NZ710814B2 - Biologically active molecules, conjugates thereof, and therapeutic uses - Google Patents

Abstract

The present disclosure relates to linker compounds that are useful in covalently linking biologically active molecules with Ligands. The disclosed compounds also relate to biologically active molecules, preferably a maytansinoid, and Ligand conjugates, wherein the biologically active molecule is linked to the Ligand through a linker. The disclosure further provides compositions comprising biologically active molecule -ligand conjugates, methods of modifying abnormal cell growth and methods of treatment using the conjugates or the compositions. ked to the Ligand through a linker. The disclosure further provides compositions comprising biologically active molecule -ligand conjugates, methods of modifying abnormal cell growth and methods of treatment using the conjugates or the compositions.

Description

Biologically Active Molecules, Conjugates Thereof, and Therapeutic Uses TECHNICAL FIELD The present disclosure provides Ligand-Biologically Active Molecule Conjugates wherein the Ligand is connected to the Biologically Active le through a linker compound. The present disclosure also provides conjugate compounds in pharmaceutical compositions for use in various therapeutic applications.

BACKGROUND OF THE INVENTION Proliferative diseases are characterized by uncontrolled growth and spread of al cells. If the spread is not lled, it can result in death. Abnormal proliferation, for example, cancer, is caused by both external s (e.g., tobacco, chemicals, radiation and infectious organisms) and internal factors ited mutations, immune system ions, the mutations that occur from metabolism). These causal factors may act together or in sequence to initiate or promote abnormal proliferation. Cancer is treated by surgery, radiation, herapy, hormones and immunotherapy. However, there is a need for more effective anti-proliferation drugs.

The ideal anti-proliferation therapy would enable targeted delivery of highly cytotoxic agents to tumor cells and would leave normal cells unaffected. Conventional chemotherapeutic treatment, with maytansine for example, is limited because of the toxic side-effects that arise from effects of the drug on non-cancerous cells. Various approaches to targeted drug delivery have been tried, including the use of conjugates of tumor targeted probes (such as antibodies or growth factors) with toxins such as pseudomonas or diphtheria toxins, which arrest the synthesis of proteins and cells. However, the side effects include reaction of the immune system due to man components of the conjugates. Further, the half-life of the drug conjugates were limited due to elimination from the circulation through renal filtration, and schematic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-targeted organs and s.

Another approach uses passive drug rs such as polymers, liposomes, and ric micelles to take age of the hyper-permeability of vascular endothelia of tumor tissue. ric drugs and macromolecules accumulate within solid tumors due to an ed permeability and retention mechanism. However, barriers of using such targeted deliveries include fast nce of foreign particles from the blood, and technological hindrances in obtaining highly rdized, pharmaceutically acceptable drug delivery systems with the necessary specificity and selectivity for binding tumor cells.

Thus, a need exists for targeted anti-proliferative nds.

SUMMARY OF THE INVENTION ] The present invention es a compound of Formula (I) wherein L is a Ligand capable of binding to a cell or cell population; a is an integer from 1 to 10; Z2 is represented by the following structural formula: –Z2A-Z2B-Z2C-Z2D–, wherein Z2A is or -C(=O)-, Z2b is C1-C10 -alkylene-, Z2C is -, and Z2d is absent; A is a natural or non-natural amino acid residue, or a peptide residue sing 2-20 amino acid residues; W is -NR4-; X is C6-C18 -arylene-; A1 is C1-C10 -alkylene-; A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, or -CH2CH2CH2CH2-; R1 is oxygen; R17 is oxygen; R4 is hydrogen; and R4a is an optionally substituted C1-C10 alkyl; wherein when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10. [0005b] The present invention also provides a compound of the following formula Z2 is represented by the following structural formula: 2B-Z2C-Z2D–, wherein Z2A is , , or -N=C=S; Z2b is C1-C10 -alkylene-, C6-C18 -arylene- or -O-C(=O)-; Z2C is -C(=O)-, -C(=S)-N(R4)-, or C1-C10 -alkylene-; and Z2d is absent or -C(=O)-; A is a natural or non-natural amino acid residue, or a e residue comprising 2-20 amino acid residues; W is -NR4-; X is C6-C18 -arylene-; A1 is C1-C10 -alkylene-; A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, or -CH2CH2CH2CH2-; R1 is oxygen or -NR4-; R17 is selected from the group consisting of oxygen and sulfur; R4 is hydrogen; and R4a is C1-C10 alkyl; wherein when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10. [0005c] The present invention also provides a compound of the following formula wherein A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH2CH2CH2-; and R4a is alkyl; n when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10. [0005d] The present invention also es a compound of the following formula wherein Ab is an antibody or an antigen binding fragment thereof; a is an integer from 1 to 10; Z2 is represented by the following structural formula: –Z2A-Z2B-Z2C-Z2D–, wherein Z2A is - C(=S)-N(R4)- or -C(=O)-; Z2b is C6-C18 -arylene- or C1-C10-alkylene-; Z2C is -N(R4)- -C(=O)-; and Z2d is absent; A is a l or non-natural amino acid residue, or a peptide residue comprising 2-20 amino acid residues; W is -NR4-; X is C6-C18 -arylene-; A1 is C1-C10 -alkylene-; A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, or -CH2CH2CH2CH2-; R1 is oxygen or -NR4-; R17 is oxygen; R4 is hydrogen; and R4a is C1-C10 alkyl; n when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10. [0005e] The present invention also provides a compound having a structure ed from the group consisting of , , , and [0005f] The present invention also provides an antibody-drug conjugate comprising an dy or antigen-binding fragment thereof conjugated to a payload of the following formula: [0005g] The t invention also provides a pharmaceutical composition comprising a therapeutically ive amount of a nd of the invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients. [0005h] The present invention also relates to use of a nd of the invention, in the manufacture of a medicament for reducing, retarding or stopping an abnormal cell growth. [0005i] The present invention also relates to use of a compound of the invention, in the manufacture of a medicament for killing a cell. [0005j] The present invention also relates to use of a compound of the invention, in the manufacture of a medicament for treatment of a medical disorder in an individual. [0005k] The present ion also relates to use of a compound of the invention, in the manufacture of a medicament for reducing tumor size, ng tumor size increase, reducing tumor proliferation, or preventing tumor proliferation in an dual in need thereof.

BRIEF DESCRIPTION The present sure relates to conjugate compounds represented by the following structural formula (I): wherein: L is absent or a ligand; further wherein: when L is a ligand, L is e of binding to a cell or cell population; a is an integer from 1 to 10; Z2 and Z1 are each independently absent or a spacer; D is a Biologically Active le; A is a natural or non-natural amino acid, or a peptide comprising 2-20 amino acids; W is absent, -O-, -S-, -CR5R6-, -NR4-; further wherein: R4, R5, and R6 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; X is absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, aryl, lkyl, and heterocyclyl are optionally tuted; and Y is absent, or a spacer.

Also described are linker-biologically active compounds represented by the following structural a (V): wherein: Z2 and Z1 are each independently absent or a spacer; D is a Biologically Active Molecule; A is a natural or non-natural amino acid, or a peptide comprising 2-20 amino acids; W is absent, -O-, -S-, -CR5R6-, -NR4-; further wherein: R4, R5, and R6 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; X is absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionally substituted; and Y is absent, or a spacer.

Also described are s ented by the following structural formula (VI).

In one ment, the linker compounds is represented by formula (VI): wherein: Z2 and Z1 are each independently absent or a spacer; A is a natural or non-natural amino acid, or a peptide sing 2-20 amino acids; W is absent, -O-, -S-, -CR5R6-, -NR4-; X is absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionally substituted; Y is absent, , or wherein A1, A3, R1 and R3 are each independently , an amino acid, a e having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)- (CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -S-C(=S)-, -C(=S)-NH-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -N(R4)-, -C(=O)- N(R4)-C(=O)-, -O-C(=O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; A4 and A5 are each independently –O-, -S-, -NR18-, -CR5R6-; R17 is selected from the group consisting of O, S, NR18, CR5R6; R18 is selected from the group ting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl, wherein alkyl, alkynyl, l, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl are optionally substituted; R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, aryl, or heterocyclyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; and x is 0, 1 or 2.

In one embodiment, the described are compounds of formula (VI), wherein Z2 is ented by the ing structural formula: -Z2A-Z2B-Z2C-Z2D-, wherein: Z2A, Z2B, Z2C and Z2D are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an l, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -O- C(=O)-N(R4), -O-C(=S)-N(R4)-, -C(=S)-N(R4)-, -N=C=S, , or wherein alkyl, alkynyl, l, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally tuted and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl.

The present disclosure also relates to pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

Also described is a method of reducing, retarding or stopping an abnormal cell growth comprising contacting the abnormal cell with a compound of formula (I), in an amount sufficient to retard, reduce or stop the abnormal cell growth, and wherein the abnormal cell growth is retarded, reduced or stopped.

Also bed is a method of killing a cell, comprising contacting the cell with a compound of a (I), in an amount sufficient to kill the cell, and wherein the cell is killed.

Also bed is a method of treatment of a medical disorder in an individual suffering from the medical disorder, sing administering to the individual an effective amount of a composition comprising a compound of formula (I).

Also described is a method of reducing tumor size, stopping tumor size increase, ng tumor proliferation, or preventing tumor eration in an individual in need thereof comprising administering to the individual an ive amount of a composition to reduce tumor size, stop tumor size se, reduce tumor proliferation, or prevent tumor proliferation, wherein the composition comprises a compound of formula (I).

The present disclosure also relates to precursor Biologically Active Moleculelinker compounds as represented by formula (V). Compounds of formula (V) provide building blocks for conjugate compounds of formula (I). In on, compounds of formula (V) may be provided as compositions, ceutical compositions and pharmaceutically acceptable salts thereof.

The present disclosure further includes the use of any of the compositions sing compounds of formula (I) and/or pharmaceutical formulations in the manufacture of a medicament for the treatment, prevention and/or amelioration of a medical disorder.

The present disclosure further es the use of any of the compositions sing compounds of formula (I) and/or ceutical formulations in the manufacture of a medicament for the treatment, prevention and/or amelioration of a tumor.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1-8 depict the results of cell viability assays in which various cancer cell lines were grown in vitro and d with serial dilutions of antibodies, free drug, or antibody-drug conjugates as shown. Percent viability was determined in accordance with the s set forth in Example 14.

Figure 1A shows the cell viability results of C4-2 cells (prostate cancer cell line) treated with compound 2, isotype control antibody conjugated to compound 3 ("Isotype l-3"), anti-PSMA dy conjugated to compound 3 ("PSMA-3"), and unconjugated anti-PSMA antibody ("PSMA").

Figure 1B shows the cell viability results of C4-2 cells (prostate cancer cell line) treated with compound 6, isotype control antibody ated to compound 7 ("Isotype Control-7"), anti-PSMA antibody conjugated to compound 7 ("PSMA-7"), and unconjugated anti-PSMA antibody ("PSMA").

Figure 1C shows the cell viability results of C4-2 cells ate cancer cell line) treated with compound 25, isotype control antibody conjugated to compound 21 ("Isotype Control-21"), anti-PSMA dy conjugated to compound 21 ("PSMA-21"), and unconjugated anti-PSMA antibody ("PSMA").

Figure 2 shows the cell viability results of PC3/hSTEAP1 cells (prostate cancer cell line expressing ous hSTEAP1) treated with compound 6, isotype control antibody conjugated to nd 7 ("Isotype Control-7"), anti-STEAP1 antibody conjugated to compound 7 ("STEAP1-7"), and unconjugated anti-STEAP1 antibody ("STEAP1").

Figure 3 shows the cell ity results of T47D cells (breast cancer cell line) treated with compound 6, isotype control antibody conjugated to compound 7 ("Isotype Control-7"), anti-PRLR antibody conjugated to compound 7 ("PRLR-7"), and ugated anti-PRLR dy ("PRLR").

Figure 4 shows the cell viability results of HEK293/hEGFRvIII cells (HEK293 cells expressing exogenous hEGFRvIII) treated with compound 6, isotype l antibody conjugated to compound 7 ("Isotype Control-7"), anti-EGFRvIII antibody conjugated to compound 7 ("EGFRvIII-7"), and unconjugated anti-EGFRvIII antibody ("EGFRvIII").

Figure 5 shows the cell viability results of MMT/hEGFRvIII cells (MMT cells expressing exogenous hEGFRvIII) treated with compound 6, isotype control antibody conjugated to compound 7 ("Isotype Control-7"), anti-EGFRvIII antibody conjugated to compound 7 ("EGFRvIII-7"), and unconjugated anti-EGFRvIII antibody ("EGFRvIII").

Figure 6 shows the cell viability results of U251/hEGFRvIII cells (U251 cells sing exogenous hEGFRvIII) treated with compound 6, isotype control dy conjugated to nd 7 ("Isotype l-7"), anti-EGFRvIII antibody conjugated to compound 7 ("EGFRvIII-7"), and unconjugated GFRvIII antibody ("EGFRvIII").

Figure 7, panels A and B show the cell viability results of HEK293 and U87MG cells, tively, treated with nds 6, 27, 29, and 31 (all unconjugated).

Figure 8, panels A-E show the cell viability results of HEK293, U251, C4-2, PC3 and MMT cells, respectively, treated with compounds 6, 9, 33 and 35 (all unconjugated).

DETAILED DESCRIPTION The references to certain embodiments made in the following description are considered rative only of the principles of the disclosure. Further, since numerous modifications and changes will readily be apparent to those skilled in the art, it is not intended to limit the disclosure to the exact construction and process shown as described herein. Accordingly, all suitable modifications and lents may be resorted to as falling within the scope of the disclosure and as defined by the claims that follow.

The words "comprise", "comprising", "include" and "including" when used in this specification and in the following claims are intended to specify the presence of the stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more additional features, integers, components, or steps thereof.

General terms used in any of the embodiments herein can be d as follows; however, the meaning stated should not be interpreted as limiting the scope of the term per The term "conjugate" as used herein refers to compound having a , linker and Biologically Active Molecule. Illustrative examples e compounds of formula (I), (III) and (IV).

The term "spacer" as used herein refers to al building blocks of the linker used to lly separate the Ligand from the Biologically Active Molecule and to allow for catabolism of the linker inside of cells. A spacer can be represented by Z1 and Z2.

The term "macrolide" as used herein refers to any Biologically Active Molecule having a macrolide ring.

The term "alkyl" as used herein refers to a hydrocarbon group having a general formula CnH2n +1. Examples of alkyl include: methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, and the like. Typical alkyl have from one to ten carbon atoms, one to nine carbon atoms, one to eight carbon atoms, one to seven carbon atoms, one to six carbon atoms, one to five carbon atoms, one to four carbon atoms, one to three carbon atoms, one to two carbon atoms or one carbon atom.

The term "aryl" as used herein refers to a monovalent or polycyclic aromatic hydrocarbon typically having 6 to 18 carbon atoms. Example aryl include phenyl (like benzene), substituted benzenes, alene, anthracene, indenyl, tetrahydronapthyl and the like.

The term "alkenyl" as used herein refers to an aliphatic linear or branched monovalent hydrocarbon radical of two or more carbon atoms with at least one site of unsaturation. Alkenyl have a general formula of R2C=CR2. Examples of alkenyl e: nyl, vinyl, allyl, and the like.

The term "alkynyl" as used herein refers to a univalent aliphatic hydrocarbon radical containing a triple bond. Typical alkynyl are from two to twenty carbon atoms (and include at least one triple bond). Examples alkynyl include ethynyl, propynyl, 1-butynyl, 2- butynyl, 1-pentynyl, hexynyl and the like.

The term "cycloalkyl" as used herein, refers to a monovalent saturated carbocyclic ring l. Typical cycloalkyl are 3 to 7 member monocyclic ring radicals. One example of a cycloalkyl is cyclohexyl.

The term "heteroaryl" as used herein, refers to a monovalent aromatic radical of or 6 membered rings. Heteroaryl es fused ring s (at least one must be aromatic) that include up to 5 to 18 atoms, containing one or more heteroatoms independently selected from nitrogen, sulfur or oxygen. Illustrative heteroaryl are nyl, triazolyl, furyl, pyrazinyl, thienyl, isoxazolyl, indazolyl, furazanyl, benzothiazolyl, quinazolinyl, and furopyridinyl.

The term "heterocyclyl" as used herein refers to saturated or lly saturated carbocyclic radical typically of 3 to 18 carbon atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorous, and sulfur. A cycyl may be a monocycle or a bicycle, for example. Example heterocyclyl are pyrolidinyl, ydrofuranyl, dihydropyranyl, thioxanyl, 2H-pyranyl, dioxanyl, dithianyl, piperidino, and the like.

The phrase "pharmaceutically able salt" as used herein refers to both organic and inorganic salts of the conjugate compounds described herein, e.g., compounds of formula (I), (III), (IV) and (V). The salts are pharmaceutically able and include: sulfate, citrate, nitrate, phosphate, ascorbate, bromide, gluconate, benzoate, oxalate, henate, and the like. Note that pharmaceutically acceptable salts herein may include more than one charged atom in its structure as well as one or more counter ion. Preparation of ate compounds herein as pharmaceutically acceptable salts is well known to one of skill in the art.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used , is not intended to include mAbs in which CDR sequences derived from the ne of another ian species have been grafted onto human FR ces.

The term "therapeutically effective amount" as used herein refers to an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for e, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

Ligands and Binding Partners The effectiveness of the conjugate compound embodiments described herein depend on the ivity of the Ligand to bind its ligand binding partner.

In one embodiment, Ligands are any molecule capable of binding with some specificity to a given binding partner within a mammal where the interaction can result in a therapeutic use. In some s the Ligand is capable of binding to a cell or cell population.

Ligands for use herein e antibodies, lymphokines, hormones, growth factors, viral ors, interleukins, or any other cell binding or peptide binding molecule or nce.

In one embodiment the Ligand is an antibody. As defined , antibody refers to monoclonal antibodies, polyclonal antibodies, dy fragments (Fab, Fab’, and F(ab)2, minibodies, diabodies, tribodies, and the like), and bispecific antibodies. Antibodies herein can be humanized using methods described in US Patent No. 6,596,541 and US Pulication No. 2012/0096572, each incorporated by reference in their entirety.

Where the Ligand is an dy, it binds to an antigen binding partner that is a polypeptide and may be a transmembrane molecule (e.g., receptor) or a growth factor.

Exemplary antigens include, but are not limited to, molecules such as renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing ; parathyroid hormone; d stimulating hormone; oteins; alpha1- antitrypsin; insulin A-chain; insulin B-chain; proinsulin; le stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting s such as factor vmc, factor IX, tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); in; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage matory protein (MlP-I- alpha); a serum albumin, such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin n; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as betalactamase; DNase; 19E; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including , TGF-β2, TGF- β3, TGF-β4, or TGF- β5; insulin-like growth factor-l and -II (IGF-l and IGF-II); 3)-IGF-l (brain IGF-l), insulin-like growth factor binding proteins, EpCAM, GD3, FLT3, PSMA, PSCA, MUCI, MUCI6, STEAP, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, lRTAI, lRTA2, lRTA3, lRTA4, lRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CDII, CDI4, CDI9, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CDI52, or an antibody which binds to one or more tumor-associated antigens or cell-surface receptors disclosed in US Publication No. 171040 or US Publication No. 2008/0305044 and incorporated in their entirety by reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon, such as interferon-alpha, -beta, and -gamma; colony stimulating factors , e.g., M-CSF, GM-CSF, and G-CSF; eukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the HIV envelope; transport proteins; homing receptors; addressins; regulatory ns; ins, such as CDlla, CDllb, CDllc, CDI8, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase- 8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, II, endoglin, Epcam, EphA2, ErbB2/Her2, ErbB3/Her3, ErbB4/Her4, ETV6-AML, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/EBNA1, HLA/k-ras, GE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, , Muc1, Muc16 (CA-125), MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, , PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, and fragments of any of the above-listed polypeptides. s may also include ankyrin repeat proteins, interferons, lymphokines such as IL-2 or IL-3, hormones like insulin and glucocorticoids, growth factors such as EGF, transferrin, ectin type III, etc.

Embodiments herein are target specific for therapeutic use. In one embodiment, Ligands are prepared to interact with and bind to antigens defined as tumor antigens, which include antigens specific for a type of tumor or antigens that are shared, pressed or modified on a ular type of tumor. Examples include: alpha-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with c myeloid leukemia, B-RAF, CLPP or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma as well as the following shared tumor-specific antigens, for example: BAGE-1, GAGE, GnTV, KK-LC-1, MAGE-A2, , TRP2-INT2.

Biologically Active Molecules Biologically Active Molecules herein include any molecules that have a therapeutic use in a mammal. In typical embodiments the molecule is beneficially delivered to a target within the mammal and in particular is beneficially delivered to and then within a cell (e.g., endocytosis) as compared to molecules released into the vascular or lymphatic systems.

In one , Biologically Active Molecules are compounds that result in the inhibition, retardation, reduction, and/or prevention of cell growth. Biologically Active Molecules can also result in cell death via necrosis or sis. Illustrative Biologically Active Molecules for use in conjugate compounds described herein include: maytansinoids (e.g., DM1, DM4, etc.), auristatins (e.g., MMAE, MMAD, MMAF, etc.), duocarmycin (e.g., MGBA), dolastatin, toxoids, and other chemotherapeutically effective drugs.

Other specific examples of ically Active les that can be used in the context of the present ion include, e.g., 1-dehydrotestosterone, 2- inodoxorubicin, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin D, anthracycline, anthramycin (AMC), bleomycin, busulfan, calicheamicins, carmustine cisplatin, colchicin, cyanomorpholino-doxorubicin, cyclophosphamide, cytarabine, alasin B, dactinomycin, daunorubicin, decarbazine, dibromomannitol, dihydroxy cin dione, doxorubicin, emetine, epirubicin, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, lomustine (CCNU), mechlorethamine, melphalan, methotrexate, mithramycin, mitomycin, mitoxantrone, morpholino-doxorubicin, procaine, propranolol, puromycin, pyrrolobenzodiazapines, sibiromycin, ozotocin, taxol, tenoposide, tetracaine, a chlorambucil, trichothecenes, tubulysin, vincristine, and stereoisomers, isosteres, analogs or derivatives of any of the foregoing.

In one ment the Biologically Active Molecule is a maytansinoid or a maytansinoid analog. Exemplary sinoids for use herein are described in Widdison et al., J. Med. Chem., 2006, 49, 4392-4408, incorporated by reference herein for all purposes.

Linker Materials The present disclosure includes a linker compound that is chemically capable of covalently linking two spaced chemical moieties. The linker spaces and links two moieties, for example, the linker can link a Ligand and a Biologically Active le. In one aspect, the linker is self immolative wherein the linker connects two or more ent chemical moieties and releases at least one of the said chemical moieties in the presence of an enzyme.

In another , the linker may be attached to other chemical moieties, including but not limited to, ical agents, biomolecules, targeting agents, detectable labels, diagnostic , and the like. In one embodiment, the linker attaches a Biologically Active le and a Ligand. In another embodiment, the linker attaches a biologically active macrolide and a Ligand. In yet r embodiment, the linker attaches a biologically active macrolide and an antibody or fragments thereof.

In one aspect, the linkers are useful to covalently link ligands with therapeutic agents and markers. In another aspect, the linkers improve chemical and/or systemic stability of the ed moieties. In another aspect, the linkers reduce in vivo toxicity of the attached es. In another aspect, the linkers improve pharmacokinetics, pharmacodynamics, and/or bioavailability of the attached moieties. In one aspect, the linkers cleave and e a ically Active Molecule at a site in or near a target cell or a cell population in a pharmacologically ive form. In one aspect, the cleavage is performed by enzymes. In one aspect, the cleavable groups on the linkers for the enzymatic cleavage include, but not limited to, peptide bonds, ester linkages, and disulfide linkages. In another aspect, the linker is cleaved through pH s.

In one embodiment, the linker nds is represented by formula (VI): wherein: Z2 and Z1 are each independently absent or a spacer; A is a natural or non-natural amino acid, or a peptide comprising 2-20 amino acids; W is absent, -O-, -S-, -CR5R6-, -NR4-; X is absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionally substituted; Y is absent, , or wherein A1, A3, R1 and R3 are each independently absent, an amino acid, a e having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)- (CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)- N(R4)-C(=O)-, -O-C(=O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; A4 and A5 are each independently –O-, -S-, -NR18-, -CR5R6-; R17 is selected from the group consisting of O, S, NR18, CR5R6; R18 is selected from the group consisting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, cyclyl, and acyl are optionally substituted; R4, R5, R6 and R8 are each ndently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; and x is 0, 1 or 2.

In one embodiment, described are nds of formula (VI), wherein Z2 is represented by the following structural formula: -Z2A-Z2B-Z2C-Z2D-, wherein: Z2A, Z2B, Z2C and Z2D are each independently , an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -C(=O)-, -N(R4)-, -C(=O)-N(R4)-C(=O)-, -O- C(=O)-N(R4), -O-C(=S)-N(R4)-, -N(R4)-, -N=C=S, -N=C=O, or wherein alkyl, l, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl.

In one embodiment, described are compounds of formula (VI), wherein Z1 is represented by the ing ural formula: -Z1A-Z1B-Z1C-Z1D-, wherein: Z1A, Z1B, Z1C and Z1D are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, C(=O)-N(R4)-C(=O)-, -O- C(=O)-N(R4), -O-C(=S)-N(R4)-, -C(=S)-N(R4)-, -N=C=S, -N=C=O, or wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, aryl, and heterocyclyl are optionally substituted and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl.

In one aspect, the sure provides compounds of a (VI), wherein A is an amino acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, ne, cysteine, and citrulline.

In another one aspect, the disclosure provides compounds of formula (VI), wherein A is a peptide selected from the group consisting of valine-citrulline, linevaline , lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- gine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine, asparagine-alanine.

In one aspect, the disclosure provides compounds of formula (VI), wherein X is an aryl selected from the group consisting of , and wherein R9, R10, R11, and R12 are each independently H, an alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, NR13R14, nitro, cyano, -OH, -O-C(=O)-R15, -C(=O)-R15, - O-R15, -C(=O)-NR13 R14; and further wherein, alkyl, cycloalkyl, aryl, heteroaryl, and cyclyl are optionally substituted; R13 and R14 are each independently H or an optionally substituted alkyl; and R15 is an optionally tuted alkyl.

According to certain embodiments, the linkers, the biologically active molecules, and other compounds of the present sure can be connected to an antibody or antigen-binding le through an ment at a particular amino acid within the antibody or antigen-binding le. Exemplary amino acid attachments that can be used in the context of the sure include, e.g., lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; ; US 5,714,586; US 2013/0101546; and US 585592), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; ; ; ; ; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., ; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, -51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non-natural amino acids (see, e.g., , and ), and acidic amino acids (see, e.g., ). Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., , , , and Shaunak et al., Nat. Chem. Biol., 2006, 2:312- 313).

According to certain other embodiments, the linkers, the biologically active molecules such as drugs can be connected to an antibody or antigen-binding molecule through an attachment at a particular amino acid within the antibody or antigen-binding le forming an antibody-drug conjugate (ADC).

Compounds In one embodiment, described are Biologically Active les and Ligand conjugates represented by the following structural formula (I): wherein: L is absent or a Ligand; further wherein: when L is a , L is e of binding to a cell or cell population; a is an integer from 1 to 10; Z2 and Z1 are each independently absent or a spacer; D is a Biologically Active Molecule; A is a natural or tural amino acid, or a peptide sing 2-20 amino acids; W is absent, -O-, -S-, -CR5R6-, -NR4-; X is absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl, lkyl, and heterocyclyl are optionally substituted; Y is absent, , or wherein A1, A3, R1 and R3 are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)- (CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)- N(R4)-C(=O)-, -O-C(=O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally tuted; A4 and A5 are each ndently –O-, -S-, -NR18-, -CR5R6-; R17 is selected from the group consisting of O, S, NR18, CR5R6; R18 is selected from the group ting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl, wherein alkyl, l, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl are optionally tuted; R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; and x is 0, 1 or 2.

In another embodiment, described are compounds where the Biologically Active le is a cytotoxic biologically active macrolide.

In yet another embodiment, described are maytansinoids as represented by formula (II) as biologically active macrolides: (II), wherein A6, A7, A8, A9 are each independently absent, an amino acid, N-alkyl amino acid, a peptide having 2-20 amino acids, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a aryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, O)-, -C(=O)-O-, -O-C(=O)- O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2- )p3-, -((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-NH-, -C(=S)-S-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, - SO2-, -NR4-, -N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, - C(=O)-N(R4)-C(=O)-, -O-C(=O)-NR4, further wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, l, alkynyl, aryl, heteroaryl, or cyclyl.

In another embodiment, the sinoid is represented by the following structural formula (II)(a): In one aspect, the disclosure provides a compound of formula (I), wherein the Ligand (L) is capable of g to a specifically targeted cell population.

In another aspect, the disclosure provides a compound of formula (I), wherein the Ligand (L) is selected from the group consisting of proteins, antibodies, fragments of dies, nucleic acids, n binding scaffolds, and carbohydrates.

In one embodiment, the disclosure provides a nd of formula (I), wherein the Ligand (L) is an antibody or a fragment thereof.

In one embodiment, the disclosure provides a compound of formula (I), wherein Ligand (L) is an antibody or nt thereof that specifically binds a tumor associated In one embodiment, the disclosure provides a compound of formula (I), wherein the antibody or a fragment thereof comprises a sulfur group that is covalently attached with In one embodiment, described are compounds of formula (I), wherein Z2 is represented by the following structural formula: -Z2A-Z2B-Z2C-Z2D-, wherein: Z2A, Z2B, Z2C and Z2D are each ndently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an l, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -N(R4)-C(=O)-, -O- C(=O)-N(R4), -O-C(=S)-N(R4)-, -C(=S)-N(R4)-, -N=C=S, -N=C=O, or wherein alkyl, alkynyl, l, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted and R4, R5, R6 and R8 are each independently H, or a tuted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl.

In one embodiment, the disclosure es compounds of formula (I), wherein the antibody or a fragment thereof comprises a sulfur group that is ntly attached with Z2A.

In one embodiment, described are compounds of formula (I), wherein Z1 is represented by the following ural formula: -Z1A-Z1B-Z1C-Z1D-, wherein: Z1A, Z1B, Z1C and Z1D are each ndently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, O)-, -O-, -O-C(=O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, C(=O)-N(R4)-C(=O)-, -O- C(=O)-N(R4), S)-N(R4)-, -C(=S)-N(R4)-, -N=C=S, -N=C=O, or wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, aryl, and heterocyclyl are optionally substituted and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl.

In one embodiment, the sure provides nds of formula (I), wherein the Biologically Active Molecule (D) is covalently attached with Z1.

In one , the sure provides compounds of formula (I), n A is an amino acid selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, e, histidine, cine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, cysteine, and citrulline.

In another aspect, the disclosure provides compounds of formula (I), wherein A is a peptide selected from the group consisting of valine-citrulline, citrulline-valine, lysinephenylalanine , phenylalanine-lysine, valine-asparagine, asparagine-valine, threonineasparagine , serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparaginephenylalanine , e-asparagine, asparagine-leucine, isoleucine-asparagine, asparagineisoleucine , glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagineglutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine, asparaginealanine.

In one aspect, the disclosure provides compounds of formula (I), wherein X is an aryl selected from the group ting of , and wherein R9, R10, R11, and R12 are each independently H, an alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, NR13R14, nitro, cyano, -OH, O)-R15, -C(=O)-R15, - C(=O)-O-R15, -C(=O)-NR13 R14; and further wherein, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; R13 and R14 are each independently H or an optionally substituted alkyl; and R15 is an optionally substituted alkyl.

In another embodiment, described are nds of formula (III): wherein: Ab is an antibody or a fragment thereof; AA1-AA2 is a peptide selected from the group consisting of valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, -asparagine, asparaginevaline , threonine-asparagine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- gine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine, asparagine-alanine; a is an integer from 1 to 10; q is 0 or an integer from 1 to 5; A3, R1 and R3 are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, O)-O-, -C(=O)-(CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, )p2-O-)p3-, -, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -OC (=O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; R17 is selected from the group ting of O, S, NR18, CR5R6; R4, R5, R6 and R8 are each independently H, or a substituted or tituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl; R9, R10, R11, and R12 are each independently H, halogen, 4, nitro, cyano, -OH, - O-C(=O)-R15, -R15, -C(=O)-O-R15, -C(=O)-NR13 R14, substituted or unsubstituted: alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl; R13 and R14 are each independently H or an optionally substituted alkyl; and R15 is an optionally substituted alkyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; x is 0, 1 or 2; and DM is represented by the following structure (e.g., compound of formula (II)(a)): (DM).

In one embodiment, the disclosure provides the compounds of formula (III) q is 4; R1 and R3 are each independently -O-, -S-, NR4, -CR5R6-; R17 is selected from the group ting of O, S, NR18, CR5R6; R18 is selected from the group consisting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, cyclyl, and acyl, wherein alkyl, alkynyl, alkenyl, lkyl, aryl, heteroaryl, heterocyclyl, and acyl are optionally substituted; R4, R5, R6, R9, R10, R11, R12 are each independently H or alkyl; and A3 is an alkyl.

In one embodiment, the disclosure provides the compounds of formula (III) represented by the following structures (III)(a) and (III)(b): (III)(a) and (III)(b) wherein Ab is an antibody or a fragment thereof.

In one embodiment, bed are compounds of formula (IV): wherein: Ab is an antibody or a fragment thereof; 2 is a peptide selected from the group consisting of valine-citrulline, citrulline-valine, lysine-phenylalanine, alanine-lysine, valine-asparagine, asparaginevaline , threonine-asparagine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine, asparagine-alanine; a is an integer from 1 to 10; q is 0 or an integer from 1 to 5; R1 is absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -OC (=O)-, -C(=O)-O-, -O-C(=O)-O-, -(CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, - (CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -SC (=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, - N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -O-C(=O)-NR4-, n alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; R4, is H, or a substituted or tituted: alkyl, alkenyl, l, aryl, heteroaryl, and heterocyclyl; R9, R10, R11, and R12 are each ndently H, halogen, NR13R14, nitro, cyano, -OH, - O-C(=O)-R15, -C(=O)-R15, -C(=O)-O-R15, -C(=O)-NR13 R14, substituted or unsubstituted: alkyl, lkyl, aryl, heteroaryl, and heterocyclyl; and DM is represented by the following structure: (DM).

In one embodiment, the disclosure provides the nds of formula (IV) wherein: q is 4; and R1 is selected from the group consisting of –O-, -S-, NR4, and -CR5R6-; and further wherein R4, R5, and R6 are each independently H or alkyl.

In one ment, the disclosure provides the compounds of formula (IV) ented by the following structure (IV)(a): (IV)(a), wherein Ab is an antibody or a fragment thereof.

In one embodiment, described is a compound of Formula (V) wherein: Z2 and Z1 are each independently absent or a spacer; D is a Biologically Active Molecule; A is a natural or non-natural amino acid, or a peptide comprising 2-20 amino acids; W is , -O-, -S-, -CR5R6-, or -NR4-; X is absent, or a substituted or unsubstituted: aryl, heteroaryl, cycloalkyl, heterocyclyl; Y is absent, , or wherein A1, A3, R1 and R3 are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an l, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -O-, -O-C(=O)-O-, -C(=O)- (CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -N(R4)-, -C(=O)- N(R4)-C(=O)-, -O-C(=O)-NR4-, wherein alkyl, l, alkenyl, cycloalkyl, aryl, aryl, and heterocyclyl are optionally substituted; A4 and A5 are each independently -O-, -S-, -NR18-, -CR5R6-; R17 is selected from the group consisting of O, S, NR18, CR5R6; R18 is ed from the group consisting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl are optionally substituted; R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; and x is 0, 1 or 2.

In one embodiment, described is a nd of formula (V), wherein: Z2 is ented by Formula (VII): -Z2A-Z2B-Z2C-Z2D- (VII), further wherein: Z2A, Z2B, Z2C and Z2D are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -O- C(=O)-N(R4), -O-C(=S)-N(R4)-, -N(R4)-, -N=C=S, -N=C=O, or wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl; Z1 is represented by Formula (VIII): -Z1A-Z1B-Z1C-Z1D- (VIII), wherein: Z1A, Z1B, Z1C and Z1D are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, O)-, -C(=O)-O-, O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -O- C(=O)-NR4-, -N=C=S, -N=C=O, or , wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, aryl, and heterocyclyl are ally substituted and R4, R5, R6, R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, l, aryl, heteroaryl, cyclyl; A is a peptide selected from the group consisting of valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonineasparagine , serine-asparagine, gine-serine, phenylalanine-asparagine, asparaginephenylalanine , leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagineisoleucine , glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, gineglutamic acid, line-asparagine, asparagine-citrulline, alanine-asparagine, asparaginealanine ; and X is an aryl selected from the group consisting of , and wherein R9, R10, R11, and R12 are each independently H, an alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, NR13R14, nitro, cyano, -OH, -O-C(=O)-R15, -C(=O)-R15, - C(=O)-O-R15, -C(=O)-NR13 R14, further wherein, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally tuted; R13 and R14 are each independently H or an ally substituted alkyl; and R15 is an optionally tuted alkyl.

The Biologically Active Molecules (D) can optionally be a tuted maytansinoid of Formula II: (II), wherein: A6, A7, A8, A9 are each independently , an amino acid, N-alkyl amino acid, a peptide having 2-20 amino acids, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a cyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, - C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, C(=O)- N(R4)-C(=O)-, O-C(=O)-NR4, further wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted, and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl.

In another embodiment, described are compounds of a (V), wherein the biologically active molecule is a optionally substituted maytansinoid represented by the following structural formula: (II), wherein: A6, A7, A8, A9 are each independently absent, an amino acid, N-alkyl amino acid, a peptide having 2-20 amino acids, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, - C(=O)-(CHx)p1, -O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, C(=O)- N(R4)-C(=O)-, O-C(=O)-NR4, further wherein alkyl, alkynyl, alkenyl, lkyl, aryl, heteroaryl, and heterocyclyl are optionally tuted, and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclyl.

In yet another embodiment, the disclosure provides compounds of formula (V), wherein the biologically active molecule is a maytansinoid represented by the following structural formula: (DM).

In one embodiment, described are compounds of formula (V) ented by the ing structures: (V)(a), (V)(b), (V)(c), (V)(d), and : (V)(a); (V)(b); (V)(c); (V)(d); and (V)(e).

In one ment, described are compounds of formula (IX): wherein: D is a Biologically Active Molecule; Y1 is wherein R3a and A3a are each independently absent, an amino acid, a peptide having ino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -OC (=O)-NR4-, wherein alkyl, alkynyl, alkenyl, lkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; and Z1 is ented by following structural formula: -Z1A-Z1B-Z1C-Z1D-, wherein: Z1A, Z1B, Z1C and Z1D are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a lkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1, -O-(CHx)p1, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, - C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, C(=O)-N(R4)-C(=O)-, -O- C(=O)-N(R4), -O-C(=S)-N(R4)-, -C(=S)-N(R4)-, -N=C=S, -N=C=O, or wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted and R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, l, aryl, heteroaryl, or heterocyclyl.

In another embodiment, described is a compound of formula (IX) wherein the Biologically Active Molecule is a cytotoxic biologically active ide. In yet another embodiment, described is a compound of formula (IX) wherein the biologically active macrolide is a sinoid. In a further embodiment, described is a compound of formula (IX) wherein the maytansinoid is represented by formula (II). In another embodiment, described is a compound of a (IX) wherein the maytansinoid is represented by formula (II)(a).

In an , the disclosure provides a compound of formula (IX) wherein IC50 of the compound is greater than about 10 nM.

In an aspect, the disclosure provides a compound of formula (IX) wherein the nd is about 10 fold less cytotoxic than the ponding nd of formula (I).

In another one embodiment, described are compounds of formula (X): wherein: Ab is an antibody or a fragment thereof; a is an integer from 1 to 10; Z2 and Z1 are each ndently absent or a spacer; A is a natural or non-natural amino acid, or a peptide sing 2-20 amino acids; W is , -O-, -S-, -CR5R6-, -NR4-; X is absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are ally substituted; Y is absent, , or wherein A1, A3, R1 and R3 are each independently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a aryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -O-C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)- (CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, - ((CH2)p2-O-)p3-, -C(=S)-, -S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, - NR4-, -N(R4)-C(=O)-N(R8)-, -C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)- N(R4)-C(=O)-, -O-C(=O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionally substituted; A4 and A5 are each ndently –O-, -S-, -NR18-, -CR5R6-; R17 is selected from the group consisting of O, S, NR18, CR5R6; R18 is selected from the group consisting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, cyclyl, and acyl, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl are optionally substituted; R4, R5, R6 and R8 are each independently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; x is 0, 1 or 2; and DM is represented by the ing structure: In one embodiment, bed is a compound of a (X) represented by the following structure (X)(a): wherein a is an integer from 1 to 10.

In another embodiment, bed is a compound of formula (XI): wherein: Ab is an antibody or a fragment f; AA1-AA2 is a peptide selected from the group consisting of valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparaginevaline , threonine-asparagine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine, asparagine-alanine; a is an integer from 1 to 10; q is 0 or an integer from 1 to 5; A3, R1 and R3 are each ndently absent, an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, an l, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -, O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-(CHx)p1-, -C(=O)-O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, -C(=S)-NH-, -S-C(=S)-, -S-C(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)- C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)-N(R4)-C(=O)-, -OC (=O)-NR4-, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and cyclyl are optionally substituted; R17 is selected from the group consisting of O, S, NR18, CR5R6; R4, R5, R6 and R8 are each ndently H, or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl; R9, R10, R11, and R12 are each independently H, halogen, NR13R14, nitro, cyano, -OH, - O-C(=O)-R15, -C(=O)-R15, -O-R15, -C(=O)-NR13 R14, substituted or unsubstituted: alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl; R13 and R14 are each independently H or an optionally substituted alkyl; and R15 is an optionally substituted alkyl; p1, p2 and p3 are each independently 0, or an integer from 1 to 100; x is 0, 1 or 2; and DM is represented by the following structure: In one embodiment, described is a nd of formula (XI) represented by the following structure (XI)(a): wherein a is an integer from 1 to 10.

In one embodiment, described are compounds of formula (I), (III), (IV) (V), and (X), wherein A is a e cleavable by a protease.

In one ment, described is a compound of formula (XI) wherein the peptide is cleavable by a protease.

In one embodiment, described are nds of formula (I), (III), (IV) (V), and (X) n A is a peptide cleavable by a protease sed in tumor tissue.

In one embodiment, described is a compound of formula (XI) wherein the peptide is cleavable by a protease expressed in tumor tissue.

In an embodiment, bed are compounds of formula (I), (III), (IV) (V), (X) wherein A is a peptide cleavable by a protease further wherein the protease is a cathepsin or a plasmin.

In an embodiment, described is the compound of formula (XI) wherein the peptide is cleavable by a protease further wherein the protease is a cathepsin or a plasmin.

Compositions ] Embodiments herein include compositions comprising conjugate nds of formula (I), (III), (IV), (V), (X), or (XI) as well as mixtures thereof. In some embodiment the compound is further represented by a compound of formula (III)(a), (III)(b), (IV)(a), (V)(a), (V)(b), (V)(c), (V)(d) (V)(e), (X)(a), or (XI)(a).

Embodiments herein include compositions comprising compounds of formula (I), (III), (IV), (V), (IX), (X), or (XI) as well as mixtures thereof.

Compositions may be pharmaceutical compositions that further e one or more pharmaceutically acceptable carriers, ts, and/or excipients. In some embodiments the pharmaceutical composition is the pharmaceutically acceptable salt of compounds of formula (I), (III), (IV), (V), (IX), (X), or (XI) or mixtures thereof. In some other embodiments the pharmaceutical composition is the pharmaceutically acceptable salt of compounds of formula (I), (III), (IV), (V), (IX), (X), or (XI) or mixtures thereof.

Suitable pharmaceutical acceptable carriers, diluents and excipients are well known in the art and can be ined by one of ordinary skill in the art as the clinical situation warrants. Examples of suitable rs, diluents and excipients include: s for maintenance of proper composition pH (e.g., citrate buffers, succinate buffers, acetate buffers, ate buffers, lactate buffers, oxalate buffers and the like), carrier proteins (e.g., human serum albumin), saline, polyols (e.g., trehalose, e, xylitol, sorbitol, and the like), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like), antimicrobials, and antioxidants.

If so desired, the pharmaceutical compositions herein may include a second or more therapeutic agent (e.g., an adjuvant to the conjugate nds of formula (I), (III), (IV), (X), and/or (XI), anti-tumor , antibiotics, anti-inflammatories, and the like). The second therapeutic agent can be included in the same composition as the compounds of formula (I), (III), (IV), (V), (IX), (X), and/or (XI), or can be administered separately from the compounds of formula (I), (III), (IV), (V), (IX), (X), and/or (XI) (by time, or type and location of administration).

One of skill in the art of ically Active Molecules will understand that each of the compounds of formula (I), (III), (IV), (V), (IX), (X), and/or (XI) can be modified in such a manner that the resulting compound still retains specificity and/or ty similar to the starting compound. In this light, the Biologically Active Molecule (D) of compounds of formula (I), (III), (IV), (V), (IX), (X), and/or (XI) can include any and all of the Biologically Active Molecules’ analogues and derivatives. In one embodiment the ically Active Molecules is a macrolide and r is maytansine or an analogue of maytansine as described in Widdison et al., J. Med. Chem., 2006, 49 (14), 4392-4408.

In one embodiment, described is a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) , (III) ,(IV), (X), (XI) including (III)(a), (III)(b) (IV)(a), (X)(a), and (XI)(a), or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or ents.

In one embodiment, described is a pharmaceutical composition comprising a therapeutically ive amount of a compound of formula (I) , (III) ,(IV), (V), (IX), (X), (XI) including (III)(a), (III)(b) (IV)(a), (V)(a), (V)(b), (V)(c), (V)(d), and (V)(e), (X)(a), and (XI)(a), or a pharmaceutically acceptable salt thereof and one or more pharmaceutically able carriers, diluents, or excipients.

In another embodiment, described is a pharmaceutical ition comprising a therapeutically effective amount of a compound of a (V) including (V)(a), (V)(b), (V)(c), (V)(d), and (V)(e), or a ceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

In another embodiment, described is a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (IX), or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, ts, or excipients.

In another embodiment, described is a pharmaceutical composition comprising a eutically effective amount of a compound of formula (V) and (IX) ing (V)(a), (V)(b), (V)(c), (V)(d), and (V)(e), or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable rs, diluents, or excipients.

Method of Use As described above, conjugate compounds of formula (I), (III), (IV), (X), and (XI) can be produced with s functional groups such that attachment of the Ligand (L) to the linker and thereby a Biologically Active Molecule form a covalent conjugate. The Ligand specially targets the ate compound to the Ligand binding partner, typically a polypeptide or other like antigen. In typical embodiment, the ate is designed to include a Ligand having a binding partner found on cells undergoing abnormal cell growth or cells involved in a proliferative disorder. singly, conjugate compounds of formula (I), (III), (IV), (X), and (XI) have been designed such that each nd’s linker is catabolized inside the cell bound by the conjugate. As such, delivery of a Biologically Active Molecule through the conjugate embodiments herein allows for delivery of Biologically Active Molecules that would normally be too toxic to administer conventionally. The embodiments herein allow for highly selective and ic delivery of these molecules to cells undergoing abnormal cell growth or cells involved in proliferative disorders (as compared to catabolism outside the cell, thereby releasing the biologically active compound into the blood or lymphatic system, for example).

As can be envisioned by one of skill in the art, the covalent conjugate compounds described herein can also be used to deliver any type of useful Biologically Active Molecule and can be selectively targeted to any type of cell population, for e, the ate may be used to deliver anti-proliferative drugs to cells undergoing abnormal growth or anti-viral drugs to cells infected with a virus, as long as the selected Ligand recognizes a proper cell g partner.

In this light, methods of use are described for the conjugate compound embodiments bed .

The pharmaceutical compositions described herein are useful in inhibiting, retarding and/or preventing abnormal cell growth or in the treatment of s proliferative disorders or disease states in mammals. In typical embodiments the mammal is a human (embodiments herein will be described in relation to humans). Other mammals include any mammal that can suffer from a detectable erative disorder, including primates, dogs, cats, horses, goats, sheep, cattle, camels, and the like. In addition, it is understood that the conjugate compounds of the pharmaceutical compositions are designed for selective targeting to the cells undergoing abnormal cell growth or for the treatment of the various proliferative disorders or disease states described herein.

As such, embodiments herein include methods of inhibiting al cell growth or treatment of a proliferative disorder in a human sing stering to the human a therapeutically effective amount of a pharmaceutical composition described herein. stration of a therapeutically effective amount of a pharmaceutical composition described herein may be effected in different ways, e.g., by enous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal, or intrabronchial administration. The pharmaceutical compositions herein may also be administered ly to an abnormal cell growth site (directly or indirectly contacting the abnormal cell ) by, for example, biolistic delivery (biolistic delivery of the ceutical compositions herein to a lung or brain tumor, for example). Dosage regiments for administration of the pharmaceutical compositions herein will be determined by the attending health care professional or other person of skill in the art as well as based on the particular clinical situation. As is well known in the pharmaceutical arts, dosages for any one human, i.e., patient, depends on a number of factors, including patient size, patient’s body surface area, patient’s age and general health, patient’s sex, the time and route of stration, and presence of a second eutic agent. In some instances the conjugate compounds of formula (I), (III), (IV), (X), and/or (XI) may be present in amounts between 1 µg and 100 mg/kg body weight per dose (note that where continuous infusion is considered as an administration route, as little as 1 pg/kg body weight per minute may be considered).

Pharmaceutical compositions can be administered one or more times a day and over a period of days, weeks, months, or years.

Treatment of erative disorder or disease, for example a tumor, includes methods of reducing a tumor size, causing necrosis or apoptosis in a tumor, killing a tumor, stopping a tumor from increasing in size and/or preventing veness or metastasis of a tumor.

Examples of medical ions that can be treated according to methods of inhibiting abnormal cell growth, or treating proliferative disorders include: malignancy of any type, e.g., cancer of the lung, colon, prostate, , pancreas, liver, ovary, skin, ma, leukemia and the like; autoimmune diseases, e.g., systemic lupus, rheumatoid arthritis, multiple sclerosis; viral infections, e.g., CMV infection, HIV infection, AIDS, Hepatitis, HPV infection; pain; mental disorders; and inflammatory diseases.

As noted above, pharmaceutical compositions described herein are also useful in the prevention or treatment of viral infections, pain, inflammatory diseases, mune diseases, and the like in a mammal.

In one embodiment, described is a method of reducing, retarding or stopping an abnormal cell growth comprising contacting the abnormal cell with a nd of formula (I), (III) (IV), (X), and/or (XI) in an amount sufficient to retard, reduce or stop the abnormal cell growth, and wherein the al cell growth is retarded, reduced or stopped.

In one embodiment, bed is a method of killing a cell, comprising contacting the cell with a compound of formula (I), (III), (IV), (X), and/or (XI) in an amount sufficient to kill the cell, and wherein the cell is killed.

In one embodiment, described is a method of killing a cell, comprising contacting the cell with a compound of formula (I), (III), (IV), (X), and/or (XI) in an amount sufficient to kill the cell, and n the cell is killed and further wherein the cell is a tumor cell.

In one embodiment, described is a method of treatment of a l disorder in an dual suffering from the medical disorder, comprising administering to the individual an effective amount of a composition sing a compound of formula (I), (III), (IV), (X), and/or (XI).

In one other embodiment, described is a method of treatment of a medical disorder in an individual ing from the medical disorder, comprising stering to the individual an effective amount of a composition comprising a compound of formula (I), (III), (IV), (V), (IX), (X), and/or (XI).

In one embodiment, the described is a method of treatment of a medical disorder in an individual ing from the medical disorder comprising administering to the individual an effective amount of a composition comprising a compound of formula (I), (III), (IV), (X), and/or (XI) and further comprising administering sequentially or consecutively an additional therapy.

In one embodiment, described are methods, wherein additional therapy is radiation therapy, chemotherapy, or a combination of both.

] In one ment, described is a method of treatment of a medical disorder in an individual suffering from the medical disorder comprising administering to the individual an effective amount of a composition comprising a compound of formula (I), (III), (IV), (X), and/or (XI) and further comprising administering sequentially or consecutively an additional therapy and administering at least one additional eutic agent.

] In one embodiment, described is a method of treatment of a medical disorder in an individual ing from the medical disorder sing administering to the individual an ive amount of a ition comprising a compound of formula (I), (III), (IV), (X), and/or (XI) and further sing administering sequentially or consecutively an additional therapy or administering at least one additional therapeutic agent.

] In one aspect, the medical disorder treated is selected from tumors, cancers, infectious es, neurodegenerative diseases, bone disorders, and cardiovascular diseases.

Embodiments herein also describe methods of preparing compounds of formula (I) from precursor or ng block compounds of formula (V). In some embodiments the compounds of formula (V) can also be used in therapeutic application where the compound of formula (V) is a pharmaceutical composition. In some embodiments compounds of formula (V) can be included in any of the compositions or pharmaceutical compositions of compound (I), (III), (IV), (IX), (X), and/or (XI).

Finally, embodiments herein may include mixtures of compounds as represented by formula (I), (III), (IV), (V), (IX), (X), and/or (XI).

Production of Conjugates The Ligand-Biologically Active Molecule conjugate compounds can be generated by any technique known to the skilled artisan. The Ligand-Biologically Active Molecule conjugate compounds se a Ligand unit, a Biologically Active Molecule, and optionally a Linker that joins the Biologically Active Molecule and the Ligand. The covalent ment of Biologically Active Molecules and/or Linkers to the Ligand can be lished using y of reactions using the amino acid residues of the Ligand, e.g., antibody, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of ne and the various moieties of the ic amino acids.

Further, conjugates in accordance with various embodiments described herein can be prepared by any known method in the art. An illustrative protocol for producing ates is provided in the Examples below. r, other known methods can be used, including, for example, protocols described in , US Patent No. 7,811,572 and US Patent No. 6,441,163, as long as the protocols are used to prepare the compounds as described herein. These nces are incorporated by reference for their intended purpose.

In one embodiment, the conjugates can be prepared by i) reacting a Ligand with Linker to form a modified Ligand-Linker compound; ii) optionally purifying the - Linker compound; iii) conjugating a ically Active Molecule, e.g., a macrolide, to the Ligand-Linker to form a conjugate of a (I), (III), (IV), (X), or (XI); and iv) purifying the conjugate.

In an alternative embodiment, the conjugates can be prepared by reacting a Biologically Active Molecule with a first component of the Linker (Z1), followed by successive reactions to build out the , including addition of Y, X, W, A and Z2, or any combination thereof.

In an alternative embodiment, the conjugates are prepared by reacting a Ligand, Linker and biologically active macrolide in a single reaction. Once the conjugates in accordance with the invention are ed they can be purified.

Identifying Cytotoxicity of Conjugate Compounds In one embodiment, the conjugate compounds described herein can be evaluated for their ability to suppress proliferation of various cancer cell lines in vitro. The in vitro cytotoxicity assays can be conducted using methods known in the art (see Widdison et al., J.

Med. Chem., 2006, 49(14), 4392-408) and as illustrated in Example 7 herein. For example, conjugate compounds can be applied to in vitro plated cancer cells for a predetermined number of days and surviving cells measured in assays by known methods. Proper controls can be utilized to ensure ty of results as can IC50 values. Examples of in vitro potency of conjugate compounds herein can be seen in Figures 1 and 2. onal in vivo efficacy can be used to confirm proposed conjugate compound potency – for e using a nude mouse model.

The above ication, examples and data provide a complete description of the cture and use of the composition of the invention. Since many embodiments of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended.

All references cited herein and in the Examples that follow are sly incorporated by reference in their entireties.

The description and Examples presented infra are provided to rate the subject invention. One of skill in the art will ize these Examples are provided by way of illustration only and are not included for the purpose of ng the invention. [00149a] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated ise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. [00149b] In the description in this specification reference may be made to t matter that is not within the scope of the claims of the current application. That subject matter should be readily fiable to a person skilled in the art and may assist in putting into ce the invention as defined in the claims of this application.

EXAMPLES Experimental Details Proton NMR spectra (for compounds that could not be detected by UV) were acquired on a Varian Inova 300 MHz ment, while mass spectra were collected on an Agilent 1100 series LC/MSD with electrospray ionization source and triple-quad ion trap analyzer. Appropriate conjugates were analyzed using a Bruker ultraFleXtreme MALDITOF-TOF mass spectrometer. All starting als and solvents were purchased commercially and used without purification, unless ise noted.

EXAMPLE 1 Step 1: MaytansinN-methyl-L-alanine (2) The title compound was prepared as a gold solid from maytansinol (1) using the methods described in U.S. Patent Application 2007/0037972 A1. MS (ESI, pos.): calc’d for C32H44ClN3O9, 649.3; found 650.6 (M+H).

Step 2: MaytansinN-methyl-L-(S)-alanine-N-[4-(amino-citrulline-valinehexanamidemaleimidyl )benzyl]carbamate (3): The product of the preceding step (2, 0.020 g, 0.031 mmol) and p-NO2-Phcarbonato-Bn-Cit-Val-maleimide (MA-VC-PAB-PNP, 0.027 g, 0.037 mmol; Concortis Biosystems) were ved in N,N-dimethylformamide (DMF, ca. 0.25 mL) in a conical vial, treated with Brockmann I basic alumina (0.10 g), the vial purged with argon, and the reaction stirred at ambient temperature for 4 days. The mixture was then filtered, the solids washed with acetonitrile/water, and filtrate purified ly on a 5u, 30x150 mm Phenomenex Gemini C18 column via HPLC (30 – 90% acetonitrile in water, 0.1% TFA in both, over 25 min, 15 mL/min). Lyophilization of the purest fractions overnight gave the title compound as a pale yellow solid (0.021 g, 55%). MS (ESI, pos.): calc’d for C61H82ClN9O17, ; found 1248.8 (M+H), 1270.7 (M+Na), 1231.5 (M-H2O+H).

EXAMPLE 2 Step 1: N-tert-Butoxycarbonyl-beta-alanine succinate ester (4) The title compound was prepared from commercial Boc-β-alanine by a method well known in the art (c.f.- on et al., J. Med. Chem., 2006, 49 (14), 4401). 1H NMR (300 MHz, CDCl3): δ 3.62 (bm, 2H), 2.88 (m, 9H), 1.47 (s, 9H).

Step 2: MaytansinN-methyl-L-(S)-alanine-Boc-β-Ala (5) ] The product of the preceding step (4, 0.45 g, 1.51 mmol) and maytansinN- methyl-L-alanine (2, 0.30 g, 0.23 mmol) were ved in 3:1 acetonitrile:water (8 mL), treated with 1M aqueous NaHCO3 (0.5 mL), and stirred at ambient temperature for 18h.

When the reaction was complete by TLC, it was then d with brine for 10 min and extracted thrice with ethyl e (EtOAc). The combined organic layers were then dried over Na2SO4, filtered, and the filtrate concentrated and dried in vacuo to a gold syrup that was purified by flash column chromatography on a 20g silica gel cartridge (0 – 10% MeOH in EtOAc over 15 min) giving the title nd as a white solid (0.084 g, 43%). MS (ESI, pos.): calc’d for C41H59ClN4O12, 834.4; found 835.2 (M+H), 857.2 (M+Na), 817.4 (M- H2O+H).

Step 3: MaytansinN-methyl-L-(S)-alanine-β-Ala (6) The product of the preceding step (5, 0.080 g, 0.095 mmol) was dissolved in a 3:1:1 e of acetonitrile/water/trifluoroacetic acid (4 mL) and stirred at ambient temperature for 26 hours. The crude reaction mixture was injected directly onto a 40g C18 silica gel column and eluted via ISCO CombiFlash (10 – 90% acetonitrile in water, 0.1% TFA in each solvent, over 18 min, 40 mL/min), and the combined pure fractions were lyophilized to give the title compound as a pale yellow solid (0.025 g, 31%). MS (ESI, pos.): calc’d for C36H51ClN4O10, 734.3; found 735.5 (M+H).

Step 4: MaytansinN-methyl-L-(S)-alanine-propanamidylN-methyl-N-[4- -citrulline-valine-hexanamidemaleimidyl)benzyl]carbamate (7) ] The product of the preceding step (6, 0.014 g, 0.019 mmol) and MA-VC-PABPNP (0.020 g, 0.027 mmol; Concortis Biosystems) were dissolved in 4:1 acetonitrile/water (2.5 mL), treated with 0.1M aqueous NaHCO3 (0.5 mL), and stirred at ambient temperature for 18 h. The reaction was purified directly by reverse-phase chromatography on C18 silica (using 0.1% TFA in acetonitrile/water gradients). Lyophilization of the final column ons gave the title compound as a white solid (0.002 g, 8%). MS (ESI, pos.): calc’d for C65H89ClN10O18, 1332.6; found 1333.9 (M+H), 1316.5 (M-H2O+H), 1355.9 (M+Na).

EXAMPLE 3 ] Step 1: 3-Methyldithio-propionic acid succinate ester (8) The title compound was prepared as a white solid from 3-mercaptopropionic acid using the methods of on et al. J. Med. Chem., 2006, 49 (14), 4392-4408. 1H NMR (300 MHz, CDCl3) δ 3.09 (m, 2H), 3.01 (m, 2H), 2.86 (s, 4H), 2.44 (s, 3H).

Step 2: sinN-methyl-L-(S)-alanine-propanamidylmethyldisulfide The product of the preceding step (8, 2.96 g, 11.9 mmol) and maytansinN- methyl-L-alanine (2, 1.54 g, 2.37 mmol) were dissolved in 4:1 acetonitrile/water (25 mL), treated with saturated aqueous NaHCO3 (2 mL), and stirred at ambient temperature for 24 hours. The reaction mixture was treated with brine, extracted thrice with EtOAc, the aqueous layer saturated with NaCl, extracted again with EtOAc, and the ed organic layers dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo to a gold syrup (ca. 4.5 g) that was purified by flash column chromatography on a 80g silica gel cartridge (0 – 100% EtOAc in hexanes over 30 min) giving the title nd as a white solid (1.14 g, 61%). MS (ESI, pos.): calc’d for C36H50ClN3O10S2, 783.3; found 784.3 (M+H), 766.6 (MH2O +H).

Step 3: MaytansinN-methyl-L-(S)-alanine-propanamidethiol (10) The title nd was prepared using a modified version of the method described by Whitesides et al. (J. Org. Chem., 1991, 56, 2648-2650). The product of the preceding step (9, 2.42 g, 3.09 mmol) was dissolved in itrile (30 mL), treated with a solution of tris(2-carboxyethyl)phosphine hydrochloride (8.23 g, 28.7 mmol) in water (30 mL), the pH raised to 3 with the addition of saturated aqueous NaHCO3 (5 mL), the flask purged with Ar, and the reaction stirred at ambient ature under a rubber septum (vented due to effervescence). After 2 hours, the reaction was treated with brine (ca. 100 mL), bubbled with Ar for 5 min (to remove the free methylmercaptan), and the phases ted. The aqueous phase was extracted twice with EtOAc, saturated with NaCl, and extracted twice more with EtOAc. The combined organic layers were then dried over Na2SO4, filtered, and the filtrate concentrated and dried in vacuo to give the title compound as a white solid (2.24 g, 98%). MS (ESI, pos.): calc’d for C35H48ClN3O10S, 737.3; found 738.3 (M+H), 720.3 (M-H2O+H).

Step 4: 4-Amino-(N-benzyloxycarbonyl)benzylamine (14) 4-Aminobenzylamine (1.00 g, 8.18 mmol) and triethylamine (1.20 mL, 8.61 mmol) were dissolved in anhydrous tetrahydrofuran (THF, 10 mL) under N2, cooled in a brine/ice bath with stirring, and d dropwise over 20 min with a solution of benzyl chloroformate (1.20 mL, 8.41 mmol) in ous THF (10 mL). After the addition was complete, the ice bath was removed and the reaction was stirred at ambient temperature for hours, then filtered over a sintered glass funnel to remove insolubles. The solids were washed with EtOAc, the filtrate evaporated in vacuo, and the residue purified by flash column chromatography on a 40g silica gel column (0 - 100% EtOAc in s, over 20 min, 40 mL/min). Evaporation of the pure mid-running ons in vacuo gave the title compound as a light yellow solid (1.47 g, 70%). MS (ESI, pos.): calc’d for C15H16N2O2, 256.1; found 256.9 (M+H), 278.9 (M+Na).

Step 5: 6-Maleimidylhexanoic acid succinate ester (20) The title nd was prepared as a colorless gum from commercial 6- aminocaproic acid by a method similar to that of Marnett et al. (J. Med. Chem., 1996, 39, 703). 1H NMR (300 MHz, CDCl 3) δ 6.72 (s, 2H), 3.56 (t, 2H, J = 7 Hz), 2.86 (s, 4H), 2.64 (t, 2H, J = 7 Hz), 1.81 (pentet, 2H, J = 8 Hz), 1.66 (m, 2H), 1.45 (m, 2H).

Step 6: Boc-valine-succinate (11) The title compound was prepared as a white solid from Boc-Val-OH by a method well known in the art (c.f.- Widdison et al., J. Med. Chem., 2006, 49 (14), 4401). 1H NMR (300 MHz, CDCl3) δ 5.03 (d, 1H, J = 10 Hz), 4.60 (dd, 1H, J = 9 Hz, 5 Hz), 2.85 (s, 4H), 2.32 (m, 1H), 1.47 (s, 9H), 1.05 (m, 6H).

Step 7: Boc-valine-citrulline (12) The product of the preceding step (11, 4.23 g, 13.5 mmol) was dissolved in acetonitrile (70 mL), treated with a solution of L-citrulline (3.20 g, 18.3 mmol) in water (30 mL) and a ted solution of NaHCO3 (18 mL), flask purged with N2, and reaction stirred at ambient temperature for 24 hours. The mixture was concentrated in vacuo to remove the itrile, washed once with EtOAc to remove nonpolar impurities, and the s layer saturated with NaCl and acidified to pH 3 with 10% HCl. The resulting cloudy mixture was extracted four times with 10% isopropanol in EtOAc, the combined organic layers dried over , and filtered. Concentration and drying of the filtrate in vacuo gave the title compound as a white solid (4.53 g, 90%). MS (ESI, neg.): calc’d for C16H30N4O6, 374.2; found 373.0 (M-H).

Step 8: Boc-valine-citrulline-aminobenzylamino-N-benzyloxycarbamate (15) The product of the preceding step (12, 3.08 g, 8.23 mmol) was dissolved in N,N- dimethylformamide (DMF, 30 mL, dried over lar sieves), treated with dicyclohexylcarbodiimide (DCC, 2.31 g, 11.2 mmol) and 1-hydroxybenzotriazole hydrate (HOBt, 1.51 g, 11.2 mmol), the flask purged with N2 and stirred at ambient temperature for 1 hour. A solution of 4-amino-(N-benzyloxycarbonyl)benzylamine (14, 2.30 g, 8.97 mmol) in DMF (15 mL) was then added, the reaction stirred another 3 days, filtered over a sintered glass funnel, and solids washed with ethyl acetate. The filtrate was washed with 1:1 water/saturated NaHCO3 (100 mL), the aqueous layer extracted thrice with 10% panol/EtOAc, and the combined organic layers washed with brine, dried over Na2SO4, and filtered. During filtration an insoluble gel formed that was ved with methanol/EtOAc. Concentration of the filtrate in vacuo gave a gummy gold gel that was treated with diethyl ether (50 mL), sonication, filtered, and suction-dried to a pale yellow solid. This was purified by flash column chromatography on a 330g silica gel column (0 - % methanol in dichloromethane, 100 mL/min) giving the title nd as a pale yellow solid (4.07 g, 81%). MS (ESI, pos.): calc’d for N6O7, 612.3; found 613.4 (M+H) Step 9: Boc-valine-citrulline-aminobenzylamine (16) The product of the preceding step (15, 3.04 g, 4.96 mmol) and 10% palladium (0) on activated charcoal (0.286 g, 0.269 mmol) were d under N2 stream with methanol (50 mL) and glacial acetic acid (0.57 mL, 9.95 mmol), the reaction bubbled a few minutes each with N2 then hydrogen, and stirred vigorously under a hydrogen balloon at ambient temperature and pressure for 1 hour. When the reaction was complete by TLC, the balloon was removed, the suspension bubbled several minutes with N2, and filtered over Celite 521.

The Celite was washed with methanol, the te evaporated to s in vacuo, and the e triturated once with diethyl ether and dried under high vacuum giving the title compound as a white solid (2.95 g, 99%). MS (ESI, pos.): calc’d for C23H38N6O5, 478.3; found 479.2 (M+H).

Step 10: Boc-valine-citrulline-aminobenzylisothiocyanate (17) The product of the preceding step (16, 0.586 g, 0.979 mmol) was dissolved in dry tetrahydrofuran (20 mL) and dry N,N-dimethylformamide (5 mL) under N2, treated with triethylamine (0.40 mL, 2.87 mmol), cooled in an ice bath, and treated dropwise with carbon disulfide (0.10 mL, 1.66 mmol) over 5 min. The reaction was warmed to ambient temperature and stirred for 2 hours, cooled again in ice, and treated with p-toluenesulfonyl chloride (0.281 g, 1.47 mmol). After warming to t temperature and stirring for 18 hours, the reaction was washed with 1:1 water/brine, ted twice with ethyl acetate, the s layer saturated with NaCl, extracted twice more with EtOAc, and the combined organic layers washed with brine, dried over Na2SO4, and filtered. The ated filtrate was purified by flash column chromatography on a 20g silica gel column (0 - 100% itrile in EtOAc, 35 mL/min) giving the title compound as a gold solid (0.391 g, 77%) after azeotroping with dichloromethane and drying under high vacuum. MS (ESI, pos.): calc’d for C24H36N6O5S, 520.3; found 521.1 (M+H).

Step 11: MaytansinN-methyl-L-(S)-alanine-propanamidylN-[4-(aminocitrulline-Boc-valine )-benzyl]-dithiocarbamate (18) The product of the preceding step (17, 0.068 g, 0.131 mmol) and maytansin N-methyl-L-(S)-alanine-propanamidethiol (10, 0.048 g, 0.065 mmol) were dissolved in dry THF (3 mL) under Ar, treated with triethylamine (0.050 mL, 0.359 mmol) via syringe, and stirred at ambient temperature under rubber septum for 18 hours. The reaction was concentrated in vacuo, dissolved in 10% isopropanol/ethyl acetate, and washed with 0.5N aq.

HCl. The aqueous layer was extracted thrice with 10% OAc, combined organic layers washed with brine, dried over Na2SO4, and filtered. The evaporated filtrate was purified by flash column chromatography on a 12g silica gel column (0 - 20% methanol in EtOAc, 30 mL/min) giving the title nd as a white solid (0.042 g, 51%). MS (ESI, pos.): calc’d for C59H84ClN9O15S2, 1257.5; found 1258.8 (M+H), 1241.5 (M-H2O+H), 1280.6 (M+Na).

] Step 12: sinN-methyl-L-(S)-alanine-propanamidylN-[4-(aminocitrulline-valine yl]-dithiocarbamate (19) The title compound was prepared as a gold solid (0.016 g, 100%) from the product of the preceding step (18, 0.014 g, 0.011 mmol) by the method of Example 2, Step 3 (compound 6). The compound was used without further purification. MS (ESI, pos.): calc’d for C54H76ClN9O13S2, 1157.5; found 1159.4 (M+H).

Step 13: MaytansinN-methyl-L-(S)-alanine-propanamidylN-[4-(aminocitrulline-valine-hexanamidemaleimidyl )benzyl]-dithiocarbamate (21) The product of the preceding step (19, 0.055 g, 0.032 mmol) was dissolved in 1:1 acetonitrile/water (4 mL), treated with 1N aq. NaHCO3 (0.5 mL) and a solution of 6- maleimidylhexanoic acid succinate ester (20, 0.070 g, 2.27 mmol) in acetonitrile (6 mL), and the flask purged with Ar under rubber septum. After the reaction d at ambient temperature for 5 hours, it was stored at -20oC for 3 days before warming again to ambient temperature and diluting with brine. The mixture was extracted thrice with ethyl acetate, combined organic layers dried over Na2SO4, and filtered. The evaporated filtrate was purified by flash column chromatography on a 12g silica gel column (0 - 20% ol in EtOAc over 18 min, 25 mL/min) giving the title compound as a pale yellow solid (0.011 g, 26%).

MS (ESI, pos.): calc’d for C64H87ClN10O16S2, 1350.5; found 1352.0 (M+H), 1334.5 (M- H2O+H), 1373.5 (M+Na).

EXAMPLE 4 Step 1: N-(4-Aminomethyl-phenyl)-acetamide hydrochloride (23) ] The title compound was prepared as a light yellow solid from 4- aminobenzylamine by the method of King et al. (J. Am. Chem. Soc., 1992, , 3033). 1H NMR (300 MHz, DMSO-d6): δ 10.18 (s, 1H), 8.36 (br s, 3H), 7.63 (d, 2H, J = 8.7 Hz), 7.41 (d, 2H, J = 8.7 Hz), 3.95 (s, 2H), 2.06 (s, 3H).

Step 2: N-(4-Isothiocyanatomethyl-phenyl)-acetamide (24) The product of the preceding step (23, 0.277 g, 1.38 mmol) was dissolved in THF (4.5 mL) and DMF (2.0 mL), cooled in ice under N2, treated with triethylamine (0.66 mL, 4.73 mmol), then treated dropwise with carbon disulfide (0.125 mL, 2.07 mmol). The reaction was warmed to ambient temperature, stirred for 3 hours, then cooled again in ice.

After treating with p-toluenesulfonyl de (0.274 g, 1.45 mmol), the reaction slowly warmed to ambient temperature while stirring for 18 hours. The e was diluted with water, acidified to pH 2 with 10% aq. HCl, and extracted thrice with EtOAc. The ed organic layers were washed with brine, dried over Na2SO4, and filtered. The evaporated filtrate was purified by flash column chromatography on a 20g silica gel column (0 - 50% acetonitrile in EtOAc over 20 min, 30 mL/min) giving the title compound as a cream-colored solid (0.157 g, 55%). 1H NMR (300 MHz, CDCl3) δ 7.55 (d, 2H, J = 8.7 Hz), 7.29 (m, 3H), 4.70 (s, 2H), 2.23 (s, 3H).

Step 3: MaytansinN-methyl-L-(S)-alanine-propanamidylN-[4- (acetamidyl)benzyl]-dithio-carbamate (25) The product of the preceding step (24, 0.093 g, 0.45 mmol) and the product of Example 3, Step 3 (10, 0.070 g, 0.095 mmol) were dissolved in acetonitrile (MeCN, 2 mL) and dry DMF (1 mL), and treated with basic alumina (activated, Brockmann I, 0.357 g). After purging the flask with argon, the reaction was stirred at ambient temperature for 2 days, filtered, and the solids washed with ol/acetonitrile. The evaporated filtrate was purified by flash column chromatography on a 12g silica gel column (0 - 50% itrile in EtOAc over 15 min, 25 mL/min) and the slower product fractions concentrated in vacuo to an impure pale yellow gum. This was purified by RP-HPLC menex Gemini C18, 30x150mm column, 30 – 90% acetonitrile in water, 0.1% TFA in both) and the pure fractions were lyophilized giving the title compound as a white solid (0.016 g, 18%). MS (ESI, pos.): calc’d for C45H58ClN5O11S2, 943.3; found 944.7 (M+H), 927.1 (M-H2O+H), 966.6 (M+Na).

EXAMPLE 5 MaytansinN-methyl-L-(S)-alanine-β-alanine (27) The title compound was prepared as a pale yellow solid from 2,5- dioxopyrrolidinyl 3-((tert-butoxycarbonyl)amino)propanoate (26) by the method of Example 2, Steps 1-3. MS (ESI, pos.): calc’d for ClN4O10, 720.3; found 721.4 (M+H).

EXAMPLE 6 MaytansinN-methyl-L-(S)-alanine--aminobutyramide (29) ] The title compound was prepared as a pale yellow solid from N-Boc-GABA-OH (28) by the method of Example 2, Steps 1-3. MS (ESI, pos.): calc’d for C36H51ClN4O10, 734.3; found 735.5 (M+H).

EXAMPLE 7 MaytansinN-methyl-L-(S)-alanine-N-Me--aminobutyramide (31) The title compound was prepared as a pale yellow solid from N-Boc-N-Me GABA-OH (30) by the method of Example 2, Steps 1-3. MS (ESI, pos.): calc’d for C37H53ClN4O10, 748.4; found 749.5 (M+H).

EXAMPLE 8 ] Step 1: MaytansinN-methyl-L-(S)-alanine-N-carboxy[3,4-dihydro(tertbutoxycarbonyl )-1H-isoquinoline] MaytanN-methyl-L-(S)-alanine (2, 0.034 g, 0.052 mmol), commercial NBoc-1 ,2,3,4-tetrahydroisoquinolinecarboxylic acid (32, 0.019 g,0.069 mmol), and 1-(3- Dimethylaminopropyl)ethylcarbodiimide hydrochloride (EDC, 0.024 g, 0.125 mmol) were weighed into a round-bottom flask with stir bar, dissolved in dichloromethane (3 mL), the flask purged with Ar and sealed with a rubber septum, and the reaction stirred at ambient temperature. After 2 days, the reaction was diluted with EtOAc and washed with dilute aq.

NaHCO3, and the aqueous layer was extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and filtered. The ated filtrate was then purified on a 12g RediSep Gold silica gel column via ISCO system (EtOAc – 5:5:1 EtOAc/DCM/MeOH over 12 mins, 30 mL/min), and the combined re fractions evaporated and dried in vacuo giving the title compound as a pale solid (0.026 g, 55%). MS (ESI, pos.): calc’d for ClN4O12, 908.4; found 909.2 (M+H), 891.2 +H).

Step 2: MaytansinN-methyl-L-(S)-alanine-N-carboxy(1,2,3,4- tetrahydroisoquinoline) (33) The title compound was prepared as a white solid (0.013 g, 52%) from the product of the preceding step (0.025 g, 0.027 mmol) by the method of Example 2, Step 3 (compound 6). MS (ESI, pos.): calc’d for C42H53ClN4O10, 808.3; found 809.2 (M+H).

EXAMPLE 9 Step 1: MaytansinN-methyl-L-(S)-alanine- N-carboxy[1-(tertbutoxycarbonyl )-piperidine] The title compound was prepared as a white solid (0.027 g, 46%) from maytan- 3-N-methyl-L-(S)-alanine (2, 0.045 g, 0.069 mmol) and commercial 1-tbutoxycarbonylpiperidinecarboxylic acid (34, 0.024 g,0.105 mmol) by the method of Example 8, Step 1. MS (ESI, pos.): calc’d for ClN4O12, 860.4; found 861.2 (M+H), 843.2 (M-H2O+H).

Step 2: MaytansinN-methyl-L-(S)-alanine- N-carboxypiperidine (35) The title compound was prepared as a white solid (0.012 g, 50%) from the product of the ing step (0.025 g, 0.029 mmol) by the method of Example 2, Step 3 (compound 6). The compound purified on a C18 column using a different gradient and modifier (20 - 80% MeCN in water, 0.05% acetic acid in both). Lyophilization of the pure fractions gave the title compound (0.008 g, 35%). MS (ESI, pos.): calc’d for C38H53ClN4O10, 760.3; found 761.2 (M+H).

E 10 Step 1: MaytansinN-methyl-L-(S)-alanine- N-methyl-beta-alanine-N-[4- (tert-butoxycarbonyl-valine-citrulline-amino)benzyloxy]-carbamate The Boc-valine-citrulline-p-aminobenzyloxy-(p-nitrophenyloxy)-carbonate (36), prepared according to WO 2005112919, (0.092 g, 0.143 mmol), the product of Example 2, Step 3 (6, 0.110 g, 0.130 mmol), and 1-Hydroxyazabenzotriazole (HOAT, 0.037 g, 0.272 mmol) were dissolved in DMF (7 mL), treated with triethylamine (0.100 mL, 0.717 mmol), and stirred at t temperature in a stoppered flask. After 18 hours, the reaction e was concentrated to an oil in vacuo, dissolved in dichloromethane, and purified on a 24g RediSep Gold column via ISCO lash (0 – 20% methanol in ethyl acetate).

Evaporation of the product fractions in vacuo then gave the title compound as a pale yellow solid (0.129 g, 80%). MS (ESI, pos.): calc’d for C60H86ClN9O17, 1239.6; found 1240.8 (M+H).

Step 2: MaytansinN-methyl-L-(S)-alanine- N-methyl-beta-alanine-N-[4- (valine-citrulline-amino)benzyloxy]-carbamate (37) The title nd was prepared as a white solid (0.074 g, 63%) from the product of the preceding step (0.128 g, 0.103 mmol) by the method of Example 2, Step 3 (compound 6). MS (ESI, pos.): calc’d for C55H78ClN9O15, 1139.5; found 1141.4 (M+H).

Step 3: MaytansinN-methyl-L-(S)-alanine- N-methyl-beta-alanine-N-[4- (4- iocyanato-phenyl}-thioureido-valine-citrulline-amino )benzyloxy]-carbamate (39) ] The product of the preceding step (37, 0.037 g, 0.029 mmol) was dissolved in tetrahydrofuran (THF, 5 mL) in a vial, treated with triethylamine (0.020 mL, 0.143 mmol), and the resulting solution added dropwise to a flask containing a stirred solution of 1,4- phenylenediisothiocyanate (38, 0.055 g, 0.286 mmol) in THF (10 mL) over 15 min. The vial was rinsed with THF (2 mL) and the solution added to the reaction flask, which was sealed with a rubber septum. After stirring at ambient temperature for 24 hours, the reaction was concentrated in vacuo to dryness, the crude product dissolve in acetonitrile, and ed over a 0.45 um PTFE membrane. The filtrate was then purified on a 30 g C18 RediSep Gold column via ISCO (20 – 80% MeCN in water, 0.05% HOAc in both solvents) and the purest fractions (by LC) combined, frozen at -78oC, and lyophilized giving the title nd as white solid (0.023 g, 59%). MS (ESI, pos.): calc’d for C63H82ClN11O15S2, 1331.5; found 1332.0 (M+H).

EXAMPLE 11 Step 1: 1-(4-Amino-butyl)-maleimide ] A solution of commercial Bocaminobutylmaleimide (0.304 g, 1.13 mmol) in dichloromethane (10 mL) was treated with trifluoroacetic acid (1.00 mL, 13.1 mmol), the flask purged with Ar, sealed with a rubber septum and bubbler vent, and stirred at ambient temperature. The reaction was complete by TLC after 18 hours, so it was concentrated in vacuo, triturated twice with diethyl ether, and dried in vacuo to a gum. This was triturated twice more with ether (while scraping with a a), decanted, and dried again in vacuo giving the title compound as a white solid (0.321 g, 100%). MS (ESI, pos.): calc’d for 2O2, 168.1; found 169.0 (M+H).

Step 2: 1-(4-Isothiocyanato-butyl)-maleimide (41) The product of the preceding step was dissolved in acetonitrile (MeCN, 3 x 40 mL) and concentrated in vacuo at 60o C via rotary evaporator. The dried product (0.650 g, 2.45 mmol) was dissolved in MeCN (75 mL) and chloroform (30 mL) in a flask, treated with triethylamine (1.0 mL, 7.35 mmol), and the resulting solution added dropwise to a flask containing hiocarbonyldi-2,2'-pyridone (0.68 g, 2.94 mmol) in chloroform (25 mL) under nitrogen over 10 min. The reaction was stirred at ambient temperature for 18 hours, the reaction was concentrated in vacuo to dryness, the crude product was dissolved in dichloromethane (DCM) and purified on a 120 g silica gel RediSep Gold column via flash column chromatography (0 – 10% MeOH in DCM). The st fractions (by LC) were combined and concentrated to dryness giving the title compound as white solid (0.26 g, 50%). MS (ESI, pos.): calc’d for 2O2S, 210.0; found 211.2 (M+H).

Step 3: MaytansinN-methyl-L-(S)-alanine- N-methyl-beta-alanine-N-[4- (4- {maleimidylbutyl}-thioureido-valine-citrulline-amino)benzyloxy]-carbamate (42) The product of Example 10, Step 2 (37, 0.029 g, 0.023 mmol) was dissolved in dry DMF (2 mL), treated with diisopropylethylamine (0.020 mL, 0.115 mmol) via dry syringe, then with a solution of product of the ing step (41, 0.026 g, 0.124 mmol) in dry DMF (2 mL). The reaction flask was purged with Ar, sealed with a rubber septum, and the on stirred at ambient temperature. After 18 hours the reaction appeared to be 80% complete by LCMS, so it was evaporated to an oil in vacuo, dissolved in MeCN/water, and purified on a 30g C18 RediSep Gold column via flash column chromatography (20 – 80% MeCN in water, 0.05% HOAc in both solvents). The st fractions by LCMS were combined, briefly rotavapped, frozen on dry ice, and lyophilized overnight giving the title compound as a white solid (0.020 g, 65%). MS (ESI, pos.): calc’d for C64H88N11O17SCl, 1349.6; found 1351.1 (M+H), 1372.9 (M+Na), 1333.6 (M-H2O+H).

EXAMPLE 12 ] Conjugate Preparation and Characterization For the initial set of ments, four antibodies were ated to various linker-drug compounds of the disclosure using the procedure below. The four antibodies used in these experiments were: (1) a PSMA antibody having the heavy and light chain variable domains of clone AB-PG1-XG1-006 as set forth in WO 2007002222A2, (2) a STEAP1 antibody having the heavy and light chain variable domains of clone mu120, expressed as a hIgG1, as set forth in WO 2008052187A2, (3) an EGFRvIII antibody having the heavy and light chain variable s of clone 131 as set forth in 075048A1, and (4) a PRLR having the heavy and light chain variable domains of clone 3N as set forth in US Application Serial No. 61/868,185; filed on August 21, 2013 (the disclosure of which is hereby incorporated by reference in its entirety). All the monoclonal antibodies were expressed in CHO cells and purified by Protein A. A nding control derived from an immunological antigen having no relation to oncology was also used.

Conjugation Method for Compounds 3, 7, 21 and 42 The dy (10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was treated with 1 mM dithiothreitol at 37° C for 30 min. After gel filtration (G-25, pH 4.5 sodium acetate), the maleimido linker payload derivative (1.2 equivalents/SH group) in DMSO (10 mg/ml) was added to the reduced antibody and the mixture adjusted to pH 7.0 with 1 M HEPES (pH 7.4). After 1 h the reaction was quenched with excess N-ethyl maleimide. The conjugates were purified by size exclusion chromatography and sterile filtered. Protein and linker payload concentrations were determined by UV spectral is. Size-exclusion HPLC established that all conjugates used were >95% monomeric, and RP-HPLC established that there was <0.5% unconjugated linker payload. Yields are reported in Table 1 based on protein. All conjugated antibodies were ed by UV for linker payload loading values according to Hamblett et al, Cancer Res., 2004 10 7063. The results are ized in Table Conjugation Method for Compound 39 To the dy (2-5 mg/ml) in 50 mM carbonate, 150 mM NaCl, pH 9.0, was added 15% by volume dimethyl acetamide. The linker payload derivative 39 (5-10 equivalents) in DMSO (10 mg/ml) was added to the antibody and the mixture incubated at 37° C for 4-12 hours. The conjugates were purified by size exclusion chromatography and sterile filtered. Protein and linker payload concentrations were determined by UV spectral analysis. Size-exclusion HPLC established that all ates used were >95% monomeric, and RP-HPLC established that there was <0.5% unconjugated linker payload. For these conjugates, the d to antibody ratio was determined by MALDI-TOF (Table 1).

Table 1 nd 252 nm (cm-1 M-1) 280 nm (cm-1 M-1) 3 32000 8500 7 50600 8100 21 44190 9460 39 -- -- Antibody 252 nm (cm-1 M-1) 280 nm (cm-1 M-1) STEAP1 87939 244276 PSMA 77652 224320 PRLR 80673 220420 II 79579 209420 Isotype Control 75113 218360 Antibody Conjugate Payload:Antibody (UV) Yield % STEAP1-7 1.4 36 PSMA-3 3.5 44 PSMA-7 3.4 60 PSMA-21 0.9 45 PRLR-7 3.0 70 EGFRvIII-7 3.4 64 EGFRvIII-39 1.3 (MALDI) 40 Isotype Control-3 3.0 48 Isotype Control-7 2.3 51 Isotype Control-21 2.3 45 Isotype Control-39 1.1 (MALDI) 40 EXAMPLE 13 In Vitro Antibody-Drug Conjugate (ADC) Cell-free Enzymatic Assays ] Cathepsin B incubation In vitro cell-free tic assay procedure was adopted from Dubowchik, et al., Bioconjugate Chem. 2002 13 855. The DAR corrected PRLR-7 and Isotype Control-7 concentration was set to 7.00 uM in 25 mM sodium acetate buffer, 1 mM EDTA, pH 5.0 and pre-incubated at 37˚C. Cathepsin B (Sigma # C8571) was activated at room temperature for minutes with 1 lent of 30 mM DTT, 15 mM EDTA to 2 equivalents of cathepsin B stock. The activated cathepsin B solution was added to the ADC solutions at a 1:750 molar ratio. Samples were incubated at 37˚C over a 24 hour period and aliquoted for either HPLC (HISEP)-UV detection or LC-MS detection vide infra.

] LC-MS detection At designated time points, a small aliquot was removed and combined with 2 equivalents by volume of cold methanol. Supernatant was recovered and analyzed by liquid chromatography-mass spectrometry (LCMS) for cathepsin B linker payload cleavage yielding nd 6 using a Merck Chromolith FastGradient RP-18e, 2x50 mm column, 10 to 90% MeCN over 5 mins, in H2O with 0.05% HOAc in both ts and a flow rate of 1 mL. The elution profile was monitored at 254 nm. All of the aliquots incubated at 37˚C with cathepsin B contained compound 6 eluting at 5.1 minute with a mass of 735 M+H (calc’d for C36H51ClN4O10, 734.3) and none of the aliquots without cathepsin B contained any 6. This was also confirmed by injection of pure compound 6 from e 2, step 3.

HPLC (HISEP)-UV detection Solutions were ed "as is" at designated time points. The following gradient method was utilized: buffer A100% 100 mM NH4OAc, pH 7.0 and buffer B 100% acetonitrile, flow rate 0.4 mL/min, from 5 to 70% buffer B, over a Supelco LC-HISEP; 150 mm x 4.6 mm, column. The elution profile was red at 280 nm and 252 nm. All ts of the cathepsin B incubated ADCs contained a species which elutes at 19.4 minute.

Pure compound 6 elutes at the identical retention time under the same gradient conditions.

The 19.4 minute species was not present in the aliquot t cathepsin B.

The results of this Example are significant in part because cathepsin B proteolysis of 6 should only occur after internalization of the ADC in the cell where the enzyme exists. Off target effects should be reduced since the antibody delivers the cytotoxic payload directly to targeted cells.

EXAMPLE 14 In Vitro Cytotoxicity Assays ] In this Example, the ability of various antibody-drug conjugates to kill antigenexpressing tumor cells in vitro was assessed.

Cells were seeded in PDL-coated 96 well plates at 375 EGFRvIII), 1500 (U251/hEGFRvIII), 2000 (HEK293/hEGFRvIII), or 3000 (C4-2, PC3/hSTEAP1, T47D, and U87-MG) cells per well in complete growth media and grown ght. For cell viability curves, ly diluted conjugates or free representative payloads were added to the cells at final concentrations ranging from 500 nM to 1 pM and incubated for 3 days. To measure viability in MMT/hEGFRvIII, U251/hEGFRvIII, HEK293/hEGFRvIII, C4-2, PC3/hSTEAP1, and U87-MG, cells were incubated with CCK8 (Dojindo) for the final 1-3 hours and the absorbance at 450nm (OD450) was determined on a Flexstation3 (Molecular Devices). To measure viability in T47D, cells were incubated on ice for 30 min in 4% formaldehye + 3ug/ml Hoechst. Images of Hoechst stained nuclei were acquired on the ImageXpress Micro XL (Molecular s) and nuclear counts were determined with the Columbus analysis software (PerkinElmer). Background OD450 levels (CCK8) or nuclear counts from digitonin (40 nM) treated cells were subtracted from all wells and ity is sed as a percentage of the untreated controls. IC50 values were determined from a fourparameter logistic equation over a 10-point response curve (GraphPad Prism). All curves and IC50 values are corrected for payload lents.

In C4-2 cells (prostate cancer line), natively expressing PSMA at 271 fold above isotype control binding, the sinoid conjugates PSMA-3, PSMA-7, and PSMA-21 possess IC50 values of 3.8, 0.5, and 8.3 nM, respectively (Figure 1). The naked PSMA antibody was devoid of any anti-proliferation activity.

In TEAP1 cells (prostate cancer line), expressing hSTEAP1 at 352 fold above isotype control binding, the maytansinoid conjugate STEAP1-7 possesses an IC50 value of 4 nM (Figure 2). The naked STEAP1 dy was devoid of any anti-proliferation activity.

In T47D cells (breast cancer line), natively expressing PRLR at 14 fold above isotype control binding, the maytansinoid conjugate PRLR-7 possesses an IC50 value of 1.0 nM e 3). The naked T47D dy was devoid of any anti-proliferation activity.

In HEK293/hEGFRvIII cells, sing hEGFRvIII at 360 fold above isotype control binding, the maytansinoid conjugate EGFRvIII-7 possesses an IC50 value of 0.4 nM (Figure 4). The naked EGFRvIII antibody was devoid of any anti-proliferation activity.

In GFRvIII cells, expressing hEGFRvIII at 280 fold above isotype control binding, the maytansinoid conjugate EGFRvIII-7 possesses an IC50 value of 0.3 nM (Figure 5). The naked EGFRvIII antibody was devoid of any anti-proliferation ty.

In U251/hEGFRvIII cells (glioblastoma cancer line), expressing hEGFRvIII at 165 fold above isotype control binding, the maytansinoid conjugate EGFRvIII-7 possesses an IC50 value of 0.3 nM (Figure 6). The naked EGFRvIII dy was devoid of any antiproliferation activity.

In vitro cytotoxicity of proposed released payloads ("free drugs") were also tested in the various cell lines described above and plotted along-side the conjugated antibodies for comparison (see closed squares (■) in Figures 1 to 6). For linker-payloads 3 and 7 the proposed released ds 2 and 6, tively, can be used in the cellular assays directly since they are stable. However, for linker-payload 21 the released payload is proposed to be the dryl compound 10. Since 10 could be a very reactive nd, which would lead to unreliable results, compound 25 was chosen to ent the released payload in these assays.

In a separate set of experiments, compound 6, along with amino s 27, 29, and 31 were assayed in HEK293 and U87MG for anti-proliferation activity (Figure 7). These compounds all had >30 nM IC50 values indicating that they are highly cytotoxic only when attached to an antibody via an riate linker. (For these experiments, background correction with digitonin was not performed).

] In yet r set of experiments, nds 6, 9, 33, and 35 were assayed in HEK293, U251, C4-2, PC3, and MMT for anti-proliferation activity (Figure 8). Amino compounds 6, 33, and 35 had varied IC50s as listed in Table 2. The trend in potency follows 9 > 33 > 35 > 6 and is consistent for the 5 cell lines assayed.

Table 2 IC50 (nM) Compound HEK293 U251 C4-2 PC3 MMT 9 0.2 0.4 1.5 0.4 0.3 33 20 15 20 30 20 50 25 55 65 60 6 200 150 200 250 250 Without being bound by any theory, the results of these experiments demonstrate that the "released" or "free drug" versions of the compounds of the present disclosure (i.e., the compounds not conjugated to an antibody) were, in most cases, substantially less cytotoxic than when conjugated to a targeting antibody. This feature of the present disclosure suggests that antibody-drug conjugates comprising the compounds of the invention will cause fewer side-effects and less unwanted toxicity since the cell killing properties will be concentrated at the site of the target antigen specifically.

EXAMPLE 15 Anti-EGFRvIII Antibody Drug Conjugates are Potent tors of Tumor Growth in in vivo EGFRvIII Positive Breast Cancer Allograft Models In this Example, two different antibody-drug conjugates of the exemplary anti- EGFRvIII antibody H1H1863N2 were tested for their ability to inhibit tumor growth in vivo.

(The amino acid sequence and various properties of H1H1863N2 are set forth in US ,963, filed on March 11, 2014, hereby orated by reference in its entirety).

H1H1863N2 comprises a heavy chain variable region (HCVR) comprising SEQ ID NO:1; a light chain variable region (LCVR) comprising SEQ ID NO:5; heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) comprising SEQ ID NOs: 2, 3 and 4, respectively; and light chain mentarity determining regions (LCDR1, LCDR2 and LCDR3) comprising SEQ ID NOs: 6, 7 and 8, respectively.

A first ADC was produced by conjugating H1H1863N2 to the sinoid DM1 via a non-cleavable MCC linker (see, e.g., US 5,208,020 and US application 20100129314) to produce "H1H1863N2-MCC-DM1." A second ADC was produced by conjugating H1H1863N2 to 7 to yield "H1H1863N2-7." When tested for cytotoxicity in vitro against MMT/EGFRvIII cells using the assay format described in Example 14, H1H1863N2- MCC-DM1 exhibited an IC50 of 12 nM whereas H1H1863N2-7 exhibited an IC50 of only 0.8 nM. Thus, in vitro, the anti-EGFRvIII ADC H1H1863N2-7 exhibited much more potent tumor cell g ability than the corresponding dy ated to DM1 via an MCC linker.

To compare the in vivo efficacy of the anti-EGFRvIII antibodies conjugated to MCC-DM1 and 7, studies were performed in immunocompromised mice g EGFRvIII positive breast cancer allografts. Briefly, tumor allografts were established by subcutaneous implantation of 0.5x106 MMT/EGFRvIII cells into the left flank of female CB17 SCID mice (Taconic, Hudson, NY). Once tumors had reached an average volume of 140 mm3 (~Day 8), mice were randomized into groups of seven, and dosed with anti-EGFRvIII ADCs using either the MCC-DM1 or 7 linker-drug format. Control reagents, including non-binding ADCs using either the MCC-DM1 or 7 linker-drug format, and PBS vehicle were also ed.

ADCs were dosed at 1 and 5 mg/kg three times over one week and thereafter monitored until an average tumor size of approximately 2000 mm3 was attained in the group administered with vehicle alone. At this point the Tumor Growth tion was calculated.

Average tumor size relative to the vehicle treated group were ated as follows: tumors were measured with calipers twice a week until the average size of the vehicle group d 1000mm3; tumor size was calculated using the formula (length x width2)/2. Tumor growth inhibition was calculated according to the following formula: (1- ((Tfinal-Tinitial)/(Cfinal-Cinitial)))*100, where T (treated group) and C (control group) represent the mean tumor mass on the day the vehicle group d 1000mm3. Results are summarized in Table 3.

Table 3: Tumor Size and Tumor Growth Inhibition Following Administration of Anti- EGFRvIII Antibody-Drug Conjugates and Controls, administered in repeat dose e Tumor Final Tumor Growth Treatment Group size at Day 8 Inhibition mm3 (mean ± SD) PBS Vehicle 2253 ± 217 0 Control-MCC-DM1 (1 mg/kg) 2827 ± 278 -27 Control-MCC-DM1 (5 mg/kg) 2402 ± 256 -7 Control-7 (1 mg/kg) 2729 ± 470 -22 Control-7 (5 mg/kg) 2787 ± 503 -25 H1H1863N2-MCC-DM1 (1 mg/kg) 931 ± 292 62 H1H1863N2-MCC-DM1 (5 mg/kg) 471 ± 227 84 H1H1863N2-7 (1 mg/kg) 679 ± 265 74 H1H1863N2-7 (5 mg/kg) 96 ± 34 102 As shown in this e, the greatest tumor inhibition was observed in mice dosed with 5 mg/kg H1H1863N2-7, where regression of the initial tumor was observed. The tumor growth inhibition of 102% resulting from treatment with 5 mg/kg H1H1863N2-7 was significantly greater relative to that ed following treatment of tumor with 5 mg/kg H1H1862N2-MCC-DM1 (83%). The superiority of the tumor growth inhibition induced by H1H1863N2-7 ed to 3N2-MCC-DM1 was maintained at the 1 mg/kg dose as well. No anti-tumor effect was observed in groups treated with Control ADC using MCCDM1 or 7.

Claims (42)

1. A compound of Formula (I) wherein L is a Ligand capable of binding to a cell or cell population; a is an integer from 1 to 10; Z2 is represented by the following structural formula: 2B-Z2C-Z2D–, wherein Z2A is or -C(=O)-, Z2b is C1-C10 -alkylene-, Z2C is -C(=O)-, and Z2d is absent; A is a natural or non-natural amino acid residue, or a peptide residue comprising 2-20 amino acid residues; W is -NR4-; X is C6-C18 -arylene-; A1 is C1-C10 ene-; A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, 3)CH2-, or -CH2CH2CH2CH2-; R1 is ; R17 is oxygen; R4 is hydrogen; and R4a is an C1-C10 alkyl; wherein when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10.

2. The compound of claim 1, wherein L is capable of binding to a specifically targeted cell population.

3. The compound of claim 1, wherein L is ed from the group consisting of proteins, antibodies, fragments of antibodies, nucleic acids, antigen binding scaffolds, and carbohydrates.

4. The compound of claim 3, wherein L is an antibody or an antigen binding fragment thereof.

5. The compound of claim 4, wherein L is an antibody or an n binding fragment thereof that specifically binds a tumor associated antigen.

6. The compound of claim 5, wherein the antibody or an antigen binding fragment thereof ses a sulfur group that is ntly attached with Z2A.

7. The compound of claim 1, wherein A is an amino acid residue selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, cysteine, and citrulline residues.

8. The compound of claim 1, n A is a valine-citrulline peptide residue.

9. The compound of claim 1, wherein X is wherein R9, R10, R11, and R12 are hydrogen.

10. The nd of claim 6 having the ing formula wherein Ab is an antibody or an n binding fragment thereof; AA2AA1 is a valine-citrulline peptide residue; q is an integer from 1 to 5; and R9, R10, R11, and R12 are hydrogen.

11. The compound of claim 10, n q is 4.

12. The compound of claim 10, having the following structure: wherein Ab is an antibody or an antigen binding fragment thereof.

13. The compound of claim 1, wherein A is a peptide residue cleavable by a protease.

14. The compound of claim 1, wherein A is a peptide residue cleavable by a protease expressed in tumor tissue.

15. The compound of claim 14, n the protease is a cathepsin or a plasmin.

16. A compound of the following formula Z2 is represented by the following structural formula: –Z2A-Z2B-Z2C-Z2D–, wherein Z2A is , , or -N=C=S; Z2b is C1-C10 -alkylene-, C6-C18 -arylene- or -O-C(=O)-; Z2C is -C(=O)-, -C(=S)-N(R4)-, or C1-C10 -alkylene-; and Z2d is absent or -C(=O)-; A is a l or non-natural amino acid residue, or a peptide residue comprising 2-20 amino acid residues; W is -NR4-; X is C6-C18 -arylene-; A1 is C1-C10 -alkylene-; A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, 3)CH2-, or -CH2CH2CH2CH2-; R1 is oxygen or -NR4-; R17 is selected from the group consisting of oxygen and ; R4 is hydrogen; and R4a is C1-C10 alkyl; wherein when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10.

17. The compound of claim 16, wherein A is a valine-citrulline peptide residue; X is wherein R9, R10, R11, and R12 are hydrogen.

18. The compound of claim 17, having the structure: ; and

19. A nd of the following formula wherein A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH2CH2CH2-; and R4a is alkyl; wherein when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10.

20. A compound of the following formula wherein Ab is an antibody or an antigen binding fragment thereof; a is an integer from 1 to 10; Z2 is represented by the following structural formula: –Z2A-Z2B-Z2C-Z2D–, wherein Z2A is - C(=S)-N(R4)- or -C(=O)-; Z2b is C6-C18 -arylene- or C1-C10-alkylene-; Z2C is -N(R4)- -; and Z2d is ; A is a natural or non-natural amino acid residue, or a peptide residue comprising 2-20 amino acid residues; W is -NR4-; X is C6-C18 -arylene-; A1 is C1-C10 ene-; A3 is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, or -CH2CH2CH2CH2-; R1 is oxygen or -NR4-; R17 is oxygen; R4 is hydrogen; and R4a is C1-C10 alkyl; wherein when A3 is -CH2CH2CH2-, then R4a is CnH2n+1 where n is 2-10.

21. The compound of claim 20 having the following structure: wherein a is an integer from 1 to 10.

22. The compound of claim 6 having the following formula wherein Ab is an antibody or an antigen binding; 1 is a -citrulline peptide residue q is an integer from 1 to 5; and R9, R10, R11, and R12 hydrogen.

23. The compound of claim 22 represented by the following structure: wherein a is an integer from 1 to 10.

24. The compound of claim 19, having the following structure

25. A compound having a structure ed from the group consisting of , , , and

26. The compound of claim 1, wherein the compound is an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof having the following formula:

27. An antibody-drug conjugate comprising an dy or antigen-binding fragment thereof conjugated to a payload of the following formula:

28. A pharmaceutical ition comprising a therapeutically effective amount of a compound of any one of claims 1 to 26 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents, or excipients.

29. Use of a compound of any one of claims 1 to 26, in the manufacture of a medicament for reducing, retarding or ng an abnormal cell growth.

30. Use of a compound of any one of claims 1 to 26, in the manufacture of a medicament for killing a cell.

31. The use of claim 30, wherein the cell is a tumor cell.

32. Use of a compound of any one of claims 1 to 26, in the manufacture of a ment for treatment of a medical disorder in an individual.

33. The use of claim 32, wherein the medicament when administered, is administered tially or consecutively with an onal therapy.

34. The use of claim 32, wherein the additional therapy is radiation therapy, chemotherapy, or a combination of both.

35. The use of claim 32, wherein the medicament when administered, is administered with at least one additional therapeutic agent.

36. The use of claim 32, wherein the l er is selected from the group ting of tumors, s, infectious diseases, neurodegenerative diseases, bone disorders, and cardiovascular diseases.

37. The use of claim 36, wherein the medical disorder is cancer.

38. Use of a compound of any one of claim 1 to 26, in the manufacture of a medicament for reducing tumor size, stopping tumor size increase, reducing tumor proliferation, or preventing tumor proliferation in an individual in need thereof.

39. The compound according to any one of claims 1 to 26, substantially as herein described, with reference to any example thereof.

40. The antibody-drug conjugate according to claim 27, substantially as herein described, with reference to any example thereof.

41. A pharmaceutical composition according to claim 28, substantially as herein described, with nce to any example thereof.

42. The use according to any one of claims 29 to 38, substantially as herein described, with reference to any example thereof.