrahydro-lHbenzo[e]pyrrolo[1,2-aJ[1,4]diazepine-10(5H)-carboxylate. I O N' CO2tBu H To a solution of 4-((14S,17S)-l-azido-17-(2-(tcrt-butoxy)-2-oxoethyl)-14-(4-((tertbutoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8- 170 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[c]pyrrolo[1,2-a][1,4]diazepine- 10(5H)-carboxylate (2.02 g, 1.94 mmol) in butanone (50 ml) was added CS2CO3 (2.50 g, 7.67 mmol) and 1,3-diiodopropane (2.50 g, 8.45 mmol). The mixture was stirred at 45 °C under dark for 36 h, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:5) to afford the title compound (2.08 g, 90% yield). MS ESI m/z calcd for C52H77IN9O15 [M+H]+ 1194.45, found 1194.95. Example 136. Synthesis of (S)-2-((S)-l-azido-14-methyl-12-oxo-3,6,9-trioxa-13- azapentadecanamido)-N-(4-(hydroxymethyl)phenyl)propanamide. HO ,n3 v/-nh2 To a solution of (14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecan-18-oic acid (3.02 g, 7.75 mmol) and (4-aminophenyl)methanol (1.05 g, 8.53 mmol) in DMA was added EDC (4.90 g, 25.52 mmol). The mixture was stirred at RT for 14 h, concentrated and purified on S1O2 column eluted with EtOAc/CH2C12 (1:8 to 1:3) to afford the title compound (3.52 g, 92% yield). MS ESI m/z calcd for C22H35IN6O7 [M+H]+ 495.25, found 495.60. Example 137. Synthesis of (HR,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-11-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate. 1). Triphosgene/THF/DIPEA MeO Na2S2O4 O A mixture of (S)-(4-(bcnzyloxy)-5-mcthoxy-2-nitrophcnyl)(2-(hydroxymethyl)-4- methylene-pyrrolidin-l-yl)methanone (3.90 g, 9.80 mmol) and Na2S2O4 (6.0 g, 34.47 mmol) in THF (60 ml) and H2O (40 ml) was stirred at room temperature for 20 h, adjusted pH to 10 with Na2CO3, concentrated, purified on C-18 short column eluted with HaO/MeOH/EtgN (from 99.4/0.5/0.2 to 50/49.8/0.2). The fractions containing the reduced amino product were pooled, concentrated, diluted with THF (50 ml), then cooled to 4-8 °C. Separately to a solution of 2-(lazido-14-methyl-12-oxo-3,6,9-trioxa-13-azapentadecanamido)-N-(4-(hydroxy-methyl)phenyl)- 171 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 propanamidc (6.70 g, 13.56 mmol) in dry THF (150 mL) was added DIPEA (3.50 g. 27.12 mmol) and a solution of triphosgene (4.10 g, 13.80 mmol) in dry THF (20 mL) at 4-8 °C. After 15 min of stirring at 4-8 °C, the solution was added dropwise to the above amino solution at 4-8 °C for 45 min. The mixture was warmed to RT and continued to stir for 2 h, concentrated, extracted with CH2CI2 (3 x 30 ml), dried over Na2SO4, evaporated and and purified on SiCL column eluted with EtOAc/CHzCL (1:10 to 1:5) to afford the title compound (7.23 g, 83% yield in two steps). MS ESI m/z calcd for C45H57IN8O12 [M+H]+ 889.40, found 889.90. Example 138. Synthesis of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-ll-hydroxy-7-methoxy-2- methylcne-5-oxo-2.3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][1,4]diazepine-10(5H)- carboxylate. To a solution of (llR,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-1l-hydroxy-7-methoxy-2-methylene-5- oxo-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-cafboxylate (3.80 g, 4.27 mmol) in dry DCM (40 mL) was added Dess-Martin periodinane (DMP) (2.80 g, 6.60 mmol) under nitrogen at room temperature. After completion of conversion, the reaction solution was added aqueous Na2SO3 and followed by aqueous NaHCOg, the mixture was stirred for further 15 minutes and extracted with DCM (3 x 20 mL). The combined organic extract was washed with brine, dried, filtered and concentrated. The residue was purified by S1O2 chromatography (DCM/EtOAc — 5/1 to 2:1) to give the title compound (2.99 g, 79% yield) as an off-white foam. MS ESI m/z calcd for C44H55N8Oi2 [M+H]+ 886.39, found 886.80. Example 139. Synthesis of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecanamido)bcnzyl 8,ll-dihydroxy-7-methoxy-2-methylene-5-oxo- 2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)-carboxylate. To a solution of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl 8-(benzyloxy)-l1-hydroxy-7-methoxy-2-methylene-5- 172 5 10 15 20 25 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 oxo-2,3,11,1la-tctrahydro-lH-bcnzo[c]pyrrolo[l,2-aJ[l,4]diazcpinc-10(5H)-carboxylatc (2.90 g, 3.27 mmol) in 40 ml of CH2O2 was added 15 ml of CH3SO3H at 0 °C. The mixture was stirred at 0 °C for 10 min then r.t. for 1 h, diluted with CH2CI2, pH adjusted with cold 1.0 N NaHCOa to 4 and filtered. The aqueous layer was extracted with CH2C12(3 x 60 ml). The organic layers were combined, dried over NajSO^ filtered, evaporated and purified on SiO2 column chromatography (CH3OH/CH2Q2 1:15 to 1:5 ) to afford 1.95 g (75% yield) of the title product. MS ESI m/z calcd for C37H48lN8Oi2 [M+HJ+ 797.34, found 797.90. Example 140. Synthesis of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-1-azido-17-(2- (tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa- 13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydrolH-benzo[e]pyrrolo[l,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-mcthoxy-2- methylene-5-oxo-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][1,4]diazepine-10(5H)- To a solution of (llS,llaS)-4-((14S,17S)-l-azido-14,17-dimethyl-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl 8,11-dihydroxy-7-methoxy-2-methylene-5-oxo- 2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (402 mg, 0.504 mmol) and (S)-4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tertbutoxycarbonyl)amino)-butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzy1 8- (3-iodopropoxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[l,2- a][l,4]diazepine-10(5H)-carboxylate (650 mg, 0.544 mmol) in butanone (50 ml) was added CS2CO3 (0.50 g, 1.53 mmol). The mixture was stirred at 45 °C under dark for 36 h, concentrated and purified on SiO2 column eluted with EtOAc/CH2C12 (1:8 to 1:3) to afford the title compound (809 mg, 86% yield). MS ESI m/z calcd for C89Hi24Ni7O27 [M+H]+ 1862.89, found 1863.45. Example 141. Synthesis of (llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-amino- 17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,1la- 173 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 hexahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,11,11a-tetrahydro-lH-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate. (llS,llaS)-4-((14S,17S)-l-Azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-azido-17-(2-(tert-butoxy)-2- oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16- diazaoctadecanamido)-benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,l1,1la-hexahydro-lHbenzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)propoxy)-ll-hydroxy-7-methoxy-2-methylene-5- oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (750 mg, 0.402 mmol) in THF (8 ml) was added Me3P (1.0 M in toluene, 2.0 ml, 2.0 mmol) at 0 - 4 °C under N2. After stirred for 5 min, the ice bath was removed and the reaction mixture was stirred at RT for 2 h. Then, water (1 ml) was added and the mixture was stirred for 10 min. The mixture was diluted with 1,4-dioxane (10 ml), concentrated and co-evaporated with dioxane/toluene to dryness to yield the crude amino product (725 mg, ~99% yield) which was used directly for next step without further purification. MS ESI m/z calcd for C89H128N13O27 [M+H]+ 1810.90, found 1811.50. Example 142. Synthesis of an asymmetrically cross-linked PBD dimer C-9. To the above crude amino compound ((llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl- 12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-lamino-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo- 174 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo- 2,3,5,10,11,1la-hexahydro-lH-benzo[e]pyrrolo[1,2-a][l,4]diazepin-8-yl)oxy)propoxy)-11- hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,1la-tetrahydro-lH-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate) in dry DMA (8 ml) was added 4,4’-(((2R,3S)-2,3-bis(3- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)succinyl)bis(azanediyl))dibutanoic acid (248.0 mg, 0.400 mmol) and EDC (500.0 mg, 2.60 mmol). The mixture was astirred for 24 h, concentrated and purified on Cis preparative HPLC (17% C18, 250 mm x 50 mm), eluted with water/CH3CN (from 80% water to 30% water in 40 min, 9 ml/min) to afford 488.1 mg (51% yield) of the C-9 product after drying under high vacuum. ESI MS m/z C115H156N19O37 [M+H]+, cacld.2395.08, found 2395.90. Example 143. Synthesis of an unsymmetrically cross-linked PBD dimer C-10. C-9 compound (465.0 mg, 0.194 mmol) was dissolved in DCM (4 ml), followed by addition of TFA (2 ml) at 0-4 °C. The reaction mixture was then stirred at RT for 1 h, diluted with toluene (5 ml), then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-3 (48.0 mg, 100% yield, 92% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-10 (373.1 mg, 85% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C106H140N19O35 [M+H]+ 2238.97, found 2239.50. Example 144. Synthesis of an asymmetrically cross-linked PBD dimer C-ll. 175 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 C-10 compound (235.0 mg, 0.105 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NaH2PO4 (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (43.0 mg, 0.084 mmol) in 4 portions in 2 h. The 5 reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-ll which was further purified by reverse phase HPLC (250 (L) mm x 50(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-ll (215.5 mg, 78% yield, 95% pure ) as a foam. ESI MS m/z: calcd for C124H174N19O44 [M+H]+ 2633.20, found 2633.85. 10 Example 145. Synthesis of an asymmetrically cross-linked PBD dimer C-12. To a solution of C-ll compound (65.0 mg, 0.0246 mmol) and 2,5,8,11,14,17,20,23- octaoxapentacosan-25-amine (15.1 mg, 0.0394 mmol) in dry DMA (2 ml) was added EDC (30.0 mg, 0.156 mmol). The reaction mixture was stirred at RT for 15 h, concentrated, purified by 15 reverse phase HPLC (250 (L) mm x 30 (d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-12 (60.2 mg, 81% yield, 95% pure by HPLC) as a foam. ESI MS m/z: calcd for Ci4iH209N20O5i [M+H]+ 2998.43, found 2999.40. Example 146. Synthesis of an asymmetrically cross-linked PBD dimer C-13. 176 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 To the crude amino compound ((llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-amino- 17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)propoxy)-ll-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)- carboxylate) (-120 mg, -0.0662 mmol) in dry THF (10 ml) was added 3,4-dibromofuran-2,5- dione (16.8 mg, 0,0661 mmol). The mixture was stirred at RT for 4 h, then EDC (50.2 mg, 0.261 mmol). The mixture was continuously stirred for 12 h, concentrated, purified by SiOi column, eluted with MeOH/CfECh (1:12 to 1:6) to afford the pure product C-13 (112.2 mg, 83% yield) as a foam. ESI MS m/z: calcd for €93^206^,3029 [M+H]+ 2046.7073, found 2046.8260. Example 147. Synthesis of an asymmetrically cross-linked PBD dimer C-14. C-13 compound (100.2 mg, 0.0489 mmol) was dissolved in DCM (4 ml), followed by addition of TFA (2 ml) at 0-4 °C. The reaction mixture was then stirred at RT for 1 h, diluted with toluene (5 ml), then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-14 (94.3 mg, 102% yield, 93% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-14 (76.6 mg, 83% yield, 96% pure ) as a foam. ESI MS m/z: calcd for C84H109Br2Ni3O27 [M+H]+ 1890.5995, found 1890.6250, 1893.6565 [M+H+2]+. Example 148. Synthesis of an asymmetrically cross-linked PBD dimer C-15. 177 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 C-14 compound (55.0 mg, 0.0291 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NalEPOi (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,ll,14,17,20,23,26-nonaoxaoctacosan-28-oate (47.2 mg, 0.0875 mmol) in 4 portions in 2 h. The reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-15 which was further purified by reverse phase HPLC (250 (L) mm x 50(d) mm, Cis column, 20-60% acctonitrilc/water in 40 min, v =8 ml/min) to afford the pure product C-15 (215.5 mg, 78% yield, 95% pure ) as a foam. ESI MS m/z: calcd for Ci03H146Br2Ni3O37 [M+H]+ 2314.8309, found 2314.8575, 2316.8705 [M+H+2]+, 2318.1445 [M+H+4]+. Example 149. Synthesis of an asymmetrically cross-linked PBD dimer C-16. To the crude amino compound ((llS,llaS)-4-((14S,17S)-l-amino-14,17-dimethyl-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)benzyl 8-(3-(((S)-10-(((4-((14S,17S)-l-amino- 17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9- trioxa-13,16-diazaoctadecanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11ahexahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepin-8-yl)oxy)propoxy)-ll-hydroxy-7-methoxy- 2-methylene-5-oxo-2,3,ll,lla-tetrahydro-lH-benzo[e]pyrrolo[l,2-a][l,4]diazepine-10(5H)- carboxylate) (~120 mg, -0.0662 mmol) in dry THF (10 ml) was added 3,4-furan-2,5-dione (6.5 mg, 0,0661 mmol). The mixture was stirred at RT for 4 h, then EDC (50.2 mg, 0.261 mmol). The mixture was continuously stirred for 12 h, concentrated, purified by SiO2column, eluted with MeOH/CH2C12 (1:12 to 1:6) to afford the pure product C-16 (107.3 mg, 86% yield) as a foam. ESIMS m/z: calcd for C93H128N13O29 [M+H]+ 1890.8941, found 1890.8990. 178 5 10 15 20 GA 03105541 2021-01-04 WO 2020/006722 PCT/CN2018/094586 Example 150. Synthesis of an asymmetrically cross-linked PBD dimer C-17. C-16 compound (85.5 mg, 0.0452 mmol) was dissolved in DCM (4 ml), followed by addition of TFA (2 ml) at 0-4 °C. The reaction mixture was then stirred at RT for 1 h, diluted with toluene (5 ml), then concentrated, and co-evaporated with DCM/toluene to dryness to afford the crude product C-17 (81.3 mg, 104% yield, 92% pure by HPLC) which was further purified by reverse phase HPLC (250 (L) mm x 20(d) mm, Cis column, 5-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-17 (67.4 mg, 86% yield, 96% pure ) as a foam. ESI MS m/z: calcd for CsÆi12N13O27 [M+H]+ 1734.7785, found 1734.8285. Example 151. Synthesis of an asymmetrically cross-linked PBD dimer C-18. C-17 compound (53.0 mg, 0.0305 mmol) was dissolved in a mixture solution of THF (3 ml) and 0.1 M, NaH2PO4 (3 ml), pH 7.5, followed by addition of N-succinimidyl 2,5,8,ll,14,17,20,23,26-nonaoxaoctacosan-28-oate (47.0 mg, 0.0874 mmol) in 4 portions in 2 h. The reaction mixture was then continued to stir at RT for 4 h, and co-evaporated with DMF (10 ml) to dryness to afford the crude product C-18 which was further purified by reverse phase HPLC (250 (L) mm x 50(d) mm, Cis column, 20-60% acetonitrile/water in 40 min, v =8 ml/min) to afford the pure product C-18 (53.25 mg, 83% yield, 95% pure ) as a foam. ESI MS m/z: calcd for C103H148N13O37 [M+H]+ 2159.0099, found 2159.0890. Example 152. Synthesis of an asymmetrically cross-linked PBD dimer C-19. 179 DEMANDE OU BREVET VOLUMINEUX LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND PLUS D’UN TOME. CECI EST LE TOME 1 DE 2 CONTENANT LES PAGES 1 À 179 NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE VOLUME THIS IS VOLUME 1 OF 2 CONTAINING PAGES 1 TO 179 NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME : NOTE POUR LE TOME / VOLUME NOTE:

Claims

What is claimed is: 1. A cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule having one of the Formula (1-01) to (1-20) below: 202 Date Reçue/Date Received 2024-05-09 (1-04), 06), 203 Date Reçue/Date Received 2024-05-09 07), 08), 09), 204 Date Reçue/Date Received 2024-05-09 (1-12), 205 Date Reçue/Date Received 2024-05-09 (1-14), 206 Date Reçue/Date Received 2024-05-09 Date Reçue/Date Received 2024-05-09 (1-19), or 208 Date Reçue/Date Received 2024-05-09 wherein V, V’ are the same or different, and are independently H, OH, -NHOH; OR5; OCORs; OCOOR5; NR5R5’, NR5COR5’, NRsNRs’NRs”; OCONR5R5’; NR5(C=NH)NR5’R5”; NR5CONR5’R5”; OCSNHR5; -SH;-SR5; SOR5; SOOR5; SO3, HSO3,HSO2, a salt of HSO3'; a salt of SO32'; a salt of -HSO2'; OSO3; NR5SOOR5’; H2S2O5; a salt of S2O52'; PO3SH3, PO2S2H2, POS3H2, PS4H2; a salt of PO3S3; a salt of PO2S23’; a salt of POS33'; a salt of PS43’; (RsO^POSRs’; HS2O3; a salt of S2Û32'; HS2O4; a salt of S2O42'; P(=S)(OR5)(S)(OH) or a salt thereof formed with a cation; - NR5OR5’; RsC(=O)NOH or a salt thereof formed with a cation; HOCH2SO2‘; a salt of HOCH2SO2'; NR5COR5’; O-glycoside; N3; CN; a halo F, Cl, Br, or I; QRsWXRs”); OP(O)(OR5)(NHR5 ); 0P(0)(NHRs)(NHR5>); PfRsXRs XRs ) triarylphosphonium; an amino acid Aa; a peptide NRsCO(AaX wherein Aa is an amino acid and t =1-100; an aminoacid-derived group; an a-, 0-, y-, or ro-aminoacid; or an unnatural aminoacid; wherein R5, R5’ and R5” are independently H, Ci-Csof al¬ kyl, alkenyl, alkinyl, heteroalkyl, aryl, arylalkyl, or carbonyl; m and m’ are independently 0-5; n is 1-30; qis 0-5; mAb is a cell-binding agent/molecule; and r, r’ and r” are independently 0-200; or a pharmaceutically acceptable salt thereof. 209 Date Reçue/Date Received 2024-05-09 2. A cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule having one of the Formula (II-01) to (11-08), (III-01) to (DI-05) and (IV-01) to (IV-11) below: (11-03), 210 Date Reçue/Date Received 2024-05-09 (11-04), (11-05), (11-06), 211 Date Reçue/Date Received 2024-05-09 (III-01), 212 Date Reçue/Date Received 2024-05-09 (III-03), 213 Date Reçue/Date Received 2024-05-09 Date Reçue/Date Received 2024-05-09 (IV-02), (IV-04), 215 Date Reçue/Date Received 2024-05-09 (IV-05), (IV-06), 07), 216 Date Reçue/Date Received 2024-05-09 217 Date Reçue/Date Received 2024-05-09 (IV-11), wherein V And V’ are as defined in claim 1; m, m’, and q are independently 0-5; m3 and n are independently 0-30; r, r’, r”, n, and r2 are independently 0-200; and mAb is the cell-binding agent/molecule; or a pharmaceutically acceptable salt thereof. 3. A cross-linked PBD dimer derivative having one of Formulas (V-01) to (V-20), (VI-01) to (VI-12), (VII-01) to (VII-06), or (VIII-Ol) to (VIII-06) below: 218 Date Reçue/Date Received 2024-05-09 219 Date Reçue/Date Received 2024-05-09 220 Date Reçue/Date Received 2024-05-09 221 Date Reçue/Date Received 2024-05-09 222 Date Reçue/Date Received 2024-05-09 223 Date Reçue/Date Received 2024-05-09 224 Date Reçue/Date Received 2024-05-09 225 Date Reçue/Date Received 2024-05-09 226 Date Reçue/Date Received 2024-05-09 227 Date Reçue/Date Received 2024-05-09 228 Date Reçue/Date Received 2024-05-09 229 Date Reçue/Date Received 2024-05-09 (VII-03) 230 Date Reçue/Date Received 2024-05-09 (vm-oi). 231 Date Reçue/Date Received 2024-05-09 (Vin-04), 232 Date Reçue/Date Received 2024-05-09 (vin-06) wherein V and V’ are as defined in claim 1; X and X’ are independently N, 0, S, alkyl, alkenyl, or ether; m, m’ and q are independently 0-5; and r, r’ and r” are independently 0-200. 4. A cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule having Formula 31,45, 57, 59,61,67,90,96, 148, 162, 176, 178, 184, 188, 209,214,224,232, 255,268,271, CC-3, CC-4, CC-5, CC-6, CC-7, CC-8, CC-10, CC-11, CC-12, CC-14, CC-15, CC-17, CC-18, CC- 20, CC-21, CC-27, or CC-29, shown below: 233 Date Reçue/Date Received 2024-05-09 Date Reçue/Date Received 2024-05-09 235 Date Reçue/Date Received 2024-05-09 236 Date Reçue/Date Received 2024-05-09 237 Date Reçue/Date Received 2024-05-09 238 Date Reçue/Date Received 2024-05-09 239 Date Reçue/Date Received 2024-05-09 240 Date Reçue/Date Received 2024-05-09 241 Date Reçue/Date Received 2024-05-09 CC-5, 242 Date Reçue/Date Received 2024-05-09 Date Reçue/Date Received 2024-05-09 244 Date Reçue/Date Received 2024-05-09 Date Reçue/Date Received 2024-05-09 , or CC-29, wherein r, n and ro are independently 0-200; n is 1-30; and mAb is a cell-binding agent/molecule; or a pharmaceutically acceptable salt thereof. 246 Date Reçue/Date Received 2024-05-09 5. A cross-linked PBD dimer derivative having Formula 30, 44, 56, 58, 60, 66, 89, 95, 147, 161, 175, 177, 183, 187, 208, 213, 223, 231, 254, 267, 270, 273, 275, 277, C-3, C-4, C-5, C-6, C- 7, C-8, C-10, C-ll, C-12, C-14, C-15, C-17, C-18, C-20, C-21, C-27, or C-29 below: 247 Date Reçue/Date Received 2024-05-09 248 Date Reçue/Date Received 2024-05-09 249 Date Reçue/Date Received 2024-05-09 250 Date Reçue/Date Received 2024-05-09 251 Date Reçue/Date Received 2024-05-09 213 223 252 Date Reçue/Date Received 2024-05-09 253 Date Reçue/Date Received 2024-05-09 254 Date Reçue/Date Received 2024-05-09 255 Date Reçue/Date Received 2024-05-09 256 Date Reçue/Date Received 2024-05-09 257 Date Reçue/Date Received 2024-05-09 258 Date Reçue/Date Received 2024-05-09 259 Date Ç.eceiVett wherein r, n, and r2 are independently 0-200. 6. The cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule or pharmaceutically acceptable salt thereof according to claim 1, 2 or 4, wherein die cell-binding agent/molecule targets a tumor cell, a virus infected cell, a microorganism infected cell, a para¬ site infected cell, an autoimmune disease cell, an activated tumor cell, a myeloid cell, an acti¬ vated T-cell, an affecting B cell, or a melanocyte. 7. The cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule or pharmaceutically acceptable salt thereof according to claim 6, wherein the tumor cell is a lym¬ phoma cell, a myeloma cell, a renal cancer cell, a breast cancer cell, a prostate cancer cell, an 260 Date Reçue/Date Received 2024-05-09 ovarian cancer cell, a colorectal cancer cell, a gastric cancer cell, a squamous cancer cell, a small cell lung cancer cell, a non-small cell lung cancer cell, a testicular cancer cell, a malignant cell, or a cell that grows and divides at an unregulated, quickened pace to cause a career. 8. A pharmaceutical composition comprising the cross-linked PBD dimer derivative conju¬ gated to a cell-binding agent/molecule or pharmaceutically acceptable salt thereof according to any one of claims 1, 2, 4, 6 and 7, and a pharmaceutically acceptable carrier, diluent, or excipient. 9. The pharmaceutical composition according to claim 8, which is either in a liquid formula or in a formulated lyophilized solid, comprising by weight: 0.01%-99% of die cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule; 0.0%-20.0% of one or more poly¬ ols; 0.0%-2.0% of one or more surfactants; 0.0% -5.0% of one or more preservatives; 0.0% -30% of one or more amino acids; 0.0% -5.0% of one or more antioxidants; 0.0% -0.3% of one or more metal chelating agents; 0.0% -30.0% of one or more buffer salts for adjusting pH of the formula¬ tion to pH 4.5 to 8.5; and 0.0% -30.0% of one or more of isotonic agent for adjusting osmotic pressure bewteen about 250 to 350 mOsm when reconstituted for administration to a patient; wherein the polyol comprises fructose, mannose, maltose, lactose, arabinose, xylose, ri¬ bose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melezitose, raffinose, manni¬ tol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, glycerol, or L-gluconate or its metallic salts; wherein the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 65, poly¬ sorbate 80, polysorbate 81, polysorbate 85, poloxamer, poly(ethylene oxide)-poly(propylene oxide), polyethylene-polypropylene, Triton, sodium dodecyl sulfate (SDS), sodium laurel sul¬ fate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myri¬ styl-, linoleyl- or stearyl-sarcosine, linoleyl-, myristyl-, or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl oleyl-taurate, do¬ decyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine, coco ampho glycinate, 261 Date Reçue/Date Received 2024-05-09 isostearyl ethylimidonium ethosulfate, polyethyl glycol, polypropyl glycol, or a copolymer of ethylene and propylene glycol; wherein the preservative comprises benzyl alcohol, octadecyldimethylbenzyl ammo¬ nium chloride, hexaméthonium chloride, benzalkonium chloride, benzéthonium chloride, phe¬ nol, butyl and benzyl alcohol, alkyl parabens, methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol; wherein the amino acid comprises arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid; wherein the antioxidant comprises ascorbic acid, glutathione, cystine or methionine; wherein the chelating agent comprises EDTA or EGTA; wherein the buffer salt comprises sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris or tromethamine hydrochloride, phosphate or sulfate; or arginine, glycine, glycylglycine, or histidine with anionic acetate, chloride, phosphate, sulfate, or succinate salts; and wherein tire tonicity agent comprises mannitol, sorbitol, sodium acetate, potassium chlo¬ ride, sodium phosphate, potassium phosphate, trisodium citrate, or sodium chloride. 10. The pharmaceutical composition according to claim 8 or 9, which is packed in a vial, bottle, pre-filled syringe, or pre-filled auto-injector syringe, in a form of a liquid or lyophilized powder/solid. 11. The cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule or pharmaceutically acceptable salt thereof of any one of claims to 1, 2, 4, 6 and 7, having in vitro, in vivo or ex vivo cell killing activity. 12. Use of the cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule or pharmaceutically acceptable salt thereof according to any one of claims 1, 2, 4, 6 and 7, or the pharmaceutical composition according to any one of claims 8 to 10, in the manufacture of a 262 Date Reçue/Date Received 2024-05-09 medicament for the treatment or prevention of a cancer, an autoimmune disease, or an infectious disease. 13. The use according to claim 12, wherein the cross-linked PBD dimer derivative conjugated to a cell-binding agent/molecule or pharmaceutically acceptable salt thereof according to any one of claims 1, 2, 4, 6 and 7, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 8 to 10, is for administration concurrently with a fur¬ ther therapeutic agent comprising a chemotherapeutic agent, a radiation therapy agent, an immu¬ notherapy agent, an autoimmune disorder agent, or an anti-infectious agent. 14. The use according to claim 13, wherein the further therapeutic agent: (1) a chemotherapeutic agent: a) an alkylating agent comprising nitrogen mustards: comprising chlorambucil, chlomaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, novembichin, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-1065, adozelesin, carzelesin, bizelesin; duocarmycin, KW-2189, CBI-TMI, or CBI dimers; benzodiaz¬ epine dimers orpyrrolobenzodiazepine (PBD) dimers, tomaymycin dimers, indolinobenzodiazepine dimers, imidazobenzothiadiazepine dimers, or oxazolidinobenzodiazepine dimers; nitro¬ soureas: comprising carmustine, lomustine, chlorozotocin, fotemustine, nimustine, or ranimustine; alkylsulphonates: comprising busulfan, treosulfan, improsulfan or piposulfan; triazenes or dacarbazine; platinum containing compounds: comprising carboplatin, cisplatin, or oxali¬ platin; aziridines, benzodopa, carboquone, meturedopa, or uredopa; ethylenimines or methylamelamines comprising altretamine, triethylenemelamine, trietylenephosphoramide, triethy¬ lenethiophosphoramide or trimethylolomelamine; b) a plant alkaloid: comprising vinca alkaloids: comprising vincristine, vinblastine, vindesine, vinorelbine, or navelbin; taxoids: comprising paclitaxel, or docetaxol; maytansinoids comprising DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine, ansamitocins, 263 Date Reçue/Date Received 2024-05-09 cryptophycins, cryptophycin 1, or cryptophycin 8; epothilones, eleutherobin, discodermolide, bryostatins, dolostatins, auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin; or spongistatin; c) a DNA topoisomerase inhibitor: comprising epipodophyllins: comprising 9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acids or retinols, teniposide, topotecan, 9-nitrocamptothecin or RFS 2000; or mitomycins; d) an antimetabolite: comprising anti-folate: DHFR inhibitors: comprising methotrexate, trimetrexate, denopterin, pteropterin, aminopterin, 4-aminopteroic acid or folic acid; IMP dehy¬ drogenase inhibitors: comprising mycophenolic acid, tiazofurin, ribavirin, or EICAR; ribonucle¬ otide reductase inhibitors: comprising hydroxyurea, ro deferoxamine; pyrimidines: comprising uracil; ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil, floxuridine, or ratitrexed; cytosines; comprising cytar¬ abine, cytosine arabinoside, or fludarabine; purines: comprising azathioprine, fludarabine, mer¬ captopurine, thiamiprine, or thioguanine; folic acid replenisher, or frolinic acid; e) a hormonal therapy: comprising receptor antagonists: anti-estrogen: comprising megestrol, raloxifene, or tamoxifen; LHRH agonists: comprising goscrclin, or leuprolide acetate; anti¬ androgens: comprising bicalutamide, flutamide, calusterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, or trilostane; retinoids/deltoids: comprising Vitamin D3; CB 1093, EB 1089 KH 1060, cholecalciferol, or ergocalciferol; photodynamic therapies: comprising verteporfin, phthalocyanine, photosensitizer Pc4, or demethoxyhypocrellin A; or cytokines: comprising Interferon-alpha, Interferon-gamma, tumor ne¬ crosis factor, or human proteins containing a TNF domain; f) a kinase inhibitor, comprising BIBW 2992, anti-EGFR/Erb2, imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080, anti-VEGFR2, mubritinib, ponatinib, bafetinib, bosutinib, cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuxi¬ mab, Trastuzumab, Ranibizumab, Panitumumab, or ispinesib; 264 Date Reçue/Date Received 2024-05-09 g) a poly (ADP-ribose) polymerase inhibitor comprising olaparib, niraparib, iniparib, talazoparib, veliparib, CEP 9722, E7016, BGB-290, or 3-aminobenzamide; h) an antibiotic, comprising an enediyne antibiotic comprising calicheamicin, calicheamicin yl , 51, al or pi ; dynemicin, comprising dynemicin A or deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, or neocarzinostatin chromophore or related chromoprotein ene¬ diyne antibiotic chromomophores, aclacinomycins, actinomycin, authramycin, azaserine, bleo¬ mycins, cactinomycin, carabicin, carminomycin, or carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin or deoxydoxorubicin, epirubicin, eribulin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivo¬ mycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin; i) a polyketide, acetogenin, bullatacin or bullatacinone; gemcitabine, epoxomicins, carfilzomib, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy, isoprenylation inhibitors or Lovastatin, dopaminergic neurotoxins or l-methyl-4-phenylpyridinium ion, cell cycle inhibitors comprising staurosporine, actinomycins comprising actinomycin D, or dactinomycin, amanitins, bleomycins comprising bleomycin A2, or bleomycin B2, peplomycin, anthracyclines comprising daunorubicin, doxorubicin adriamycin, idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDR inhibitors or verapamil, Ca2+ATPase inhibitors or thapsigargin, histone deacetylase inhibitors comprising Vorinostat, Romidepsin, Panobinostat, Valproic acid, Mocetinostat, Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, or Trichostatin A; Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate, Di¬ sulfiram, Salinosporamide A.; anti-adrenals, comprising aminoglutethimide, mitotane, or trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflomithine (DFMO), elfomithine; elliptinium acetate, etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; 265 Date Reçue/Date Received 2024-05-09 rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes comprising T-2 toxin, verrucarin A, roridin A or anguidine; urethane, siRNA, or antisense drugs; or (2) an anti-autoimmune disease agent: cyclosporine, cyclosporine A, aminocaproic acid, az¬ athioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids com¬ prising amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propio¬ nate, fluocortolone danazol, dexamethasone, Triamcinolone acetonide, beclometasone dipropio¬ nate, DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, or tacrolimus; or (3) an anti-infectious disease agents comprising: a) aminoglycosides: amikacin, astromicin, gentamicin, netilmicin, sisomicin, isepamicin, hygromycin B, kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, tobramycin, neomy¬ cin, framycetin, paromomycin, ribostamycin, netilmicin, spectinomycin, streptomycin, tobramy¬ cin, or verdamicin; b) amphenicols: azidamfenicol, chloramphenicol, florfenicol, or thiamphenicol; c) ansamycins: geldanamycin, or herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem, cilastatin, meropenem, or panipenem; e) cephems: carbacephem, loracarbef, cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin, cefoxitin, cefotetan, cefmetazole, oxacephem flomoxef, or latamoxef; f) glycopeptides: bleomycin, vancomycin, oritavancin, telavancin, teicoplanin, dalbavancin, or ramoplanin; 266 Date Reçue/Date Received 2024-05-09 g) glycylcyclines: tigecycline; h) p-Lactamase inhibitors: penam, sulbactam, tazobactam, clavam, or clavulanic acid; i) lincosamides: clindamycin, or lincomycin; j) lipopeptides: daptomycin, A54145, or calcium-dependent antibiotics; k) macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide, telithromycin, cethromycin, midecamycin, miocamycin, oleandomycin, rifamycin, rifampin, rifabutin, rifapentine, rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus, troleandomycin, or telithromycin; 1) monobactams: aztreonam, or tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin, carindacillin, cioxacillin, dicloxacillin, epicillin, flucioxacillin, mecillinam, pivmecillinam, mezlocillin, meti¬ cillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, pipe¬ racillin, propicillin, sulbenicillin, temocillin, or ticarcillin; o) polypeptides: bacitracin, colistin, or polymyxin B; p) quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, di¬ floxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, nor¬ floxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, or trovafloxacin; q) streptogramins: pristinamycin, or quinupristin/dalfopristin; r) sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sul¬ fasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole, or co-trimoxazole; s) steroid antibacterials: fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline, or glycylcyclines comprising tigecycline; or 267 Date Reçue/Date Received 2024-05-09 u) antibiotics comprising annonacin, arsphenamine, bactoprenol inhibitors, Bacitracin, DADAL/AR inhibitors, cycloserine, dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors, fosfomycin, nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin, rifampin, tazobactam tinidazole, or uvaricin; or (4) an anti-viral drug, comprising: a) entry/fusion inhibitor: aplaviroc, maraviroc, vicriviroc, gp41 enfuvirtide, PRO 140, or CD4 ibalizumab; b) integrase inhibitor: raltegravir, elvitegravir, or globoidnan A; c) maturation inhibitor: bevirimat, or vivecon; d) neuraminidase inhibitor: oseltamivir, zanamivir, or peramivir; e) nucleoside & nucleotide: abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine, elvucitabine, emtricitabine, entecavir, famciclovir, fluorouracil, 3’-fluoro-2’,3’-dideoxythymidine or 3’-fluoro-2’,3’-dideoxyguanosine, fomivirsen, ganciclovir, idoxuridine, lamivudine, 1-nucleosides comprising /M-thymidine or ^-1- 2’-deoxycytidine, penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), taribavirin, virarnidine, telbivudine, tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine, or zidovudine; f) non-nucleoside: amantadine, ateviridine, capravirine, diarylpyrimidines, etravirine, rilpivirine, delavirdine, docosanol, emivirine, efavirenz, foscamet, phosphonoformic acid, imiquimod, interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205, peginter¬ feron alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod, or tromantadine; g) protease inhibitor: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfmavir, pleconaril, ritonavir, saquinavir, telaprevir, or tipranavir; or h) anti-virus drug: abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate, foscamet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin, or seliciclib; or (5) pharmaceutically acceptable salts, acids, hydrate or hydrated salt; or an optical isomer, racemate, diastereomer or enantiomer of any of the above drugs. 268 Date Reçue/Date Received 2024-05-09 15. The use according to claim 13 or 14, wherein the further therapeutic agent comprises one or more of the following agents: Abatacept, abemaciclib, Abiraterone acetate, Abraxane, Acetaminophen/hydrocodone, Acalabrutinib, aducanumab, Adalimumab, ADXS31-142, ADXS-HER2, afatinib dimaleate, aldesleukin, alectinib, alemtuzumab, Alitretinoin, ado-trastuzumab emtansine, Amphetamine/ dextroamphetamine, anastrozole, Aripiprazole, anthracyclines, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin, Avelumab, Axicabtagene ciloleucel, axitinib, belinostat, BCG Live, Bevacizumab, bexarotene, blinatumomab, Bortezomib, bosutinib, brentuximab vedotin, brigatinib, Budesonide, Budesonide/ formoterol, Buprenorphine, Cabazitaxel, Cabozantinib, capmatinib. Capecitabine, carfilzomib, chimeric antigen receptor-engineered T (CAR-T) cells, Celecoxib, ceritinib, Cetuximab, Chidamide, Ciclosporin, Cinacalcet, crizotinib, Cobimetinib, Cosentyx, crizotinib, CTL019, Dabigatran, dabrafenib, dacarbazine, daclizumab, dacomotinib, daptomycin, Daratumumab, Darbepoetin alfa, Darunavir, dasatinib, denileukin diftitox, Denosumab, Depakote, Dexlansoprazole, Dexmethylphenidate, Dexamethasone, DigniCap Cooling System, Dinutuximab, Doxycycline, Duloxetine, Duvelisib, durvalumab, elotuzumab, Emtricibine/Rilpivirine/Tenofovir, disoproxil fumarate, Emtricitbine/tenofovir/efavirenz, Enoxaparin, ensartinib, Enzalutamide, Epoetin alfa, erlotinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, exemestane, everolimus, exenatide ER, Ezetimibe, Ezetimibe/simvastatin, Fenofibrate, Filgrastim, fingolimod, Fluticasone propionate, Fluticasone/salmeterol, fulvestrant, gazyva, gefitinib, Glatiramer, Goserelin acetate, Icotinib, hnatinib, Ibritumomab tiuxetan, ibrutinib, idelalisib, ifosfamide, Infliximab, imiquimod, ImmuCyst, Immuno BCG, iniparib, Insulin aspart, Insulin detemir, Insulin glargine, Insulin lispro, Interferon alfa, Interferon alfa-lb, Inter¬ feron alfa-2a, Interferon alfa-2b, Interferon beta, Interferon beta la, Interferon beta lb, Interferon gamma-la, lapatinib, Ipilimumab, Ipratropium bromide/ salbutamol, Ixazomib, Kanuma, Lanreotide acetate, lenalidomide, lenaliomide, lenvatinib mesylate, letrozole, Levothyroxine, Levothy¬ roxine, Lidocaine, Linezolid, Liraglutide, Lisdexamfetamine, LN-144, lorlatinib, Memantine, Methylphenidate, Metoprolol, Mekinist, mericitabine/ Rilpivirine/Tenofovir, Modafinil, Mometasone, Mycidac-C, Necitumumab, neratinib, Nilotinib, niraparib, Nivolumab, ofatumumab, obinutuzumab, olaparib, Olmesartan, Olmesartan/ hydrochiorothiazide, Omalizumab, Omega-3 269 Date Reçue/Date Received 2024-05-09 fatty acid ethyl esters, Oncorine, Oseltamivir, Osimertinib, Oxycodone, palbociclib, Palivizumab, panitumumab, panobinostat, pazopanib, pembrolizumab, PD-1 antibody, PD-L1 antibody, Pemetrexed, pertuzumab, Pneumococcal conjugate vaccine, pomalidomide, Pregabalin, ProscaVax, Propranolol, Quetiapine, Rabeprazole, radium 223 chloride, Raloxifene, Raltegravir, ramucirumab, Ranibizumab, regorafenib, ribociclib, Rituximab, Rivaroxaban, romidepsin, Rosuvastatin, ruxolitinib phosphate, Salbutamol, savolitinib, semaglutide, Sevelamer, Sildenafil, siltuximab, Sipuleucel-T, Sitagliptin, Sitagliptin/metformin, Solifenacin, solanezumab, Sonidegib, Sorafenib, Sunitinib, tacrolimus, tacrimus, Tadalafil, tamoxifen, Tafinlar, Talimogene laherparepvec, talazoparib, Telaprevir, talazoparib, Temozolomide, temsirolimus, Tenofovir/ emtricitabine, tenofovir disoproxil fumarate, Testosterone gel, Thalidomide, TICE BCG, Tiotropium bromide, Tisagenlecleucel, toremifene, trametinib, Trastuzumab, Trabectedin (ecteinascidin 743), trametinib, tremelimumab, Trifluridine/tipiracil, Tretinoin, Uro-BCG, Ustekinumab, Valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, ziv-aflibercept, and Zostavax, or a pharmaceutically acceptable salt thereof, or any combination thereof. 270 Date Reçue/Date Received 2024-05-09