HK1203945B - Pyrimido[4,5-b]indole derivatives and use thereof in the expansion of hematopoietic stem cells - Google Patents

Description

Pyrimido [4,5-b ] indole derivatives and their use in expansion of hematopoietic stem cells

Reference to related applications

This application claims the benefit of priority from U.S. provisional application No. 61/591,521 filed on 27/1/2012. The entire contents of this application are incorporated herein by reference.

Technical Field

The present invention relates to pyrimido [4,5-b ] indole derivatives. Furthermore, the present invention relates to the use of pyrimido [4,5-b ] indole derivatives for the expansion of hematopoietic stem cells. Furthermore, the present invention relates to the treatment of diseases comprising hematopoietic stem cells.

Background

The main sources of Hematopoietic Stem Cells (HSCs) are bone marrow and Umbilical Cord Blood (UCB). HSCs are used at the site of transplantation (autologous or allogeneic), which constitutes one of the most effective therapeutic strategies for achieving a cure in patients with hematological malignancies, bone marrow failure anemias (bone marrow failure conditions), a variety of congenital diseases of global interest (e.g., sickle cell anemia and thalassemia), and autoimmune diseases such as lupus. However, the opportunity for such life-saving or life-improving treatments is not available to thousands of people worldwide due to the inability to expand these cells ex vivo sufficiently to make the procedure safe and successful. More specifically, one would forego the opportunity for transplantation for every 3 patients because no Human Leukocyte Antigen (HLA) identical donors were found. Another portion of patients will not simply obtain a transplant because too few HSCs are available for successful transplant grafting (i.e., cord blood or autologous). The safety and efficacy of bone marrow transplantation depends directly on the number of HSCs and the progenitor cells available for engraftment. The more infusions that can be injected, the more rapid the blood system recovers and the smaller the window of risk of infection due to the absence of granulocytes or the window of risk of bleeding due to the absence of platelets. The challenge of providing adequate HSCs is further escalated, with non-myelosuppressive conditioning being preferred as in the context of gene therapy for major inherited blood diseases (the major genetic cause of morbidity and mortality worldwide).

In adults, HSCs are predominantly present in the bone marrow and must be mobilized in order to enter the circulation before being collected by apheresis for autologous or allogeneic Hematopoietic Stem Cell Transplantation (HSCT). The collection of sufficient numbers of CD34+ cells, a surrogate marker for (HSCs) is of prime importance, as the dose of CD34+ cells affects the success and rate of hematopoietic recovery. Several reports indicate that higher injected CD34+ cell doses are independent predictors of improved survival.

The two most commonly used mobilization regimens are granulocyte colony stimulating factor (G-CSF) and G-CSF-enhanced chemotherapy. CXCR4 antagonists, when administered with G-CSF, increased HSC mobilization approved by the U.S. Food and Drug Administration (FDA) and the canadian health agency 2011 in 2008. However, plerixafor is contraindicated in patients with leukemia because of the mobilization of leukemia cells. It is estimated that a sufficient number of CD34+ cells/kg affected up to 15% of patients (varying between diseases) cannot be obtained with the currently used mobilization protocol. Often, the use of autologous HSCT in hematological malignancies is limited by the fact that normal and cancer stem cells are present in the bone marrow and, thus, may be mobilized.

Allogeneic HSCT with BM or mPBSC is another transplantation alternative. However, about one-third to one-fourth of patients eligible for this type of transplantation cannot find a suitable donor. For those that acquire transplants, relapse or graft rejection due to graft-versus-host disease; and the risk of long-term immunodeficiency, there is a high frequency of transplant-related mortality. Alternatively, cord blood has been shown to be a valid option in allogeneic HSCT. However, typically, a single CB unit provides insufficient HSCs for an adult patient for rapid and efficient hematopoietic recovery.

Cytokine-mediated short-term maintenance or even modest increases in vitro conditions that support murine or human HSC numbers as measured by murine reconstitution assays are often accompanied by more robust increases in the subsequent types of progenitor cell populations. More significant increases in murine and human HSCs have recently been described in cultures containing other factors such as Fibroblast Growth Factor (FGF), insulin growth factor binding protein, angiopoietin-like growth factor and pleiotrophin. However, these latter reports are solitary and await independent certification to date. Short-term increases in HSCs obtained in vitro using standard cytokines are also unavoidable, followed by eventual HSC depletion.

Alternative strategies for human HSC expansion involve their culture using either stromal elements or soluble morphogenic ligands (e.g., stimulating Notch, Wnt and Hedgehog pathways), targeted manipulation of specific intracellular signaling pathways (PGE2, ROS, p38 and MAPK inhibitors), or manipulation of specific transcription factors (e.g., Hox, Hlf). Other clinical approaches for ex vivo expansion of HSCs include the use of: i) purine derivatives (StemRegenin 1) (SR1), arene receptor antagonists (Boitano, AEet al, "Aryl hydrocarbon receptor antagonists progress the expansion of humane chemometric materials cells" Science 329: 1345-1348.2010); ii) mangosteen alcohol (Garcinol), histone acetyltransferase inhibitors (Nishino, T et al, "Ex vivo expansion of human hematogicous cells by Garcinol, a tensinthihibitor of hormone acetyl transferase" PLoSONE 6(9): e 24298.2011); and iii) NR-101, a non-peptidyl small molecule c-MPL agonist (Nishino et al, "Exvivo expansion of human hematotic stem cells by a small-molecule-polypeptide agonist c-MPL" exp. Hem. 2009; 37: 1364-. The characterization of SR1 provides proof of the principle that Low Molecular Weight (LMW) compounds have the ability to promote HSC expansion.

Clinical studies emphasize the importance of not only the persistence of the transplant that needs to be performed, but also the need to minimize the time that useful levels of granulocytes appear after transplantation, which, in turn, depends on the number of short-term re-proliferations of the infused cells. No useful acceleration of hematopoietic recovery by transplantation of expanded bone marrow or cord blood cells in culture with cytokines has been clinically demonstrated to date compared to untreated cells. Early results with experiments with cells expanded with mobilized Notch ligands first showed a potential clinical utility for any (even modest) progenitor cell expansion strategy (Delaney et al, "Notch-media expansion of human cord blood promoter cells capable of expressing rapid myeloid recovery" nat. Med.16(2): 232-236.2010). However, this approach is limited by the need to utilize the mobilized Delta-1 fusion protein during the ex vivo expansion step and by the lack of documented effects on stem cells (the effect appears to be limited to more differentiated progenitor cells). Other approaches in clinical trials include: i) StemEx, a combination of UCB cells cultured with the copper chelator Tetraethylpentamine (TEPA) and cytokines using co-infusion of untreated UCB cells; the stage I results show that there is no improvement in time to neutrophil or platelet engraftment compared to previous reports (de Lima M et al, "Transplantation of ex vivo expanded clone cells using the linker chemor grafted cysteine: a phase l/llclinical trial" Bone Marrow transplant.2008; 41: 771-); and 16-16 dimethyl prostaglandin E2(PGE2) for improving the homing of UCBT in the stage i test.

Thus, new strategies for increasing the expansion of hematopoietic stem and progenitor cells are needed. Certain pyrimido [4,5-b ] indole derivatives known in the art for use in that regard; for example, in WO 2003/037898; WO 2004/058764; WO 1998/042708; WO 1997/002266; WO 2000/066585; WO 1993/020078; WO 2006/116733; WO 2008/055233; WO 2010/006032; WO 1995/019970; WO 2005/037825; and WO 2009/004329. However, these documents do not disclose pyrimido [4,5-b ] indole derivatives according to the present invention or their use in the expansion of hematopoietic stem and progenitor cells.

Drawings

FIG. 1: the compounds do not act via the arene (AhR) pathway. Mobilized peripheral blood CD34(+) cells were cultured for 12 hours using DMSO, SR1[ AhR antagonist 1000nM ], and Compound 1[500nM ], harvested and subjected to real-time quantitative RT-PCR of AhR-responsive genes (CYP1B1 and AhRR). Compound 1, unlike SR1, did not inhibit AhR-downstream target genes, indicating that its function is independent of the AhR pathway.

FIG. 2: the effect of compound 1 is reversible. Mobilized peripheral blood CD34(+) cells cultured for 7 days with compound 1 (shown in green) or vehicle (DMSO, shown in blue) were washed and re-seeded in fresh medium with or without compound supplementation (solid vs dashed lines, respectively). Cells were cultured for an additional 8 days and the percentage of CD34+ CD45RA "was measured. When compound 1 was washed away, a rapid decrease in the percentage of CD34+ CD45 RA-was observed, indicating that its effect was reversible.

FIG. 3: flt3, SCF, and TPO are required for compound 1 mediated stem cell expansion. Mobilized peripheral blood CD34(+) cells were cultured for 7 days in the presence or absence of growth factor (Flt3+ SCF + TPO). In the absence of any growth factor, the CD34+ CD45 RA-cell counts were low, indicating their need for the observed effect.

FIG. 4: A) compound 1 reduced the differentiation of CD34+ mobilized peripheral blood cells. Compound 1 was able to expand CD34+ cell populations compared to DMSO-treated control cells as determined by FACS analysis. SR1 remained expanded cultured cells CD34+ in synergy with compound 1, indicating that inhibition of differentiation with the combination was even more significant. B) Compound 1 reduced the differentiation of CD34+ cord blood cells. C) Compound 40 reduced the differentiation of CD34+ cord blood cells. D) Inhibition of CD34+ cell differentiation by compound 40 exhibited a dose-dependent effect.

FIG. 5: A) compound 1 ex vivo expanded mobilized peripheral blood-derived CD34+ and CD34+ CD45 RA-cell populations. Although the total cell count was the same in DMSO and compound 1 treated cells, the CD34+ population was more than twice that of DMSO. Likewise, the CD34+ CD45 RA-population in compound 1 treated group was more than three times higher than in DMSO. This observation is enhanced with co-processing with SR 1. B) Compound 1 enhanced the expansion of cord blood-derived CD34+ and CD34+ CDRA-cells during 7-day incubation. C) Compound 1 showed a positive effect on the in vitro expansion of cord blood-derived CD34+ and CD34+ CDRA-cells during 12-day incubation.

FIG. 6: CD34+ mPB cells were cultured for ten days in the presence of vehicle (DMSO), SR1, compound 1, and a combination of compound 1 and SR 1. Results of 50,000 and 500,000 treated cells were transplanted into NSG mice, and bone marrow analysis was performed 13 weeks after transplantation to evaluate human hematopoietic transplant survival. CD34+ mPB cell transplantation with compound 1 treatment was better than DMSO treatment at any given cell dose. Interestingly, the percentage of human CD45+ cells in the BM was highest in NSG mice, which received cells treated with the combination (compound 1+ SR1) compared to individual compounds.

FIG. 7: A) compound 1 expanded cells are capable of reconstituting human hematopoietic cells in NSG mice. Results of 5,000CD34+ CB cells treated with vehicle (DMSO), SR1, compound 1, and the combination were transplanted into immunocompromised NSG mice. Bone marrow analysis showed human CD45+ engraftment in all treatment groups except DMSO after 8 weeks post-transplantation. Cells treated with the combination (compound 1+ SR1) showed the highest level of engraftment. B) Compound 40 prevented the loss of human hematopoietic cells capable of engrafting NSG mouse bone marrow during 12 days in vitro incubation.

FIG. 8: primary cell phenotype in a bioreactor using a fed-batch culture method as measured by flow cytometry (CD 34)⁺、CD34⁺CD90⁺And CD34⁺CD45RA⁺) Amplification of (3). FB control-fed without compound 40 and Cpd 40-fed with compound 40.

Disclosure of Invention

The inventors have discovered certain pyrimido [4,5-b ] indole derivatives. These compounds are useful for expanding hematopoietic stem cell populations, particularly human hematopoietic stem cell populations. The chemicals are also useful in the treatment of diseases involving hematopoietic stem cells.

According to one aspect, the present invention provides compounds of the following general formulae I, II, III, IV, V and VI:

substituents in the above formulae I, II, III, IV, V and VI, i.e. Z, W, L, Li, X_i、R¹、R²、R³、R⁴And m is as defined herein below.

According to one aspect, the present invention provides pharmaceutical compositions comprising compounds of formulae I, II, III, IV, V, and VI.

According to one aspect, the present invention provides the use of compounds of formulae I, II, III, IV, V and VI for the expansion of hematopoietic stem cells. In an embodiment of the invention, the hematopoietic stem cells are human cells.

According to one aspect, the present invention provides a method for increasing hematopoietic stem or progenitor cells, the method comprising culturing a starting population of cells in the presence of a compound of formula I, II, III, IV, V, or VI. In embodiments of the invention, the starting cell population is in vivo, in vitro or ex vivo. Also, in embodiments of the invention, the starting cell population comprises CD34+ cells harvested from mobilized peripheral blood (mPB), Bone Marrow (BM), or Umbilical Cord Blood (UCB). Furthermore, in an embodiment of the method according to the invention, optionally the culturing of the starting cell population is performed in the presence of a compound of general formulae I, II, III, IV, V and VI, together with at least one cell expansion factor as a biological or further small molecule.

According to one aspect, the invention provides a cell population expanded according to the method of the invention, more particularly a cell population expanded using a compound according to the invention. In an embodiment, the present invention provides hematopoietic stem cells expanded according to the method of the present invention, more specifically, hematopoietic stem cells expanded using the compound according to the present invention.

According to one aspect, the present invention provides a method of treating a hematopoietic disorder/malignancy, an autoimmune disease and/or an inherited immunodeficient disease in a subject, the method comprising administering to a subject in need of such treatment hematopoietic stem cells expanded with a compound of formula I, II, III, IV, V or VI or a compound of formula I, II, III, IV, V or VI.

In embodiments of the invention, the hematopoietic disorder/malignancy, autoimmune disease and/or inherited immunodeficiency disorder comprises anemia of bone marrow failure, a variety of congenital diseases of global interest (e.g., sickle cell anemia and thalassemia), lupus, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome, multiple myeloma, non-hodgkin's lymphoma, hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, fanconi anemia, thalassemia, sickle cell anemia, wiskott-aldrich syndrome, congenital defects of metabolism (e.g., gaucher disease, among others).

According to one aspect, the present invention provides a kit for augmenting stem or progenitor cells or for expanding hematopoietic stem cells, the kit comprising a compound of formula I, II, III, IV, V, or VI and instructions for use. In an embodiment of the invention, the kit comprises at least one cell expansion factor, which is a biological or another small molecule.

Detailed Description

The inventors have discovered certain pyrimido [4,5-b ] indole derivatives. These compounds are useful for expanding hematopoietic stem cell populations, in particular, human hematopoietic stem cell populations. The compounds are also useful in the treatment of diseases involving hematopoietic stem cells.

The compounds according to the invention have the general formula I, II, III, IV, V or VI shown below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

In the formulae I, II, III, IV, V and VI, the substituents are as defined in the following summary.

Z is: 1) -P (O) (OR)¹)(OR¹)、2)-C(O)OR¹、3)-C(O)NHR¹、4)-C(O)N(R¹)R¹、5)-C(O)R¹、6)-CN、7)-SR¹、8)-S(O)₂NH₂、9)-S(O)₂NHR¹、10)-S(O)₂N(R¹)R¹、11)-S(O)R¹、12)-S(O)₂R¹13), L, 14) optionally with 1,2, or 3R^AOr R¹-benzyl substituted with substituents, 15) optionally substituted with a linkerOne or more R attached to either or both of L and heteroaryl^AOr R¹-L-heteroaryl substituted with a substituent, 16) optionally with one or more R attached to either or both of L and the heterocyclic group^AOr R¹a-L-heterocyclyl substituted with a substituent, 17) optionally with one or more R attached to either or both of L and heteroaryl^AOr R¹-L-aryl substituted with a substituent, 18) optionally with one or more R^AOr R¹A heteroaryl group substituted with a substituent, or 19) optionally substituted with one or more R^AOr R¹-aryl substituted with a substituent. In this list, if it is not present, each substituent is optionally attached to the L group; and when (R) is to be¹) And R¹When attached to a nitrogen atom, optionally they are taken together with the nitrogen atom to form a 3 to 7-membered ring, optionally containing one or more other heteroatoms selected from N, O and S, optionally with one or more R¹Or R^AA substituted ring.

W is H, halogen or a group attached to the pyrimidoindole core of the molecule through N, O, S or a C atom. Optionally, W comprises at least one moiety which is a saturated, unsaturated, linear, branched and/or cyclic alkyl and/or heteroalkyl group having from 1 to 20 carbon atoms. Also, optionally, the moiety includes at least one other heteroatom, which is N, O or S. As the skilled person will appreciate, W in the chemical structure of the compounds according to the invention may belong to a number of classes of chemical groups commonly used in the art.

More specifically, W is: 1) -H, 2) -halogen, 3) -OR¹、4)-L-OH、5)-L-OR¹、6)-SR¹、7)-CN、8)-P(O)(OR¹)(OR¹)、9)-NHR¹、10)-N(R¹)R¹、11)-L-NH₂、12)-L-NHR¹、13)-L-N(R¹)R¹、14)-L-SR¹、15)-L-S(O)R¹、16)-L-S(O)₂R¹、17)-L-P(O)(OR¹)(OR¹)、18)-C(O)OR¹、19)-C(O)NH₂、20)-C(O)NHR¹、21)-C(O)N(R¹)R¹、22)-NHC(O)R¹、23)-NR¹C(O)R¹、24)-NHC(O)OR¹、25)-NR¹C(O)OR¹、26)-OC(O)NH₂、27)-OC(O)NHR¹、28)-OC(O)N(R¹)R¹、29)-OC(O)R¹、30)-C(O)R¹、31)-NHC(O)NH₂、32)-NHC(O)NHR¹、33)-NHC(O)N(R¹)R¹、34)-NR¹C(O)NH₂、35)-NR¹C(O)NHR¹、36)-NR¹C(O)N(R¹)R¹、37)-NHS(O)₂R¹、38)-NR¹S(O)₂R¹、39)-S(O)₂NH₂、40)-S(O)₂NHR¹、41)-S(O)₂N(R¹)R¹、42)-S(O)R¹、43)-S(O)₂R¹、44)-OS(O)₂R¹、45)-S(O)₂OR¹46) optionally with 1,2 or 3R^AOr R¹Substituent-substituted-benzyl, 47) optionally with one or more R attached to either or both of L and heteroaryl^AOr R¹-L-heteroaryl substituted with a substituent, 48) optionally with one or more R attached to either or both of L and the heterocyclic group^AOr R¹a-L-heterocyclyl substituted with a substituent, 49) optionally with one or more R attached to either or both of L and aryl^AOr R¹substituent-substituted-L-aryl, 50) -L-NR¹(R¹)、51)-L-)₂NR¹、52)-L-(N(R¹)-L)_n-N(R¹)R¹、53)-L-(N(R¹)-L)_n-heteroaryl, optionally with one or more R attached to either or both of L and heteroaryl^AOr R¹Substituent substituted heteroaryl, 54) -L- (N (R)¹)-L)_n-heterocyclyl, optionally with one or more R attached to either or both of L and the heterocyclyl^AOr R¹Said heterocyclyl, 55) -L- (N (R) is substituted by a substituent¹)-L)_n-aryl radicalOptionally with one or more R attached to either or both of L and heteroaryl^AOr R¹The aryl is substituted by substituent 56) -O-L-N (R)¹)R¹57) -O-L-heteroaryl, optionally with one or more R attached to either or both of L and heteroaryl^AOr R¹(iii) substitution of said heteroaryl, 58) -O-L-heterocyclyl with a substituent, optionally with one or more R attached to either or both of L and the heterocyclyl^AOr R¹(iii) substituent(s) substituted for said heterocyclyl, 59) -O-L-aryl, optionally with one or more R attached to either or both of L and aryl^AOr R¹Substituent substituted aryl, 60) -O-L)₂-NR¹、61)-O-L-(N(R¹)-L)_n-N(R¹)R¹、62)-O-L-(N(R¹)-L)_n-heteroaryl, optionally with one or more R attached to either or both of L and heteroaryl^AOr R¹Substituent substituted heteroaryl, 63) -O-L- (N (R)¹)-L)_n-heterocyclyl, optionally with one or more R attached to either or both of L and the heterocyclyl^AOr R¹Said heterocyclyl, 64) -O-L- (N (R) is substituted by a substituent¹)-L)_n-aryl, optionally with one or more R^AOr R¹(iii) substitution of said aryl, 65) -S-L-heteroaryl with a substituent, optionally with one or more R^AOr R¹(iii) substitution of said heteroaryl, 66) -S-L-heterocyclyl with a substituent, optionally with one or more R^AOr R¹(iii) substitution of said heterocyclyl, 67) -S-L-aryl with a substituent, optionally with one or more R attached to either or both of L and aryl^AOr R¹Substituent substituted aryl, 68) -S-L)₂NR¹、69)-S-L-(N(R¹)-L)_n-N(R¹)R¹、70)-S-L-(N(R¹)-L)_n-heteroaryl, optionally with one or more R^AOr R¹Said heteroaryl being substituted by a substituent, 71) -S-L- (N (R)¹)-L)_n-heterocyclyl, optionally with one or more R^ASaid heterocyclyl being substituted by substituents 72) -S-L- (N (R)¹)-L)_n-aryl, optionally with one or more R^ASubstituent substituted for said aryl, 73) -NR¹(R¹)、74)-(N(R¹)-L)_n-N(R¹)R¹、75)-N(R¹)L)₂-NR¹、76)-(N(R¹)-L)_n-N(R¹)R^A、77)-(N(R¹)-L)_n-heteroaryl, optionally with one or more R^AOr R¹Substituents said heteroaryl, 78) - (N (R)¹)-L)_n-heterocyclyl, optionally with one or more R^AOr R¹Said heterocyclic group, 79) - (N (R) is substituted by a substituent¹)-L)_n-aryl, optionally with one or more R^AOr R¹(iii) substitution of said aryl, 80) -heteroaryl with a substituent, optionally with one or more R^AThe heteroaryl, or 81) -aryl is substituted with a substituent, optionally with one or more R^AThe aryl group is substituted by a substituent. In this list, if it is not present, each substituent is optionally attached to the L group; and, when two R are¹When present on the same nitrogen atom, then each R¹The substituents are independently selected from R described hereinafter¹A list of values; and n is an integer equal to any of 0,1, 2, 3,4, or 5; and when (R) is to be¹) And R¹When attached to a nitrogen atom, optionally they are taken together with the nitrogen atom to form a 3 to 7-membered ring, optionally containing one or more other heteroatoms selected from N, O and S, optionally with one or more R¹Or R^AA substituted ring.

L is: 1) -C_1-6Alkyl, 2) -C_2-6Alkenyl, 3) -C_2-6Alkynyl, 4) -C_3-7Cycloalkyl, 5) -C_3-7Cycloalkenyl, 6) heterocyclyl, 7) -C_1-6alkyl-C_3-7Cycloalkyl, 8) -C_1-6Alkyl-heterocyclyl, 9) aryl, or 10) heteroaryl. In this list, optionally with one or two R^AThe substituents are respectively and independently substituted alkyl, alkenyl, alkynyl and cycloalkylCycloalkenyl, heterocyclyl, aryl and heteroaryl groups.

R¹The method comprises the following steps: 1) -H, 2) -C_1-6Alkyl, 3) -C_2-6Alkenyl, 4) -C_2-6Alkynyl, 5) -C_3-7Cycloalkyl, 6) -C_3-7Cycloalkenyl radical, 7) -C_1-5Perfluorinated, 8) -heterocyclyl, 9) -aryl, 10) -heteroaryl, 11) -benzyl, or 12)5- [ (3aS,4S,6aR) -2-oxohexahydro-1H-thiophene [3,4-d ]]Imidazol-4-yl]A pentanoyl group. In this list, optionally 1,2 or 3R^AOr R¹The substituents each independently substitute an alkyl, alkenyl, alkynyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl, and benzyl group.

R²The method comprises the following steps: 1) -H, 2) -C_1-6Alkyl, 3) -SR¹、4)-C(O)R¹、5)-S(O)R¹、6)-S(O)₂R¹7) optionally with 1,2 or 3R^AOr R¹Substituted-benzyl, 8) optionally with one or more R attached to either or both of L and heteroaryl^AOr R¹-L-heteroaryl substituted with a substituent, 9) optionally with one or more R attached to either or both of L and the heterocyclic group^AOr R¹a-L-heterocyclyl substituted with a substituent, 10) optionally substituted with one or more R attached to either or both of L and aryl^AOr R¹-L-aryl substituted with a substituent, 11) optionally with one or more R^AOr R¹A heteroaryl group substituted with a substituent, or 12) optionally substituted with one or more R^AOr R¹-aryl substituted with a substituent. In this list, if it is not present, each substituent is optionally attached to the L group.

R^AThe method comprises the following steps: 1) -halogen, 2) -CF₃、3)-OH、4)-OR¹、5)-L-OH、6)-L-OR¹、7)-OCF₃、8)-SH、9)-SR¹、10)-CN、11)-NO₂、12)-NH₂、13)-NHR¹、14)-NR¹R¹、15)-L-NH₂、16)-L-NHR¹、17)-L-NR⁴R¹、18)-L-SR¹、19)-L-S(O)R¹、20)-L-S(O)₂R¹、21)-C(O)OH、22)-C(O)OR¹、23)-C(O)NH₂、24)-C(O)NHR¹、25)-C(O)N(R¹)R¹、26)-NHC(O)R¹、27)-NR¹C(O)R¹、28)-NHC(O)OR¹、29)-NR¹C(O)OR¹、30)-OC(O)NH₂、31)-OC(O)NHR¹、32)-OC(O)N(R¹)R¹、33)-OC(O)R¹、34)-C(O)R¹、35)-NHC(O)NH₂、36)-NHC(O)NHR¹、37)-NHC(O)N(R¹)R¹、38)-NR¹C(O)NH₂、39)-NR¹C(O)NHR¹、40)-NR¹C(O)N(R¹)R¹、41)-NHS(O)₂R¹、42)-NR¹S(O)₂R¹、43)-S(O)₂NH₂、44)-S(O)₂NHR¹、45)-S(O)₂N(R¹)R¹、46)-S(O)R¹、47)-S(O)₂R¹、48)-OS(O)₂R¹、49)-S(O)₂OR¹50) -benzyl, 51) -N₃Or 52) -C (-N ═ N-) (CF)₃). In this list, optionally with 1,2 or 3R^AOr R¹The substituent is substituted benzyl.

In an embodiment of the invention, the compounds have the general formula IIA, IIB, IIC, IVA or VIA shown below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

In an embodiment of the present invention according to the compounds of formula IIA above, R¹W and R²Each as defined above.

In an embodiment of the present invention according to the compounds of formula IIB described above, W and R²Each as defined aboveAnd Het is optionally with one or more R as defined herein above¹Or R^AA substituted 3 to 7-membered heterocyclic ring.

In an embodiment of the present invention according to the compounds of the above formula IIC, W and R²Each as defined above; r⁵And R⁶Are the same or different and are each independently L as defined above, or they are taken together with C to form a 5 to 7-membered ring optionally containing one or more heteroatoms selected from N, O and S, and optionally with one or more R¹Or R^AA substituted ring. In a further embodiment, the ring is a 5-membered ring and the heteroatom is a nitrogen atom. In still further embodiments, the ring comprises four nitrogen atoms. In still further embodiments, R²Is benzyl.

In an embodiment of the present invention according to the compounds of formula IVA described above, W, L, R¹And R²Each as defined above. And, m, Li, R³And R⁴Each as defined above.

In an embodiment of the present invention of the compounds according to the above general formula VIA, Z is CO₂Me or 2-methyl-2H-tetrazol-5-yl; r²Is benzyl, 3-thienylmethyl or 3-pyridylmethyl; and W is NH-L-N (R)¹)R¹Wherein L is C_2-4Alkyl and R¹Is C_1-4Alkyl or (R)¹) And R¹Taken together with the nitrogen atom to which they are attached to form a 3 to 7-membered ring, optionally containing one or more additional heteroatoms selected from N, O and S, optionally with one or more R¹Or R^AA substituted ring.

In an embodiment of the invention, the compounds of the invention are compounds 1 to 55 described in table 2 herein below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

In a further embodiment of the invention, the compounds of the invention have the formula described in table 2 herein below. Salts or prodrugs of such compounds are also within the scope of the compounds according to the invention.

Defining:

unless otherwise stated, the following definitions apply:

the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the term "comprising" means that a list of elements following the word "comprising" is required or mandatory, however, other elements are optional and may or may not be present.

As used herein, the term "consisting of …" refers to anything that includes and is limited to following the phrase "consisting of …". Thus, the phrase "consisting of …" indicates that the listed elements are required or mandatory, and indicates that no other elements are possible.

As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group containing branched and straight chains having the specified number of carbon atoms, e.g., C_1-6C in alkyl_1-6Defined as comprising groups having 1,2, 3,4, 5, or 6 carbons in a linear or branched saturated arrangement. C as defined above_1-6Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, and hexyl.

As used herein, the term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, e.g., C_3-7C in cycloalkyl₃-C₇Defined as comprising groups having 3,4, 5,6, or 7 carbons in a monocyclic saturated arrangement. C as defined above₃-C₇Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

As used herein, the term "alkenyl" refers to an unsaturated straight or branched chain hydrocarbon group having the specified number of carbon atoms therein, and wherein at least two carbon atoms are bonded to each other by a double bond, and having E or Z domain chemistry and combinations thereof. For example, C₂-C₆C in alkenyl₂-C₆Defined as comprising groups having 2, 3,4, 5, or 6 carbons in a linear or branched arrangement with at least two carbon atoms bonded together by a double bond. C₂-C₆Examples of alkenyl groups include, but are not limited to, ethenyl (ethenyl), 1-propenyl, 2-propenyl, 1-butenyl, and the like.

As used herein, the term "alkynyl" refers to an unsaturated, straight chain hydrocarbon group having the specified number of carbon atoms therein and wherein at least two carbon atoms are joined together by a triple bond. For example, C₂-C₄Alkynyl is defined as including groups having 2, 3, or 4 carbon atoms in the chain, at least two of which are joined together by three bonds. Examples of such alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

As used herein, the term "cycloalkenyl" refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, e.g., C₃-C₇C in cycloalkenyl₃-C₇Defined as comprising groups having 3,4, 5,6, or 7 carbons in a single ring arrangement. C defined above₃-C₇Examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

As used herein, the term "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

As used herein, the term "haloalkyl" refers to an alkyl group as defined above, wherein each hydrogen atom may be substituted sequentially by a halogen atom. Examples of haloalkyl groups include, but are not limited to, CH₂F、CHF₂And CH₃。

As used herein, the term "aryl", alone or in combination with another radical, refers to a carbocyclic aromatic monocyclic group containing 6 carbon atoms, which may be further fused to a second 5-or 6-membered carbocyclic group, which may be aromatic, saturated or unsaturated. Examples of aryl groups include, but are not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl, tetrahydronaphthyl, and the like. The aryl group may be attached to another group at the appropriate position on the cycloalkyl or aromatic ring.

As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system of up to 10 atoms in which at least one ring is aromatic and contains 1 to 4 heteroatoms selected from the group consisting of O, N and S. The heteroaryl group may be attached via a ring carbon atom or a heteroatom. Examples of heteroaryl groups include, but are not limited to, thienyl, benzimidazolyl, benzo [ b ] thienyl, furyl, benzofuryl, pyranyl, isobenzofuryl, benzopyranyl, xanthenyl, 2H-pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (indolizinyl), isoindolyl, 3H-indolyl, indazolyl, purinyl, 4H-quinolyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl (napthylidinyl), quinoxalinyl, quinazolinyl, cinnolinyl (cinnolinyl), pteridinyl, isothiazolyl, isobenzodihydropyranyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl, thiazolo [4,5-b- ] -pyridine, tetrazolyl, oxadiazolyl (oxadiazolyl), oxadizyl, and mixtures thereof, Thiadiazolyl, thienyl and fluorescein derivatives.

As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" refers to a 3,4, 5,6 or 7-membered non-aromatic ring system containing 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, 3, 5-dimethylpiperidinyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, and the like, wherein the attachment to the ring can be on a nitrogen or carbon atom of the ring as described below.

As used herein, the term "optionally substituted with one or more substituents" or its equivalent, the term "optionally substituted with at least one substituent" means that the subsequently described event of a circumstance may or may not occur, and means that the description includes instances where the event or circumstance occurs and instances where it does not. This definition refers to from zero to five substituents.

As used herein, the term "subject" or "patient" refers to human and non-human mammals such as primates, cats, dogs, pigs, cows, sheep, goats, horses, rabbits, rats, mice, and the like.

If the substituents themselves are not compatible with the synthetic methods described herein, the substituents may be protected with an appropriate Protecting Group (PG) that is stable to the reaction conditions used in these methods. The protecting group may be removed at an appropriate point in the reaction sequence of the process to provide the desired intermediate or target compound. Suitable protecting groups and methods for protecting and deprotecting various substituents using such suitable protecting groups are well known to those skilled in the art; available from T.Greene and P.Wuts, "Protecting Groups in Chemical Synthesis" (4th ed.), John Wiley&Examples of this are found in Sons, NY (2007), the entire contents of which are incorporated herein by reference. Examples of protecting groups used throughout include, but are not limited to, Fmoc, Bn, Boc, CBz and COCF₃. In certain embodiments, substituents that are reactive under the reaction conditions used in the methods described herein may be specifically selected. In these cases, the reaction conditions convert the selected substituent to another substituent that is useful in an intermediate compound in the methods described herein or is a desired substituent in the target compound.

The term "pharmaceutically acceptable salts" as used herein refers to acid and base addition salts.

As used herein, the term "pharmaceutically acceptable acid addition salts" refers to those salts that retain the biological potency and properties of the free base, which are not biologically or otherwise undesirable, and which are formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

As used herein, the term "pharmaceutically acceptable base addition salts" refers to those salts that retain the biological potency and properties of the free acid, which are not biologically or otherwise undesirable. These salts can be prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted ammoniums including naturally occurring substituted ammoniums, cyclic ammoniums, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like.

The compounds according to the invention or their pharmaceutically acceptable salts may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms and may be defined according to absolute stereochemistry, such as (R) -or (S) -, or such as (D) -or (L) -for amino acids. The present invention is intended to encompass all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R) -and (S) -, or (D) -and (L) -isomers can be prepared using chiral synthetic monomers or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. Racemic mixtures can be prepared and thereafter separated into individual optical isomers or these optical isomers can be prepared by chiral synthesis. Enantiomers can be resolved by methods known to those skilled in the art, for example, by formation of diastereomeric salts, which can then be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer-specific reagent. It will also be appreciated by those skilled in the art that the desired enantiomer may be converted to another chemical entity by separation techniques, and then additional steps may be required to form the desired enantiomeric form. One enantiomer may be converted to another alternatively specific enantiomer by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by asymmetric transformation.

Certain compounds according to the present invention may exist as a mixture of epimers. Epimers refer to diastereomers which have the opposite configuration at only one of the two or more stereocenters present in the respective compounds.

The compounds according to the invention may be present in zwitterionic form and the invention encompasses both the zwitterionic forms of these compounds and mixtures thereof.

In addition, the compounds according to the invention can also be present in both aqueous and anhydrous form. A hydrate comprising a compound of any of the formulae described herein. In a further embodiment, the compound according to any of the formulae described herein is a monohydrate. In an embodiment of the invention, the compounds described herein comprise about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less, by weight, of water. In other embodiments, the compounds described herein comprise about 0.1% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, or about 6% or more water by weight.

It may be convenient or desirable to prepare, purify, and/or handle the compounds in prodrug form. Thus, as used herein, the term "prodrug" refers to a compound that, when metabolized (e.g., in vivo), produces the desired active compound. Prodrugs are typically inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic performance. Unless otherwise indicated, reference to a particular compound also includes prodrugs thereof.

As used herein, the term "EC 50" refers to a concentration that causes a 50% increase in CD34+ CD45 RA-cell count compared to vehicle culture (DMSO).

As used herein, the term "hematopoietic stem cells" or "HSCs" refers to cells that have a pluripotency that allows them to differentiate into functional mature cells such as granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, megakaryocyte-producing platelets, platelets), and monocytes (e.g., monocytes, macrophages), and cells that are capable of regeneration while maintaining their pluripotency.

HSCs are part of the starting cell population. Alternatively, the cells of hematopoietic origin are obtained from the body or an organ of the body containing the cells. Such sources include unfractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph, and spleen. All of the above mentioned natural or unfractionated blood products can be enriched for cells having hematopoietic stem cell characteristics in a manner known to the person skilled in the art.

As used herein, the term "starting cell population" refers to a population of cells that identifies HSCs that comprise HSCs harvested from one of the aforementioned sources, as is known in the art. The starting cell population may be enriched in CD34+ cells, meaning that the cell population is selected based on the presence of the cell surface marker CD34 +. For example, CD34+ cells can be detected and counted using flow cytometry and fluorescently labeled anti-CD 34 antibody. In addition, the starting cell population can be used directly for expansion or freezing and storage for later time point applications.

During hematopoiesis, HSCs first branch into the progenitor stage, into the myeloid lineage and lymphoid lineage, and then differentiate into myeloid stem cells (mixed colony forming cells, CFU-GEMM) and into lymphoid stem cells, respectively. Further, the myeloid-like stem cells are differentiated into erythrocytes via erythroid burst forming cells (BFU-E) and erythroid colony forming cells (CFU-E), into thrombocytes via megakaryocyte colony forming cells (CFU-MEG), into monocytes, neutrophils and basophils via granulocyte-macrophage colony forming cells (CFU-GM), and into eosinophils via eosinophil colony forming cells (CFU-Eo), while the lymphoid stem cells are differentiated into T cells via T-lymphoid progenitors and into B cells via B-lymphoid progenitors. These myeloid stem cells and the various hematopoietic progenitor cells derived from them are identified by the performance of their clones formed on soft agar, semi-solid methylcellulose medium, etc., in the presence of various cytokines.

The invention also encompasses the use of a compound according to the invention and defined herein, or a salt thereof, in the manufacture of a medicament for the treatment of a patient suffering from the following non-limiting list of diseases: autologous or allogeneic transplantation or treatment of a subject (or patient) suffering from the above mentioned diseases or autoimmune diseases. Examples of hematological malignancies/diseases and congenital diseases can include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplasia and signs, multiple myeloma, non-hodgkin's lymphoma, hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, fanconi anemia, thalassemia, sickle cell anemia, wiskott-aldrich syndrome, congenital metabolic defects (e.g., gaucher disease, among others). Examples of immunological diseases that may benefit from transplantation are many and include multiple sclerosis, lupus, certain forms or arthritis, severe combined immunodeficiency, and the like.

Thus, the present invention comprises administering HSCs expanded with a compound according to the invention to a patient suffering from any one of the above mentioned diseases/malignancies.

In addition, the compounds and compositions as described may be used in the following non-limiting sites: autologous or allogeneic transplantation or treatment of subjects (or patients) suffering from the above mentioned diseases or autoimmune diseases. Examples of hematologic malignancies/diseases and congenital diseases can include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome, multiple myeloma, non-hodgkin's lymphoma, hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, fanconi anemia, thalassemia, sickle cell anemia, wiskott-aldrich syndrome, inborn errors of metabolism (e.g., gaucher disease, among others). Examples of immunological diseases that may benefit from transplantation are numerous and include multiple sclerosis, lupus, certain forms or arthritis, severe combined immunodeficiency, and the like.

Thus, the present invention comprises administering HSCs expanded with a compound according to the invention to a patient suffering from any one of the above mentioned diseases/malignancies.

Also encompassed by the present invention are cell populations obtained after expansion using the methods according to the present invention and described herein. Hematopoietic stem and progenitor cells can be harvested from adult, cord blood, fetal, or embryonic sources. Cell expansion using the methods of the invention can result in an increase in the number of progenitor cells, which is useful, for example, in accelerating the time to neutrophil and platelet engraftment. Such a method comprises: the starting population comprising HSCs is cultured using an agent capable of increasing the number of HSCs. The starting population may be enriched for the cell surface marker of interest or a combination thereof (e.g., CD34+, CD34+ CD45RA + /).

Methods for expansion of HSCs

Accordingly, the present invention relates to a method for expanding hematopoietic stem cells comprising (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo under suitable conditions for expanding hematopoietic stem cells.

Accordingly, the present invention relates to a method for expanding hematopoietic stem cells comprising (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing said starting cell population ex vivo under suitable conditions for expanding hematopoietic stem cells.

In a specific embodiment, the method for expanding hematopoietic stem cells comprises (a) providing a starting cell population comprising hematopoietic stem cells and (b) culturing the starting cell population ex vivo in the presence of a compound or composition of the invention.

The cell population is first subjected to an enrichment or purification step comprising negative and/or positive selection of cells based on a specific cell marker in order to provide a starting cell population. The methods for isolating the starting cell population based on specific cell markers may utilize Fluorescence Activated Cell Sorting (FACS) also known as flow cytometry or antibody or ligand-bound solid or insoluble matrices that interact with specific cell surface markers. For example, the cells can be contacted with a solid matrix containing the antibody (e.g., glass beads, flasks, magnetic particles), and any unbound cells removed. When a solid matrix comprising magnetic or paramagnetic glass beads is used, cells bound to the glass beads can be easily separated by a magnetic separator.

In one embodiment, the starting cell population is enriched in CD34+ cells. Methods for enriching a blood cell population in CD34+ cells include kits commercialized by Miltenyi Biotec (CD34+ direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) or by Baxter (Isolex 3000).

Cord blood from a single fetus is often insufficient in volume to treat an adult or older child. One advantage of the amplification method using the compounds or compositions of the invention is that it enables the production of a sufficient amount of hematopoietic stem cells from only one cord blood unit.

Thus, in one embodiment, the starting cell population is derived from neonatal cord blood cells that are already enriched in CD34+ cells. In a related embodiment, the starting cell population is derived from one or two cord blood units.

In another embodiment, the starting cell population is derived from human mobilized peripheral blood cells already enriched in CD34+ cells. In a related embodiment, the starting cell population is derived from human mobilized peripheral blood cells isolated from only one patient.

Preferably, the starting cell population may contain at least 50% CD34+ cells, in certain embodiments, more than 90% CD34+ cells.

The culture conditions of the starting cell population for hematopoietic stem cell expansion will vary depending on the starting cell population, the desired final cell number, and the desired final proportion of HSCs.

In a specific embodiment, in particular, using a starting cell population from cord blood cells enriched in CD34+ cells, culture conditions include the use of other cell expansion factors, cytokines and growth factors, commonly known in the art, for HSC expansion. Such cytokines and growth factors may be biological molecules or small molecules and they include, but are not limited to, IL-1, IL-3, IL-6, IL-11, G-CSF, GM-CSF, SCF, FIT3-L, Thrombopoietin (TPO), erythropoietin, and analogs thereof. As used herein, "analogs" include any structural variant of cytokines and growth factors that are biologically active as naturally occurring forms, including, but not limited to, variants that have enhanced or reduced biological activity when compared to naturally occurring forms or cytokine receptor agonists such as agonist antibodies to the TPO receptor (e.g., VB22B sc (fv)2, as detailed in patent publication WO 2007/145227, etc.). The cytokine and growth factor combination is selected to expand HSCs and progenitor cells while limiting the production of terminally differentiated cells. In a specific embodiment, one or more cytokines and growth factors are selected from the group consisting of SCF, FH3-L, and TPO.

Human IL6 or interleukin-6, also known as B-cell stimulating factor 2 and commercially available, has been described by (Kishimoto, Ann. review of 1mm.23: 12005). (Smith, M Aetal, ACTAHaematology, 105,3:143,2001) human SCF or stem cell factor, also known as c-kit ligand, mast cell growth factor or Steel factor, has been described and is commercially available. Flt3-L or FLT-3 ligand, also known as FL, is a factor that binds to the Flt 3-receptor. (Hannum C, Nature 368(6472):643-8) has been described and is commercially available. TPO or thrombopoietin, also known as megakaryocyte growth factor (MGDF) or c-MpI ligand, has been described (Kaushansky K (2006); N.Engl. J.Med.354(19):2034-45) and is commercially available.

The above-mentioned chemical components and biological components are used not only by adding them to the culture medium but also by fixing them onto the surface of the substrate or support for culture, specifically, by dissolving the components to be used in an appropriate solvent, coating the substrate or support with the resultant solution, and then washing off the excess components. Such ingredients used may be added to a substrate or support that is initially coated with a substance that binds to the ingredient.

The expansion of HSC may be carried out in a natural, semi-synthetic or synthetic medium according to the composition, and according to the shape, it may be a solid, semi-solid or liquid medium, and for the culture of hematopoietic stem cells and/or hematopoietic progenitor cellsAny nutrient medium fed, supplemented with a mixture of cell expansion factors as described above. Such media typically include sodium, potassium, calcium, magnesium, phosphorus, chloride, amino acids, vitamins, cytokines, hormones, antibiotics, serum, fatty acids, sugars, and the like. In the culture, other chemical or biological components may be incorporated singly or in combination, as the case requires. Such components incorporated in the medium may be fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, thioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various growth factors, and the like. Examples of such basal media suitable for methods of expanding HSCs include, but are not limited to, StemSpan^TMSerum-free expansion Medium (Vancouver, Stem cell technology, Canada), StemSpan^TMH3000-defined Medium (Vancouver, Stem cell technology, Canada), CellGro^TMSCGM (CellGenix, Frisbee, Germany), StemPro^TM-34SFM (Invitrogen), Dulbecco's Modified Eagle's Medium (DMEM), Ham's nutrient mixture H12 mixture F12, McCoy's 5A Medium, Eagle's Minimal Essential Medium (EMEM), aMEM Medium (a modified eagle's minimal essential Medium), RPMI1640 medium, Eschoft modified Dulbecco's Medium (IMDM), StemPro34(Invitrogen), X-VIVO 10(Cambrex), X-VIVO 15(Cambrex), and Stemline II (Sigma-Aldrich).

In one embodiment, the compound or composition of the invention is administered during the method of expansion of the starting cell population at a concentration suitable for HSC expansion. In a specific embodiment, the compound or composition is administered at a concentration comprised between 1 and 3000nmol or, for example, between 1 and 100 nmol.

In a particular embodiment, wherein the starting cell population consists essentially of CD34+ rich cells from one or both cord blood units, or from mobilized PB cells or from harvested bone marrow, the cells are grown under conditions for HSC expansion, e.g., between 2 to 21 days and/or until the indicated fold expanded and characteristic cell population is obtained. In a specific embodiment, the cells are grown ex vivo for no more than 21 days, 12 days, 10 days, or 7 days under conditions for HSC expansion.

The cell population can then be washed to remove the compound or composition of the invention and/or any other components of the cell culture, and resuspended in an appropriate cell suspension medium for short-term use or resuspended in a long-term storage medium, e.g., a medium suitable for cryopreservation.

Alternatively, after preliminary coating with extracellular matrix or cell adhesion molecules, HSC and/or hematopoietic progenitor cells can be cultured in culture vessels commonly used for animal cell culture, such as cover medium, flasks, plastic bags, Teflon^TMAnd (4) a bag. The materials used for such coating may be collagen L to XIX, fibronectin, vitronectin, laminin 1 to 12, nitrogen, tenascin, thrombospondin, von willebrand factor, osteopontin, fibrinogen, various elastin, various proteoglycans, various cadherins, desmocollin (desmocolin), desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, artificial basement membrane, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, alginate gel, hydrogel or fragments thereof. Such coating material may be a recombinant material with artificially modified amino acid sequences. Hematopoietic stem and/or progenitor cells can be cultured by using a bioreactor that can mechanically control the composition of the medium, pH, etc. and achieve high density culture (Schwartz R M, proc. natl. acad. sci. u.s.a.,88:6760,1991; Koller M R, Bone Marrow Transplant,21:653,1998; Koller, M R, Blood,82:378,1993; astorii G, Bone Marrow Transplant,35:1101,2005).

The invention further provides a cell population having expanded HSCs, obtainable or obtained by the expansion method described above. In a specific embodiment, such a population of cells is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition.

The invention further provides a cell population with expanded HSCs or a composition thereof for autologous or allogeneic stem cell transplantation in a mammalian subject.

For example, a subject as referred to herein is a bone marrow donor or an individual who has or is at risk of depleted or limited blood cell levels. Optionally, the subject is a bone marrow donor prior to bone marrow harvest or a bone marrow donor after bone marrow harvest. Optionally, the subject is a recipient of a bone marrow transplant. The methods described herein are particularly useful in subjects with limited bone marrow reserves, such as the above grade subjects or subjects previously exposed to an immunodeficiency therapy or a spinal cord transplantation therapy, such as chemotherapy (e.g., for the treatment of leukemia or lymphoma). Optionally, the subject has or is at risk of developing a reduced blood cell level as compared to a control blood cell level. As used herein, the term control blood cell level refers to the average level of blood cells of a subject prior to an event that alters the subject's blood cell level or in the actual absence of said event. Events that alter the subject's blood cell levels include, for example, anemia, trauma, chemotherapy, bone marrow transplantation, and radiation therapy. For example, the subject has anemia or blood loss due to, for example, trauma.

In addition to hematopoietic stem cells and/or hematopoietic progenitor cells expanded by the methods of the invention, the graft may also be a composition containing a buffer solution, an antibiotic, a drug.

For example, the expanded HSC population or the composition comprising a cell population with expanded HSCs is administered to the subject prior to, concurrently with, or following chemotherapy, radiation therapy, or bone marrow transplantation. Optionally, the subject consumes, e.g., bone marrow associated with an innate, genetic or acquired syndrome characterized by marrow depletion or depleted bone marrow. Thus, optionally, the subject is a subject in need of hematopoiesis. Optionally, the subject is a bone marrow donor or a subject with or at risk of depleting bone marrow.

Hematopoietic stem cell processing is useful as a complementary treatment to chemotherapy or radiation therapy. For example, HSCs are pooled into the peripheral blood and then isolated from a subject who will undergo chemotherapy, and after treatment, the cells are returned. Thus, a subject is a subject who is undergoing or is expected to undergo an immune cell depleting therapy such as chemotherapy, radiation therapy or is acting as a donor for a bone marrow transplant. Bone marrow is one of the most productive tissues in the body and, therefore, is the organ that is often first damaged by chemotherapeutic drugs and radiation. As a result, blood cell production is rapidly destroyed during chemotherapy or radiation treatment, and chemotherapy or radiation must be terminated to allow hematopoietic cells to replenish the blood cell supply before the patient is treated again with chemotherapy. Thus, as described herein, HSCs or blood cells produced by the methods described herein are optionally administered to such a subject in need of additional blood cells.

HSCs expanded by the compounds or compositions of the invention described above are provided in combination with a therapeutic agent capable of enhancing the proliferation of HSCs in vivo, in vitro, or ex vivo (e.g., small molecules, antibodies, etc.) and optionally, at least one pharmaceutically acceptable excipient or carrier. A therapeutic agent capable of enhancing HSC proliferation means: agonist antibodies to the TPO receptor (e.g., VB22B sc (fv)2, described in detail in patent publication WO 2007/145227, etc.); cytokines such as SCF, IL-6, Flt-3 ligand, TPO or TPO mimetic (e.g., as described in WO/2007/022269; WO/2007/009120; WO/2004/054515; WO/2003/103686; WO/2002/085343; WO/2002/049413; WO/2001/089457; WO/2001/039773; WO/2001/034585; WO/2001/021180; WO/2001/021180; WO/2001/017349; WO/2000/066112; WO/2000/035446; WO/2000/028987; WO/2008/028645, etc.); granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); prostaglandin or a prostaglandin receptor agonist (e.g., prostaglandin E2 receptor-1 (EP-1) agonist, prostaglandin E2 receptor-2 (EP-2) agonist, prostaglandin E2 receptor-3 (EP-3) agonist, and prostaglandin E2 receptor-4 (EP-4) agonist, as detailed in patent publication WO/2008/073748); tetraethylpentamine (TEPA); notch-ligand (Delta-1); and/or a WNT agonist. In addition, culturing stem cells with Mesenchymal Stem Cells (MSCs) prevents Graft Versus Host Disease (GVHD) and may aid in stem cell expansion.

Pharmaceutically acceptable refers to materials that are not biologically or otherwise undesirable, i.e., the materials can be administered to a subject or cell without causing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition contained therein. The carrier or excipient is selected to minimize degradation of the active ingredient and to minimize adverse side effects on the subject or cell.

The compositions are formulated in any conventional manner for use in the methods described herein. Administration is via any route known to be effective by the skilled person. For example, the composition is administered orally, parenterally (e.g., intravenous injection), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, intranasally, or topically.

The preferred method of administration is intravenous injection. The number of cells infused will take into account factors such as sex, age, weight, type of disease or disorder, stage of disorder, percentage of cells desired in the cell population, and the amount of cells needed to produce a therapeutic benefit. In one embodiment, the compositions are administered by intravenous injection and include at least ≧ 0.3x10⁵CD34⁺/kg or > 2x 10⁶CD34⁺Cord blood and 2.5X 10⁵CD34⁺/kg or more bone marrow or mobilized peripheral blood cells. In a specific embodiment, the injected cells are all cells derived from cord blood cells expanded from a single fetus.

For example, in the case of treatment of leukemia, the expanded hematopoietic stem cells and/or hematopoietic progenitor cells can be injected by instillation into a patient who is pre-treated with an anticancer drug, systemic irradiation, or an immunosuppressive drug, for the elimination of cancer cells or for the facilitation of donor cell engraftment. Suitably, the disease to be treated, the pre-treatment and the cell transplantation method are selected by the responsible person. Such engraftment of the transplanted hematopoietic stem cells and/or hematopoietic progenitor cells in the recipient, restoration of hematopoietic function, side effects of transplantation, and the presence of therapeutic effects of transplantation can be judged according to ordinary tests used in transplantation therapy.

As described above, the present invention makes it possible to expand hematopoietic stem cells and/or hematopoietic progenitor cells, and to perform transplantation therapy safely and easily in a short period of time by using expanded HSCs.

Also provided herein are kits comprising one or more containers filled with one or more of the ingredients described herein. Optionally, such kits include solutions and buffers as needed or desired. Optionally, the kit comprises an expanded population of stem cells produced by the methods described above or a container or composition that can contain the expanded population for producing HSCs. In particular, the invention provides a kit for expanding ex vivo hematopoietic stem cells, comprising a compound as defined in the summary of the invention and instructions for the use of such a compound in a method of HSC expansion, and optionally, one or more cell expansion factors, or a medium for cell growth, in particular, for HSC growth as described above. The kit may further comprise antibodies for monitoring the growth of cells, such as anti-CD 34, anti-CD 38, and/or anti-CD 45RA antibodies. In a specific embodiment, such a kit further comprises one or more cell expansion factors selected from the group consisting of IL6, FLT3-L, SCF and TPO. Optionally, associated with such a package or kit is instructions for use.

In vivo application: also provided are kits for providing an effective amount of a compound of the invention to increase HSCs in a subject, including one or more doses of the compound for use over a period of time, wherein the total number of doses of the compound of the invention in the kit is equal to an effective amount sufficient to increase HSCs in the subject. The period of time is from about one day to several days or weeks or months. Thus, a period of time is from at least about 5,6, 7, 8, 10, 12, 14, 20, 21, 30, or 60 days or more or any number of days between one day and 180 days.

Biological experiments

Screening experiments:

to identify new putative agonists of HSC self-renewal, we adapted a high throughput-based-screening assay to examine a pool of small molecule compounds (5280 low molecular weight compounds) on primary human mobilized CD34+ cells. It is understood that the same approach applies to CD34+ cells from various sources known to those skilled in the art to isolate CD34+ cells. Monocytes were stained with mouse anti-human CD34+ APC (from BD mab) and subsequently magnetically labeled with anti-APC magnetic microspheres (MicroBeads) (from MACS, Miltenyi Biotec). Magnetically labeled cells were retained using an AutoMACS column. Our studies are based on the fact that mobilized peripheral blood-derived CD34+ CD45 RA-cells cultured in media supplemented with interleukin-6, thrombopoietin, Flt-3 ligand, and stem cell factor will promote expansion of Monocytes (MNC) with a reduction in the CD34+ CD45 RA-population and HSC depletion. Thus, low molecular weight compounds that prevent this loss can act as agonists of HSC expansion.

2000CD34+ cells/well were cultured in 50. mu.l of medium containing 1 μm of test compound or 0.1% DMSO (vehicle) in 384-well plates. The proportion of CD34+ CD45 RA-cells was determined at the beginning of the experiment and after 7-day incubation. Six of the 5280 compounds of different chemical backgrounds examined first, promoted CD34+ CD45 RA-cell expansion, and seventeen (17) enhanced differentiation, as determined by a proportional increase in CD34+ CD45 RA-cells compared to control (DMSO). In the second screen, the CD34+ CD45 RA-cell population was again analyzed for six compound-promoted expansion. Four of these six compounds act as arene receptor (AhR) antagonists, showing a mechanism of action (identical to SR1) that promotes ex vivo expansion of huCD34+ cells. During the 7-day incubation period, the remaining two compounds (identified as not arene receptor (AhR) antagonists) were shown to promote the expansion of MNCs comprising CD34+ cells. One of those two remaining compounds was identified as compound 1 (table 2).

The following biological experiments were conducted to evaluate the effect of the compounds of the present invention on hematopoietic stem cell expansion. Culture medium: in the presence of vehicle (DMSO), positive control (SR1), or a compound or combination of compounds of the invention, the culture medium used consisted of serum-free medium supplemented with the following recombinant cytokines: interleukin-6, thrombopoietin, Flt-3 ligand, and stem cell factor, each at a final concentration of 100 ng/ml. Cell culture: the initially harvested CD34+ cells were more than 90% pure as determined by flow cytometry. The CD34+ CD45 RA-subpopulation reached a purity higher than 70%. Cells were seeded at 40,000 cells/ml and 5% CO at 37 ℃₂Incubate for 7 to 12 days. For long-term culture, 200,000CD34+ cells/ml of mobilized PB were inoculated using serum-free medium supplemented with interleukin-6, thrombopoietin, Flt-3 ligand, and stem cell factor, each at a final concentration of 100ng/ml, in the presence of 500nM of vehicle (DMSO), a positive control, or a compound of the invention. After 10 days of ex vivo culture, compound 1 (table 2) promoted more than 7-fold expansion of MNCs, 5-fold increase of CD34+ cells over the input value (day 0), and almost 4-fold increase over the value used for the medium determination. After 10 days of ex vivo culture, compound 1-treated cells retained high levels of CD34 amplification (65.8 ± 5.5%) compared to cells cultured with vehicle (DMSO) (22.8 ± 0.9%). Furthermore, only compound 1-treated cells retained the highest expression of the CD34+ CD45 RA-population (24.8 ± 0.9%) compared to vehicle-treated (4.7 ± 0.4%). The number of CD34+ CD45 RA-cultured with Compound 1 was almost 3-fold increased compared to the vehicle, and 7-fold increased over the input number. Finally, the compounds of the invention were analyzed in a dose-responsive format (concentrations ranging from 1nM to 5000nM) to determine the effective concentration that produced a 50% increase in the number of CD34+ CD45RA "compared to vehicle conditions. The results are shown in table 2.

Compound 1 does not act via the aromatic hydrocarbon (AhR) pathway (FIG. 1)

We next demonstrated that the effect of compound 1 on primitive CD34+ CD45 RA-primitive hematopoietic cells was rapidly reversible in culture. This effect is best shown in figure 2, where CD34+ mobilized peripheral blood cells were cultured for up to 7 days in the presence of compound 1. At that time, compound 1 was removed by washing the cells. The dashed green line shows that the proportion of CD34+ CD45 RA-cells decreased proportionally to the culture immediately following the control (DMSO: solid blue line and dashed line), whereas cells fed in the presence of compound 1 retained more of the original phenotype throughout the 2-week culture (solid green line). These results clearly indicate that within 2 days of no compound exposure, as seen in control cultures, the cells have acquired a differentiation marker. Thus, the effect of compound 1 on naive human cells is rapidly reversible.

Compound 1 is not a mitogen (mitogen) (FIG. 3)

We also show that compound 1 is unable to independently trigger cell proliferation in the absence of growth factors. These results, shown in figure 3, indicate that compound 1 was unable to induce cell proliferation in the absence of any of the 3 listed growth factors, i.e., Flt3, TPO, and SCF, similar to that observed with antagonists of the arene receptor (SR 1). This indicates that, similar to SR1, the effect of compound 1 on the ex vivo feeding of primitive HSC phenotypes is not due to mitogenic effects on this population, but rather to the effects on the prevention of cell differentiation.

Both Compound 1 and Compound 40 prevent cell differentiation and act synergistically with AhR antagonists (FIG. 4)

We also evaluated the effect of compound 1 and one of its potent derivatives, compound 40, on cell differentiation using cytological analysis and flow cytometry. Both types of studies showed that compound 1 and its derivative compound 40 prevented cell differentiation. The FACS results are shown in fig. 4. Figure 4A is a graph showing the effect of compound 1 on the differentiation of mobilized peripheral blood cells. After 7 days of amplification, relatively pure (> 85% CD34+) mobilized peripheral blood was exposed to control (DMSO) or compound 1 or SR1 or compound 1+ SR 1. The results strongly indicate that in control cultures, cells rapidly released CD34 cell surface expression, however, this effect was partially abolished by introducing optimal levels of SR1 and compound 1. Interestingly, SR1 and compound 1 played a synergistic role in maintaining CD34 expression on the cell surface. These observations were repeated for the cord blood sample in fig. 4B and with compound 40 of fig. 4C.

In addition to this, we show that the effect of compound 1 or compound 40 on cells with more primitive phenotypes is most significant. For example, CD34+ CD45 RA-cells (top left quarter of the bottom panels of fig. 4A to 4C) were more in cultures supplemented with compound 1 or 40 than they were in SR1 or in control cultures. The additive effect of compound 1 or compound 40 plus SR1 was again observed in these cultures.

Fig. 4D provides a dose-response curve indicating the efficacy of compound 40 in preventing the disappearance of CD34 marker on the surface of the original human HSC-enriched population. The expression of CD34 was recorded as a function of different doses of the compound.

Compounds 1 and 40 ex vivo expansion of human HSC phenotypes (FIG. 5)

Figure 5 shows that not only compound 1 but also compound 40 expanded human HSC phenotypes ex vivo in both short-term and long-term culture. Figure 5A shows that total cell technology increased approximately 20-fold in input in cultures initiated with CD34+ mobilized peripheral blood (mPB) and fed for 12 days. The level of amplification was also the same, whether culture was initiated with compound 1, SR1, compound 1+ SR1, or control DMSO. Most significantly, the effect of compound 1 on a larger subpopulation of CD34+ CD45RA cells was observed, the population expanded approximately 10-fold in the presence of compound 1 and approximately fifteen-fold in the presence of compound 1+ SR 1. This observation, together with the results presented in figure 4, strongly suggests that compound 1 has no effect on cell proliferation, but prevents differentiation of CD34+ CD45RA cells, causing their pure expansion at the expense of more mature cells. The results of figure 5B indicate that compound 1 will cause a 30-40-fold expansion of CD34+ CD45 RA-cord blood cells over a seven day cycle, whereas these cells expand approximately fifteen-fold in the presence of DMSO (control). Figure 5C shows similar results, but the time of incubation was extended to twelve days and compound 1 was replaced by compound 40. Again, as indicated in the left panel, the total cell expansion was the same regardless of whether the cells were exposed to DMSO (control), SR1, compound 40, or compound 40 plus SR 1. Even more impressively, more of the original CD34+ CD45 RA-cells were expanded 80-fold at 12 days in culture supplemented with compound 40, whereas these cells were expanded slightly less than 20-fold in culture initiated with SR1, indicating the superiority of this compound over an arene inhibitor antagonist.

Taken together, these results show that compound 1 and compound 40 are directed against CD34⁺And more primitive CD34⁺CD45RA^-The expansion of the population has a major impact, both with CD34+ derived from mobilized peripheral blood or cord blood cells.

The expanded cells were tested for functionality ex vivo using a conventional culture colony forming unit (CFU-C) assay. Untreated cells or cells incubated with DMSO, positive controls or compounds of the invention were seeded in methylcellulose medium under conventional conditions. As an example, compound 1 (table 2, example 1) expanded a number of multipotent hematopoietic progenitor cells. Methylcellulose cultures of 1000CD34+ mPB cells treated with compound 1 for 10 days resulted in a 5-fold increase of multipotent granulocytes, erythrocytes, macrophages and megakaryocytes (GEMM clones) over the input cells and a 10-fold increase compared to control cells. This suggests that compound 1 described herein also promotes the expansion of pluripotent progenitor cells.

The effect of compound 1 on cultured mPB HSCs was evaluated using the NSG mouse model.

Next, we evaluated the effect of compound 1 and compound 40 on cord blood and mobilized peripheral blood human HSCs expanded ex vivo for ten to twelve days and introduced into the NSG mouse model. The purpose of these experiments was to demonstrate that the effect of our compounds on the primitive HSC phenotype shown in fig. 4 and 5 was also observed on long term re-infused bone marrow stem/progenitor cells. Figure 6 shows the reconstitution of mouse bone marrow of human cells evaluated thirteen weeks after transplantation. For mobilized blood, 50,000 and 500,000 cell outcomes are presented in fig. 6A. As shown here, the HSC agonist SR1 was consistently better than DMSO (control) in expanding human stem cells as evaluated in the NSG mouse model. Again, compound 1 appeared to outperform SR1 in these experiments. As seen in vitro culture, compound 1 and SR1 showed synergistic effects in these assays (fig. 6).

Effect of Compounds 1 and 40 on cultured umbilical Cord Blood (CB) HSC assessed using the NSG mouse model (FIG. 7)

Figure 7 shows the effect on cultured cord blood human HSCs evaluated in vivo in an NSG mouse model. The results in fig. 7A indicate that compound 1 has a significant effect on the reconstitution activity of human cells when compared to control cultures. These experiments were done in short term culture, i.e., 7 days. Most importantly, figure 7B indicates that compound 40 has a fairly important effect of 10% of the mean level of reconstitution when using 1500CD34+ cells compared to 2% for the DMSO control. The effect of compound 40 in this assay was greater than that of compound 1 (fig. 7A) probably due to the longer incubation period used in the assay described in fig. 7B. In the next section, a longer incubation period (12 to 16 days) was used to provide a more definitive in vivo test.

Effect of Compounds 1 and 40 on cultured umbilical Cord Blood (CB) HSC assessed using the NSG mouse model (FIG. 8)

Zandstra et al recently demonstrated that a new method called fed-batch culture optimized in vitro conditions that caused expansion of human HSCs (U.S. Pat. No. US7,795,024). We wanted to demonstrate whether the compounds of the invention work with these previously optimized fed-batch culture conditions. For these studies, we followed the evaluation of expansion of cord blood-derived Hematopoietic Stem Cells (HSCs) using fed-batch culture + compound 4012 days or 16 days in vitro culture. HSC numbers were assessed based on engraftment of human cells into immunodeficient mice.

As shown in figure 8, up to at least 16 days, the addition of compound 40 (Cpd40) provided a major effect on the expansion of all tested populations containing CD34+ CD45 RA-cells. This effect was most pronounced in the 12-16 day time point, clearly demonstrating the synergistic effect of fed-batch culture (FB in fig. 8) and compound 40.

In addition, freshly enriched CD34+ cells and 12 or 16 day expanded cells were transplanted into female NOD/SCID/IL-2R yc-Null (NSG) mice, which had been irradiated 24h sublethally (250 rads) prior to transplantation. Cells were injected intravenously via tail vein. At defined time points (3 weeks, 9 weeks, and 16 weeks), animals were sacrificed and bone marrow was harvested from both tibias and both femurs. Bone marrow was emptied of red blood cells and evaluated by flow cytometry to quantify engraftment of human cells. Cells were scored as graft positive if ≧ 0.5% of the cells were positive for human CD45 and human HLA-ABC. At each time point of evaluation, some animals were shipped for independent histological analysis.

As shown in table 1 below, there was a dose response of the grafts for each condition at the late 16-week time point. Limiting dilution analysis showed the highest expansion of HSCs (18.4-fold) by fed-batch plus compound 4012 days of culture. This is significantly higher (8.1-fold) than the amplification produced by fed-batch (fed-batch) control 12-day culture. Both conditions produced higher amplification with the 12 day culture compared to the 16 day culture. These results provide clear evidence of expansion of pure human HSCs in vitro in the presence of compound 40 and its activity in fed-on culture conditions (figure 8+ table 1).

Table 1: summary of 16 week limiting dilution grafting data

Pure HSC expansion (# HSC 0 days)/(# HSC 12 or 16 days); measured using Limiting Dilution Analysis (LDA) in NSG mice at 16 weeks.

Synthesis method

The synthetic methods outlined below relate to embodiments of the present invention in which the substituent Z is at the 7-position of the pyrimidoindole nucleus. As the skilled person will appreciate, a similar synthetic method may be performed with variations apparent to the skilled person for embodiments of the invention wherein the substituent Z is at a different position, such as, for example, at the 5, 8 or 6-position, in particular at the 6-position.

Scheme 1 describes the synthesis of common precursors (1-VI) to the compounds of the present invention. In the first step, the aryl fluoride 1-I is treated with alkyl cyanoacetate 1-II in the presence of a base such as, but not limited to, sodium hydride. The synthesized product 1-III is then treated with a reducing agent such as, but not limited to, zinc powder in acetic acid to provide the aminoindole 1-IV, which is converted to the pyrimidine 1-V upon treatment with formamide and ammonium formate. Compounds 1-V are treated with reagents such as phosphorus oxychloride or phosphorus tribromooxyboride to provide reaction intermediates 1-VI, which are treated with amines 1-VII to provide compounds 1-VIII of the present invention.

Scheme 1

Scheme 2 describes the preparation of compounds 2-V. The reaction of 4, 6-dichloro-5-iodopyrimidine 2-II and the corresponding phenol, aniline or thiophenol 2-I is carried out with or without a base (Morsin m.et al chemistry-aeuropen Journal,2009, vol.15, #6, pp.1468-1477) or palladium catalyst to give intermediate 2-III. The synthetic intermediate 2-III was converted to the tricyclic adduct 2-IV using Pd (OAc)2 (Zhang M. et al. tetrahedron letters,2002, vol.43, p.8235). Finally, compounds 2-V of the present invention were obtained according to example 1, herein outlined below.

Scheme 2

Scheme 3: compound 4-II (Tully W. R. et al. journal of Medicinal Chemistry,1991, vol.34, p.2060) was treated with hydroxylamine and then with dimethylacetamide dimethyl acetal. This gave compound 5-I.

Scheme 3

Scheme 4: intermediate 1B (example 1 herein below) was treated with propionitrile in HCl/dioxane, followed by base treatment to afford methyl 2-ethyl-4-hydroxy-9H-pyrimido [4,5-B ] indole-7-carboxylate (6-I). Then, according to the procedure described for example 1, compound 6-II was obtained.

Scheme 4

Scheme 5: starting from methyl 3-fluoro-4-nitrobenzoate 7-I, and following the procedure of example 1, compound 7-II was obtained.

Scheme 5

General of

The reported HPLC retention times are for reverse phase HPLC (Agilent, 1200 series) solvent A: MeOH: H using the following conditions₂TFA (5: 95: 0.05); solvent B MeOH H₂TFA (95: 5: 0.05); flow rate: 3.0 mL/min; gradient 0 to 100% in 2.0 min; column: ZorbaxC18, 3.5 microns, 4.6x 30 mm; the wavelength is 220 nm.

Mass spectra were recorded on a 6210G1969A LC/MSD TOF spectrometer from agilent technologies or on a Quadrupole LC/MS Model G6120B from agilent technologies using the following conditions: solvent A: AcCN: H₂O: HCOOH (5: 95: 0.05); solvent B: AcCN: H₂O: HCOOH (95: 5: 0.05); gradient 0 to 100% in 2.0 min; flow rate: 0.3 mL/min; column: ZorbaxC18, 3.5 microns, 2.1x 30 mm; the wavelength is 220 nm.

Experimental procedures

Example 1

4- ((3- (Piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b ] indole-7-carboxylic acid methyl ester

Intermediate 1A

4- (1-cyano-2-ethoxy-2-oxoethyl) -3-nitrobenzoic acid methyl ester

Ethyl 2-cyanoacetate (10.9mL, 102mmol) was added slowly to a 60% suspension of sodium hydride (4.10g, 102mmol) in N, N-dimethylformamide (125mL) at 0 ℃ to give a grey suspension. The mixture was stirred at 0 ℃ for 15 minutes and a solution of methyl 4-fluoro-3-nitrobenzoate (10.2g, 51mmol) in N, N-dimethylformamide (125mL) was added. The resultant dark red mixture was stirred at 0 ℃ for 30 minutes and at room temperature for 3 hours. The reaction mixture was diluted with 1N HCl (40mL) and ethyl acetate (40 mL). The separated aqueous layers were extracted with ethyl acetate (3X 50 mL). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a residue (26g), which was purified by flash-chromatography (starting with 100% hexane and adding stepwiseEthyl acetate was added in increments to include 100% ethyl acetate) to provide 14.9g of the title compound. LCMS M/z 291.0(M-H)^-Retention time (on analytical HPLC) 1.76 min.

Intermediate 1B

3-Ethyl-6-methyl-2-amino-1H-indole-3, 6-dicarboxylic acid ester

In a 500mL round bottom flask, in acetic acid (255mL), methyl 4- (1-cyano-2-ethoxy-2-oxoethyl) -3-nitrobenzoate (14.9g, 51.0mmol) and zinc powder (16.7g, 255mmol) were added to give a grey suspension. The addition of zinc was completed over 35 minutes at room temperature under nitrogen atmosphere, which was quite exothermic. The mixture was heated at 100 ℃ for 15 hours. The mixture was allowed to cool, filtered through celite and rinsed with ethyl acetate. Evaporation afforded a residue, which was triturated in dichloromethane-hexane, and after filtration, afforded 6.3g of the title compound. LCMS M/z263.2(M + H)⁺Retention time (on analytical HPLC) 1.90 min.

Intermediate 1C

4-hydroxy-9H-pyrimido [4,5-b ] indole-7-carboxylic acid methyl ester

In a 100mL round bottom flask, 3-ethyl 6-methyl 2-amino-1H-indole-3, 6-dicarboxylate (1.1g, 4.19mmol), ammonium formate (0.53g, 8.39mmol), and formamide (16.7mL, 419mmol) were added to produce a tan suspension, which was heated to 165 ℃ for 12 hours. The mixture was allowed to cool to room temperature and water was added. The synthesized precipitate was filtered, air dried and dried under high vacuum overnight to provide 1.1g of the title compound. LCMS M/z 244.2(M + H)⁺Retention time (in)Analytical HPLC) ═ 1.51 minutes.

Intermediate 1D

4-chloro-9H-pyrimido [4,5-b ] indole-7-carboxylic acid methyl ester

In a 100mL round-bottom flask, 4-hydroxy-9H-pyrimido [4,5-b ] was placed]A mixture of methyl indole-7-carboxylate (1.1g, 4.5mmol) and phosphorus oxychloride (15mL, 161mmol) was heated to 90 deg.C, heated for 16 hours, cooled to room temperature, and evaporated under reduced pressure. The residue was suspended in dichloromethane (20mL) and filtered through celite. Evaporation afforded the title compound (360mg) as an orange solid. LCMS M/z 262.0(M + H)⁺Retention time (on analytical HPLC) 2.02 min.

Example 1

4- ((3- (Piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b ] indole-7-carboxylic acid methyl ester

In a 2-5mL microwave glass vial, 4-chloro-9H-pyrimido [4,5-b ] in methanol (2mL) was added]Indole-7-carboxylic acid methyl ester (86mg, 0.33mmol), triethylamine (0.09mL, 0.66mmol) and 3- (piperidin-1-yl) propan-1-amine (0.078mL, 0.49mmol) and in a microwave reactor, the mixture was heated to 140 ℃ for 15 minutes. The mixture was allowed to cool to room temperature and evaporated under reduced pressure. The starting material was dissolved in N, N-dimethylformamide and purified on a reverse phase Zorbax SB-C18 column 21.2x 100mm and eluted with MeOH-water-0.1% TFA. Gradient: isocratic 20% for 4 min, then, over 15min, to a 100% MeOH gradient. The title compound was obtained as the trifluoroacetate salt (prepared according to standard procedures known to those skilled in the art fromFrom bases and HCl salts). LCMS M/z 368.2(M + H)⁺Retention time (on analytical HPLC) 1.38 min.

Example 14

7- (1-methyl-1H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b ] indol-4-amine and 7- (2-methyl-2H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b ] indol-4-amine

Intermediate 14A

4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b ] indole-7-carbonitrile

Intermediate 2A was prepared according to the procedure described in example 1 starting from 4-fluoro-3-nitrobenzonitrile.

Intermediate 14B

N- (3- (piperidin-1-yl) propyl) -7- (2H-tetrazol-5-yl) -9H-pyrimido [4,5-b ] indol-4-amine

In a 2-5mL microwave glass vial, 4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4, 5-b) was added to (trifluoromethyl) benzene (2mL)]Indole-7-carbonitrile (47.5mg, 0.142mmol) and tri-n-butyltin azide (409. mu.l, 1.491mmol) to give a tan suspension. The glass vial was placed in a microwave and heated to 180 ℃ for 30 minutes. The mixture was concentrated to dryness and MeOH (3mL) followed by HCl 4M in dioxane (1.07mL, 4.26mmol) was added to obtainA yellow solution was obtained. To the synthesized solution, diethyl ether (3mL) was added. Stirred at 20 ℃ for 16 hours. The solid obtained was collected on a Buchner. The cake was washed with diethyl ether (3 × 1mL) and hexane (3 × 1mL), and the solid was dried under high vacuum at 30 ℃ until constant weight to provide 56mg of the title compound as the HCl salt. LCMS M/z 378.2(M + H)⁺Retention time (on analytical HPLC) 1.30 min.

Example 14

7- (1-methyl-1H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b ] indol-4-amine and 7- (2-methyl-2H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b ] indol-4-amine

In a 25mL round bottom flask, to tetrahydrofuran (2mL) and methanol (0.5mL) was added N- (3- (piperidin-1-yl) propyl) -7- (2H-tetrazol-5-yl) -9H-pyrimido [4, 5-b)]Indol-4-amine hydrochloride (43mg, 0.10mmol) and N, N-diisopropylethylamine (36. mu.l, 0.21mmol) to give a tan suspension. Then, 2M hexane solution (260. mu.l, 0.52mmol) of trimethylsilylated diazomethane (Trimethylsilyldiazomethane) was added. The resulting thin yellow suspension was stirred at 20 ℃ for 3 hours, and acetic acid (59. mu.l, 1.04mmol) was added. Stirring was continued for 30 minutes and the solvent removed in vacuo to provide a residue which was purified by flash-chromatography (starting with 100% dichloromethane and adding dichloromethane: methanol: 28% wt. aqueous amine hydroxide (90: 10: 1) incrementally to include 100% dichloromethane: methanol: 28% wt. aqueous amine hydroxide (90: 10: 1)). The first eluted product obtained was 7- (2-methyl-2H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4, 5-b)]Indol-4-amine (19 mg). LCMS M/z 392.2(M + H)⁺Retention time (on analytical HPLC) 1.44 min. The second eluted product was 7- (1-methyl-1H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b]Indol-4-amine (5 mg). LCMS M/z 392.2(M + H)⁺Retention time (on analytical HPLC) 1.27 min.

Example 15

NaH (3.41g, 85mmol) was added portionwise to a cold solution of 2-cyanoacetamide (7.18g, 85mmol) in DMF (53 mL). After 30 minutes at room temperature, a solution of methyl 4-fluoro-3-nitrobenzoate (8.5g, 42.7mmol) in 15mL of DMF was added dropwise. After 3 hours, a mixture of ice, water and 12ml hcl (10%) was added. The synthesized solid was filtered, washed with water and dried under high vacuum overnight to give 9.1g of methyl 4- (2-amino-1-cyano-2-oxoethyl) -3-nitrobenzoate:¹H NMR(400MHz,DMSO-d₆)δppm3.93(s,3H)5.78(s,1H)7.77(s,1H)7.91(d,J＝7.83Hz,1H)8.04(s,1H)8.39(dd,J＝8.02,1.76Hz,1H)8.56(d,J＝1.56Hz,1H)。

ferric chloride hexahydrate (1.540g, 5.70mmol) and zinc (1.242g, 19.00mmol) were added to a solution of the above prepared crude cyano-amine based compound (0.5g, 1.900mmol) in DMF (4.75mL) and water (4.75mL) to give a yellow suspension. After the exotherm, the mixture was heated to 100 ℃, heated for 45 minutes, and then slowly cooled to 20 ℃, and stirred for 22 hours. The solid was filtered, washed with DMF (3X 3mL) and the filtrate diluted with water (40mL) while stirring at 0 ℃. The solid was filtered and the cake was washed with water (2X 5 mL). The solids usually contain impurities. The aqueous layer was extracted with EtOAc (3X 50mL) and the combined organic layers were washed with water (50mL) and then brine (30 mL). Over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to give 287mg of solid as brown, which was treated with acetone (6mL) to give a solid suspension, which was diluted with hexane (5 mL). The solid was then collected and dried under high vacuum at 40 c until constant weight,to give intermediate 15B 2-amino-3-carbamoyl-1H-indole-6-carboxylic acid methyl ester (162mg, 36.6% yield) as an off-white solid:¹H NMR(400MHz,DMSO-d₆)δppm 3.80(s,3H)6.62(br.s.,2H)7.04-7.18(m,2H)7.53-7.63(m,2H)7.72(s,1H)10.80(s,1H)；MS m/z 232.2(M+H)⁺(ii) a Hplcca.96%, RT 1.37 min.

A mixture of intermediate 15B (0.100g, 0.429mmol), methyl 2- (pyridin-3-yl) acetate (0.130g, 0.858mmol) and a 25% solution of sodium methoxide in MeOH (0.196mL) in methanol (0.954mL) was placed in a microwave oven and heated to 140 deg.C for 45 minutes. After cooling, AcOH (0.050mL, 0.879mmol) was added, and the resulting slurry was stirred at 20 ℃ for 1 hour. The solid was filtered, washed with MeOH (3 × 0.5mL), and dried under high vacuum at 20 ℃ until constant weight to yield intermediate 15C as a brown solid: 4-hydroxy-2- (pyridine-3-methylene) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (82mg, 57.2% yield):¹H NMR(400MHz,DMSO-d₆)δppm 3.87(s,3H)4.09(s,2H)7.34-7.40(m,1H)7.79(dt,J＝8.1,1.8Hz,1H)7.83(dd,J＝8.2,1.2Hz,1H)7.99(d,J＝0.8Hz,1H)8.02(d,J＝8.2Hz,1H)8.48(dd,J＝4.9,1.4Hz,1H)8.60(d,J＝2.0Hz,1H)12.49(br.s.,2H):MS m/z 335.2(M+H)⁺(ii) a HPLC 95.2% @220nm and 92.8% @254nm, RT ═ 1.42 min.

Reacting 4-hydroxy-2- (pyridine-3-methylene) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (0.050g, 0.150mmol) in POCl₃The mixture in (0.948mL, 10.17mmol) was placed in a glass vial and heated to 175 ℃ in a microwave oven for 15 minutes. After cooling, the reaction mixture was poured into ice water (19mL), then basified to pH8 by slow addition of 50% aqueous NaOH (2.7mL), and finally diluted with EtOAc (20mL), the solid was filtered (first harvest of chloride) and the aqueous layer was extracted with EtOAc (20mL) and over anhydrous MgSO₄The combined organic layers were dried, filtered and concentrated to dryness to provide an additional 35mg harvest of the desired chloride derivative. Directly in the next step using 4-chloro-2- (pyridine-3-methylene) -9H-pyrimido [4,5-b]Isolated from a combination of indole-7-carboxylic acid methyl ester (53mg, 100 yield)And (6) harvesting.

Reacting 4-chloro-2- (pyridine-3-methylene) -9H-pyrimido [4,5-b]A mixture of methyl indole-7-carboxylate (0.053g, 0.150mmol), 3- (piperidin-1-yl) propan-1-amine (0.072mL, 0.451mmol) and triethylamine (0.063mL, 0.451mmol) in MeOH (2.5mL) was placed in a glass vial and heated to 140 deg.C in a microwave oven for 15 minutes. After cooling and evaporation of the solvent, the residue was purified by flash chromatography to yield 23mg of a yellow oil and a solid, using CH₃CN (3mL) diluted and stirred for 30 min. Filtration of solids and utilization of CH₃CN (2 × 0.5mL) and then dried under high vacuum at 30 ℃ until constant weight to provide the compound of example 15 as a tan solid: 4- ((3- (piperidin-1-yl) propyl) amino) -2- (pyridin-3-ylidene) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (11mg, 16% yield): 1H NMR (400MHz, DMSO-d)₆)δppm 1.31-1.44(m,2H)1.44-1.56(m,4H)1.71-1.86(m,2H)2.17-2.47(m,6H)3.56-3.66(m,2H)3.88(s,3H)4.08(s,2H)7.28-7.34(m,1H)7.43(t,J＝5.5Hz,1H)7.76(dt,J＝7.8,2.0Hz,1H)7.81(dd,J＝8.2,1.4Hz,1H)7.99(d,J＝1.4Hz,1H)8.35(d,J＝8.2Hz,1H)8.41(dd,J＝4.7,1.6Hz,1H)8.59(d,J＝2.0Hz,1H)12.08(s,1H)；MS m/z 459.2(M+H)⁺(ii) a HPLC > 99.5%, RT ═ 1.43 min.

Example 22

2- ((benzyloxy) (phenyl) methyl) -4-chloro-9H-pyrimido [4, 5-b)]A mixture of indole-7-carboxylic acid methyl ester (prepared as described in example 5, 0.228g, 0.498mmol), 3- (piperidin-1-yl) -1-amine (0.158mL, 0.996mmol) and triethylamine (0.173mL, 1.245mmol) in MeOH (3.8mL) was placed in a microwave oven and heated to 140 deg.C for 30 min. After cooling to room temperature, the mixture was concentrated to dryness and the residue was purified by flash chromatography to provide 2- ((benzyloxy) (phenyl) methyl) -4- (b) (r) methyl) 4-as a pale yellow solid(3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (172mg, 61.3% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.29-1.42(m,2H)1.42-1.56(m,4H)1.72-1.91(m,2H)2.18-2.47(m,6H)3.66(tt,J＝13.2,6.6Hz,2H)3.88(s,3H)4.54(d,J＝11.7Hz,1H)4.64(d,J＝12.1Hz,1H)5.50(s,1H)7.21-7.43(m,8H)7.51(t,J＝5.9Hz,1H)7.57(d,J＝7.0Hz,2H)7.82(dd,J＝8.2,1.2Hz,1H)8.00(d,J＝1.2Hz,1H)8.38(d,J＝8.2Hz,1H)12.22(s,1H)；HRMS m/z 564.2979(M+H)⁺(ii) a HPLC 99.6%, RT 2.02 min.

In methanol, under hydrogen, in the presence of Pd-C10% wt. (50% wet weight) (0.159g, 0.075mmol) and ammonium formate (0.235g, 3.73mmol) in the derivative 2- ((benzyloxy) (phenyl) methyl) -4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4, 5-b)]Hydrogenolysis of benzyl group on methyl indole-7-carboxylate (0.042g, 0.075 mmol). After stirring at 55 ℃ for 26 hours, the reaction mixture was filtered on celite, washed with MeOH, and concentrated to dryness on a rotary evaporator to give 133mg of residue, which was purified by RP HPLC using a Zorbax SB-C18 column 21.2x 150mm, eluting with MeOH-water-0.1% TFA, to afford 21.9mg of example 22 as a white solid TFA salt (50% yield): 2- (hydroxy (phenyl) methyl) -4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester:¹H NMR(400MHz,DMSO-d₆)δppm 1.30-1.43(m,1H)1.52-1.73(m,3H)1.79(br.d,J＝14.5Hz,2H)1.96-2.09(m,2H)2.54(s,1H)2.74-2.89(m,2H)3.11(dt,J＝10.4,5.4Hz,2H)3.38(d,J＝12.1Hz,2H)3.71-3.77(m,2H)3.88(s,3H)5.63(s,1H)7.19-7.26(m,1H)7.27-7.35(m,2H)7.48-7.56(m,2H)7.62(t,J＝5.9Hz,1H)7.85(dd,J＝8.2,1.4Hz,1H)8.03(d,J＝1.4Hz,1H)8.38(d,J＝8.2Hz,1H)8.92(br.s.,1H)12.21(s,1H)；HRMS m/z474.2511(M+H)⁺(ii) a HPLC > 99%, RT ═ 1.68 min.

Des-Martin periodinane reagent (22.67mg, 0.053mmol) was added to 2- (hydroxy (phenyl) methyl) -4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4, 5-b)]Indole-7-carboxylic acid methyl ester (compound of example 22) and TFA (15.7mg, 0.027mmol) in a mixture of DCM (1000. mu.L, 15.54mmol) to yield a solutionA light orange solution resulted. After 1h, the solvent was evaporated and the residue was purified by flash chromatography to afford example 23 as a bright yellow solid: 2-benzoyl-4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (10mg, 79% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.27-1.36(m,2H)1.36-1.48(m,4H)1.74-1.88(m,2H)2.14-2.44(m,6H)3.52-3.68(m,2H)3.91(s,3H)7.54(t,J＝7.8Hz,2H)7.69(t,J＝7.4Hz,1H)7.76(t,J＝5.1Hz,1H)7.90(dd,J＝8.2,1.4Hz,1H)7.94(d,J＝7.0Hz,2H)8.10(d,J＝1.4Hz,1H)8.52(d,J＝8.2Hz,1H)12.43(s,1H)；HRMS m/z 472.2342(M+H)⁺(ii) a HPLC 97.1% @220nm and 98.9% @254nm, RT ═ 1.86 min.

Example 25

Hydroxylamine hydrochloride (0.08g, 1.2mmol) was added to 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b ] in MeOH (4.00mL) and pyridine (0.666mL)]Indole-7-carboxylic acid methyl ester (compound of example 33) (0.285g, 0.515mmol) to give a yellow solution. After heating at 60 ℃ for 5 days, the mixture was concentrated to dryness, and the residue was dissolved in DCM (75mL) and MeOH (15mL), and saturated NaHCO was used₃The solution was washed (20 mL). By means of CH₂Cl₂The aqueous layer was extracted twice with a mixture of (50mL) and MeOH (10mL), and over anhydrous MgSO₄The combined organic layers were dried, filtered and concentrated to dryness to give 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- (isonitroso) ethyl) benzyl) -9H-pyrimido [4, 5-b) as an off-white solid]Indole-7-carboxylic acid methyl ester (293mg, 100% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.32-1.43(m,2H)1.44-1.56(m,4H)1.76-1.87(m,2H)2.23-2.44(m,6H)3.57-3.67(m,2H)3.88(s,3H)4.04-4.12(m,2H)7.25-7.33(m,1H)7.34-7.46(m,2H)7.50(m,J＝7.6,4.1Hz,1H)7.55(d,J＝7.4Hz,1H)7.81(dd,J8.2,1.2Hz,1H)7.99(d, J ═ 1.2Hz,1H)8.36(d, J ═ 8.2Hz,1H)12.07(d, J ═ 3.5Hz, 1H); MS M/z 569.2(M + H) +; HPLC > 95%, RT ═ 1.88 min.

P-toluenesulfonyl chloride (0.048g, 0.251mmol) was added portionwise to 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- (isonitroso) ethyl) benzyl) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (0.130g, 0.229mmol), 4-dimethylaminopyridine (2.79mg, 0.023mmol) and triethylamine (0.038mL, 0.274mmol) in a cold mixture of DCM (10.00mL) to give a white suspension. After 1 hour at room temperature, the amber solution was diluted with DCM (10mL) and washed with water (3X 10 mL). Over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to dryness to give 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- ((tosyl) imino) ethyl) benzyl) -9H-pyrimido [4,5-b ] as a tan foam]Indole-7-carboxylic acid methyl ester (188mg, 99% yield): MS M/z 723.2(M + H) +; HPLC > 89%, RT ═ 2.09 min.

To a solution of 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- ((tosyl) imino) ethyl) benzyl) -9H-pyrimido [4,5-b ] cooled to-78 deg.C]To a solution of methyl indole-7-carboxylate (0.188g, 0.260mmol) in DCM (5.00mL) was added ammonia (1.689mL, 78mmol) and the tube sealed and warmed to 20 ℃. The reaction mixture was blue over time and after 3.5 hours it was cooled again to-78 ℃ and then slowly warmed to 20 ℃ using a septum + nitrogen outlet to evaporate most of the ammonia. After 3.5 hours, the reaction mixture was filtered on Buchner to remove most of the p-toluenesulfonic acid ammonium salt, the solid washed with DCM (3 × 1.5mL) and the filtrate concentrated to dryness to give a yellow foam, which was purified by flash chromatography to provide 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (3- (trifluoromethyl) diazacyclopropan-3-ylbenzyl) -9H-pyrimido [4, 5-b) as a white foam]Indole-7-carboxylic acid methyl ester (115mg, 78% yield):¹H NMR(400MHz,DMSO-d₆) δ ppm 1.31-1.43(m,2H)1.50 (quintuple, J ═ 5.3Hz,4H)1.80(dt, J ═ 14.1,7.0Hz,2H)2.32(m, J ═ 6.7,6.7Hz,6H)3.58-3.67(m,2H)3.88(s,3H)3.93(br.d, J ═ 7.4Hz,1H)4.05(br.d,J＝8.6Hz,1H)4.07(s,2H)7.32-7.43(m,3H)7.47(m,J＝6.3Hz,1H)7.59(s,1H)7.81(dd,J＝8.4,1.2Hz,1H)7.99(d,J＝1.2Hz,1H)8.35(d,J＝8.4Hz,1H)12.07(s,1H)；MS m/z 568.2(M+H)⁺(ii) a HPLC > 94%, RT ═ 1.72 min.

Iodine (0.028g, 0.111mmol) was added to 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (3- (trifluoromethyl) diazepan-3-ylbenzyl) -9H-pyrimido [4, 5-b)]A mixture of methyl indole-7-carboxylate (0.060g, 0.106mmol) and triethylamine (0.044mL, 0.317mmol) in DCM (2mL) to give a yellow solution. After 15 minutes, the solvent was evaporated under reduced pressure to give a residue which was purified by flash chromatography to give 95mg of a yellow foam. The foam was dissolved in DCM (15mL) and sat₃(10mL) washing. Over anhydrous MgSO₄The upper organic layer was dried, filtered and concentrated to dryness to provide the compound of example 25 as a pale yellow solid: 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (3- (trifluoromethyl) -3H-diazacyclopropan-3-ylbenzyl) -9H-pyrimido [4, 5-b)]Indole-7-carboxylic acid methyl ester (50mg, 84% yield): 1H NMR (400MHz, DMSO-d6) δ ppm1.36(m, J ═ 5.1Hz,2H)1.48 (quintuple, J ═ 5.5Hz,4H)1.76 (quintuple, J ═ 7.0Hz,2H)2.30(br.t, J ═ 6.5,6.5Hz,6H)3.54-3.67(m,2H)3.88(s,3H)4.09(s,2H)7.14(d, J ═ 7.8Hz,1H)7.26(s,1H)7.38-7.47(m,2H)7.52(d, J ═ 7.4Hz,1H)7.81(d, J ═ 8.2, 1H) 7.06 (s,1H)8.35(d, J ═ 8.06, 1H) 8.06 (s,1H)8.35 (J ═ 8.12H); HRMS M/z566.2497(M + H)⁺(ii) a HPLC 94.5% @220nm and 92.9% @254nm, RT ═ 2.05 min.

Examples 33 and 34

Charging CO into 2- (3-bromobenzyl) -4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4, 5-b)]Indole-7-carboxylic acid methyl ester (0.090 g, 0.168mmol, as prepared for example 15), triethylsilane (0.054mL, 0.336mmol) and PdCl₂(dppf)(6.14mg，839 μmol) and the mixture was heated at 95 ℃ overnight. The crude mixture was purified by preparative HPLC to yield 44mg of carbonylation product as a solid: 1H NMR (400MHz, DMSO-d6) δ ppm 1.28-1.40(m,1H)1.50-1.72(m,3H)1.73-1.84(m,2H)1.95-2.06(m,2H)2.74-2.87(m,2H)3.03-3.12(m,2H)3.33-3.41(m,2H)3.88(s,3H)4.20(s,2H)7.46-7.60(m,2H)7.73(d, J7.83 Hz,1H)7.79(d, J ═ 7.43Hz,1H)7.84(dd, J ═ 8.22,1.57Hz,1H)7.91(s,1H)8.01(d, J ═ 1.17, 1H)8.92 (d, 1.8.92, 1H) 8.61 (br, 1H)8.00 (H), 1.12H) 7.7.60 (m, 2H).

Trimethyl (trifluoromethyl) silane (0.7mL, 3.5mmol) was added to 2- (3-formylbenzyl) -4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b ] cooled to 0 deg.C]Indole-7-carboxylic acid methyl ester (0.260g, 0.535mmol) and cesium fluoride (5.69mg, 0.037 mmol). After stirring at room temperature for 2 days, concentrated HCl (0.5mL) was added to 2mL of water and stirred for 15 min. The mixture was diluted with ethyl acetate and solid Na was used₂CO₃Neutralization, phase separation and extraction of the aqueous layer 2 times with EA. The combined organic layers were washed with water over anhydrous MgSO₄Dry, filter and remove solvent to give a residue, which is purified by preparative HPLC to give 126mg of the corresponding TFA salt: 1H NMR (400MHz, DMSO-d6) δ ppm1.24-1.43(m,1H)1.49-1.73(m,4H)1.73-1.82(m,2H)1.97-2.07(m,2H)2.80(q, J ═ 11.70Hz,2H)3.02-3.12(m,2H)3.36-3.42(m,2H)3.88(s,3H)4.10(s,2H)5.12(q, J ═ 7.17Hz,1H)6.81(br.s.,1H)7.30-7.35(m,2H)7.36-7.42(m,1H)7.48-7.58(m,2H)7.84(dd, J ═ 8.41, 1.37H) 1.01 (m, 8H) 1.95H, 1H) 1.95 (1H) 1.17Hz,1H) 1.17 Hz; MSm/z 554.2(M + H) +; HPLC RT 2.142 min.

Daiss-Martin periodinane (56.5mg, 0.133mmol) was added to 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- ((hydroxyethyl) benzyl) -9H-pyrimido [4,5-b ] in DCM (753 μ L)]Indole-7-carboxylic acid methyl ester (20mg, 0.036mmol) to give a white suspension. After stirring for 1 hour at 20 ℃ the mixture was purified by flash chromatography to give example 33, 4- ((3- (piperidin-1-yl) propyl) amino) -2- (3- (2,2, 2-trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester: in DMSO-d6¹H NMR is consistent with the desired product, but is more complex due to the presence of the hydrate form; HRMS M/z554.2384(M + H)⁺(ii) a HPLC > 95%, RT ═ 1.76 and 1.87 min (ketone + hydrate).

Example 35

Intermediate 35B: n2 isomer precursors

A mixture of p-fluorobenzonitrile (5g, 41.3mmol), dibutyltin oxide (2.055g, 8.26mmol), and trimethylsilylazide (8.22mL, 61.9mmol) in toluene (165mL) was heated to 100 deg.C and stirred for 16.5 h. After cooling to room temperature, the organic layer was extracted with NaOH 1M (83mL), and the aqueous layer was washed with EtOAc (2X 85 mL). The aqueous layer was acidified to pH2 using HCl 2M (41.3 mL). The aqueous mixture was extracted twice with EtOAc (200mL then 100mL) and the combined organic layers were washed with brine (60mL) over anhydrous MgSO₄Dried, filtered and concentrated to dryness to afford intermediate (5- (4-fluorophenyl) -2H-tetrazole, 6.61g, 98% yield) as a white solid:¹H NMR(400MHz,DMSO-d₆)δppm 7.42-7.53(m,2H)8.04-8.14(m,2H)；MS m/z 165.2(M+H)⁺(ii) a HPLC > 99.5%, RT ═ 1.96 min.

5- (4-fluorophenyl) -2H-tetrazole (6.61g, 40.3mmol), K₂CO₃A mixture of (6.68g, 48.3mmol), and methyl iodide (3.02mL, 48.3mmol) in acetonitrile (115mL) was heated to reflux (actual 82 ℃ C.) for one hour. After cooling, the mixture was concentrated to dryness and the residue was partitioned between water (75mL) and EtOAc (100 mL). The layers were separated and worked up with EtOAc (50mL)The organic layer was extracted and the combined organic layer was washed with water (50mL) and brine (50 mL). Over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to give 9.5g of a colorless oil which solidified on standing. The residue was purified by flash chromatography to yield 2 major products: intermediate 35B as the N2 isomer of white solid: 5- (4-fluorophenyl) -2-methyl-2H-tetrazole (5.09g, 70.9% yield): no NOE was observed between methyl and aromatic protons at 4.42 ppm;¹H NMR(400MHz,DMSO-d₆)δppm 4.42(s,3H)7.33-7.45(m,2H)8.03-8.14(m,2H)；MSm/z 179.2(M+H)⁺(ii) a HPLC > 99.5%, RT ═ 1.75 min.

N1 isomer as white solid: 5- (4-fluorophenyl) -1-methyl-1H-tetrazole (1.87g, 26.1% yield): the NOE observed between methyl at 4.16ppm and two aromatic protons at 7.89-7.97ppm confirms the structure;¹H NMR(400MHz,DMSO-d₆)δppm 4.16(s,3H)7.43-7.53(m,2H)7.89-7.97(m,2H)；MS m/z179.2(M+H)⁺(ii) a HPLC > 99.5%, RT ═ 1.29 min.

Intermediates 35C and D

A solution of intermediate 35B (5- (4-fluorophenyl) -2-methyl-2H-tetrazole, 1g, 5.61mmol) in sulfuric acid (16.45mL, 309mmol) is cooled to 0 deg.C, then fuming nitric acid (0.288mL, 6.17mmol) is added dropwise. After 2.5 hours, more fuming nitric acid (0.065mL, 1.403mmol) was added and the mixture was allowed to heat to 20 ℃. After 5 hours, the mixture was poured into 2: 1 ice-water mixture (150mL), resulting in the formation of a white suspension. After 30 minutes, the solid was filtered, washed with water (4 × 10mL until neutral pH of wash), dried under high vacuum at 25 ℃ until constant weight: 5- (4-fluoro-3-nitrophenyl) -2-methyl-2H-tetrazole as an off-white solid (1.16g, 93% yield):¹H NMR(400MHz,DMSO-d₆)δppm 4.47(s,3H)7.81(dd,J＝11.2,8.8Hz,1H)8.44(ddd,J＝8.7,4.2,2.3Hz,1H)8.68(dd,J＝7.2,2.2Hz,1H)；MS m/z 224.2(M+H)⁺(ii) a HPLC 98.3%, RT 1.72 min.

A solution of 2-cyanoacetamide (0.888g, 10.56mmol) in DMF (2.268mL) was added to a suspension of sodium hydride 60% wt in mineral oil (0.443g, 11.08mmol) in DMF (5.67mL) to give a grey suspension. After cooling to 0 deg.C (note: hydrogen evolution), the resultant mixture was stirred at 0 deg.C for 30 minutes. Then, a solution of 5- (4-fluoro-3-nitrophenyl) -2-methyl-2H-tetrazole (1.15g, 5.15mmol) in DMF (2.3mL) was added to give a dark purple solution. After 3 hours, the reaction mixture was poured slowly into an ice-water mixture (33.0mL) and concentrated HCl (0.952 mL). The resulting yellow slurry was stirred for 30 minutes, the solid was filtered, washed with water (3 × 5mL) and then with hexane (2 × 5mL), dried under high vacuum at 40 ℃ until constant weight to give 2-cyano-2- (4- (2-methyl-2H-tetrazol-5-yl) -2-nitrophenyl) acetamide (1.41g, 95% yield) as a yellow solid:¹H NMR(400MHz,DMSO-d₆)δppm4.49(s,3H)5.77(s,1H)7.77(s,1H)7.95(d,J＝8.2Hz,1H)8.03(s,1H)8.51(dd,J＝8.2,1.8Hz,1H)8.70(d,J＝1.8Hz,1H)；MS m/z 288.1(M+H)⁺(ii) a HPLC 96.4% @220nm, RT ═ 1.31 min.

Iron chloride hexahydrate (2.82g, 10.44mmol) and zinc (2.276g, 34.8mmol) were added portionwise to a mixture of 2-cyano-2- (4- (2-methyl-2H-tetrazol-5-yl) -2-nitrophenyl) acetamide (1g, 3.48mmol) in DMF (4.75mL) and water (4.75mL) to give a yellow suspension which was heated to 100 ℃ for 1.25H. The mixture was then cooled to 20 ℃, diluted with MeOH (50.0mL), filtered over celite and concentrated under reduced pressure to the actual 20mL (to remove most of the MeOH). The mixture was then diluted with water (50mL) and EtOAc (100mL), vigorously stirred and filtered. The aqueous layer was extracted with EtOAc (2X 50mL) and with saturated NaHCO₃The combined organic layers were washed with brine (30mL) (50 mL). Over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to give 489mg of a purple solid which was purified by flash chromatography to give a purple solid 2-amino-6- (2-methyl-2H-tetrazole-5-Yl) -1H-indole-3-carboxamide (356mg, 39.7% yield):¹H NMR(400MHz,DMSO-d₆)δppm 4.38(s,3H)6.57(s,2H)7.01(s,2H)7.61-7.69(m,2H)7.81(s,1H)10.77(s,1H)；MS m/z 258.2(M+H)⁺(ii) a HPLC ca.78%, RT 1.34 min.

A mixture of intermediate 35D (2-amino-6- (2-methyl-2H-tetrazol-5-yl) -1H-indole-3-carboxamide, 0.35g, 1.361mmol), methyl 2-phenyl acetate (0.288mL, 2.041mmol), and sodium methoxide 25% wt in MeOH (0.467mL) in a microwave tube was placed in a microwave oven and heated to 140 deg.C for one hour. After cooling to room temperature, diluted with water (1mL) and AcOH (4mL), the mixture was stirred for 30 minutes to allow crystallization. The solid was filtered, washed with MeOH (5X 1mL), and dried under high vacuum at 40 deg.C until constant weight to provide 2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b ] as a brown solid]Indol-4-ol (220mg, 45.2% yield).¹H NMR(400MHz,DMSO-d₆)δppm 4.03(s,2H)4.43(s,3H)7.24-7.29(m,1H)7.34(t,J＝7.8Hz,2H)7.37-7.43(m,2H)7.92(dd,J＝8.0,1.4Hz,1H)8.04-8.10(m,2H)12.38(s,1H)12.47(s,1H)；MS m/z 358.2(M+H)⁺(ii) a HPLC 82.9%, RT 1.89 min.

In a 2-5mL microwave vial, the crude product 2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b ] is added]Indol-4-ol (0.220g, 0.616mmol) and POCl₃(3.90mL, 41.9mmol) to give a brown suspension. The glass vial was placed in a microwave oven and heated to 175 ℃, for 15 minutes, then allowed to cool. The reaction mixture was then poured into a water and ice mixture (80mL), basified to pH8 by the slow addition of NaOH 50% wt (11mL) and then EtOAc (80 mL). Some solids were filtered and the layers were separated. The aqueous layer was extracted with EtOAc (80mL) and over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to dryness to give the corresponding chloro derivative: 2-benzyl-4-chloro-7- (2-methyl) as brown solidradical-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b]Indole (189mg, 82% yield).¹H NMR(400MHz,DMSO-d₆)δppm 4.31(s,2H)4.46(s,3H)7.20-7.26(m,1H)7.28-7.39(m,4H)8.09(dd,J＝8.2,1.2Hz,1H)8.21-8.25(m,1H)8.39(d,J＝8.2Hz,1H)12.93(s,1H)；MS m/z 376.2(M+H)⁺(ii) a HPLC 95.6%, RT 2.30 min.

2-benzyl-4-chloro-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b ] prepared as described above was heated in a microwave oven at 140 deg.C]Indole (0.050mg, 0.133mmol) and Et₃A mixture of N (0.037mL, 0.266mmol) and 3- (piperidin-1-yl) propan-1-amine (0.033mL, 0.200mmol) in MeOH (0.6mL) was heated for 25 minutes. After cooling and evaporation of the solvent, the residue was purified by RP-HPLC (MeOH-water (0.5% TFA) 20% to 100% MeOH) to afford 55mg of example 35: 2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -N- (3- (piperidin-1-yl) propyl) -9H-pyrimido [4,5-b]Indol-4-amine 2,2, 2-trifluoroacetate:¹H NMR(400MHz,DMSO-d₆) δ ppm 1.27-1.41(m,1H)1.53-1.72(m,3H)1.79(d, J ═ 13.69Hz,2H)1.98-2.09(m,2H)2.75-2.87(m,2H)3.09(dt, J ═ 10.27,5.23Hz,2H)3.39(d, J ═ 11.35Hz,2H)3.69(q, J ═ 5.87Hz,2H)4.09(s,2H)4.44(s,3H)7.18-7.24(m,1H)7.31(t, J ═ 7.63Hz,2H)7.35-7.42(m,2H)7.51(br.s.,1H)7.93(dd, J ═ 8.1H) 1.53-1.72(m,3H)1.79(d, J ═ 13.8H) 1.17, 1H) 1.17H (d, 1.9H) 1.9 Hz, 1.9H) 4.9 (d, 2H); HPLC 99% at 254nm, Rt 2.063 min; HRMS M/z 482.2817(M + H) +.

Example 36 (methyl oxadiazole from cyanide)

Hydroxylamine hydrochloride (32.7mg, 0.471mmol) was added to example 37 (2-benzyl-4- (3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4, 5-b)]Indole-7 nitrile, 50mg, 0.118mmol) in EtOH (1.5mL) followed by DIPEA (84. mu.L, 0.483mmol) to give a light yellow suspension. Stirring at room temperature for 2.5 days and at 75 deg.CAfter stirring for 6 hours, the solvent was evaporated and water (3mL) was added and after stirring for 30min, the solid was collected, washed with water (3 × 1mL) and the solid material was dried under high vacuum at 35 ℃ until constant weight to give (Z) -2-benzyl-N' -hydroxy 4- (3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b as a tan solid]Indole-7 carboximidamide-HCl (53mg, 0.107mmol, 91% yield);¹H NMR(400MHz,DMSO-d₆)δppm 1.58-1.85(m,6H)1.98-2.10(m,2H)2.69-2.90(m,2H)2.94-3.15(m,2H)3.34-3.46(m,2H)3.59-3.74(m,2H)4.05(s,2H)5.84(br.s.,2H)7.15-7.24(m,1H)7.29(t,J＝7.4Hz,3H)7.37(d,J＝7.4Hz,2H)7.55(dd,J＝8.2,1.2Hz,1H)7.72(d,J＝1.2Hz,1H)8.25(d,J＝8.6Hz,1H)9.47(d,J＝7.4Hz,1H)9.56(s,1H)11.88(s,1H)；HRMS m/z 458.2662(M+H)⁺HPLC 95.4% @220nm and 97.4% @254nm, RT ═ 1.40 min.

Anhydrous acetic acid (0.917mL, 9.72mmol) was added to (Z) -2-benzyl-N' -hydroxy 4- (3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b]Indole-7 carboximidamide, HCl (0.040g, 0.081mmol) to give a tan suspension, and the mixture was heated to 140 ℃ by microwave for 30 minutes. The solvent was then evaporated and the residue purified by flash chromatography to give 36mg (85% yield) of 1- (2-benzyl-7- (5-methyl-1, 2, 4-oxadiazol-3-yl) -4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b ] as a tan solid]Indol-9-yl) ethanone, which was immediately dissolved in methanol (2.3mL) and treated with DBU (0.021mL, 0.138 mmol). The resulting yellow solution was heated to reflux for 30 minutes, then cooled to 0 ℃ while stirring for 1 hour. The solid was filtered and washed with cold MeOH (2 × 0.5mL), dried under high vacuum at 40 ℃ until constant weight to provide example 36 as a tan solid: 2-benzyl-7- (5-methyl-1, 2, 4-oxadiazol-3-yl) -N- (3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b]Indol-4-amine (20mg, 51.3% yield):¹H NMR(400MHz,DMSO-d₆) δ ppm 1.38(m, J ═ 4.7Hz,2H)1.50 (quintuple, J ═ 5.5Hz,4H)1.80 (quintuple, J ═ 6.9Hz,2H)2.18-2.45(m,6H)2.67(s,3H)3.57-3.70(m,2H)4.04(s,2H)7.15-7.22(m,1H)7.27(m, J ═ 7.6,7.6Hz,2H)7.34(t, J ═ 7.6Hz,2H)5.9Hz,1H)7.36-7.40(m,2H)7.83(dd,J＝8.2,1.4Hz,1H)8.01(d,J＝1.4Hz,1H)8.39(d,J＝8.2Hz,1H)12.02(br.s.,1H)；HRMS m/z 482.2663(M+H)⁺HPLC 99.3%, RT ═ 1.79 min.

Example 37

An orange mixture of 2-amino-6-cyano-1H-indole-3-carboxamide (0.172g, 0.859mmol), methyl 2-phenyl acetate (0.303mL, 2.148mmol) and sodium methoxide 30% wt in MeOH (0.403mL, 2.148mmol) in methanol (2.82mL) was heated at 140 ℃ for 45 minutes in a microwave tube. Then, methyl 2-phenylacetate (0.151mL, 1.074mmol) and 30% wt (0.201mL, 1.074mmol) of sodium methoxide in MeOH of a new load were added and the glass vial was placed in a microwave and heated again at 140 ℃ for 45 minutes. Then, after cooling to room temperature, AcOH (0.197mL, 3.44mmol) was added and the resulting slurry was stirred at 20 ℃ for 1 hour. The solid was filtered, washed with MeOH (3 × 1mL) and dried under high vacuum at 20 ℃ until constant weight to yield intermediate 37B as a tan solid: 2-benzyl-4-hydroxy-9H-pyrimido [4,5-b]Indole-7-carbonitrile (182mg, 70.5% yield):¹H NMR(400MHz,DMSO-d₆)δppm4.03(s,2H)7.22-7.29(m,1H)7.30-7.36(m,2H)7.36-7.43(m,2H)7.58(dd,J＝8.2,1.4Hz,1H)7.82-7.87(m,1H)8.05(d,J＝8.2Hz,1H)12.59(br.s.,2H)；MS m/z 301.2(M+H)⁺(ii) a HPLC 94.2% @220nm and 91.3% @254 nm; RT ═ 1.90 min.

2-benzyl-4-hydroxy-9H-pyrimido [4,5-b ]]A red mixture of indole-7-carbonitrile (intermediate 37B, 0.180g, 0.599mmol) and phosphorus oxychloride (3.63mL, 39.0mmol) was heated to 95 deg.C and stirred for 16 h. After concentration to dryness on a rotary evaporator, the resultant dark red foam is suspended in saturated NaHCO₃(10mL) and stirred for 30 minutes. The solid was collected and washed with water (3X 1mL) and dried under high vacuum at 40 deg.CUntil constant weight to yield intermediate 37C as a tan solid: 2-benzyl-4-chloro-9H-pyrimido [4,5-b]Indole-7-carbonitrile (190mg, 99% yield), which was used immediately for the next step: MS M/z 319.2(M + H)⁺(ii) a HPLC 95.0% @220nm and 92.3% @254nm, RT ═ 2.28 min.

2-benzyl-4-chloro-9H-pyrimido [4,5-b ] is treated by microwave]A mixture of indole-7-carbonitrile (intermediate 37C, 0.190g, 0.596mmol), 3- (piperidin-1-yl) propan-1-amine (0.142mL, 0.894mmol) and triethylamine (0.208mL, 1.490mmol) in MeOH (4.50mL) was heated to 140 deg.C for 30 min. Then, it was concentrated to dryness to give 346mg of orange solid, which was purified by flash chromatography to give 176mg of yellow solid, which was suspended in ether (7mL) and stirred at 20 ℃ for 1 hour. The solid was filtered, washed with diethyl ether (3 × 1mL), and dried under high vacuum at 30 ℃ until constant weight to provide 2-benzyl-4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b ] of example 37 as a pale yellow solid]Indole-7-carbonitrile (172mg, 68.0% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.30-1.43(m,2H)1.43-1.57(m,4H)1.80(m,J＝5.5Hz,2H)2.18-2.47(m,6H)3.62(q,J＝6.4Hz,2H)4.04(s,2H)7.15-7.22(m,1H)7.27(m,J＝7.4,7.4Hz,2H)7.33-7.39(m,2H)7.47(t,J＝5.7Hz,1H)7.62(dd,J＝8.2,1.2Hz,1H)7.79(d,J＝1.2Hz,1H)8.43(d,J＝8.2Hz,1H)12.19(s,1H)；HRMS m/z 425.2448(M+H)⁺(ii) a HPLC > 99%, RT ═ 1.68 min.

Example 43

In a 2-5mL microwave glass vial, 2-benzyl-4-chloro-9H-pyrimido [4,5-b ] is added]Indole-7-carboxylic acid methyl ester (0.100g, 0.284mmol), (E) -1- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) 3-butan-1-yl) piperidine (0.113g, 0.426mmol), potassium carbonate (0.106g, 0.768mmol), and Pd (Ph3P)4(0.05g, 0.044 mmol). With N₂The glass vial was purged (3 vacuum + backfill cycle). DME (2.84mL) and water (0.398mL) were added, and N was added₂The glass vial was rinsed (with vacuum + back fill) and then heated to 110 ℃ while stirring for 24 hours. After cooling, the mixture was concentrated to dryness under reduced pressure, and the residue was purified by flash chromatography to give (E) -methyl 2-benzyl-4 (4- (piperidin-1-yl)) 1-butan-1-yl-9H-pyrimido [4, 5-b) as a pale yellow solid]Indole-7-carboxylate (55mg, 0.121mmol, 42.6% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.41(s,2H)1.50-1.61(m,4H)2.30-2.47(m,4H)2.53-2.59(m,2H)2.59-2.70(m,2H)3.91(s,3H)4.27(s,2H)7.16-7.24(m,1H)7.29(t,J＝7.6Hz,2H)7.34-7.43(m,4H)7.88(dd,J＝8.2,1.4Hz,1H)8.07(d,J＝1.4Hz,1H)8.41(d,J＝8.2Hz,1H)12.48(s,1H)；HRMS m/z 455.2442(M+H)⁺(ii) a HPLC 100% @220nm and 99.4% @254nm, RT 1.84 min.

Reacting (E) -methyl-2-benzyl-4 (4- (piperidin-1-yl)) 1-butan-1-yl) -9H-pyrimido [4,5-b ] with hydrogen]Indole-7-carboxylate (20mg, 0.044mmol) and a mixture of Pd-C10% wt. (50% wet) (23.41mg) in MeOH (2mL) and THF (2mL) were treated for 17 hours. The reaction mixture was diluted with DCM (3mL), filtered, washed with MeOH (2 × 2mL) and then DCM (2 × 2mL) and concentrated to dryness to give 19mg of a light yellow solid which was purified by flash chromatography to give a white solid (14mg) using CH₃CN (2mL) treated the white solid. After stirring the white suspension at 20 ℃ for 1 hour, the solid is filtered, using CH₃CN (1X 1mL) was washed and dried under high vacuum at 40 ℃ until constant weight to provide the compound of example 43, 2-benzyl-4 (4- (piperidin-1-yl)) butyl) -9H-pyrimido [4,5-b ] as a white solid]Indole-7-carboxylic acid methyl ester (14.4mg, 71.7% yield):¹H NMR(400MHz,DMSO-d₆) δ ppm1.36(m, J ═ 5.5Hz,2H)1.45 (quintuple, J ═ 5.5Hz,4H)1.57 (quintuple, J ═ 7.3Hz,2H)1.83(dt, J ═ 14.9,7.4Hz, 2H))2.18-2.31(m,6H)3.20-3.28(m,2H)3.91(s,3H)4.26(s,2H)7.16-7.22(m,1H)7.28(t,J＝7.4Hz,2H)7.32-7.38(m,2H)7.90(dd,J＝8.2,1.2Hz,1H)8.09(d,J＝1.2Hz,1H)8.25(d,J＝8.2Hz,1H)12.48(br.s.,1H)；HRMS m/z 457.2598(M+H)⁺(ii) a HPLC > 99.5%, RT ═ 1.75 min.

Example 44

2-benzyl-4-chloro-9H-pyrimido [4,5-b ] was heated at 140 ℃ in a microwave oven]Indole-7-carboxylate (0.100g, 0.284mmol), Et₃A mixture of N (0.079mL, 0.569mmol) and tert-butyl (3-aminopropyl) (methyl) carbamic acid methyl ester (0.080g, 0.426mmol) in MeOH (1mL) was heated for 40 min. The solvent was removed under reduced pressure and the residue was purified by flash chromatography to give 0.092mg of crude Boc derivative, which was used directly in the next step. TFA (1.0ml, 12.98mmol) was added dropwise to 2-benzyl-4 ((3-tert-butoxycarbonyl) (methyl) amino) propyl) amino) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (0.092g, 0.183mmol) in cold suspension and the mixture allowed to warm to room temperature over 30 minutes. After dilution with toluene, the solvent was removed under reduced pressure, then the residue was diluted with EtOAc to yield 85mg of a solid used directly in the next step: HRMS M/z 404.2091(M + H) +.

Heating 2-benzyl-4 ((3-methylamino) propyl) amino) -9H-pyrimido [4,5-b ] in acetone (0.2mL) at 70 deg.C]Indole-7-carboxylate methyl 2,2, 2-trifluoroacetate (0.020g, 0.039mmol), sodium carbonate (8.81mg, 0.083mmol), sodium iodide (1.448mg, 9.66. mu. mol) and a mixture of 2- (2- (2-chloroethoxy) ethoxy) ethanol (6.46. mu.l, 0.044 mmol). After 15 hours, a second portion of 2- (2- (2-chloroethoxy) ethoxy) ethanol (6.46 μ l, 0.044mmol) was added and the mixture was heated again at 70 ℃ for 15 hours. After cooling to room temperature, the mixture was diluted with EtOAc, washed with water, over anhydrous MgSO₄Dry, filter, and evaporate the solvent to give a residue, which is purified by flash chromatography to provide 9mg of example 44:¹H NMR(400MHz,DMSO-d₆)δppm 1.74-1.85(m,2H)2.25(br.s.,3H)2.55(br.s.,2H)3.32-3.36(m,4H)3.39-3.46(m,6H)3.51(t,J＝5.87Hz,2H)3.64(q,J＝6.52Hz,2H)3.88(s,3H)4.04(s,2H)4.53(br.s.,1H)7.18(t,J＝7.40Hz,1H)7.28(t,J＝7.63Hz,2H)7.37(d,J＝7.04Hz,2H)7.55(t,J＝5.28Hz,1H)7.82(dd,J＝8.22,1.17Hz,1H)7.99(s,1H)8.27(d,J＝8.22Hz,1H)12.05(s,1H)；HRMS m/z536.2855(M+H)⁺(ii) a HPLC RT 2.035 min.

Examples 45 and 51

In a 2-5mL microwave glass vial, 2-benzyl-4-chloro-9H-pyrimido [4,5-b ] in MeOH (2.000mL, 49.4mmol) is added]Indole-7-carboxylic acid methyl ester (0.050g, 0.142mmol), and N1- (3-aminopropyl) -N1-methylpropane-1, 3-diamine (0.115 mL, 0.711mmol) to give a tan suspension. The glass vial was placed in a microwave and heated to 140 ℃ for 30 min. After 30 minutes, the mixture is concentrated to dryness on a rotary evaporator and the residue is purified by flash chromatography and separated from CH₃CN was lyophilized to provide two different products: example 45 as a mono-N-alkylated product: 4- ((3- ((3-aminopropyl) (methyl) amino) propyl) amino) 2-benzyl-9H-pyrimido [4, 5-b) as a white solid]Indole-7-carboxylic acid methyl ester (44mg, 67.2% yield);¹H NMR(400MHz,DMSO-d₆)δppm 1.44(dt,J＝13.8,6.6Hz,2H)1.72(dt,J＝13.7,6.8Hz,2H)2.11(s,3H)2.24-2.30(m,2H)2.33(t,J＝6.7Hz,2H)2.47(br.s.,2H)3.51-3.61(m,2H)3.76-3.85(m,3H)3.97(s,2H)7.08-7.15(m,1H)7.17-7.24(m,2H)7.27-7.34(m,2H)7.50(t,J＝5.3Hz,1H)7.76(dd,J＝8.2,1.6Hz,1H)7.92(d,J＝1.6Hz,1H)8.20(d,J＝8.2Hz,1H)；MS m/z 461.2(M+H)⁺(ii) a HPLC > 99%, RT ═ 1.63 min.

Example 51 as the di-alkylated product: 4, 4' - (((methylurondiyl) bis (propane-3, 1-diyl)) bis (urondiyl)) bis (2-benzyl-9H-pyrimido [4, 5-b) as a pale yellow solid]Indole-7-carboxylic acid dimethyl ester) (3.7mg, 6.71% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.18-1.29(m,4H)1.79-1.91(m,4H)2.25(s,3H)3.58-3.71(m,4H)3.84(s,6H)3.97(s,4H)7.07-7.16(m,2H)7.22(t,J＝7.4Hz,4H)7.29-7.34(m,4H)7.53(t,J＝5.3Hz,2H)7.77(dd,J＝8.2,1.4Hz,2H)7.96(d,J＝1.4Hz,2H)8.22(d,J＝8.2Hz,2H)12.01(s,2H)；MS m/z 776.3(M+H)⁺(ii) a HPLC 94.6% @220nm and 93.8% @254nm, RT ═ 2.01 min.

Example 52

2, 5-dioxopyrrolidin-1-yl-5- ((3aS, 4S,6aR) -2-oxohexahydro-1H-thiophene [3,4-d ]]Imidazol-4-yl) pentanoate (12.01mg, 0.035mmol) was added to 4- ((3- ((3-aminopropyl) (methyl) amino) propyl) amino) 2-benzyl-9H-pyrimido [4, 5-b)]A solution of methyl indole-7-carboxylate (example 45, 15mg, 0.033mmol) and triethylamine (6.81. mu.L, 0.049mmol) in DMF (750. mu.L, 9.69mmol) gave a light yellow solution. After stirring for 1 hour at 20 ℃, the mixture was concentrated to dryness and the residue was purified by flash chromatography to give a pale yellow foam. Suspend the foam in Et₂O (1mL) and stirred for 30min, and the solid was collected using Et₂O (2 × 0.5mL) was washed and dried under high vacuum at 20 ℃ until constant weight to give the compound of example 52 as a yellow solid: 2-benzyl-4- ((3- (methyl) (3- (5- ((3aS, 4S,6aR) -2-oxohexahydro-1H-thiophene [3, 4-d)]Imidazol-4-yl) pentaneamino) propyl) amino) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (17mg, 76% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.17-1.34(m,3H)1.36-1.51(m,2H)1.51-1.64(m,3H)1.78(dt,J＝13.5,6.6Hz,2H)2.02(t,J＝7.4Hz,2H)2.17(s,3H)2.32(t,J＝7.0Hz,2H)2.40(t,J＝6.7Hz,2H)2.55(d,J＝12.3Hz,1H)2.77(dd,J＝12.3,5.1Hz,1H)2.99-3.11(m,3H)3.58-3.68(m,2H)3.88(s,3H)4.04(s,2H)4.08(m,J＝4.9,4.9,2.3Hz,1H)4.26(dd,J＝7.6,5.3Hz,1H)6.34(s,1H)6.40(s,1H)7.14-7.22(m,1H)7.27(t,J＝7.4Hz,2H)7.37(d,J＝7.0Hz,2H)7.54(t,J＝5.5Hz,1H)7.74(t,J＝5.5Hz,1H)7.82(dd,J＝8.2,1.6Hz,1H)7.99(d,J＝1.6Hz,1H)8.27(d,J＝8.2Hz,1H)12.05(s,1H)；MS m/z 687.3(M+H)⁺(ii) a HPLC > 99.5%, RT ═ 1.70 min.

Example 53

Intermediate 53A was prepared from commercial ethyl 2 (3-tolyl) acetate. This was then converted to intermediate 53B as described for examples 15, 45 and 47. Then, an aziridine moiety was developed according to the description provided for example 25. The final step is based on example 52.

A mixture of ethyl 2- (m-tolyl) acetate (4.8g, 26.9mmol), NBS (5.27g, 29.6mmol) and benzoyl peroxide (0.110g, 0.454mmol) was placed in CCl₄(28mL) under reflux. After 5 hours, the reaction was cooled to 5 ℃, filtered and the solvent removed. Purification by flash chromatography using ethyl acetate-hexane yielded 3.8g of the corresponding bromobenzyl derivative:¹h NMR (400MHz, DMSO-d6) δ ppm 1.19(t, J ═ 6.70Hz,3H)3.67(s,2H)4.09(q, J ═ 6.70Hz,2H)4.69(s,2H)7.15-7.25(m,1H)7.27-7.42(m, 3H); the material was used directly in the next step.

A mixture of ethyl 2- (3- (bromomethyl) phenyl) acetate (8.11g, 31.5mmol) and 4-methylmorpholine 4-oxide hydrate (5.54g, 47.3mmol) in 1, 4-dioxane (110mL) was heated to 100 ℃ for 1.5 hours. After cooling to room temperature, the volume of solvent was reduced to half, and then Et was used₂EtOAc (1: 1, 120mL) diluteThe aqueous layer was washed with water (50mL) and extracted with EtOAc (60 mL). The combined organic layers were washed with water (2X 50mL) and then brine (50 mL). Over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to give 4.72g of a pale yellow oil, which was purified by flash chromatography to provide 2- (3-formylphenyl) acetate as a pale yellow oil (2.91g, 48% yield):¹H NMR(400MHz,DMSO-d6)δppm 1.19(t,J＝7.0Hz,3H)3.81(s,2H)4.09(q,J＝7.0Hz,2H)7.52-7.65(m,2H)7.79-7.85(m,2H)10.00(s,1H)；MS m/z 207.2(M+H)⁺(ii) a HPLC 99%, RT 1.67 min.

Trimethyl (trifluoromethyl) silane (3.13mL, 21.20mmol) was added to a mixture of ethyl 2- (3-formylphenyl) acetate (2.91g, 15.14mmol) and cesium fluoride (0.161g, 1.060mmol) in DMF (20.19mL) cooled to 0-5 ℃. After stirring for 1.5 hours, a solution of TBAF 1M in THF (15.14mL) was added. The resulting yellow solution was stirred at 0-5 ℃ and after 30 minutes the mixture was poured into water (150mL) and extracted with MTBE (1X 150mL, then 2X 100 mL). The combined organic layers were washed with water (1X 150mL, then 1X 100mL) and brine (100mL), and then, over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to give 3.84g of a light orange oil, purified by flash chromatography to give ethyl 2- (3- (2,2, 2-trifluoro-1-hydroxyethyl) phenyl) acetate as a colourless oil (663mg, 16% yield):¹H NMR(400MHz,DMSO-d6)δppm 1.17(t,J＝7.0Hz,3H)3.68(s,2H)4.08(q,J＝7.0Hz,2H)5.13(q,J＝7.4Hz,1H)6.82(s,1H)7.24-7.31(m,1H)7.37(t,J＝7.2Hz,3H)；MS m/z263.1(M+H)⁺(ii) a HPLC 93.7% @220nm, RT ═ 1.82 min.

Benzyl bromide (0.330mL, 2.78mmol) was added to ethyl 2- (3- (2,2, 2-trifluoro-1-hydroxyethyl) phenyl) acetate (0.648g, 2.471mmol) and K₂CO₃(1.059g, 7.66mmol) in acetonitrile (17.00mL) and the mixture was stirred while heating to reflux (75-80 ℃ C.) for 24 hours. After cooling and concentration to dryness, the residue was purified by flash chromatography to yield intermediate 53A: 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) as a colorless oil) Phenyl) acetic acid ethyl ester (686mg, 79% yield): MS M/z 353.2(M + H)⁺(ii) a HPLC 99.7%, RT 2.19 min.

A mixture of intermediate 15B (0.310g, 1.329mmol), ethyl 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) phenyl) acetate (0.679g, 1.927mmol) and sodium methoxide 30% wt in intermediate 53A and MeOH (0.524mL) in methanol (3.23mL) was added to give a thin brown suspension which was heated to 140 ℃ for 1 hour in a microwave apparatus. After cooling and dilution with MeOH (0.75mL) and AcOH (0.167mL, 2.92mmol), the resulting suspension was stirred at 20 ℃ for 2 h. Then, the solid was collected and washed with MeOH (4 × 0.5mL), then dried under high vacuum at 40 ℃ until constant weight to provide 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -4-hydroxy-9H-pyrimido [4,5-b ] as a tan solid]Indole-7-carboxylic acid methyl ester (399mg, 57.6% yield):¹H NMR(400MHz,DMSO-d6)δppm 3.87(s,3H)4.09(s,2H)4.45-4.57(m,2H)5.23(q,J＝7.2Hz,1H)7.20-7.29(m,5H)7.36-7.51(m,3H)7.52(s,1H)7.84(dd,J＝8.2,1.4Hz,1H)8.01(d,J＝1.4Hz,1H)8.03(d,J＝8.2Hz,1H)12.46(br.s.,1H)12.56(br.s.,1H)；MS m/z 522.2(M+H)⁺(ii) a HPLC 92.7% @220nm and 91.7% @254nm, RT ═ 2.20 min.

Reacting 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -4-hydroxy-9H-pyrimido [4,5-b]A mixture of methyl indole-7-carboxylate (0.395g, 0.757mmol) in phosphorus oxychloride (6mL, 64.4mmol) was heated to 90 deg.C for 2 hours, then after cooling, it was concentrated to dryness to give 650mg of a brown foam which was suspended in saturated NaHCO₃(15mL) and stirred for 1 hour. The solid was filtered, washed with water (3 × 2mL) and dried under high vacuum at 40 ℃ until constant weight to provide 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -4-chloro-9H-pyrimido [4,5-b ] as a tan solid]Indole-7-carboxylic acid methyl ester (375mg, 92% yield) and was also used in the next step: MS M/z540.2(M + H)⁺(ii) a HPLC 92%, RT 2.51 min.

In a microwave oven, 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -4-chloro-substituted benzene9H-pyrimido [4,5-b]A mixture of methyl indole-7-carboxylate (0.375g, 0.695mmol) and N1- (3-aminopropyl) -N1-methylpropane-1, 3-diamine (0.784mL, 4.86mmol) in MeOH (9.83mL, 243mmol) was heated to 140 deg.C for 30 min. The resulting brown oil was then purified by flash chromatography after concentration to dryness under reduced pressure to provide 4- ((3- ((3-aminopropyl) (methyl) amino) propyl) amino) -2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -9H-pyrimido [4, 5-b) as a yellow foam]Indole-7-carboxylic acid methyl ester:¹h NMR (400MHz, DMSO-d6) δ ppm 1.48(dt, J ═ 14.0,6.9Hz,2H)1.75 (quintuple, J ═ 6.7Hz,2H)2.14(s,3H)2.24-2.42(m,4H)2.52-2.60(m,2H)3.61(q, J ═ 6.3Hz,2H)3.88(s,3H)4.10(s,2H)4.49(s,2H)5.17(q, J ═ 7.0Hz,1H)7.18-7.30(m,5H)7.31-7.36(m,1H)7.39(t, J ═ 7.6Hz,1H)7.44-7.53(m,2H)7.58(t, J ═ 3.5, J ═ 8.84 (J, 8H) (J ═ 1.8, 8H) 4.8 (1H) 1.8 Hz, 8 (J ═ 8H) 1.8 Hz; MS M/z649.3(M + H)⁺(ii) a HPLC 97.6% @220nm and 95.5% @254nm, RT ═ 1.96 min.

A solution of di-tert-butyl-sodium bicarbonate (0.124mL, 0.533mmol) in DCM (1mL) was slowly added to 4- ((3- ((3-aminopropyl) (methyl) amino) propyl) amino) -2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -9H-pyrimido [4,5-b ]]Indole-7-carboxylic acid methyl ester (0.288g, 0.444mmol) and triethylamine (0.074mL, 0.533mmol) in a mixture of DCM (3mL) and MeOH (2mL) to give a yellow solution. After stirring for 45 min at 20 ℃ the solution was concentrated to dryness to give 373mg of yellow foam which was purified by flash chromatography to afford intermediate 53B, 2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -9H-pyrimido [4,5-B ] as a yellow foam]Indole-7-carboxylic acid methyl ester (306mg, 92% yield):¹H NMR(400MHz,DMSO-d6)δppm 1.33(s,9H)1.52(dt,J＝14.1,7.0Hz,2H)1.67-1.81(m,2H)2.12(s,3H)2.28(t,J＝7.2Hz,2H)2.34(t,J＝6.8Hz,2H)2.92(q,J＝6.7Hz,2H)3.54-3.67(m,2H)3.88(s,3H)4.10(s,2H)4.49(s,2H)5.17(q,J＝6.8Hz,1H)6.76(t,J＝5.5Hz,1H)7.16-7.30(m,5H)7.31-7.36(m,1H)7.39(t,J＝7.6Hz,1H)7.43-7.51(m,2H)7.53(t,J＝5.3Hz,1H)7.83(dd,J＝8.4,1.4Hz,1H)8.00(d,J＝1.4Hz,1H)8.27(d,J＝8.2Hz,1H)12.06(s,1H)；MS m/z 749.3(M+H)⁺(ii) a HPLC 97.7%, RT 2.06 min.

2- (3- (1- (benzyloxy) -2,2, 2-trifluoroethyl) benzyl) -4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -9H-pyrimido [4,5-b ] amino]A mixture of methyl indole-7-carboxylate (0.306g, 0.409mmol) and Pd-C10% wt (50% wet) (0.304g,0.143mmol) in MeOH (9.92mL) was treated for 22 h. The reaction mixture was then filtered over celite, the cake was washed with MeOH (2 × 10mL) and with DCM: MeOH (1: 1,2 × 10mL) and then concentrated to dryness on a rotary evaporator to give 226mg of oil, which was purified by flash chromatography to provide 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3-2,2, 2-trifluoro-1-hydroxyethyl) benzyl) -9H-pyrimido [4,5-b ] as a white foam]Indole-7-carboxylic acid methyl ester (202mg, 75% yield):¹H NMR(400MHz,DMSO-d6)δppm 1.33(s,9H)1.48-1.62(m,2H)1.71-1.85(m,2H)2.16(s,3H)2.25-2.35(m,2H)2.39(t,J＝6.8Hz,2H)2.86-3.00(m,2H)3.56-3.70(m,2H)3.88(s,3H)4.06(s,2H)5.01-5.14(m,1H)6.77(m,J＝6.3Hz,2H)7.30(d,J＝4.7Hz,2H)7.36-7.42(m,1H)7.49(s,1H)7.51-7.57(m,1H)7.82(dd,J＝8.2,1.2Hz,1H)7.99(d,J＝1.2Hz,1H)8.26(d,J＝8.2Hz,1H)12.06(br.s.,1H)；MS m/z 659.2(M+H)⁺(ii) a HPLC > 97%, RT ═ 1.90 min.

4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3-2,2, 2-trifluoro-1-hydroxyethyl) benzyl) -9H-pyrimido [4,5-b ] at 20 ℃]A mixture of methyl indole-7-carboxylate (0.200g, 0.304mmol) and dess-Martin periodinane reagent (0.567g, 1.336mmol) in DCM (7.50mL) was stirred for one hour. After evaporation to dryness, the residue was purified by flash chromatography to provide 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2, 2-trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b ] as a yellow foam]Indole-7-carboxylic acid methyl ester (181mg, 91% yield): in DMSO-d6¹H NMR is consistent with the desired product, but is more complex due to the presence of the hydrate form: MS M/z 657.3(M + H)⁺；HPLC 96.0% @220nm and 95.3% @254nm, RT 1.87 and 1.96 (ketone + hydrate) min.

In a microwave oven, 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2, 2-trifluoroacetyl) benzyl) -9H-pyrimido [4,5-b ] is]A mixture of methyl indole-7-carboxylate (0.180g, 0.274mmol), hydroxylamine hydrochloride (0.023g, 0.329mmol) and pyridine (0.355mL) in MeOH (1.9mL) was heated to 65 deg.C for 48 h. After concentration of the reaction mixture to dryness, saturated NaHCO was used₃(15mL) the residue was washed with a solution of anhydrous MgSO₄The organic layer was dried, filtered and concentrated to dryness to provide 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- (isonitroso) ethyl) benzyl) -9H-pyrimido [4, 5-b) as a pale yellow foam]Indole-7-carboxylic acid methyl ester (184mg, 100% yield): MS M/z 672.3(M + H)⁺(ii) a HPLC 96.0% @220nm and 91.4% @254nm, RT ═ 2.01 min.

Ts-Cl (0.060g, 0.315mmol) was added portionwise to 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- (isonitroso) ethyl) benzyl) -9H-pyrimido [4,5-b]Methyl indole-7-carboxylate (0.184g, 0.274mmol), DMAP (3.35mg, 0.027mmol) and triethylamine (0.048mL, 0.342mmol) in DCM (12mL) to give a tan solution. After 1 hour, the reaction mixture was diluted with DCM (12mL), washed with water (3X 12mL), and over anhydrous MgSO₄The organic layer was dried, filtered and concentrated to dryness to provide 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- ((tosyl) imino) ethyl) benzyl) -9H-pyrimido [4,5-b ] as a tan foam]Indole-7-carboxylic acid methyl ester (217mg, 96% yield): MS M/z 826.2(M + H)⁺(ii) a HPLC 93.2% @220nm and 91.3% @254nm, RT ═ 2.20 min.

Reacting 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (2,2, 2-trifluoro-1- ((tosyl) imino) ethyl) benzyl) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (0.217g, 0.263 mm)ol) solution in DCM (5.07mL) was cooled to-78 deg.C and ammonia (1.7mL, 79mmol) was compressed into a sealed tube. The mixture was allowed to warm slowly to 20 ℃ and stirred for 3 hours. After cooling down again to-78 ℃, the sealed tube was assembled with a septum having a gas outlet and slowly warmed to 20 ℃ to evaporate the ammonia. After 3 hours, the mixture was concentrated to dryness and then purified by flash chromatography to provide 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (3-trifluoromethyl) overlapping acridin-3-yl) benzyl) -9H-pyrimido [4,5-b ] as a white foam]Indole-7-carboxylic acid methyl ester (139mg, 79% yield):¹H NMR(400MHz,DMSO-d6)δppm 1.34(s,9H)1.52-1.62(m,2H)1.72-1.89(m,2H)2.08-2.25(m,3H)2.30-2.44(m,4H)2.94(q,J＝6.4Hz,2H)3.64(q,J＝6.5Hz,2H)3.88(s,3H)3.93(d,J＝8.4Hz,1H)4.04(d,J＝8.4Hz,1H)4.08(s,2H)6.78(br.s.,1H)7.31-7.41(m,2H)7.44-7.50(m,1H)7.54(t,J＝5.5Hz,1H)7.59(s,1H)7.83(dd,J＝8.4,1.4Hz,1H)7.99(d,J＝1.4Hz,1H)8.27(d,J＝8.4Hz,1H)12.07(s,1H)；MS m/z 671.4(M+H)⁺(ii) a HPLC 98.6% @220nm and 96.4% @254nm, RT ═ 1.91 min.

Iodine (27.8mg, 0.110mmol) was added to a 5mL round bottom flask, stored protected from light and pre-filled with 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (3-trifluoromethyl) overlapping acridin-3-yl) benzyl) -9H-pyrimido [4,5-b]A mixture of methyl indole-7-carboxylate (70mg, 0.104mmol) and triethylamine (43.6. mu.L, 0.313mmol) in DCM (2mL) to give a light yellow solution. After stirring for 15min at 20 ℃, the solvent was evaporated and the residue was purified by flash chromatography to give 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (3-trifluoromethyl) -3H-diazepin-3-yl) benzyl) -9H-pyrimido [4,5-b ] as a pale yellow foam]Indole-7-carboxylic acid methyl ester (65mg, 0.097mmol, 93% yield):¹H NMR(400MHz,DMSO-d6)δppm 1.33(s,9H)1.53(dt,J＝13.8,7.0Hz,2H)1.70-1.82(m,2H)2.15(br.s.,3H)2.24-2.34(m,2H)2.34-2.42(m,2H)2.87-2.98(m,2H)3.55-3.66(m,2H)3.88(s,3H)4.10(s,2H)6.76(br.s.,1H)7.14(d,J＝8.2Hz,1H)7.27(s,1H)7.43(t,J＝7.8Hz,1H)7.49-7.58(m,2H)7.83(dd,J＝8.2,1.4Hz,1H)7.99(d,J＝1.4Hz,1H)8.27(d,J＝8.2Hz,1H)12.06(s,1H)；MSm/z 669.2(M+H)⁺(ii) a HPLC 97.6% @220nm and 97.3% @254nm, RT ═ 2.18 min.

Trifluoroacetic acid (0.400mL, 5.19mmol) was added to 4- ((3- ((3- ((tert-butoxycarbonyl) amino) propyl) (methyl) amino) propyl) amino) -2- (3- (3-trifluoromethyl) -3H-diazacyclopropan-3-yl) benzyl) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (0.064g, 0.096mmol) in DCM (4mL) to give a pale yellow solution. After stirring for 30min at 20 ℃, the reaction mixture was diluted with DCM (15mL) and saturated NaHCO₃Washed (10mL) and the aqueous layer was extracted backwards with DCM (10 mL). Over anhydrous MgSO₄The combined organic layers were dried, filtered and concentrated to dryness to provide intermediate 53C: 4- ((3- ((3-aminopropyl (methyl) amino) propyl) amino) -2- (3- (3-trifluoromethyl) -3H-diazacyclopropan-3-yl) benzyl) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (45mg, 83% yield): HRMS M/z 569.2601(M + H)⁺(ii) a HPLC 97.1% @220nm and 96.9% @254nm, RT ═ 1.96 min.

2, 5-dioxopyrrolidin-1-yl-5- ((3aS, 4S,6aR) -2-oxohexahydro-1H-thiophene [3,4-d ]]Imidazol-4-yl) pentanoate (29.2mg, 0.085mmol) was added to 4- ((3- ((3-aminopropyl (methyl) amino) propyl) amino) -2- (3- (3-trifluoromethyl) -3H-diazacyclopropan-3-yl) benzyl) -9H-pyrimido [4,5-b]A mixture of methyl indole-7-carboxylate (45mg, 0.079mmol) and triethylamine (16.55. mu.L, 0.119mmol) in DMF (750. mu.L) gave a yellow solution. After stirring at 20 ℃ for 30min, the reaction mixture was concentrated to a light orange oil under high vacuum and the residue was purified by flash chromatography to yield 56mg of a white solid, starting from CH₃CN lyophilize it to provide the compound of example 53 as a white solid: 4- ((3- (methyl (3- (5- ((3aS, 4S,6aR) -2-Oxohexahydro-1H-thiophene [3, 4-d)]Imidazol-4-yl) pentaneamino) propyl) amino) -2- (3- (3-trifluoromethyl) -3H-diazacyclopropan-3-yl) benzyl) -9H-pyrimido [4,5-b]Indole-7-carboxylic acid methyl ester (51mg, 0.064mmol, 81% yield):¹H NMR(400MHz,DMSO-d₆)δppm 1.18-1.34(m,3H)1.35-1.50(m,3H)1.50-1.64(m,3H)1.70-1.82(m,2H)2.02(t,J＝7.4Hz,2H)2.15(s,3H)2.26-2.34(m,2H)2.37(t,J＝6.7Hz,2H)2.55(d,J＝12.5Hz,1H)2.77(dd,J＝12.1,5.1Hz,1H)2.99-3.10(m,3H)3.56-3.66(m,2H)3.88(s,3H)4.03-4.14(m,1H)4.10(s,2H)4.26(dd,J＝7.6,5.3Hz,1H)6.34(s,1H)6.39(s,1H)7.14(d,J＝7.4Hz,1H)7.28(s,1H)7.44(t,J＝7.8Hz,1H)7.52(d,J＝7.8Hz,1H)7.56(t,J＝5.3Hz,1H)7.73(t,J＝5.5Hz,1H)7.83(dd,J＝8.2,1.4Hz,1H)8.00(d,J＝1.4Hz,1H)8.28(d,J＝8.2Hz,1H)12.07(s,1H)；HRMS m/z 795.3368(M+H)⁺(ii) a HPLC 95.4% @220nm and 96.2% @254nm, RT ═ 2.06 min.

Example 55

2-benzyl-4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4, 5-b) in methylamine 2M]A solution of methyl indole-7-carboxylate (example 27, 0.030g, 0.066mmol) in MeOH (10.00mL) was placed in a sealed tube and heated to 110 ℃ for 66 hours, then the mixture was cooled to 20 ℃, concentrated to dryness and purified by flash chromatography to provide 28mg of a colorless oil, which was suspended in ether (2 mL). After stirring the resulting suspension for 2 hours, the solid was collected on a Buchner, the cake was washed with diethyl ether (2 x 0.5mL), and the product was dried under high vacuum at 40 ℃ until constant weight to provide example 55 as a white solid: methyl 2-benzyl-N-methyl-4- ((3- (piperidin-1-yl) propyl) amino) -9H-pyrimido [4,5-b]Indole-7-carboxamide (23mg, 77% yield):¹H NMR(400MHz,DMSO-d₆) δ ppm 1.38(m, J ═ 5.1Hz,2H)1.50 (quintuple, J ═ 5.5Hz,4H)1.80 (quintuple, J ═ 7.0Hz,2H)2.22-2.41(m,6H)2.81(d, J ═ 4.3Hz,3H)3.58-3.68(m,2H)4.03(s,2H)7.15-7.21(m,1H)7.27(m, J ═ 7.4,7.4Hz,3H)7.34-7.40(m,2H)7.70(dd, J ═ 8.2,1.4Hz,1H)7.90(d, J ═ 1.4Hz,1H)8.27(d, J ═ 8.2, 1H)8.46, 1H (q ═ 8.46, 1H) 8.11H (s, 1H); HRMS M/z 457.2708(M + H)⁺(ii) a HPLC > 99.5% @220nm and 98.9% @254nm, RT 1.53 min.

The reported HPLC retention times were used for reverse phase HPLC (Agilent, 1200 series) solvent A with the following conditions MeOH: H₂TFA (5: 95: 0.05); solvent B MeOH H₂TFA (95: 5: 0.05); flow rate: 3.0 mL/min; gradient 0 to 100% B in 2.0 min; column: ZorbaxC18, 3.5 microns, 4.6x 30 mm; the wavelength is 220 nm.

TABLE 2 Structure, analytical HPLC retention time, LCMS data and biological data for the examples

EC50 was defined as the concentration that resulted in a 50% increase in CD34+ CD45 RA-cell count compared to vehicle medium (DMSO). EC₅₀A is more than 1000 nM; b is 500 nM; c250-; d100-; e ═ 100 nM; f ═ compound showing > 1.3 fold amplification.

And (3) analyzing in vitro functions:

the ex vivo functionality of the expanded cells was examined using a conventional cultured colony forming unit (CFU-C) assay. Untreated cells or cells incubated with DMSO, positive controls or compounds of the invention were inoculated into methylcellulose medium under conventional conditions. As an example, compound 1 (table 2, example 1) expanded a number of multipotent hematopoietic progenitor cells. Methylcellulose culture of 1000CD34+ mPB cells treated with compound 1 for 10 days resulted in a 5-fold increase in multipotent granulocyte erythrocytes, macrophages and megakaryocytes (GEMM clones) over the input cells, and a 10-fold increase compared to control cells. This indicates that compound 1 promotes the expansion of pluripotent progenitor cells.

In vivo functional analysis:

CD34+ mPB cells cultured with the compounds of the present invention were grafted with immunodeficiency strain NOD Severe Combined immunodeficiency Gamma (NSG) mice. Results of 2,000,000 and 500,000CD34+ mPB cells incubated for 10 days with compound 1 (table 2, example 1) or vehicle control conditions were transplanted into NSG mice. Human hematopoietic reconstitution in NSG bone marrow was examined 8 weeks after transplantation using an antibody to human CD 45. Cells treated with compound 1, but not with the vehicle, were able to engraft NSG mice. In addition, reconstitution of human bone marrow and lymphoid compartments was also demonstrated, as bone marrow cells were positive for human CD33+ and CD19+, respectively. These results show that CD34+ mPB amplified with compound 1 not only helped grafting but also retained the multipotent population recovery potential in vivo.

Combination of compounds:

CD34+ mPB cells grafted immunodeficiency strain NOD severe combined immunodeficiency gamma (NSG) mice cultured with the compounds of the invention. Results of 2,000,000 and 500,000CD34+ mPB cells incubated for 10 days with compound 1 (table 2, example 1) or vehicle control conditions were transplanted into NSG mice. Human hematopoietic reconstitution in NSG bone marrow was examined 8 weeks after transplantation using an antibody to human CD 45. Cells treated with compound 1, but not with the vehicle, were able to engraft NSG mice. In addition, reconstitution of human bone marrow and lymphoid compartments was also demonstrated, as bone marrow cells were positive for human CD33+ and CD19+, respectively. These results indicate that CD34+ mPB amplified with compound 1 not only helped grafting but also retained the multipotent population recovery potential in vivo.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the scope of the present disclosure and appended claims.

Claims (28)

1. A compound of formula II

Or a salt thereof,

wherein:

z is-C (O) OMe, -C (O) OEt, CN,or-C (O) NHMe

W is

R²Is a group of formulae-H, -Me,

with the proviso that the compound of the formula II is not

2. A compound of formula IIA

Or a salt thereof,

wherein R is¹Is Me or Et and W and R²Each according to the definition in claim 1.

3. A compound of the general formula IIB

Or a salt thereof,

wherein W and R²Each as defined in claim 1,and Het is

4. A compound according to any one of claims 1 to 3, wherein R²Is benzyl.

5. The compound of claim 1, wherein:

z is CO₂Me or 2-methyl-2H-tetrazol-5-yl;

R²is benzyl or H.

6. The compound of claim 5, wherein R²Is H.

7. The compound of claim 5, wherein W is

8. The compound of claim 7, wherein Z is

9. The compound of claim 8, wherein R²Is H.

10. A compound, which compound is:

or salts of compounds 1-18, salts of compounds 21-25, salts of compounds 33-34, salts of compounds 37-40, salts of compounds 42-48, and salts of compounds 50-55.

11. A compound, which compound is:

12. a compound, which compound is:

or a salt thereof.

13. A pharmaceutical composition comprising a compound or salt thereof as defined in any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. Use of an agent comprising a compound or salt thereof as defined in any one of claims 1 to 12 in the manufacture of a medicament for increasing stem and/or progenitor cells, wherein a starting cell population is contacted with the agent in vitro or ex vivo.

15. The use of claim 14, which is ex vivo.

16. The use of claim 14 or 15, comprising contacting the starting cell population with at least one cell expansion factor.

17. The use of claim 14 or 15, wherein the starting cell population comprises CD34+ cells harvested from mobilized peripheral blood (mPB), Bone Marrow (BM) or Umbilical Cord Blood (UCB).

18. Use of at least one compound as defined in any one of claims 1 to 12, or a salt thereof, optionally together with at least one cell expansion factor, for the manufacture of a medicament for expanding hematopoietic stem cells, wherein a starting cell population is cultured in vitro or ex vivo in the presence of at least one said compound or salt thereof.

19. The pharmaceutical composition of claim 13, which is suitable for intravenous injection.

20. A kit for increasing stem and/or progenitor cells comprising a compound or salt thereof as defined in any one of claims 1 to 12 and instructions for use.

21. A kit for expanding hematopoietic stem cells comprising a compound or salt thereof as defined in any one of claims 1 to 12 and instructions for use, optionally the kit comprising at least one cell expansion factor.

22. The compound of claim 12, wherein the salt of the compound isThe hydrobromide salt of (1).

23. The use of claim 14 or 18, wherein the starting cell population consists of one or two cord blood unit purified CD34+ cells.

24. The use of claim 23, wherein the starting cell population is cultured in the presence of the compound for 2 to 21 days.

25. The use of claim 14 or 18, said compound being administered to said starting cell population at a concentration between 1nM and 3000 nM.

26. The use of claim 16 or 18, wherein the cell expansion factor is interleukin-3 (IL-3), granulocyte macrophage colony stimulating factor (GM-CSF), Thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), Stem Cell Factor (SCF), interleukin-6 (IL-6), or a combination thereof.

27. The use of claim 26, wherein the cell expansion factor is SCF, FLT3-L, TPO, IL-6, or a combination thereof.

28. The use according to claim 18, wherein said at least one cell expansion factor is StemRegenin 1(SR 1).