Peptide Drug Conjugates

Abstract

The present invention relates to compounds of formula (I) which are peptidase-enhanced cytotoxic agents. The present invention also relates to pharmaceutical compositions containing said compounds, and to the use of said compounds or pharmaceutical compositions as medicaments for the prevention or treatment of cancers, particularly multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma. [Formula 1] TIFF2024523411000113.tif52170

Description

[Technical field]

[0001] This invention relates to peptide drug conjugates (PDCs) and the use of such compounds in the treatment of diseases such as cancer. [Background technology]

[0002] Increased expression of various hydrolases, such as peptidases, esterases and proteases, has been described in several human malignant tumors, especially those characterized by rapid growth and aggressiveness (Pharmacol Ther. 1999; 83; 67-123). Zn, which is widely expressed in mammalian cells, 2+ Aminopeptidase N (APN, also known as CD13), a CD13-dependent membrane-bound ectopeptidase, plays an important role in cancer development, including processes such as tumor cell invasion, differentiation, proliferation, apoptosis, motility, and angiogenesis (Cancer Lett. 2006; 243; 135-43; Curr Med Chem. 2007; 14; 639-47; Curr Med Chem. 2008; 15; 2850-65). The multiple functions of APN have led to it being called a "moonlighting ectoenzyme" (Trends Mol Med. 2008; 14; 361-71). Taken together, these capabilities suggest that APN may be a therapeutic target in the treatment of cancer. Various approaches are being used to develop new drugs directed against APN, including enzyme inhibitors and APN-targeting carrier constructs (Cancer Sci. 2011; 102; 501-8).

[0003] One class of drugs that may be used in the treatment of cancer that utilizes hydrolytic enzymes such as peptidases, esterases, and proteases present in malignant tumors are peptide drug conjugates (PDCs).We have found a new group of peptide drug conjugates that are useful in the treatment of cancer. Summary of the Invention

[0004] The invention relates to a compound of formula (I) as defined in claim 1 or a pharma- ceutically acceptable salt, ester, amide or carbamate thereof, including the salts of such esters, amides or carbamates.

[0005] The inventors have found that the compound of formula (I) is a potent anti-cancer agent. In particular, the inventors have demonstrated in an in vitro cytotoxicity assay that the compound of formula (I) exhibits excellent in vitro cytotoxicity against various hematological cancer cell lines. The inventors have also found that the compound of formula (I) is effective in reducing tumor growth in an in ovo xenograft model of lymphoma in chicken embryos. Furthermore, the inventors have found that the compound of formula (I) is readily hydrolyzed in cancer cells, preferentially sequestered and retained in the cells, and forms metabolites that exhibit potent alkylating activity.

[0006] The present invention further provides for a pharmaceutical composition comprising a compound of the present invention together with a pharma- ceutically acceptable carrier.The pharmaceutical composition may optionally contain one or more additional therapeutic agents, such as steroids, checkpoint inhibitors, nuclear transport inhibitors, anti-apoptotic inhibitors, cell therapy (including adoptive cell therapy), bispecific T cell inducers (BiTEs), immunomodulatory imid drugs (IMiDs), proteasome inhibitors (PIs), histone deacetylase (HDAC) inhibitors, peptide drug conjugates (PDCs), alkylating agents, or DNA intercalators.

[0007] The invention also provides a compound or pharmaceutical composition of the invention for use as a medicament.In addition, the invention also provides a compound or pharmaceutical composition of the invention for use in the prevention or treatment of cancer, such as multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

[0008] The invention also provides a method of treating a patient comprising administering a pharma- ceutically effective amount of a compound or pharmaceutical composition of the invention.

[0009] The invention also provides the use of a compound of the invention in the manufacture of a medicament for preventing or treating cancer.

[0010] There is further provided another compound of the invention as defined in claim 25. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of measuring the intracellular and extracellular concentrations of a metabolic compound of the present invention (Example compound 18, identified after treatment with Example compound 1 of the present invention, FIG. 1(A)), bendamustine (FIG. 1(A)), and melphalan (FIG. 1(B)) as comparative controls following in vitro treatment of MM.1S cells. [Diagram 2] FIG. 2 shows the effect of Example Compound 1 on tumor growth of SU-DHL-4 human lymphoma cell line xenografts in a chicken embryo in ovo model. [Diagram 3] 3(A)-(G) show the results of measuring the intracellular and extracellular concentrations of the compound of the present invention and its metabolites after treatment of MM.1S cells in vitro. [Figure 4] 4(A)-(C) show the results of assays examining DNA damage caused by compounds of the invention and controls. [Diagram 5] 5(A) and (B) show the results of an assay examining DNA fragmentation caused by compounds of the invention and controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention The inventors have found new PDCs that are highly cytotoxic to human cancer cells, particularly human blood cancer cells. As described in the Examples section, various example compounds of the present invention were synthesized and tested for cytotoxicity against several blood cancer cell lines in in vitro cytotoxicity assays. The inventors have found that the compounds of the present invention are highly potent and exhibit selectivity against blood cancer cells, as shown by the low cytotoxicity of the compounds against the fibroblast cell line BJ. The inventors have found that example compound 1 is particularly effective in inhibiting tumor growth in an in ovo xenograft model in chicken embryos using the human lymphoma cell line SU-DHL-4.

[0013] In another experiment, the inventors found that the example compounds of the present invention were easily hydrolyzed in MM.1S cancer cells to form metabolites that were sequestered and retained in the cells and had strong alkylating activity, demonstrating that the compounds of the present invention are new PDCs that are effective in the prevention or treatment of cancer, particularly blood cancer.

[0014] The present invention relates to a compound represented by formula (I) [ka] (wherein W1, W2, W3 and W4 are each CH, or one of W1, W2, W3 and W4 is N and the others are CH). The present invention relates to a compound represented by the formula:

[0015] In some embodiments, W2, W3 and W4 are each CH and W1 is N.

[0016] It is immediately understood that the -N(CH2CH2Cl)2 group is attached to the ring backbone of the molecule through a carbon atom of the ring. In such a W group, there is no H of CH.

[0017] In a preferred embodiment, W1, W2, W3 and W4 are each CH, and the compound of formula (I) has formula (Ia): [ka] The compound is represented by the formula:

[0018] In a preferred embodiment, the compound of formula (I) has the formula (Ib) [ka] It is a compound represented by the formula:

[0019] In the compound of formula (I), X is C 1-6 For example, X is C 1-4 Alkylene, C 1-3 Alkylene, C 1-2 X may be an alkylene, a C2 alkylene or a C1 alkylene. X may be a straight or branched alkylene. The alkylene of X is in the imidazole ring of the compound of the present invention and in the peptide (i.e., R 3 ) to form a bond. Alkylene linkers of X having 1 or 2 carbon atoms have been found to be particularly effective. Thus, in a preferred embodiment, X is a C1 alkylene (i.e., -CH2-) or a linear C2 alkylene (i.e., -CH2-CH2-).

[0020] In the compound represented by formula (I), R 1 C, which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen 1-4 alkyl, and halogen (e.g., H, C 1-4 (selected from the group consisting of alkyl and halogen). For example, R 1 may be selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, F, Cl, Br, and I. Optionally, these groups may be substituted with one, two, or three substituents independently selected from halogen. In certain embodiments, R 1is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, F and Cl. 1 is H.

[0021] In the compound represented by formula (I), R 2 is H, phenyl optionally substituted with 1, 2 or 3 substituents independently selected from halogen, and C optionally substituted with 1, 2 or 3 substituents independently selected from halogen. 1-6 For example, R 2 is H, phenyl optionally substituted with 1, 2 or 3 F or Cl, and C optionally substituted with 1, 2 or 3 F or Cl. 1-4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl). 2 is phenyl optionally substituted with 1, 2 or 3 substituents independently selected from halogen, and C 1-6 C, optionally substituted with 1, 2 or 3 substituents selected from, for example, halogen; 1-4 In certain embodiments, R 2 is the unsubstituted C 1-2 It is alkyl (eg methyl) or unsubstituted phenyl.

[0022] In the compound represented by formula (I), R 3 is represented by the formula (II) [ka] Or formula (III) [ka] It is a group represented by the following formula:

[0023] In a preferred embodiment, R 3 is represented by the formula (IIa) [ka] or formula (IIIa) [ka] It is a group represented by the following formula:

[0024] For the avoidance of doubt, in formulae (II), (III), (IIa) and (IIIa), [ka] indicates the point of attachment of the group of formula (II), (III), (IIa) or (IIIa) to the compound of formula (I), (Ia) or (IIa).

[0025] In formulae (II), (IIa), (III) and (IIIa), R 4 is N(R c )(R d ) and formula (IV) [ka] The group consisting of the groups represented by:

[0026] In a preferred embodiment, R 4 is N(R c )(R d ) and formula (IVa) [ka] The group consisting of the groups represented by:

[0027] For the avoidance of doubt, in formula (IV) and (IVa) [ka] indicates the point of attachment of a group of formula (IV) or (IVa) to a compound of formula (II), (III), (IIa) or (IIIa).

[0028] In the compound represented by formula (I), R 4 is a group represented by formula (IV) or formula (IVa), R 5 is R b It is.

[0029] R b -OH, -N(R e )(R f ), and halogens, -OH, -CN, -N(R e )(R f ), -C 6-10 -OC, which may be substituted with one or more substituents selected from aryl, or a 3- to 12-membered heterocycle having one or more O, N or S atoms and optionally substituted with 1, 2 or 3 halogen atoms; 1-6 alkyl, optionally interrupted by 1, 2 or 3 O, N or S atoms.

[0030] For example, R 4 is a group represented by formula (IV) or formula (IVa), R 5 (i.e. R b ) is -OH and -OC 1-4 alkyl (e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy or tert-butoxy). 4 is a group represented by formula (IV) or formula (IVa), R 5 -OH and -OC 1-3 alkyl (e.g., methoxy, ethoxy, propoxy, or isopropoxy). More preferably, R 4 is a group represented by formula (IV) or formula (IVa), R 5 is selected from the group consisting of methoxy, ethoxy or isopropoxy.

[0031] In the compound represented by formula (I), R 4 N(R c )(R d ), then R 5 is R b and formula (V) [ka] The group consisting of the groups represented by:

[0032] In a preferred embodiment, R 4 N(R c )(R d ), then R 5 -OH, -OC 1-6 Alkyl or formula (Va) [ka] It is a group represented by the following formula:

[0033] For the avoidance of doubt, in formulas (V) and (Va), [ka] indicates the point of attachment of a group of formula (V) or (Va) to a compound of formula (II), (III), (IIa) or (IIIa).

[0034] In some embodiments, R 4 is N(R c )(R d ) and R 5 (i.e. R b ) is OH, -OC 1-4 alkyl (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy or tert-butoxy), or R 5 may be selected from the groups represented by formula (V) and formula (Va). 4 N(R c )(R d ), then R 5 -OH, -OC 1-3More preferably, R is selected from the group consisting of alkyl (e.g., methoxy, ethoxy, propoxy or isopropoxy) and a group represented by formula (Va). 4 N(R c )(R d ), then R 5 is selected from the group consisting of methoxy, ethoxy or isopropoxy.

[0035] The inventors have 4 But N(R c )(R d ), a group represented by formula (IV) or formula (IVa), 5 (i.e. R b ) is -OC 1-3 It has been found that compounds of formula (I) that are alkyl (e.g., methoxy, ethoxy, or isopropoxy) are particularly cytotoxic to cancer cells. Thus, in certain preferred embodiments, in compounds of formula (I), R 5 Ha-OC 1-3 alkyl (e.g., methoxy, ethoxy, or isopropoxy). That is, R 4 But N(R c )(R d ), when R is a group represented by formula (IV) or formula (IVa), 5 -OC 1-3 It is preferably alkyl (eg methoxy, ethoxy or isopropoxy).

[0036] In certain embodiments, R 5 is -OH.

[0037] In the compound represented by formula (I), each R a , H, C 1-6 alkyl, -CH2-phenyl or -CH2-heterocyclyl, wherein the heterocyclyl is a 3- to 12-membered heterocyclyl having 1, 2, 3 and 4 heteroatoms selected from N, O and S, and the C 1-6 Alkyl is -OH, -OC 1-6and optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of alkyl, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 and halogen, wherein said phenyl or heterocyclyl is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of -NH2, -OH, -OC 1-6 Each R may be substituted with one, two, or three substituents independently selected from the group consisting of alkyl and -NO2. a -H, -C 1-6 Independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl and -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3 or 4 N or S atoms, wherein C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen (e.g., F, Cl, Br or I); phenyl may be substituted with halogen (e.g., F, Cl, Br or I), -NH2, -OH, -OC 1-6 Each R may be substituted with one, two or three substituents independently selected from the group consisting of alkyl and -NO2. a , H, C 1-4 Each R is independently selected from the group consisting of alkyl and -CH-phenyl, where alkyl or phenyl may be optionally substituted with 1 or 2 halogens (e.g., F or Cl). For example, each R a may be independently methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, -CH-phenyl, -CH-fluorophenyl, -CH-chlorophenyl, -CH-difluorophenyl, or -CH-dichlorophenyl. a may independently be isopropyl, isobutyl, sec-butyl, -CH2-phenyl, or -CH2-fluorophenyl (i.e., 2-fluorobenzyl, 3-fluorobenzyl, or 4-fluorobenzyl).

[0038] In the compound represented by formula (I), R b -OH, -N(R e)(R f ), and halogens, -OH, -CN, -N(R e )(R f ), -C 6-10 -OC, which may be substituted with one or more substituents selected from aryl, or a 3- to 12-membered heterocycle having one or more O, N or S atoms and optionally substituted with 1, 2 or 3 halogen atoms; 1-6 In certain embodiments, R is selected from the group consisting of alkyl, which may be interrupted by 1, 2, or 3 O, N, or S atoms. b is -OH or -OC 1-6 In another preferred embodiment, R b is -OH or -OC 1-6 Alkyl, e.g. -OC 1-6 Preferably, R b Ha-OC 1-4 For example, R b may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, or sec-butoxy. In certain preferred embodiments, R b is methoxy, ethoxy or isopropoxy. More preferably, R b is ethoxy.

[0039] In certain embodiments, R b is -OH.

[0040] R c and R d are H and -C, respectively. 1-6 Alkyl, -C(O)C 1-6 In preferred compounds, R is independently selected from the group consisting of alkyl and -CH-phenyl, said alkyl or said phenyl being optionally substituted with 1, 2 or 3 substituents selected from halogen. c is H and R d -H, -C 1-6 Alkyl and -C(O)C 1-6alkyl, which may be substituted with 1, 2 or 3 substituents selected from halogen. For example, R c is H and R d -H, -C 1-4 Alkyl and C(O)C 1-4 For example, R c is H and R d is selected from H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, -C(O)methyl, -C(O)ethyl, -C(O)propyl, -C(O)isopropyl, -C(O)butyl, -C(O)isobutyl and -C(O)sec-butyl. c is H and R d is selected from H, methyl and -C(O)methyl. c is H and R d is H.

[0041] In the compound represented by formula (I), R e and R f are H and -C, respectively. 1-6 alkyl, optionally substituted with 1, 2 or 3 substituents selected from halogen; or R e and R f together with the nitrogen atom to which it is attached form a 4-, 5- or 6-membered heterocyclic ring which may be substituted with 1, 2 or 3 substituents selected from among halogen. e and R f are each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and sec-butyl. For example, R e and R f are each independently selected from the group consisting of H and methyl. e and R f are all H, all methyl, or one H and the other methyl.

[0042] In certain embodiments, the compound of the present invention is a compound of formula (Ia), or a pharma- ceutically acceptable salt, ester, amide, or carbamate thereof, including the salts of such esters, amides, or carbamates, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 C optionally substituted with 1, 2 or 3 F 1-2 alkyl or phenyl optionally substituted with 1, 2 or 3 F, e.g., R 2 is the unsubstituted C 1-2 may be alkyl or unsubstituted phenyl; R 3 is a group represented by formula (IIa) or formula (IIIa); R 4 is N(R c )(R d ) and a group represented by formula (IVa); R 4 is a group represented by formula (IVa), R 5 (i.e. R b ) is -OC 1-4 is alkyl, R 4 N(R c )(R d ), then R 5 Ha-OC 1-4 alkyl or a group represented by formula (Va); Each R a -H, -C 1-6 is independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3 or 4 N or S atoms; 1-6 The alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen, and the phenyl may be substituted with halogen, -NH2, -OH, -OC 1-6optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2; R b Ha-OC 1-6 is alkyl; R c is H and R d -H, -C 1-4 Alkyl and -C(O)C 1-4 is selected from alkyl.

[0043] In another preferred embodiment, the compound of the present invention is a compound of formula (Ia), or a pharma- ceutically acceptable salt, ester, amide, or carbamate thereof, including the salts of such esters, amides, or carbamates, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 is methyl or phenyl; R 3 is a group represented by formula (IIa) or formula (IIIa); R 4 is N(R c )(R d ) or a group represented by formula (IVa); R 5 (i.e. R b ) is -OC 1-3 is alkyl; Each R a -H, -C 1-6 independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3 or 4 N or S atoms, wherein C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen; phenyl may be substituted with halogen, NH2, -OH, -OC 1-6It may be substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2.

[0044] The inventors have disclosed a compound of formula (Ia) or a pharma- ceutically acceptable salt, ester, amide or carbamate thereof, including the salts of such esters, amides or carbamates, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 is methyl or phenyl; R 3 is a group represented by formula (IIa) or formula (IIIa); R 4 is N(R c )(R d ) or a group represented by formula (IVa); R 5 (i.e. R b ) is -OC 1-3 is alkyl; Each R a found that compounds independently selected from the group consisting of isopropyl, isobutyl, and -CH2-phenyl optionally substituted with one F (e.g., 2-fluorobenzyl, 3-fluorobenzyl, or 4-fluorobenzyl) were particularly cytotoxic to hematological cancer cells in in vitro cytotoxicity assays and in a chicken embryo in ovo xenograft model of lymphoma.

[0045] In another preferred embodiment, the compound of the present invention is a compound of formula (Ia), or a pharma- ceutically acceptable salt, ester, amide, or carbamate thereof, including the salts of such esters, amides, or carbamates, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2C optionally substituted with 1, 2 or 3 F 1-2 phenyl optionally substituted with alkyl or 1, 2 or 3 F, e.g., R 2 is the unsubstituted C 1-2 may be alkyl or unsubstituted phenyl; R 3 is a group represented by formula (IIa); R 4 is NH2; R 5 (i.e. R b ) is -OC 1-4 is alkyl; Each R a -H, -C 1-6 independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3 or 4 N or S atoms, wherein C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen; phenyl may be substituted with halogen, -NH2, -OH, -OC 1-6 It may be substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2.

[0046] In another preferred embodiment, the compound of the present invention is a compound of formula (Ia), or a pharma- ceutically acceptable salt, ester, amide, or carbamate thereof, including the salts of such esters, amides, or carbamates, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 C optionally substituted with 1, 2 or 3 F 1-2 phenyl optionally substituted with alkyl or 1, 2 or 3 F, e.g., R 2 is the unsubstituted C 1-2alkyl or unsubstituted phenyl; R 3 is a group represented by formula (IIIa); R 4 is NH2; R 5 Ha-OC 1-4 is alkyl; Each R a -H, -C 1-6 independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3 or 4 N or S atoms, wherein C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen; phenyl may be substituted with halogen, -NH2, -OH, -OC 1-6 It may be substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2.

[0047] In another preferred embodiment, the compound of the present invention is a compound of formula (Ia), or a pharma- ceutically acceptable salt, ester, amide, or carbamate thereof, including the salts of such esters, amides, or carbamates, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 C optionally substituted with 1, 2 or 3 F 1-2 phenyl optionally substituted with alkyl or 1, 2 or 3 F, e.g., R 2 is the unsubstituted C 1-2 alkyl or unsubstituted phenyl; R 3 is a group represented by formula (IIIa); R 4 is a group represented by formula (IVa); R 5 Ha-OC1-4 is alkyl; Each R a -H, -C 1-6 Independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl, or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3, or 4 N or S atoms, where -C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen; phenyl may be substituted with halogen, -NH2, -OH, -OC 1-6 It may be substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2.

[0048] In certain preferred embodiments, the compounds of the invention are (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 1); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example compound 2); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid ethyl ester (Example compound 3); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 4); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example compound 5); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid ethyl ester (Example compound 6); (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 7); (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 8); (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 9); (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester (Example compound 10); (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester (Example compound 11); (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester (Example compound 12); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 13); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid methyl ester (Example compound 14); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid isopropyl ester (Example compound 15); (2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 16); (2S)-2-[[(2R)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 21); (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example compound 22); (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 23); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 2-morpholinoethyl ester (Example compound 24); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 2-isopropoxyethyl ester (Example compound 25); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid isopropyl ester (Example compound 26); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid methyl ester (Example compound 27); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 3-(dimethylamino)propyl ester (Example compound 28); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (2-methoxy-1-methyl-ethyl) ester (Example compound 29); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,N,4-trimethyl-pentanamide (Example compound 30); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,4-dimethyl-pentanamide (Example compound 31); (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 32); (2S)-2-[[(2S)-2-acetamido-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example compound 34); (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 35); (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example compound 36); (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 38); (2R)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 45); (2S)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 46); (2R)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 47); (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 48); and (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 50) or a pharma- ceutically acceptable salt, amide or carbamate thereof, including salts of such amides or carbamates.

[0049] The compounds of the present invention can be prepared by methods known to those skilled in the art of organic chemistry. Representative procedures for the preparation of compounds of formula (I) are described in the Examples section.

[0050] In some embodiments, the compounds of the present invention may contain isotopic atoms. In this specification, an isotopic atom is an atom of an element that is not the most common isotope of naturally occurring hydrogen. Deuterium is a safe and stable isotope of hydrogen. In some embodiments, the compounds of the present invention have more deuterium than the natural abundance of deuterium. The natural abundance of deuterium is 0.0156 mol%, where mol% is the percentage of deuterium to the total moles of hydrogen in a sample. Thus, in one mole of natural hydrogen, 0.156 mmol is deuterium, or 6.022 x 10 23 A sample of natural hydrogen atoms has 9.39 × 10 19There are deuterium atoms present, or 6413 naturally occurring hydrogen atoms contain 1 deuterium atom. The level of deuterium above the natural abundance of deuterium may be at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% deuterium. In certain embodiments, at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% deuterium is present in the compounds of the invention. Procedures for producing deuterated compounds are known in the art. See, for example, Sajiki, New Horizons of Process Chemistry (2017), Springer, pg 29-40 and Hanson, The Organic Chemistry of Isotopic Labelling (2011), Chapter 3, RSC Publishing.

[0051] Depending on the substituents present in the compounds of the present invention, the compounds can form esters, amides, carbamates and / or salts. Salts of the compounds of the present invention suitable for use in medicine are those in which the counterions are pharma- ceutically acceptable. However, salts with pharma-ceutically unacceptable counterions are included within the scope of the present invention, for example, for use as intermediates in the preparation of the compounds of the present invention and their pharma-ceutically acceptable salts and physiologically functional derivatives. The term "physiologically functional derivatives" refers to chemical derivatives of the compounds of the present invention that have the same physiological function as the compounds of the present invention, for example, by being convertible thereto in the body. Esters, amides and carbamates are examples of physiologically functional derivatives.

[0052] Salt forms suitable for use in the present invention include those formed with organic or inorganic acids or bases. In particular, suitable salts formed with the acids of the present invention include salts formed with inorganic acids, strong organic carboxylic acids such as alkane carboxylic acids having 1 to 4 carbon atoms, unsubstituted or substituted, for example, by halogens, saturated or unsaturated dicarboxylic acids, hydroxycarboxylic acids or amino acids, or organic sulfonic acids such as (C1-C4) alkyl or aryl sulfonic acids, unsubstituted or substituted, for example, by halogens. Pharmaceutically acceptable acid addition salts include those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, isethionic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid, and glutamic acid, lysine, and arginine. Other acids, such as oxalic acid, are not themselves pharma- ceutical acceptable, but may be useful as intermediates in obtaining the compounds of the invention and their pharma- ceutical acceptable acid addition salts.

[0053] Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts (e.g. potassium and sodium salts), alkaline earth metal salts (e.g. calcium and magnesium salts), and salts with organic bases, such as, for example, dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di- or tri-lower alkylamines (e.g. ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl or dimethyl-propylamine), or mono-, di- or trihydroxy lower alkylamines (e.g. mono-, di- or triethanolamine). Corresponding internal salts may also be formed.

[0054] Preferred salts of the compounds of the invention include acid addition salts such as those formed with hydrochloric acid, hydrobromic acid, acetic acid, p-toluenesulfonic acid, tartaric acid, sulfuric acid, succinic acid, phosphoric acid, oxalic acid, nitric acid, methanesulfonic acid, malic acid, maleic acid and citric acid. More preferably, the salts of the compounds of the invention are hydrochloride salts (i.e., addition salts formed with hydrochloric acid).

[0055] Compounds that are inactive themselves but are converted to active drugs upon administration to a recipient are known as "prodrugs". For example, prodrugs may be converted in the body, for example by hydrolysis in the blood, into active forms that have medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series (1976);"Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985 and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987.

[0056] The compounds of claim 1 or claim 25 (and their corresponding dependent claims) of the present invention may be provided in the form of a prodrug. Examples of prodrugs include esters, amides and carbamates.

[0057] The compounds of the present invention may have suitable groups converted to esters, amides, or carbamates. Thus, representative ester and amide groups formed with acid groups in the compounds of the present invention include -COOR G , -CONR G 2. -SO2OR G or -SO2N(R G )2, and in the compound of the present invention, an OH group or an -NHR GRepresentative ester, amide and carbamate groups formed from the group -OC(O)R G , -NR G C(O)R G , -NR G CO2R G , -OSO2R G and -NR G SO2R G where R G is C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl and C 3-8 Cycloalkyl C 1-8 Alkyl, haloC 1-8 Alkyl, dihaloC 1-8 Alkyl, trihalo C 1-8 Alkyl, phenyl and phenyl C 1-4 alkyl; more preferably, R G is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl and C 3-8 Cycloalkyl C 1-6 alkyl.

[0058] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are known as "solvates." For example, a complex with water is known as a "hydrate." The complex can incorporate the solvent in stoichiometric or non-stoichiometric amounts. Solvates are described in Water-Insoluble Drug Formulation, 2 ndedn, R. Lui, CRC Press, page 553 and Byrn et al., Pharm. Res., 12(7), 1995, 945-954. Before going into solution, the compounds of the present invention, as well as their esters, amides, carbamates and / or salts, may be in the form of solvates. Solvates of the compounds of the present invention suitable for use as pharmaceuticals are those in which the solvent is pharma-ceutically acceptable. For example, hydrates are pharma-ceutically acceptable solvates.

[0059] The inventors have 5 Ga-OC 1~6 Alkyl, Formula (V) [ka] and formula (Va) [ka] and wherein the compound is selected from the group consisting of R b But -N(R e )(R f ) and -OC 1~6 It has been found that compounds of the present invention, in which the group is selected from alkyl, are readily hydrolyzed in cancer cells to form metabolites that are preferentially sequestered and retained within the cells and have potent alkylating activity.

[0060] Thus, the present invention also relates to a metabolite, said metabolite having a structure of formula (I), formula (Ia) or formula (Ib) or a salt thereof, wherein X is C 1-6 is alkylene; W1, W2, W3 and W4 are each CH or one of W1, W2, W3 and W4 is N and the others are CH; R 1 C, which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen 1-4 selected from the group consisting of alkyl, and halogen; R 2is H, phenyl optionally substituted with 1, 2 or 3 substituents independently selected from halogen, and C optionally substituted with 1, 2 or 3 substituents independently selected from halogen. 1-6 selected from the group consisting of alkyl; R 3 is a group represented by formula (II), formula (III), formula (IIa) or formula (IIIa); R 4 is N(R c )(R d ), a group represented by formula (IV) or formula (IVa); R 4 is a group represented by formula (IV) or formula (IVa), R 5 is -OH and R 4 N(R c )(R d ), then R 5 is -OH, a group represented by formula (V) or formula (Va); Each R a , H, C 1-6 alkyl, -CH2-phenyl or -CH2-heterocyclyl, wherein the heterocyclyl is a 3- to 12-membered heterocyclyl having 1, 2, 3 and 4 heteroatoms selected from N, O and S, and the -C 1-6 Alkyl is -OH, -OC 1-6 and optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of alkyl, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 and halogen, wherein said phenyl or said heterocyclyl is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of halogen, -NH2, -OH, -OC 1-6 optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2; R b is OH.

[0061] Preferred radicals of the metabolite compounds are as described above for compounds of formula (I), (Ia) and (Ib). For example, in certain embodiments, the metabolite has a structure of formula (I), (Ia) or (Ib) or is a salt thereof, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 is methyl or phenyl; R 3 is a group represented by formula (IIa) or formula (IIIa); R 4 is N(R c )(R d ) or a group represented by formula (IVa); R 4 is a group represented by formula (IVa), R 5 is -OH, R 4 N(R c )(R d ), then R 5 is -OH or a group represented by formula (Va); Each R a -H, -C 1-6 Independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl, or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3, or 4 N or S atoms, where -C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen; phenyl may be substituted with halogen, -NH2, -OH, -OC 1-6 optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of alkyl and -NO2; R b is OH.

[0062] In certain embodiments, the metabolite has a structure according to Formula (I), Formula (Ia) or Formula (Ib), or a salt thereof, wherein: X is -C 1-2 alkylene (i.e., -CH- or -CH-CH-); R 1 is H; R 2 is methyl or phenyl; R 3 is a group represented by formula (IIa) or formula (IIIa); R 4 is NH2 or a group represented by formula (IVa); R 5 is -OH; Each R a -H, -C 1-6 independently selected from the group consisting of alkyl, -CH2-indolyl, -CH2-phenyl or -CH2-heteroaryl, wherein said heteroaryl is a 5-membered heteroaryl having 1, 2, 3 or 4 N or S atoms, wherein C 1-6 Alkyl may be substituted with -OH, -NH2, -NHC(=NH)NH2, -C(O)OH, -C(O)NH2, -SH, -SCH3 or halogen; phenyl may be substituted with halogen, -NH2, -OH, -OC 1-6 Each R may be substituted with one, two or three substituents independently selected from the group consisting of alkyl and -NO2. a are independently selected from the group consisting of isopropyl, isobutyl, and -CH2-phenyl optionally substituted with one F (e.g., 2-fluorobenzyl, 3-fluorobenzyl, or 4-fluorobenzyl).

[0063] In certain preferred embodiments, the metabolite is (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid (Example compound 19); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 20); (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid (Example compound 33); (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid (Example compound 37); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid (Example compound 39); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 40); 2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid (Example compound 42); (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid (Example compound 43); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid (Example compound 44); and (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 51) or a salt thereof.

[0064] The inventors have further found that the compounds of the present invention are readily hydrolyzed in cancer cells to form more advanced metabolites that are preferentially sequestered and retained in the cells and have potent alkylating activity. Thus, the present invention also relates to metabolites of the structure represented by formula (I), formula (Ia) or formula (Ib), wherein: X is C 1-6 is alkylene; W1, W2, W3 and W4 are each CH or one of W1, W2, W3 and W4 is N and the others are CH; R 1 C, which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen 1-4 selected from the group consisting of alkyl, and halogen (e.g., F or Cl); R 2 is H, phenyl optionally substituted with 1, 2 or 3 substituents independently selected from halogen (e.g., F or Cl), and C optionally substituted with 1, 2 or 3 substituents independently selected from halogen (e.g., F or Cl). 1-6 selected from the group consisting of alkyl; R 3 is represented by formula (VIa) [ka] (In the formula, R c and R d are H and -C, respectively. 1-6 Alkyl, -C(O)C 1-6 independently selected from the group consisting of alkyl and -CH-phenyl, said alkyl or said phenyl being optionally substituted with 1, 2 or 3 substituents selected from halogen (preferably R c and R d is H); R g H and -C 1-6 alkyl, optionally substituted with 1, 2 or 3 substituents selected from halogen (preferably R g is H) It is a group represented by the following formula:

[0065] For the avoidance of doubt, in formula (VI) and (VIa) [ka] indicates the point of attachment of the group of formula (VI) or (VIa) to the compound of formula (I), (Ia) or (Ib).

[0066] In certain embodiments, the metabolite has a structure according to Formula (I), Formula (Ia) or Formula (Ib), or a salt thereof, wherein: X is C 1-6 is alkylene; R 1 -H, -C 1-4 selected from the group consisting of alkyl and halogen (e.g., F or Cl); R 2 is H, phenyl substituted with 1, 2 or 3 halogens (e.g., F or Cl), and C substituted with 1, 2 or 3 halogens (e.g., F or Cl). 1-6 Alkyl (e.g., phenyl substituted with 1, 2 or 3 halogens (e.g., F or Cl) and C substituted with 1, 2 or 3 halogens (e.g., F or Cl) 1-6 alkyl); R 3 is a group represented by formula (VIa); R c and R d is H.

[0067] In other preferred aspects, the metabolite has a structure according to Formula (I), Formula (Ia) or Formula (Ib), or a salt thereof, wherein: X is a C alkylene (i.e., -CH-) or a C alkylene (i.e., -CH-CH-); R 1 -H, -C 1-4 selected from the group consisting of alkyl and halogen (e.g., F or Cl); R 2is H, phenyl optionally substituted with 1, 2 or 3 halogens (e.g., F or Cl), and C optionally substituted with 1, 2 or 3 halogens (e.g., F or Cl). 1-6 selected from the group consisting of alkyl; R 3 is a group represented by formula (VIa); R c and R d is H.

[0068] Preferably, the compound is of formula (Ia) or (Ib).

[0069] The present invention therefore also relates to compounds of the structure of formula (I), (Ia) or (Ib), wherein X is C 2-6 is alkylene; W1, W2, W3 and W4 are each CH or one of W1, W2, W3 and W4 is N and the others are CH; R 1 C, which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen 1-4 selected from the group consisting of alkyl, and halogen (e.g., F or Cl); R 2 is H, phenyl optionally substituted with 1, 2 or 3 substituents independently selected from halogen (e.g., F or Cl), and -C optionally substituted with 1, 2 or 3 substituents independently selected from halogen (e.g., F or Cl). 1-6 selected from the group consisting of alkyl; R 3 is represented by formula (VI) [ka] (In the formula, R c and R d are H and -C, respectively. 1-6 Alkyl, -C(O)C 1-6and -CH-phenyl, wherein said alkyl or said phenyl is optionally substituted with 1, 2 or 3 substituents selected from halogen (e.g., R c and R d is H); R g H and -C 1-6 alkyl, which may be substituted with 1, 2 or 3 substituents selected from halogen (e.g., R g is H) It is a group represented by the following formula:

[0070] The more metabolized compounds can be used as medicines. Thus, the present invention relates to a compound of formula (I) for use as a medicine, for example for the treatment of cancer selected from the group consisting of multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma, for example multiple myeloma, acute myeloid leukemia or lymphoma. [ka] (In the formula, X is C 1-6 is alkylene; W1, W2, W3 and W4 are each CH or one of W1, W2, W3 and W4 is N and the others are CH; R 1 C, which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen 1-4 selected from the group consisting of alkyl, and halogen; R 2 is H, phenyl optionally substituted with 1, 2 or 3 substituents independently selected from halogen, and C optionally substituted with 1, 2 or 3 substituents independently selected from halogen. 1-6 selected from the group consisting of alkyl; R 3 is represented by formula (VI) [ka] (In the formula, R cand R d are H and -C, respectively. 1-6 Alkyl, -C(O)C 1-6 independently selected from the group consisting of alkyl and -CH2-phenyl, said alkyl or said phenyl being optionally substituted with 1, 2 or 3 substituents selected from among halogen; R g H and -C 1-6 alkyl, said alkyl being optionally substituted with 1, 2 or 3 substituents selected from halogen; is a group represented by or a salt or solvate thereof.

[0071] Preferred embodiments of these compounds for use as pharmaceuticals are as described above in relation to other aspects of the invention.

[0072] In certain preferred embodiments, the more advanced metabolites are (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid (Example compound 17); (S)-2-amino-4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid (Example compound 18); (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example compound 38); (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoic acid (Example compound 41); or (2S)-2-Amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid (Example compound 49) Or a salt thereof.

[0073] The metabolites described herein may be isolated from cells treated with one or more compounds of formula (I) and / or may be prepared using standard organic chemistry techniques. Exemplary procedures for preparing the metabolites of the invention are described in the Examples section. For the avoidance of doubt, the metabolites described herein are further examples of compounds of the invention and may be used in the form of compositions and / or as medicaments in the same manner as described herein for other compounds of formula (I).

[0074] The following definitions apply to terms used throughout this specification, unless otherwise limited in specific instances.

[0075] As used herein, the term "alkyl" refers to both straight-chain and branched-chain saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, pentyl and hexyl groups. Among the unbranched alkyl groups, methyl, ethyl, n-propyl, isopropyl and n-butyl groups are preferred. Among the branched alkyl groups, t-butyl, i-butyl, 1-ethylpropyl and 1-ethylbutyl groups can be mentioned.

[0076] As used herein, the term "cycloalkyl" refers to a saturated ring system. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0077] In this specification the term "halogen" means fluorine, chlorine, bromine or iodine, with fluorine, chlorine and bromine being preferred, and fluorine and chlorine being especially preferred.

[0078] In this specification, the term "heteroaryl containing 1, 2, 3 or 4 nitrogen, oxygen or sulfur atoms" refers to an aromatic cyclic group of carbon atoms in which 1, 2, 3 or 4 carbon atoms are replaced by 1, 2, 3 or 4 heteroatoms independently selected from nitrogen, oxygen and sulfur (preferably nitrogen and sulfur). Examples of 5-membered heteroaryl containing 1, 2 or 3 nitrogen and / or sulfur atoms include thiophene, thiazole, isothiazole, pyrrole, pyrroline, pyrazole, pyrazoline, imidazole, imidazoline, triazole and thiadiazole.

[0079] Pharmaceutical Compositions The compounds, compositions and pharmaceutical compositions of the present invention can be used to prevent and / or treat cancer, reducing tumor growth and / or killing tumor cells. Thus, the compounds of the present invention can be used to cure and / or prolong the survival of cancer patients. This invention is particularly useful for the prevention and / or treatment of carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed cancers, such as multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

[0080] The compound of the present invention can be administered alone, but is preferably present in a composition, particularly a pharmaceutical composition.The pharmaceutical composition includes those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous injection, intramuscular, intravascular (bolus or infusion), intravitreal and intraosseous), intraperitoneal, transmucosal, transdermal, rectal and topical (including cutaneous, buccal, sublingual and intraocular) administration, although the most suitable route may depend, for example, on the condition and disorder of the subject being treated.

[0081] Pharmaceutical compositions of the present invention suitable for oral administration can be provided as discrete dosage units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient, as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion. The compounds of the present invention can also be provided as a bolus, electuary or paste. Various pharmacologic carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences by EW Martin. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42;2S, 1988.

[0082] Pharmaceutical compositions of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may include antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the patient's blood, and aqueous and non-aqueous sterile suspensions, which may include suspending and thickening agents. Preferably, the formulations may be presented in unit dose or divided dose containers, such as sealed ampoules and vials. The formulations may be stored in a lyophilized state, requiring only the addition of sterile liquid carriers, such as saline or water for injection, immediately prior to use. Ready-to-administer injection and infusion solutions and suspensions may be prepared from sterile powders, granules or other dry compositions. Representative compositions for parenteral administration include injectable solutions or suspensions, which may include suitable non-toxic parenterally acceptable solutes, diluents or solvents, such as, for example, mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, synthetic mono- or diglycerides, oleic acid or other suitable fatty acids, including Cremophor®, dispersing or wetting agents and suspending agents.

[0083] Pharmaceutical compositions of the invention suitable for nasal administration, administration as an aerosol, or inhalation include saline, which may contain, for example, benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other solubilizing or dispersing agents known in the art.

[0084] Pharmaceutical compositions of the invention suitable for rectal administration can be presented as a suppository with conventional carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycols. Such carriers are normally solid at ordinary temperatures, but liquefy and / or melt in the rectum to release the drug.

[0085] Pharmaceutical compositions of the invention suitable for topical administration in the mouth, e.g., buccal or sublingual, include lozenges comprising the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).

[0086] Preferred unit dosage compositions are those containing a probing or therapeutic dose, or an appropriate fraction thereof, of a compound of the present invention.

[0087] In a preferred embodiment, the compositions of the present invention consist essentially of a compound of the present invention and at least one pharma- ceutically acceptable excipient.

[0088] It should be understood that the compositions used in the present invention may contain, in addition to the ingredients specifically mentioned above, other agents conventional in the art having regard to the type of composition.

[0089] The compositions of the invention may also include one or more other therapeutic agents. Examples of other therapeutic agents that may be present in the compositions of the invention include steroids (prednisone and dexamethasone), checkpoint inhibitors (e.g., CTLA-4 inhibitors and PD-1 / PD-L1 inhibitors), nuclear transport inhibitors (e.g., selinexor), anti-apoptotic inhibitors (e.g., venetoclax), adoptive cell therapy (e.g., tumor infiltrating lymphocyte (TIL) therapy, natural killer (NK) cell therapy, chimeric antigen receptor (CAR) T cell therapy, and engineered T cell receptor therapy), bispecific T cell inducers (BiTEs), immunomodulatory imines, and the like. These include, but are not limited to, interferon-dependent inhibitors (IMiDs) (e.g., thalidomide, lenalidomide, and pomalidomide), proteasome inhibitors (PIs) (e.g., bortezomib, carfilzomib, ixazomib, and marizomib), histone deacetylase (HDAC) inhibitors (e.g., panobinostat), peptide drug conjugates (PDCs) other than the compounds of the present invention (e.g., melflufen), alkylating agents (e.g., melphalan, cyclophosphamide), and DNA intercalators (e.g., doxorubicin).

[0090] treatment As previously explained, the compounds of the present invention are peptide drug conjugates (PDCs). The present invention relates to a compound of the present invention or a composition comprising a compound of the present invention together with a pharma- ceutically acceptable carrier for use as a medicament.

[0091] In particular, the compounds and compositions of the present invention are used to prevent and / or treat cancer. Specific examples of cancers that can be prevented or treated by administering the compounds or compositions of the present invention include carcinoma, sarcoma, myeloma, leukemia, lymphoma, or mixed cancers. Representative cancers that can be prevented or treated by administering the compounds or compositions of the present invention include hematological / blood cell cancers such as leukemia (e.g., acute lymphoblastic leukemia, including acute lymphoblastic leukemia in adults and children; acute myeloid leukemia, including acute myeloid leukemia in adults and children; chronic lymphocytic leukemia, such as B-cell chronic lymphocytic leukemia; chronic myeloid leukemia and hairy cell leukemia); lymphomas (e.g., AIDS-related lymphoma; cutaneous T-cell lymphoma; Hodgkin's lymphoma, including adult and childhood Hodgkin's lymphoma and Hodgkin's lymphoma in pregnancy; adult and childhood non-Hodgkin's lymphoma, including ... These include, but are not limited to, mycosis fungoides; Sézary syndrome; Waldenstrom's macroglobulinemia; primary mediastinal large B-cell lymphoma; mantle cell lymphoma; diffuse large B-cell lymphoma and primary central nervous system lymphoma); other blood cancers (e.g., chronic myeloproliferative disorders; multiple myeloma / plasma cell neoplasms; myelodysplastic syndromes and myelodysplastic / myeloproliferative disorders); osteosarcoma, ovarian cancer, breast cancer; lung cancer; glioblastoma; retinoblastoma and metastases of these cancers.

[0092] The inventors have found that the compounds of the invention are particularly cytotoxic to hematological cancer cells and are therefore particularly useful in the prevention or treatment of one or more of the above-mentioned hematological cancers.

[0093] In addition, the compounds can be used to treat solid tumors. The compounds and compositions of the present invention are also useful in methods of preventing or treating diseases or disorders, comprising administering the compounds of the present invention or the compositions of the present invention to a subject in need thereof. Thus, the compounds or compositions of the present invention can be administered to a subject suffering from or at risk of developing cancer, particularly hematological cancer.

[0094] The compounds of the invention are also used in the manufacture of a medicament, particularly for administration to a subject suffering from or at risk of developing cancer, particularly a hematological cancer.

[0095] The amount of the compound of the invention required to achieve a therapeutic effect will vary depending on the route of administration and the characteristics of the subject being treated, such as the species, age, weight, sex, physical condition, the disease or condition and its severity, and other medical and physical factors. A physician of ordinary skill can readily determine and administer the effective amount of the compound of the invention required to prevent or treat the disease or condition.

[0096] The compounds of the invention or salts and / or solvates thereof can be administered as a single dose daily (including several times a day), every other day, every third day, every week, every two weeks, every three weeks, every four weeks, or even higher, depending on the subject and the disease or disorder being treated.

[0097] Preferably, the compound of the present invention or the metabolite of the present invention or its salt and / or solvate can be administered in an amount of about 1 mg to 150 mg per administration (excluding the mass of counterions or solvents), for example, 1 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or 150 mg. Alternatively, the compound of the present invention or its salt and / or solvate can be administered in a single high dose (excluding the mass of counterions or solvents). The single high dose can be about 150 mg to 800 mg, for example, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg.

[0098] The compounds of the present invention can be used as the only active ingredient in this invention, but can also be used in combination with one or more other therapeutic agents, and the use of such combinations provides one of the preferred aspects of the present invention. Such other therapeutic agents can be drugs useful for preventing or treating cancer, or other pharmacologic active substances. Such drugs are known in the art. Suitable examples of other therapeutic agents for use in this invention are described in this specification.

[0099] The one or more other therapeutic agents can be used simultaneously, sequentially or separately with the administration of the compounds of the present invention. The individual components of such combinations can be administered separately at different times during the course of treatment or concurrently in divided or single combination forms.

[0100] Preferred unit dosage compositions for use in the present invention are those containing an effective dose, or an appropriate fraction thereof, of a compound of the present invention.

[0101] kit The present invention relates to kits comprising a compound of formula (I), one or more pharma- ceutically acceptable excipients, and, optionally, one or more other therapeutic agents, examples of which include those described herein as suitable for use in the invention and which may optionally be present in a pharmaceutical composition of the invention as other therapeutic agents.

[0102] The kit of the present invention is used for the prevention and / or treatment of cancer, particularly blood cancer.

[0103] In certain embodiments, the kits include one or more containers and may also include sampling devices, such as bottles, bags (such as intravenous fluid bags), vials, syringes, and test tubes. Other elements may include needles, diluents, washing reagents, and buffers. For practical purposes, the kits may include at least one container containing a pharma- ceutically acceptable organic solvent or a pharma-ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution.

[0104] Preferably, the kits of the invention include instructions, e.g., instructions instructing a user to mix a specified amount of a compound or composition of the invention with a specified amount of a physiologically acceptable aqueous solvent or diluent, a pharma- ceutically acceptable organic solvent, and / or one or more other therapeutic agents (optionally). Such instructions may also provide guidance regarding storage conditions and / or administration.

[0105] For the avoidance of doubt, the compound or composition of the invention, the physiologically acceptable aqueous solvent or diluent (optional), one or more pharma- ceutically acceptable organic solvents (optional), and one or more other therapeutic agents (optional) are present in the kit of the invention in forms and amounts suitable for the manufacture of pharmaceutical formulations of the invention. Those skilled in the art can readily determine the amounts of the compound or composition of the invention, the physiologically acceptable aqueous solvent or diluent, the pharma-ceutically acceptable organic solvent, and one or more other therapeutic agents (optional) suitable for use in the invention.

[0106] Equivalent The invention has been described broadly and generically herein. Those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are representative, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or uses for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the embodiments described above are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, combinations of two or more such features, systems, articles, materials, kits, and / or methods are within the scope of the invention, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent. Furthermore, species and subgenerics narrower than those falling within the scope of the generic disclosure also form part of the invention. This includes a negative limitation that removes subject matter from the general description of the invention or from a genus by proviso, regardless of whether the removed material was specifically described in the specification.

[0107] Incorporation by Reference The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entireties as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from such articles, patents, patent applications, or other physical and electronic documents.

[0108] The following examples illustrate the invention. EXAMPLES

[0109] Example compounds: The compounds of the present invention can be prepared by methods known to those skilled in the art. Other reaction schemes, solvents, temperatures and reaction conditions can be readily devised by those skilled in the art.

[0110] Intermediates 1-4 were synthesized according to General Scheme 1. First, an amidation reaction was carried out between the corresponding protected amino acid and aniline. The resulting product was cyclized under acidic conditions to form a benzimidazole heterocycle, giving intermediate 1 (n=1) and intermediate 3 (n=0). BOC protection and saponification were carried out to give intermediate 3 (n=1) and intermediate 4 (n=0).

[0111] [ka]

[0112] Peptide coupling between the intermediates (2 or 4) and the corresponding protected amino acids using TCFH afforded the desired dipeptides. Reduction of the nitro group was achieved using Pd / C under pressurized hydrogen atmosphere. The resulting anilines were alkylated with ethylene oxide under acidic conditions. Mesylation followed by chlorine displacement afforded the nitrogen mustard moiety. Finally, the BOC protecting group was removed with HCl or TFA to afford the desired analogs (Scheme 2).

[0113] Intermediates 2 and 4 can also be synthesized according to Scheme 13.

[0114] [ka]

[0115] Example compounds 1-6 were synthesized according to the synthetic route shown in Scheme 2. Example compounds 13-15, 19, 20, 22-36, 39, 40-42 and 44-51 were synthesized as detailed below. A similar strategy was applied to the synthesis of Example compounds 7-12, which have a benzimidazole substituent at the C-terminus of the dipeptide (Scheme 3). Example compounds 16, 21, 37 and 43 were synthesized as detailed below.

[0116] [ka]

[0117] Materials and Methods General Method A Preparative HPLC was performed using a Kinetex XB C column with 0.1% TFA / CH3CN in Milli-Q water (acidic conditions) (flow rate 25 mL / min, 15 min gradient) as the mobile phase. 18 The analysis was performed on a Gilson HPLC system equipped with a (5 μm, 21 × 100 mm) column. Fractionation was based on UV signal. The analysis was performed on a Kinetex XB C column using 0.1% TFA / CH3CN in Milli-Q water (acidic conditions) as the mobile phase. 18 (2.6 μm, 3.0 × 50 mm) column or a Kinetex EVO C column with 10 mM NH4HCO3 (pH 10) / CH3CN (basic conditions) in Milli-Q water as the mobile phase. 18 The method was performed on an Agilent Series 1100 system using a (2.6 μm, 3.0 × 50 mm) column (flow rate 1 mL / min). Electrospray ionization mass spectrometry (ESI-MS) was performed using an Agilent 1100 Series liquid chromatography / mass selective detector (MSD) to obtain the pseudomolecular [M+H] of the target molecule. + ions were obtained.

[0118] Nuclear magnetic resonance (NMR) spectra were measured at 25° C. on a Varian Inova 600 MHz equipped with a 5 mm triple resonance probe.

[0119] General Method B In some examples, general method B was used. Flash chromatography was performed on silica gel 60A, 40-63 μm. Preparative HPLC was performed on a Gilson system equipped with a UV detector using an ACE5 C8Prep, 100×21.2 mm column. Analytical HPLC-MS was performed on an Agilent 1100 Series Liquid Chromatography / Mass Selective Detector (MSD) (single quadrupole) equipped with an electrospray interface and a UV diode array detector. Analyses were performed on an ACE3 C8 (3.0×50 mm) column with a 3 min gradient of acetonitrile in 0.1% TFA in water (flow rate 1 mL / min) or on an Xbridge C 18 (3.0×50 mm) column was used, with a 3 min gradient of acetonitrile in 10 mM ammonium bicarbonate (flow rate 1 mL / min). 1 H-NMR spectra were measured at 25 °C on a Bruker 400 MHz.

[0120] Common general methods Where example compounds were prepared as the hydrochloride salt, hydrogen chloride (eg 4M in 1,4-dioxane) was added to the combined pure fractions from the preparative chromatography in order to replace the trifluoroacetic acid with hydrochloric acid.

[0121] Compounds were named using Biovia, Dotmatics and PerkinElmer (ChemDraw) software. Additionally, commercial or trivial names were used for commercially available starting materials and reagents.

[0122] Spectra were processed using MestReNova provided by Mestrelab Research SL. Chemical shifts are reported in ppm (δ) using residual solvent as internal standard. Peak multiplicities, given in Hz, are as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; t, triplet; dt, triplet of doublets; q, quartet; dq, quartet of doublets; p, pentet; h, heptet; m, multiplet; br s, broad singlet. Some low-field protons (approximately 14.5-14.9 ppm) may be outside the range and are not necessarily included in the multiplet.

[0123] Intermediate 1 (2S)-2-Amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester [ka]

[0124] To a solution of (4S)-4-(tert-butoxycarbonylamino)-5-methoxy-5-oxo-pentanoic acid (7.46 g, 28.6 mmol), N1-methyl-4-nitro-benzene-1,2-diamine (5.01 g, 30.0 mmol), 1-methylimidazole (4.78 mL, 60.0 mmol) in MeCN (220 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphoric acid (8.81 g, 31.4 mmol) at room temperature [slightly exothermic]. The mixture was stirred at room temperature for 1 h and the volatiles were evaporated under reduced pressure. The crude product was dissolved in DCM (400 mL) and washed with 2N HCl:water 1:4 solution (200 mL). The organic phase was separated, dried over MgSO4, filtered and concentrated to give a brown solid. The crude product (2S)-2-(tert-butoxycarbonylamino)-5-[2-(methylamino)-5-nitro-anilino]-5-oxo-pentanoic acid methyl ester (13.4 g, 28.4 mmol, 99% yield) was used in the next step without further purification. HPLC purity: 87%; MS (ESI+) m / z = 354 [M+H-tBu] + .

[0125] To a solution of (2S)-2-(tert-butoxycarbonylamino)-5-[2-(methylamino)-5-nitro-anilino]-5-oxo-pentanoic acid methyl ester (11.7 g, 28.5 mmol) in EtOH (400 mL) was added hydrochloric acid (12.0 mol / L, 42.3 mL, 507 mmol) and the mixture was stirred at 70 °C overnight. The reaction was evaporated under reduced pressure and water (500 mL) was added. The aqueous phase was washed with EtOAc (2 × 200 mL). The organic phase was discarded. The aqueous phase was mixed with EtOAc (500 mL) and solid K2CO3 was added until pH 9-10. The aqueous phase was extracted with EtOAc (2 × 300 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure to give the crude product (2S)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester (Intermediate 1), which was used in the next step without further purification. HPLC purity: 83%; MS (ESI + ) m / z = 307 [M+H] + .

[0126] Intermediate 2 (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoate lithium [ka]

[0127] To a solution of crude (2S)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester (intermediate 1) (5.73 g, 18.7 mmol) in THF (100 mL) were added triethylamine (3.13 mL, 22.4 mmol) and di-tert-butyl dicarbonate (4.49 g, 20.6 mmol) and the mixture was stirred at room temperature overnight. The crude product was concentrated under reduced pressure, dissolved in EtOAc (200 mL) and washed with 0.5 N HCl (200 mL). The two phases were separated and the aqueous phase was extracted with EtOAc (2×50 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. Et2O (150 mL) was added and a solid started to precipitate. The flask was cooled to 0° C. and left for 30 min. The solid was collected by filtration and washed with cold Et2O (3 x 50 mL). The solid was dried under vacuum to give (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester as a pale yellow solid (4.70 g, 11.6 mmol, 55% yield for three steps). HPLC purity: 96%, MS (ESI+) m / z = 407 [M+H]. + .

[0128] To a solution of (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester (1.47 g, 3.62 mmol) in THF (20 mL) and MeOH (20 mL) was added water (5 mL) and LiOH·H2O (0.159 g, 3.80 mmol) and the mixture was stirred at room temperature overnight. The crude product (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid lithium (Intermediate 2) was used in the next step without further purification. HPLC purity: 100%; MS (ESI+) m / z = 379 [M(carboxylic acid)+H] + .

[0129] Intermediate 3 (2S)-2-Amino-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester [ka]

[0130] Intermediate 3 was synthesized using the same procedure as Intermediate 1, except that (3S)-3-(tert-butoxycarbonylamino)-4-methoxy-4-oxo-butanoic acid was used as the starting material:

[0131] Step 1 The crude product (2S)-2-(tert-butoxycarbonylamino)-4-[2-(methylamino)-5-nitro-anilino]-4-oxo-butanoic acid methyl ester was isolated as a brown solid (71% yield). HPLC purity: 71%; m / z = 341 (M+H-tBu) + .

[0132] Step 2 (2S)-2-Amino-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester (Intermediate 3) was isolated as a yellowish orange solid (98% yield). HPLC purity: 91%; m / z = 293 (M+H) + .

[0133] Intermediate 4 (2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid lithium salt [ka]

[0134] Intermediate 4 was synthesized following the same procedure as Intermediate 1, except that (2S)-2-amino-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester (Intermediate 3) was used as the starting material.

[0135] Step 1 The crude product (2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester was isolated as a brown solid (83% yield). HPLC purity: 97%; m / z = 393 [M+H] + .

[0136] Step 2 (2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl) lithium propanoate (Intermediate 4) was isolated as a yellowish orange solid (quantitative). HPLC purity: 91%; m / z = 365 [M(carboxylic acid)+H] + .

[0137] Example 1 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 1) [ka]

[0138] Step 1 (2S)-2-amino-4-methyl-pentanoic acid ethyl ester; hydrochloride (0.581 g, 2.97 mmol), (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoate lithium (intermediate 2) (1.04 g, 2.70 mmol) and 1-methylimidazole (0.452 mL, 5.67 mmol) in MeCN (30 mL) were added [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphoric acid (0.834 g, 2.97 mmol) at room temperature. The mixture was stirred at room temperature for 1 h. The reaction was quenched with citric acid solution (10% aq.) and the volatiles were evaporated. The crude product was diluted with water and extracted with EtOAc (x3). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was purified by silica chromatography (DCM-->DCM / MeOH (10%)) to give (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester as a pale yellow foam (1.27 g, 2.44 mmol, 90% yield). HPLC purity: 96%; MS (ESI+) m / z = 520 [M+H] + .

[0139] Step 2 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (1.27 g, 0.00244 mol) was dissolved in EtOH (30 mL) and placed in a H reactor. Pd / C (10%, 0.130 g, 0.000122 mol) was added and the reactor was sealed. A nitrogen purge was performed. Hydrogen gas was charged to the reactor at 3 bar and the reaction was stirred at 35° C. for 1.5 h. The hydrogen gas was vented with a nitrogen purge. The catalyst was removed by filtration through Celite. The solvent was evaporated and the crude product (2S)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester was isolated (0.670 g, 1.37 mmol, 85% yield) and used in the next step without further purification. HPLC purity: 93%; MS (ESI+) m / z = 490 [M+H] + .

[0140] Step 3 (2S)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (670 mg, 0.00137 mol) was dissolved in acetic acid (5 mL), oxirane (2.30 mol / L, 5.95 mL, 0.0137 mol) was added and the reaction was stirred at room temperature for 24 hours. The volatiles were removed in vacuo and the crude material was dissolved in DCM and washed with a saturated aqueous solution of NaHCO3. The organic phase was washed with water and brine, passed through a phase separator and concentrated. The crude product was purified by silica chromatography (DCM-->DCM / MeOH (10%)) to give (2S)-2-[[(2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester as a pale white foam (0.340 g, 0.589 mmol, 43% yield). HPLC purity: 96%; MS (ESI+) m / z = 578 [M+H]+ .

[0141] Step 4 To a solution of (2S)-2-[[(2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (340 mg, 0.589 mmol) and triethylamine (0.238 g, 2.35 mmol) in DCM (10 mL) was added mesyl chloride (0.141 mL, 1.82 mmol). The reaction mixture was stirred at room temperature for 20 min and then washed with a mixture of NaHCO3(sat):water (2 / 5) (20 mL), 0.3 M HCl (20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure.

[0142] The crude mixture (mesylated intermediate) and lithium chloride (0.374 g, 8.83 mmol) in DMF (10 mL) was heated at 60 °C for 2.5 h. The reaction mixture was diluted with a mixture of toluene (40 mL) and EtOAc (40 mL), then washed with brine (3 × 50 mL), dried over MgSO4, filtered and the solvent was evaporated to give the crude residue. The crude product was purified by flash chromatography using DCM-->DCM / MeOH (5%) as eluent. The desired compound, (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester, was obtained as a pale yellow foam (0.308 g, 0.481 mmol, 82% yield). HPLC of mesylated intermediate: MS (ESI+) m / z = 734 [M+H] + HPLC purity of desired product: 96%; MS (ESI+) m / z = 614 [M+H] + .

[0143] Step 5: To a solution of (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (308 mg, 0.476 mmol) in EtOH (15 mL) was added hydrochloric acid in dioxane (4 M) (4.00 mol / L, 1.79 mL, 7.14 mmol) and the mixture was stirred at room temperature overnight. The solvent was evaporated to give the crude product, which was stirred in Et2O. The resulting off-white solid was filtered, washed with Et2O (2 x 10 mL), EtOAc (2 mL) and dried in vacuum to give the final product (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride salt (Example compound 1) as an off-white solid (0.170 g, 0.272 mmol, 57% yield). HPLC purity: 95%. MS (ESI+) m / z = 514 [M+H] + .

[0144] 1H NMR (600 MHz, DMSO-d6) δ 14.86 (br s, 1H), 9.31 (d, J = 7.0 Hz, 1H), 8.60 (d, J = 5.3 Hz, 3H), 7.75 (d, J = 9.2 Hz, 1H), 7.15 (dd, J = 9.4, 2.4 Hz, 1H), 6.93 (d, J = 2.4 Hz, 1H), 4.34 - 4.27 (m, 1H), 4.15 - 4.06 (m, 3H), 3.91 (s, 3H), 3.83 (t, J = 6.8 Hz, 4H), 3.78 (t, J = 6.9Hz, 4H), 3.43 - 3.30 (m, 2H), 2.41 - 2.25 (m, 2H), 1.81 - 1.71 (m, 1H), 1.64 (ddd, J = 13.7, 10.4, 5.1 Hz, 1H), 1.54 (ddd, J = 13.8, 9.2, 4.7 Hz, 1H), 1.18 (t, J = 7.1 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H).

[0145] Example 1 was also synthesized according to the procedure described in Example 46.

[0146] Example 2 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride (Example compound 2) [ka]

[0147] The title compound was prepared in five steps by the methods described in Example 1.

[0148] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester was isolated as a white solid (5.14 g, 8.99 mmol, 86% yield). HPLC purity: 99%; MS (ESI+) m / z = 572 [M+H] + .

[0149] 1 H NMR (600 MHz, DMSO-d6, presence of rotamers) δ 8.44 (d, J = 2.3 Hz, 1H), 8.37 (d, J = 7.5 Hz, 1H), 8.15 (dd, J = 8.9, 2.2 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.26 (dd, J = 8.4, 5.6 Hz, 2H), 7.10 - 7.01 (m, 3H), 4.51 - 4.44 (m, 1H), 4.10 - 4.04 (m, 1H), 4.02 (t, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.06 - 2.92 (m, 2H), 2.92 - 2.83 (m, 2H), 2.13 - 2.02 (m, 2H), 1.37 (s, 8H, major rotamer), 1.21 (s, 1H, minor rotamer), 1.08 (t, J = 7.1 Hz, 3H).

[0150] Step 2 The crude product (2S)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester was isolated as a light yellow foam (4.83 g, 0.00892 mol, 93% yield). The crude product was used directly in the next step. HPLC purity: 96%; MS (ESI+) m / z = 542 [M+H] + .

[0151] 1H NMR (600 MHz, DMSO-d6, presence of rotamers) δ 8.49 (d, J = 7.5 Hz, 1H), 7.28 (dd, J = 8.5, 5.7 Hz, 2H), 7.14 - 7.03 (m, 4H), 6.69 (d, J = 2.0 Hz, 1H), 6.53 (dd, J = 8.5, 2.1 Hz, 1H), 4.68 (s, 2H), 4.49 - 4.41 (m, 1H), 4.07 - 3.98 (m, 3H), 3.58 (s, 3H), 3.07 - 2.94 (m, 2H), 2.82 - 2.70 (m, 2H), 2.08 - 1.98 (m, 1H), 1.98 - 1.87 (m, 1H), 1.37 (s, 8H, major rotamer), 1.19 (s, 1H, minor rotamer), 1.13 - 1.03 (m, 3H).

[0152] Step 3 The desired product, (2S)-2-[[(2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester, was isolated as a white solid (3.00 g, 4.76 mmol, 54% yield). HPLC purity: 100%, MS (ESI+) m / z = 630 [M+H] + .

[0153] 1H NMR (600 MHz, DMSO-d6, presence of rotamers) δ 8.47 - 8.43 (m, 1H), 7.30 - 7.23 (m, 3H), 7.13 (d, J = 8.1 Hz, 1H), 7.06 (t, J = 8.8 Hz, 2H), 6.82 (d, J = 2.4 Hz, 1H), 6.73 - 6.69 (m, 1H), 4.71 (t, J = 5.4 Hz, 2H), 4.46 (q, J = 7.4 Hz, 1H), 4.07 - 3.98 (m, 3H), 3.61 (s, 3H), 3.53 (q, J = 6.2 Hz, 4H), 3.42 - 3.35 (m, 4H), 3.07 - 2.93 (m, 2H), 2.80 - 2.74 (m, 2H), 2.09 - 2.00 (m, 1H), 2.00 - 1.92 (m, 1H), 1.37 (s, 8H, major rotamer), 1.20 (s, 1H, minor rotamer), 1.11 - 1.05 (m, 3H).

[0154] Step 4 The crude product was purified on a Buchi Auto-Flash system (80 g column, DCM / MeOH, 100:0 to 92:8) to give (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester as a light yellow solid (1.60 g, 2.40 mmol, 50% yield). HPLC purity: 97%, MS (ESI+) m / z = 666 [M+H] + .

[0155] Step 5 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride salt (Example compound 2) was isolated as an off-white solid (1.46 g, 2.28 mmol, 95% yield). HPLC purity: 97%, MS (ESI+) m / z = 566 [M+H]+ .

[0156] 1 H NMR (600 MHz, DMSO-d6) δ 14.75 (br s, 1H), 9.42 - 9.38 (m, 1H), 8.56 (s, 3H), 7.75 (d, J = 9.2 Hz, 1H), 7.35 (dd, J = 8.5, 5.6 Hz, 2H), 7.18 - 7.09 (m, 3H), 6.93 (d, J = 2.3 Hz, 1H), 4.52 (q, J = 7.1 Hz, 1H), 4.11 - 4.00 (m, 3H), 3.90 (s, 3H), 3.85 - 3.82 (m, 4H), 3.79 - 3.76 (m, 4H), 3.41 - 3.26 (m, 2H), 3.10 - 3.01 (m, 2H), 2.36 - 2.27 (m, 2H), 1.11 (t, J = 7.1 Hz, 3H).

[0157] Example 3 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid ethyl ester; dihydrochloride (Example compound 3) [ka]

[0158] The title compound was prepared in five steps by the methods described in Example 1.

[0159] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoyl]amino]-3-methyl-butanoic acid ethyl ester was isolated as a pale yellow foam (1.27 g, 2.27 mmol, 84% yield). HPLC purity: 93%, MS (ESI+) m / z = 506 [M+H] + .

[0160] Step 2 The crude product (2S)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-methyl-butanoic acid ethyl ester was obtained as a beige solid (1.2 g, 97% yield) and used in the next step without further purification. HPLC purity: 97%, MS (ESI+) m / z = 476 [M+H] + .

[0161] Step 3 (2S)-2-[[(2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-methyl-butanoic acid ethyl ester was obtained as a brown oil (1.38 g, 98% yield) which solidified upon standing. The compound was used in the next step without further purification. HPLC purity: 98%, MS (ESI+) m / z = 564 [M+H] + .

[0162] Step 4 The desired compound, (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-methyl-butanoic acid ethyl ester, was obtained as a brown oil (836 mg, 65% yield). HPLC purity: 93%, MS (ESI+) m / z = 600 [M+H] + HPLC of mesylated intermediate: MS (ESI+) m / z = 720 [M+H] + .

[0163] Step 5: (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-3-methyl-butanoic acid ethyl ester (93% purity, 216 mg, 0.334 mmol) in EtOH (2 mL) was added with hydrochloric acid in dioxane (4.00 mol / L, 0.836 mL, 3.34 mmol) and stirred at 25° C. for 3 h. The crude product was evaporated and purified by preparative chromatography (acidic buffer). The fractions were kept in ice bath until LC-MS analysis. HCl in dioxane (4M, 1 mL) was added and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid ethyl ester; dihydrochloride (Example compound 3) as a pale pink solid (54 mg, 28% yield). HPLC purity: 98%, MS (ESI+) m / z = 500 [M+H] + .

[0164] 1 H NMR (600 MHz, DMSO-d6) δ 14.78 (s, 1H), 9.07 (d, J = 7.3 Hz, 1H), 8.67 - 8.50 (m, 3H), 7.75 (d, J = 9.1 Hz, 1H), 7.15 (d, J = 9.2 Hz, 1H), 6.93 (d, J = 2.4 Hz, 1H), 4.21 (dd, J = 7.3, 5.5 Hz, 2H), 4.18 - 4.07 (m, 2H), 3.90 (s, 3H), 3.83 (t, J = 6.7 Hz, 4H), 3.78 (t, J = 6.2 Hz, 4H), 3.33 (dq, J = 24.7, 8.5 Hz, 2H), 2.40 - 2.22 (m, 2H), 2.14 (ddt, J = 14.0, 12.5, 6.9 Hz, 1H), 1.19 (t, J = 7.1 Hz, 3H), 0.97 (dd, J = 6.9, 5.4 Hz, 6H).

[0165] Example 4 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 4) [ka]

[0166] The title compound was prepared in five steps by the methods described in Example 1.

[0167] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoyl]amino]-4-methyl-pentanoic acid ethyl ester (1.36 g, 2.42 mmol, 89% yield) was isolated as a pale yellow foam. HPLC purity: 90%, MS (ESI+) m / z = 506 [M+H] + .

[0168] Step 2 The crude product (2S)-2-[[(2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)propanoyl]amino]-4-methyl-pentanoic acid ethyl ester was isolated as a yellow oil (1.47 g, 2.78 mmol, 100% yield) and used directly in the next step without further purification. HPLC purity: 90%, MS (ESI+) m / z = 476 [M+H] + .

[0169] Step 3 The crude product (2S)-2-[[(2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-4-methyl-pentanoic acid ethyl ester was obtained as a brown solid (1.50 g, 2.42 mmol, 86% yield). HPLC purity: 91%, MS (ESI+) m / z = 564 [M+H] + .

[0170] Step 4 (2S)-2-[[(2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-4-methyl-pentanoic acid ethyl ester was isolated as a yellow oil (0.550 g, 0.870 mmol, 33% yield). HPLC purity: 95%, MS (ESI+) m / z = 600 [M+H] + HPLC of mesylated intermediate: MS (ESI+) m / z = 720 [M+H] + .

[0171] Step 5 To a solution of (2S)-2-[[(2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-4-methyl-pentanoic acid ethyl ester (95%, 200 mg, 0.316 mmol) in EtOH (15 mL) was added hydrochloric acid in dioxane (4 M) (4.00 mol / L, 1.19 mL, 4.75 mmol) and the mixture was stirred at room temperature overnight. The crude product was obtained by evaporation of the solvent and purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 4) as a white solid (0.112 g, 0.189 mmol, 60% yield). HPLC purity: 100%, MS (ESI+) m / z = 500 [M+H] + .

[0172] 1H NMR (600 MHz, DMSO-d6) δ 14.75 (br s, 1H), 9.29 (dd, J = 7.8, 2.5 Hz, 1H), 8.86 (s, 3H), 7.78 (d, J = 9.2 Hz, 1H), 7.18 (dd, J = 9.4, 2.4 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 4.49 (d, J = 6.8 Hz, 1H), 4.34 (ddd, J = 10.0, 7.5, 4.9 Hz, 1H), 4.06 - 4.01 (m, 2H), 3.99 (s, 3H), 3.90 - 3.74 (m, 9H), 3.65 (dd, J = 15.4, 6.6 Hz, 1H), 1.72 (ddd, J = 11.9, 7.9, 5.9 Hz, 1H), 1.56 (ddd, J = 13.7, 10.0, 5.2 Hz, 1H), 1.50 (ddd, J = 13.9, 9.0, 5.0 Hz, 1H), 1.15 (t, J = 7.1 Hz, 3H), 0.88 (dd, J = 16.2, 6.6 Hz, 6H).

[0173] Example 5 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride (Example compound 5) [ka]

[0174] The title compound was prepared in five steps by the methods described in Example 1.

[0175] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester was isolated as a light yellow foam (1.36 g, 2.19 mmol, 81% yield). HPLC purity: 90%, MS (ESI+) m / z = 558 [M+H] + .

[0176] Step 2 The crude product (2S)-2-[[(2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester was obtained as an off-white solid (954 mg, 73% yield) and used in the next step without further purification. HPLC purity: 98%, MS (ESI+) m / z = 528 [M+H] + .

[0177] Step 3 The crude product (2S)-2-[[(2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester was obtained as a brown oil (964 mg, 85% yield) which solidified upon standing. The compound was used in the next step without further purification. HPLC purity: 96%, MS (ESI+) m / z = 616 [M+H] + .

[0178] Step 4 (2S)-2-[[(2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester was isolated as a yellow oil (0.635 g, 0.924 mmol, 69% yield). HPLC purity: 95%, MS (ESI+) m / z = 652 [M+H] +HPLC mesylated intermediate: MS (ESI+) m / z = 772 [M+H] + .

[0179] Step 5 The crude product was purified by preparative HPLC (acidic conditions; flow rate 25 mL / min, gradient from 12% B to 42% B in 15 min, fractions collected based on UV signal at 239 nm). 4N HCl in dioxane (1 mL) was added to the combined fractions and lyophilized to give (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride salt (Example compound 5) as an off-white solid (78 mg, 0.121 mmol, 13% yield). HPLC purity: 97%, MS (ESI+) m / z = 552 [M+H] + .

[0180] 1 H NMR (600 MHz, DMSO-d6) δ 14.66 (br s, 1H), 9.31 (d, J = 7.5 Hz, 1H), 8.79 (s, 3H), 7.81 - 7.67 (m, 1H), 7.33 - 7.28 (m, 2H), 7.15 (d, J = 9.2 Hz, 1H), 7.12 - 7.06 (m, 2H), 6.88 (d, J = 2.4 Hz, 1H), 4.59 (p, J = 8.4, 7.7 Hz, 1H), 4.41 (s, 1H), 4.01 (dddd, J = 13.6, 10.8, 7.2, 3.8Hz, 2H), 3.95 (s, 3H), 3.84 (ddt, J = 23.1, 12.2, 6.2 Hz, 4H), 3.77 (ddd, J = 9.3, 7.2, 4.8 Hz, 5H), 3.63 (s, 1H), 3.05 - 2.95 (m, 2H), 1.10 (t, J = 7.1 Hz, 3H).

[0181] Example 6 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid ethyl ester; dihydrochloride (Example compound 6) [ka]

[0182] The title compound was prepared in five steps by the methods described in Example 1.

[0183] Step 1 The desired product, (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoyl]amino]-3-methyl-butanoic acid ethyl ester, was isolated as a light yellow foam (0.992 g, 1.82 mmol, 67% yield). HPLC purity: 90%, MS (ESI+) m / z = 492 [M+H] + .

[0184] Step 2 The crude product (2S)-2-[[(2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-methyl-butanoic acid ethyl ester was obtained as a brown oil (931 mg, 98% yield) and used in the next step without further purification. HPLC purity: 98%, MS (ESI+) m / z = 462 [M+H] + .

[0185] Step 3 The crude product (2S)-2-[[(2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-methyl-butanoic acid ethyl ester was obtained as a yellow oil (0.848 g, 1.39 mmol, 70% yield) and used in the next step without further purification. HPLC purity: 90%, MS (ESI+) m / z = 550 [M+H] + .

[0186] Step 4 (2S)-2-[[(2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3-methyl-butanoic acid ethyl ester was isolated as a yellow oil (0.210 g, 0.340 mmol, 22% yield). HPLC purity: 95%, MS (ESI+) m / z = 586 [M+H] + HPLC mesylated intermediate: MS (ESI+) m / z = 706 [M+H] + .

[0187] Step 5 The crude product was purified by preparative HPLC (flow rate 25 mL / min, gradient from 8% B to 38% B in 15 min, fractions collected based on UV signal at 239 nm) to give (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid ethyl ester; dihydrochloride salt (Example compound 6) as an off-white solid (85 mg, 0.147 mmol, 43% yield). HPLC purity: 97%, MS (ESI+) m / z = 486 [M+H] + .

[0188] 1H NMR (600 MHz, DMSO-d6) δ 14.77 (br s, 1H), 9.04 (d, J = 7.9 Hz, 1H), 8.85 (s, 3H), 7.78 (d, J = 9.2 Hz, 1H), 7.17 (dd, J = 9.6, 2.3 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 4.59 (s, 1H), 4.26 (dd, J = 7.9, 5.5 Hz, 1H), 4.07 - 3.99 (m, 2H), 3.98 (s, 3H), 3.89 - 3.75 (m, 9H), 3.59 (dd, J = 15.5, 6.8 Hz, 1H), 2.12 - 2.03 (m, 1H), 1.15 (t, J = 7.1 Hz, 3H), 0.92 (d, J = 6.8 Hz, 6H).

[0189] Example 7 (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 7) [ka]

[0190] Step 1 To a solution of (2S)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester (intermediate 1) (1.00 g, 3.26 mmol), (2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoic acid; hydrate (0.814 g, 3.26 mmol) and 1-methylimidazole (0.546 mL, 6.86 mmol) in MeCN (30 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphoric acid (1.01 g, 3.59 mmol) at room temperature. The mixture was stirred at room temperature for 1 h. The reaction was quenched with citric acid solution (10% aq.) and the volatiles were evaporated. The crude product was diluted with water and extracted with EtOAc (x3). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was purified by silica chromatography (DCM-->DCM / MeOH (10%)) to give (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester as a pale yellow foam (90%, 1.47 g, 2.54 mmol, 78% yield). HPLC purity: 90%; MS (ESI+) m / z = 520 [M+H] + .

[0191] Step 2 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester (1.47 g, 0.00283 mol) was dissolved in EtOH (30 mL) and placed in a H reactor. Pd / C (10%, 0.151 g, 0.000141 mol) was added and the reactor was sealed. A nitrogen purge was performed. Hydrogen gas was charged to the reactor at 3 bar and the reaction was stirred at 35° C. for 1.5 h. The hydrogen gas was vented with a nitrogen purge. The catalyst was removed by filtration through Celite. The solvent was evaporated to give the crude product (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester as a brown foam (1.20 g, 2.21 mmol, 78% yield) which was used in the next step without further purification. HPLC purity: 90%; MS (ESI+) m / z = 490 [M+H] + .

[0192] Step 3 (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (1.26 g, 2.45 mmol, 95% purity) was dissolved in acetic acid (5 mL). Oxirane (2.3 M, 10.6 mL) was added and the reaction was stirred at room temperature for 24 hours. The volatiles were removed in vacuo and the crude material was dissolved in DCM and washed with a saturated aqueous solution of NaHCO3. The organic phase was washed with water and brine, passed through a phase separator cartridge and concentrated to give (2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester as a brown oil (1.38 g, 93% yield) which solidified on standing. The product was used in the next step without further purification. HPLC purity: 95%; MS (ESI+) m / z = 578 [M+H] + .

[0193] Step 4 To a solution of (2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (1.38 g, 2.27 mmol, 95% purity) and triethylamine (1.27 mL, 9.08 mmol) in DCM (50 mL) was added mesyl chloride (0.544 mL, 7.03 mmol) and the mixture was stirred at room temperature for 10 min. The reaction mixture was washed with a mixture of NaHCO3 (sat):water (2:5) (100 mL), 0.3 M HCl (100 mL) and brine (100 mL). The organic phase was passed through a phase separation cartridge and evaporated under reduced pressure. The crude product (2S)-4-[5-[bis(2-methylsulfonyloxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester was used immediately in the next step without further purification.

[0194] (2S)-4-[5-[bis(2-methylsulfonyloxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (1.82 g, 1.64 mmol, 66% purity) was dissolved in DMF (5 mL). LiCl (1.04 g, 24.6 mmol) was added and the reaction was heated at 60 °C for 1 h. The reaction mixture was diluted with a mixture of toluene (10 mL) and EtOAc (10 mL) and then washed with brine (3 × 20 mL), dried over MgSO4, filtered and evaporated. The resulting crude product was purified by flash chromatography using a 0-20% gradient of ethyl acetate in DCM. Fractions containing the desired compound were combined and concentrated to give (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester as a brown oil (652 mg, 62% yield). HPLC of the mesylated intermediate: MS (ESI+) m / z = 734 [M+H] + HPLC purity of desired product: 96%; MS (ESI+) m / z = 614 [M+H] + .

[0195] Step 5: To a solution of (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (205 mg, 0.316 mmol, 95% purity) in EtOH (2 mL) was added hydrochloric acid in dioxane (4.00 mol / L, 1.19 mL, 4.75 mmol) and the mixture was stirred at 25° C. for 3 h. The crude product was obtained by evaporation of the solvent and purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 7% B to 37% B in 15 min, fractions collected based on UV signal at 235 nm). Fractions were kept on ice until LC-MS analysis. HCl in dioxane (4M, 1 mL) was added and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 7) as a yellow oil (50 mg, 26% yield). HPLC purity: 95%, MS (ESI+) m / z = 514 [M+H] + .

[0196] 1H NMR (600 MHz, DMSO-d6) δ 14.41 (s, 1H), 9.04 (d, J = 7.9 Hz, 1H), 8.22 (d, J = 5.3 Hz, 3H), 7.74 (d, J = 9.2 Hz, 1H), 7.15 (dd, J = 9.4, 2.3 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 4.51 (ddd, J = 9.6, 8.0, 4.9 Hz, 2H), 4.10 (d, J = 7.1 Hz, 2H), 3.88 (s, 3H), 3.84 (t, J = 6.7 Hz, 4H), 3.78 (t, J = 6.9 Hz, 4H), 3.18 (d, J = 8.6 Hz, 2H), 2.42 - 2.32 (m, 1H), 2.19 - 2.09 (m, 1H), 1.70 (dp, J = 13.4, 6.8 Hz, 1H), 1.61 (dt, J = 13.9, 7.0 Hz, 1H), 1.55 (dt, J = 14.1, 7.5 Hz, 1H), 1.19 (t, J = 7.1 Hz, 3H), 0.92 (dd, J = 16.4, 6.5 Hz, 6H).

[0197] Example 8 (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 8) [ka]

[0198] The title compound was prepared in 5 steps by the methods described in Example 7.

[0199] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester was isolated as a pale yellow foam (1.87 g, 2.94 mmol, 90% yield). HPLC purity: 90%, MS (ESI+) m / z = 572 [M+H] + .

[0200] Step 2 The crude product (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]butanoic acid ethyl ester was obtained as a light yellow solid (1.78 g, 90% yield) and used in the next step without further purification. HPLC purity: 90%, MS (ESI+) m / z = 542 [M+H] + .

[0201] Step 3 The crude product (2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]butanoic acid ethyl ester was obtained as a light brown solid (1.74 g, 86% yield). The compound was used in the next step without further purification. HPLC purity: 92%, MS (ESI+) m / z = 630 [M+H] + .

[0202] Step 4 (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]butanoic acid ethyl ester was isolated as a brown oil (628 mg, 37% yield). HPLC purity: 92%, MS (ESI+) m / z = 666 [M+H] + HPLC mesylated intermediate: MS (ESI+) m / z = 786 [M+H]+ .

[0203] Step 5 The crude product was purified by preparative HPLC (acidic conditions; flow rate 25 mL / min, gradient from 11% B to 40% B in 15 min. Fractions were collected based on UV signal at 235 nm). HCl in dioxane (4M, 1 mL) was added to the combined fractions and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride salt (Example compound 8) as an off-white solid (35 mg, 23% yield). HPLC purity: 95%, MS (ESI+) m / z = 566 [M+H] + .

[0204] 1 H NMR (600 MHz, DMSO-d6) δ 14.61 (s, 1H), 9.57 (d, J = 7.6 Hz, 1H), 8.40 (d, J = 5.5 Hz, 3H), 7.75 (d, J = 9.2 Hz, 1H), 7.45 - 7.36 (m, 2H), 7.15 (dtd, J = 9.4, 7.4, 6.9, 2.3 Hz, 3H), 6.91 (d, J = 2.4 Hz, 1H), 4.44 (ddd, J = 9.5, 7.6, 4.3 Hz, 1H), 4.22 (d, J = 6.8 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.83 (t, J = 6.8 Hz, 4H), 3.78 (t, J = 6.1 Hz, 4H), 3.36 - 3.25 (m, 2H), 3.19 (dd, J = 14.0, 5.7 Hz, 1H), 2.98 (dd, J = 14.0, 8.0 Hz, 1H), 2.40 - 2.31 (m, 1H), 2.17 (dtd, J = 14.6, 9.5, 5.5 Hz, 1H), 1.18 (t, J = 7.1 Hz, 3H).

[0205] Example 9 (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 9) [ka]

[0206] The title compound was prepared in 5 steps by the methods described in Example 7.

[0207] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester was isolated as a light yellow foam (0.757 g, 1.35 mmol, 41% yield). HPLC purity: 90%, MS (ESI+) m / z = 506 [M+H] + .

[0208] Step 2 The crude product (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]butanoic acid ethyl ester was obtained as a light brown solid (0.642 g, 1.28 mmol, 86% yield) and used in the next step without further purification. HPLC purity: 95%, MS (ESI+) m / z = 476 [M+H] + .

[0209] Step 3 The crude product (2S)-4-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]butanoic acid ethyl ester was obtained as a brown oil (0.685 g, 1.09 mmol, 85% yield) which solidified upon standing. The compound was used in the next step without further purification. HPLC purity: 90%, MS (ESI+) m / z = 564 [M+H] + .

[0210] Step 4 (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]butanoic acid ethyl ester was isolated as a light brown oil (0.402 g, 0.623 mmol, 58% yield). HPLC purity: 93%, MS (ESI+) m / z = 600 [M+H] + HPLC mesylated intermediate: MS (ESI+) m / z = 720 [M+H] + .

[0211] Step 5 The crude product was evaporated and purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 7% B to 36% B in 15 min. Fractions were collected based on UV signal at 235 nm). The resulting fractions were placed in an ice bath, HCl in dioxane (4 M, 1 mL) was added, and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride salt (Example compound 9) as an off-white solid (12 mg, 6.3% yield). HPLC purity: 95%, MS (ESI+) m / z = 500 [M+H] + .

[0212] 1H NMR (600 MHz, DMSO-d6) δ 14.70 (s, 1H), 9.37 (d, J = 7.6 Hz, 1H), 8.44 (d, J = 5.7 Hz, 3H), 7.75 (d, J = 9.2 Hz, 1H), 7.15 (dd, J = 9.3, 2.4 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 4.46 (ddd, J = 10.0, 7.6, 4.0 Hz, 1H), 4.10 (d, J = 7.1 Hz, 2H), 3.96 (s, 3H), 3.83 (t, J = 6.6 Hz, 4H), 3.80 - 3.77 (m, 4H), 3.74 - 3.63 (m, 1H), 3.52 - 3.41 (m, 1H), 3.31 (ddd, J = 15.9, 9.6, 6.8 Hz, 1H), 2.37 (dt, J = 13.3, 4.9 Hz, 1H), 2.20 - 2.10 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.04 - 0.94 (m, 6H).

[0213] Example 10 (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride (Example compound 10) [ka]

[0214] The title compound was prepared in 5 steps by the methods described in Example 7.

[0215] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester was isolated as a light yellow foam (1.68 g, 2.99 mmol, 87% yield). HPLC purity: 90%, MS (ESI+) m / z = 506 [M+H] +.

[0216] Step 2 The crude product (2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]propanoic acid ethyl ester was obtained as a brown oil (1.50 g, 2.90 mmol, 87% yield) and used in the next step without further purification. HPLC purity: 92%, MS (ESI+) m / z = 476 [M+H] + .

[0217] Step 3 The crude product (2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]propanoic acid ethyl ester (1.20 g, 1.60 mmol, 52% yield) was obtained as a brown solid. HPLC purity: 75%, MS (ESI+) m / z = 564 [M+H] + .

[0218] Step 4 (2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-methyl-pentanoyl]amino]propanoic acid ethyl ester was isolated as a yellow oil (0.430 g, 0.680 mmol, 32% yield). HPLC purity: 95%, MS (ESI+) m / z = 600 [M+H] + HPLC mesylated intermediate: MS (ESI+) m / z = 720 [M+H] + .

[0219] Step 5 The crude product was purified by preparative HPLC (acidic conditions; flow rate 25 mL / min, gradient from 6% B to 36% B in 15 min. Fractions were collected based on UV signal at 237 nm). 4N HCl in dioxane (1 mL) was added to the combined fractions and lyophilized to give (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride salt (Example compound 10) as a white solid (0.102 g, 0.169 mmol, 53% yield). HPLC purity: 95%, MS (ESI+) m / z = 500 [M+H] + .

[0220] 1 H NMR (600 MHz, DMSO-d6) δ 14.85 (br s, 1H), 9.56 (d, J = 7.7 Hz, 1H), 8.40 (s, 3H), 7.76 (d, J = 9.2 Hz, 1H), 7.16 (d, J = 9.2 Hz, 1H), 6.95 (d, J = 2.5 Hz, 1H), 5.02 (q, J = 7.6 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.97 (s, 3H), 3.87 - 3.73 (m, 9H), 3.67 (dd, J = 15.8, 6.1 Hz, 1H), 3.58 (dd, J = 15.6, 8.2 Hz, 1H), 1.64 (dt, J = 13.4, 6.7 Hz, 1H), 1.52 (hept, J = 6.8 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H), 0.87 (dd, J = 9.5, 6.5 Hz, 6H).

[0221] Example 11 (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride (Example compound 11) [ka]

[0222] The title compound was prepared in 5 steps by the methods described in Example 7.

[0223] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester was isolated as a light yellow solid (0.794 g, 1.14 mmol, 29% yield). HPLC purity: 80%, MS (ESI+) m / z = 558 [M+H] + .

[0224] Step 2 The crude product (2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]propanoic acid ethyl ester was obtained as a light brown solid (772 mg, 93% yield) and used in the next step without further purification. HPLC purity: 91%, MS (ESI+) m / z = 528 [M+H] + .

[0225] Step 3 The crude product (2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]propanoic acid ethyl ester was obtained as a brown oil (817 mg, 91% yield) which solidified upon standing. The compound was used in the next step without further purification. HPLC purity: 95%, MS (ESI+) m / z = 616 [M+H] + .

[0226] Step 4 (2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]propanoic acid ethyl ester was isolated as a brown oil (449 mg, 51% yield). HPLC purity: 86%, MS (ESI+) m / z = 652 [M+H] + HPLC mesylated intermediate: MS (ESI+) m / z = 772 [M+H] + .

[0227] Step 5 The crude product was purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 10% B to 40% B in 15 min, fractions collected based on UV signal at 237 nm). HCl in dioxane (4 M, 1 mL) was added to the combined fractions and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride salt (Example compound 11) as an off-white solid (12 mg, 15% yield). HPLC purity: 96%, MS (ESI+) m / z = 552 [M+H] + .

[0228] 1H NMR (600 MHz, DMSO-d6) δ 14.73 (s, 1H), 9.64 (s, 1H), 8.32 (s, 3H), 7.77 - 7.67 (m, 1H), 7.33 (dt, J = 9.6, 4.9 Hz, 2H), 7.14 (t, J = 8.7 Hz, 3H), 6.94 (d, J = 2.4 Hz, 1H), 4.98 (q, J = 7.5 Hz, 1H), 4.12 (dtt, J = 10.8, 6.7, 3.7 Hz, 3H), 3.96 (s, 3H), 3.83 (t, J = 6.8 Hz, 4H), 3.77 (t, J = 6.4 Hz, 4H), 3.14 (dd, J = 14.1, 6.0 Hz, 1H), 2.97 (dd, J = 14.1, 7.8 Hz, 1H), 1.13 (t, J = 7.1 Hz, 3H), 0.87 - 0.81 (m, 2H).

[0229] Example 12 (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride (Example compound 12) [ka]

[0230] The title compound was prepared in 5 steps by the methods described in Example 7.

[0231] Step 1 (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester was isolated as a yellow solid (0.428 g, 0.784 mmol, 23% yield). HPLC purity: 90%, MS (ESI+) m / z = 492 [M+H] + .

[0232] Step 2 The crude product (2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoic acid ethyl ester was obtained as a yellow solid (393 mg, 93% yield) and used in the next step without further purification. HPLC purity: 95%, MS (ESI+) m / z = 462 [M+H] + .

[0233] Step 3 The crude product (2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoic acid ethyl ester was obtained as a brown solid (0.305 g, 0.499 mmol, 58% yield). The compound was used in the next step without further purification. HPLC purity: 90%, MS (ESI+) m / z = 550 [M+H] + .

[0234] Step 4 (2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoic acid ethyl ester was isolated as a yellow oil (0.168 g, 0.272 mmol, 37% yield). HPLC purity: 95%, MS (ESI+) m / z = 586 [M+H] + HPLC mesylated intermediate: MS (ESI+) m / z = 706 [M+H] + .

[0235] Step 5 The crude product was evaporated and purified by preparative HPLC (acidic conditions; flow rate 25 mL / min, gradient from 5% B to 34% B in 15 min. Fractions were collected based on UV signal at 238 nm). HCl in dioxane (4 M, 1 mL) was added to the combined fractions and the solution was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride salt (Example compound 12) (45 mg, 0.0724 mmol, 27% yield). HPLC purity: 90%, MS (ESI+) m / z = 486 [M+H] + .

[0236] 1 H NMR (600 MHz, DMSO-d6) δ 13.82 (br s, 1H), 8.51 (s, 1H), 7.49 - 7.30 (m, 3H), 6.85 (d, J = 9.2 Hz, 1H), 6.25 (d, J = 9.3 Hz, 1H), 6.05 (d, J = 2.5 Hz, 1H), 4.15 (q, J = 7.4 Hz, 1H), 3.22 (q, J = 7.1 Hz, 2H), 3.06 (s, 3H), 2.96 - 2.86 (m, 8H), 2.80 (dt, J = 9.3, 4.1 Hz, 2H), 2.66 (d, J = 8.2 Hz, 1H), 1.22 (dq, J = 13.4, 6.7 Hz, 1H), 0.23 (t, J = 7.1 Hz, 3H), 0.11 - 0.00 (m, 6H).

[0237] Example 13 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; hydrochloride (Example compound 13) [ka]

[0238] Example 13 was carried out according to Scheme 4 below. [ka]

[0239] Scheme 4: a) TEA, MeOH, 60°C, b) Sodium dithionite, EtOH, 80°C, c) HATU, TEA, DMF, rt, d) 12M HCl, EtOH, 70°C, e) BOC2O, TEA, THF, rt, f) LiOH-H2O, 1,4-dioxane, water, 50°C, g) HATU, TEA, DMF, rt, h) Pd / C10%, hydrogen gas atmosphere, EtOH, rt, i) NaBH3CN, TFA, EtOH, rt, j) TFA, DCM followed by 12M HCl, rt.

[0240] 2,4-Dinitro-N-phenyl-aniline (Compound 13-3) TEA (6.19 mL, 44.4 mmol) was added to 1-chloro-2,4-dinitro-benzene (3.00 g, 14.8 mmol, compound 13-2) and aniline (1.35 mL, 14.8 mmol, compound 13-1) in MeOH (150 mL). The reaction was stirred at 60° C. overnight. The reaction was removed from the heat to give a light brick-colored solid. The solid was filtered off and washed with ice-cold MeOH to give the title compound (3.26 g, 12.54 mmol, 85% yield). HPLC purity: 95%, MS (ESI+) m / z = 260 [M+H] + .

[0241] 4-Nitro-N1-phenyl-benzene-1,2-diamine (compound 13-4) Sodium dithionite (6.57 g, 37.7 mmol) was added to 2,4-dinitro-N-phenyl-aniline (3.26 g, 12.6 mmol) in a solvent mixture of EtOH (120 mL) and water (30 mL) at 80° C. The reaction was stirred for 1 h. The organic solvent was evaporated and more water was added. The compound was extracted with DCM. The aqueous phase was discarded. The DCM phase contained solids which were filtered off. The filtrate was washed with brine, dried over MgSO4, filtered and the solvent was evaporated to give the title compound (1.25 g, 5.45 mmol, 43% yield). HPLC purity: 95%, MS (ESI+) m / z = 230 [M+H] + .

[0242] (2S)-5-(2-anilino-5-nitro-anilino)-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid benzyl ester (compound 13-5) HATU (2.28 g, 6.00 mmol) was added to (4S)-5-benzyloxy-4-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (1.93 g, 5.73 mmol) and TEA (2.28 mL, 16.4 mmol) in DMF (15 mL). To this mixture was added 4-nitro-N1-phenyl-benzene-1,2-diamine (1.25 g, 5.45 mmol). The reaction was stirred overnight. Toluene (70 mL) was added and the organic phase was washed with water (100 mL) and brine (60 mL). The organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (2.90 g, 5.45 mmol, 97% yield). HPLC purity: 88%, MS (ESI+) m / z = 449 [M+H-BOC] + This material was used as is in the next step.

[0243] (2S)-2-Amino-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid benzyl ester (compound 13-6) To a solution of (2S)-5-(2-anilino-5-nitro-anilino)-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid benzyl ester (2.99 g, 5.45 mmol) in EtOH (37 mL) was added 12 M HCl (8.18 mL, 98.1 mmol) and the mixture was stirred at 70° C. for 40 min. The crude product was diluted with water (200 mL) and carefully neutralized to pH 7 with 4 M NaOH. The product was extracted with ethyl acetate (3×100 mL), the organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (2.35 g, 5.45 mol, quantitative). HPLC purity: 65%, MS (ESI+) m / z = 431 [M+H] + This material was used as is in the next step.

[0244] (2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid benzyl ester (compound 13-7) To (2S)-2-amino-4-(6-nitro-3-phenyl-imidazo[4,5-b]pyridin-2-yl)butanoic acid benzyl ester (2.35 g, 0.00545 mol) in THF (25 mL) was added TEA (2.28 mL, 0.0163 mol) and di-tert-butyl dicarbonate (1.43 g, 6.54 mmol). The mixture was stirred at room temperature over the weekend. To remove excess BOC2O, imidazole (0.111 g, 1.63 mol) was added and the reaction was stirred for an additional hour. The solvent was evaporated and the residue was dissolved in ethyl acetate. The organic phase was washed with 5% citric acid, dried over MgSO4, filtered and the solvent was evaporated. The crude product was subjected to flash chromatography on silica eluting with 20-30% ethyl acetate in p-ether to give the title compound (2.21 g, 4.17 mmol, 76% yield). HPLC purity: 77%, MS (ESI+) m / z = 531 [M+H] + .

[0245] (2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid (compound 13-8) To a solution of (2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid benzyl ester (2.21 g, 4.17 mmol) in 1,4-dioxane (30 mL) containing water (10 mL) was added LiOH-H2O (0.699 g, 16.7 mmol). The reaction was stirred at 50° C. for 40 min, diluted with ethyl acetate at room temperature and washed with 5% citric acid. The organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (1.83 g, 0.00415 mol, quantitative). HPLC purity: 80%, MS (ESI+) m / z = 441 [M+H] + This material was used as is in the next step.

[0246] (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 13-9) HATU (1.74 g, 0.00457 mol) was added to a mixture of (2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid (1.83 g, 0.00415 mol), (2S)-2-amino-4-methyl-pentanoic acid ethyl ester; hydrochloride (0.894 g, 0.00457 mol) and triethylamine (2.32 mL, 0.0166 mol) in DMF (30 mL). The reaction was stirred for 1 h. Toluene was added and the mixture was washed with 5% citric acid and brine. The organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (1.82 g, 0.00312 mol, 75% yield). HPLC purity: 95%, MS (ESI+) m / z =582 [M+H] + This material was used as is in the next step.

[0247] (2S)-2-[[(2S)-4-(5-amino-1-phenyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 13-10) A mixture of (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (1.81 g, 0.00311 mol) and Pd / C 10% (0.331 g, 0.00311 mol) in ethanol (90 mL) was stirred under hydrogen gas atmosphere for 4 h. The solid material was filtered and the solvent was evaporated to give the title compound (1.71 g, 0.00311 mol, quantitative). This material was used directly in the next step. HPLC purity: 93%, MS (ESI+) m / z = 552 [M+H] + .

[0248] 1 H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.5 Hz, 2H), 7.55 - 7.49 (m, 1H), 7.43 (d, J = 7.0 Hz, 2H), 6.92 (d, J = 8.2 Hz, 1H), 6.80 - 6.75 (m, 2H), 6.52 (dd, J = 8.5, 2.1 Hz, 1H), 4.81 (s, 2H), 4.24 (ddd, J = 9.9, 7.6, 5.0 Hz, 1H), 4.09 - 3.96 (m, 3H), 2.77 - 2.64 (m, 2H), 2.20 - 2.09 (m, 1H), 1.99 - 1.87 (m, 1H), 1.70 - 1.44 (m, 3H), 1.33 (s, 9H), 1.14 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.5 Hz, 3H).

[0249] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 13-11) NaBH3CN (0.137 g, 0.00218 mol) was added to a slurried mixture of (2S)-2-[[(2S)-4-(5-amino-1-phenyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (0.300 g, 0.000544 mol), 2-chloroacetaldehyde (50.0% in water, 0.344 mL, 0.00272 mol) and TFA (0.242 mL, 3.26 mmol) in EtOH (6 mL) at room temperature. After 20 min, 2-chloroacetaldehyde (50% in water, 0.344 mL, 2.72 mmol) and NaBH3CN (0.137 g, 2.18 mmol) were further added. After 10 min, the final 2-chloroacetaldehyde (50% in water, 0.344 mL, 2.72 mmol), NaBH3CN (0.137 g, 2.18 mmol) and TFA (0.242 mL, 3.26 mmol) were added. Ethyl acetate was added to the mixture and the organic phase was washed with saturated sodium bicarbonate solution and brine. The compound was subjected to flash chromatography twice on silica eluting first with 2% EtOH in DCM and then 20-30% ethyl acetate in petroleum ether to give the title compound (0.158 g, 0.00234 mol, 43% yield). HPLC purity: 97%, MS (ESI+) m / z = 676 [M+H] + .

[0250] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; hydrochloride (Example compound 13) (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (190 mg, 0.281 mmol) was treated with TFA (2 mL) in DCM (2 mL). The solvent was evaporated and the crude residue was purified by several injections on a preparative HPLC on a Gilson system equipped with a UV detector using an ACE5 C8Prep, 100 x 21.2 mm column (0.1% TFA in water) 25-50% acetonitrile gradient. Pure fractions were pooled to give 750 mL of solution. 12 M HCl (3 mL) was added to the ice-cold solution to give a 0.05 M HC1 solution. The solvent was removed by lyophilization to give the title compound (Example compound 13) (92 mg, 0.159 mmol, 53% yield). MS (ESI+) m / z 576 [M+H] + .

[0251] 1 H NMR (400 MHz, DMSO-d6) δ 9.04 (d, J = 7.1 Hz, 1H), 8.54 - 8.42 (m, 3H), 7.73 - 7.62 (m, 4H), 7.16 - 7.01 (m, 3H), 4.28 - 4.20 (m, 2H), 4.06 - 3.97 (m, 3H), 3.88 - 3.73 (m, 8H), 3.14 (t, J = 7.2 Hz, 2H), 2.41 - 2.24 (m, 2H), 1.76 - 1.65 (m, 1H), 1.61 - 1.46 (m, 2H), 1.13 (t, J = 7.1 Hz, 3H), 0.89 (dd, J = 18.0, 6.5 Hz, 6H).

[0252] Example 14 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid methyl ester; dihydrochloride (Example compound 14) [ka]

[0253] (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 4) (11 mg, 0.0182 mmol) was dissolved in MeOH (2 mL). One drop of HCl was added and the reaction mixture was stirred at 70° C. overnight. The sample was purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 10% B to 40% B in 15 min. Fractions were collected based on the UV signal at 238 nm). HCl in dioxane (4M, 0.01 mL) was added to the combined fractions and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid methyl ester; dihydrochloride salt (Example compound 14) as a yellow solid (3.5 mg, 0.00619 mmol, 34% yield). HPLC purity: 99%; MS (ESI+) m / z = 486 [M+H] + .

[0254] 1 H NMR (600 MHz, DMSO-d6) δ 14.8 (s, 1H), 9.09 (d, J = 7.6 Hz, 1H), 8.59 (s, 3H), 7.62 (s, 1H), 6.89 (d, J = 2.4 Hz, 1H), 6.54 (s, 1H), 4.37 (dt, J = 31.5, 7.6 Hz, 2H), 3.93 - 3.70 (m, 11H), 3.54 (s, 3H), 2.51 (t, J = 5.6 Hz, 1H), 1.63 (dq, J = 12.5, 6.3 Hz, 1H), 1.56 - 1.45 (m, 2H), 1.31 - 1.17 (m, 1H), 0.85 (dd, J = 13.8, 6.5 Hz, 6H).

[0255] Example 15 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid isopropyl ester; dihydrochloride (Example compound 15) [ka]

[0256] (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 4) (10 mg, 0.0166 mmol) was dissolved in isopropyl alcohol (2 mL). One drop of HCl was added and the reaction mixture was stirred at 70° C. overnight. The sample was purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 11% B to 42% B in 15 min. Fractions were collected based on the UV signal at 239 nm). HCl in dioxane (4M, 0.01 mL) was added to the combined fractions and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid isopropyl ester; dihydrochloride salt (Example compound 15) as a yellow solid (4.8 mg, 0.00793 mmol, 48% yield). HPLC purity: 97%; MS (ESI+) m / z = 514 [M+H] + .

[0257] 1H NMR (600 MHz, DMSO-d6) δ 14.60 (s, 1H), 9.14 (d, J = 7.6 Hz, 1H), 8.68 (s, 3H), 7.71 (s, 1H), 7.11 (s, 1H), 6.89 (d, J = 2.4 Hz, 1H), 4.83 (h, J = 6.3 Hz, 1H), 4.44 (t, J = 6.9 Hz, 1H), 4.28 (ddd, J = 9.8, 7.6, 5.2 Hz, 1H), 3.91 (s, 3H), 3.81 (dt, J = 12.4, 6.9 Hz, 4H), 3.75 (dd, J = 9.9, 4.8 Hz, 4H), 3.58 (s, 2H), 1.68 (ddt, J = 15.3, 12.9, 6.6 Hz, 1H), 1.50 (qdd, J = 13.8, 9.3, 5.3 Hz, 2H), 1.14 (dd, J = 36.1, 6.3 Hz, 6H), 0.86 (dd, J = 17.3, 6.6 Hz, 6H).

[0258] Example 16 (2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 16) Example 16 was carried out according to Scheme 5 below. [ka]

[0259] (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 16-1) To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoic acid (0.700 g, 0.00247 mol) in DMF (5.0 mL) was added HATU (0.940 g, 0.00247 mol) and DIEA (0.423 mL, 0.00247 mol) at room temperature, followed by (2S)-2-amino-4-methyl-pentanoic acid methyl ester (0.395 g, 0.00272 mol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with H2O and extracted with DCM. The organic phase was dried (Na2SO4) and concentrated in vacuo below 20 °C. The crude product was purified on a Buchi Auto-Flash system (10 g column, EtOAc / petroleum ether) to give (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid methyl ester as a white solid (0.420 g, 1.02 mmol, 41% yield). HPLC purity: 75%; MS (ESI+) m / z = 411 [M+H] + .

[0260] (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid (compound 16-2) To a solution of (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid methyl ester (420 mg, 1.02 mmol) in THF (1.5 mL) was added a solution of LiOH·HO (47.2 mg, 1.13 mmol) in water (0.5 mL). The mixture was stirred at room temperature for 1 h. The precipitated solid was filtered and dried to give (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid as a white solid (350 mg, 0.883 mmol, 86% yield). HPLC purity: 85%; MS (ESI+) m / z = 341 [M+H-tBu] + .

[0261] (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (compound 16-3) To a solution of (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid (95.0 mg, 0.240 mmol) in DMF (3.0 mL) was added HATU (91.1 mg, 0.240 mmol), DIEA (0.0820 mL, 0.479 mmol) and (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 38) (125 mg, 0.264 mmol) at room temperature. The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with H2O and extracted with DCM using a phase separator. The organic phase was dried (Na2SO4) and concentrated in vacuo below 20 °C. The crude product was purified by preparative HPLC (acidic buffer; flow rate 25 mL / min, gradient from 31% B to 57% B in 15 min. Fractions collected based on UV signal at 235 nm) to give (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]butanoic acid ethyl ester as an off-white solid (80.0 mg, 0.103 mmol, 43% yield). HPLC purity: 100%; MS (ESI+) m / z = 779 [M+H] + .

[0262] (2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 16) To (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (80.0 mg, 0.103 mmol) in EtOH (1 mL) was added HCl in dioxane (4.00 mol / L, 0.128 mL, 0.513 mmol) and the reaction mixture was stirred overnight. After completion of the reaction, the solvent was evaporated at about 15° C. and immediately subjected to preparative HPLC (acidic buffer; flow rate 25 mL / min, gradient from 12% B to 44% B in 15 min. Fractions were collected based on UV signal at 235 nm). All pure HPLC fractions were kept on a Mivacuum to evaporate acetonitrile, and HCl in dioxane (4M, 0.01 mL) was added to the resulting fractions. The mixture was immediately frozen in an ice bath with dry ice and placed in a freeze dryer to give (2S)-2-[[(2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride salt (Example compound 16) as a white powder (6.00 mg, 0.00797 mmol, 8% yield). HPLC purity: 95%; MS (ESI+) m / z = 679 [M+H] + .

[0263] 1H NMR (600 MHz, DMSO-d6) δ 14.4 (br s, 1H), 8.80 - 8.71 (m, 2H), 8.24 - 8.01 (m, 3H), 7.36 - 7.25 (m, 2H), 7.18 - 7.08 (m, 2H), 6.97 - 6.82 (m, 1H), 4.53 - 4.32 (m, 2H), 4.19 - 3.99 (m, 3H), 3.90 - 3.67 (m, 12H), 3.09 (m, 4H), 2.88 (dd, J = 14.2, 8.3 Hz, 1H), 2.38 -2.26 (m, 1H), 2.21 - 2.09 (m, 1H), 1.67 (dq, J = 13.5, 6.6 Hz, 1H), 1.55 - 1.43 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H).

[0264] Example 17 (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid; dihydrochloride (Example compound 17) Example 17 was carried out according to Scheme 6 below. [ka]

[0265] (2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (compound 17-1) (2S)-2-(tert-butoxycarbonylamino)-3-(1-methyl-5-nitro-benzimidazol-2-yl)propanoic acid ethyl ester (Scheme 1) (573 mg, 1.42 mmol) was dissolved in EtOH (10 mL) and placed in a H reactor. 10% Pd / C (75.4 mg) was added and the reactor was sealed. Nitrogen purging was performed. Hydrogen gas was charged to the reactor at 3 bar and the reaction was stirred at 35° C. for 3 h. Hydrogen gas was evacuated with a nitrogen purge. The catalyst was removed by filtration through Celite. The solvent was evaporated to give the crude product (2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester as a yellow solid (421 mg, 1.13 mmol, 74% yield) which was used in the next step without further purification. HPLC purity: 90%; MS (ESI+) m / z = 363 [M+H] + .

[0266] (2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (compound 17-2) (2S)-3-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (421 mg, 1.13 mmol) was dissolved in acetic acid (5 mL). Oxirane (2.3 M, 4.55 mL) was added and the reaction was stirred at room temperature for 24 h. The solvent was evaporated and the crude material was dissolved in DCM and washed with a saturated aqueous solution of NaHCO3. The organic phase was washed with water and brine and passed through a phase separator. The material was concentrated to give (2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester as a brown oil (0.421 g, 0.888 mmol, 85% yield) which was sufficiently pure for use in the next step. HPLC purity: 95%; MS (ESI+) m / z = 451 [M+H] + .

[0267] (2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (compound 17-3) To a solution of (2S)-3-[5-[bis(2-hydroxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (421 g, 0.888 mmol, 95% purity) and triethylamine (0.495 mL, 3.55 mmol) in DCM (10 mL) was added methanesulfonic acid chloride (0.213 mL, 2.75 mmol) and the reaction mixture was stirred at room temperature for 20 min. The reaction mixture was washed with a mixture of NaHCO3 (sat):water (2:5) (50 mL), 0.3 M HCl (50 mL) and brine (50 mL). The organic phase was subjected to a phase separator and evaporated under reduced pressure. The crude product (2S)-3-[5-[bis(2-methylsulfonyloxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester was used immediately in the next step without further purification.

[0268] (2S)-3-[5-[bis(2-methylsulfonyloxyethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (521 mg, 0.79 mmol, 92% purity) was dissolved in DMF (5 mL). LiCl (0.502 g, 11.9 mmol) was added and the reaction was heated at 60 °C for 2 h. The reaction mixture was diluted with a mixture of toluene (10 mL) and EtOAc (10 mL) and then washed with brine (3 × 20 mL), dried over MgSO4, filtered and evaporated. The resulting crude product was purified by flash chromatography using a 0-20% gradient of ethyl acetate in DCM. Fractions containing the target compound were combined and concentrated to give (2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester as a brown oil (375 mg, 0.692 mmol, 88% yield). HPLC purity: 90%; MS (ESI+) m / z = 487 [M+H] + . HPLC purity (mesylated intermediate): 95%; MS (ESI+) m / z = 607 [M+H] + .

[0269] (2S)-2-Amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride (compound 17-4) To a solution of (2S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)propanoic acid ethyl ester (375 mg, 0.692 mmol, 90% purity) in EtOH (5 mL) was added hydrochloric acid in dioxane (4.00 mol / L, 1.73 mL, 6.92 mmol) and stirred at 25° C. for 3 h. The solvent was evaporated under reduced pressure to give (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride salt as an off-white solid (0.307 g, 0.660 mmol, 95% yield). HPLC purity: 99%; MS (ESI+) m / z = 387 [M+H] + .

[0270] (2S)-2-Amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid; dihydrochloride (Example compound 17) (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester (30 mg, 0.064 mmol) and 5N HCl (0.025 mL) were stirred at room temperature for 4 hours. The temperature was slowly increased to 60° C. The temperature was increased to 100° C. and stirred for 30 minutes. The reaction was allowed to cool to room temperature and the volatiles were removed in vacuo. The crude product was purified by preparative HPLC (acidic buffer; flow rate 25 mL / min, gradient from 1% B to 28% B in 15 min, fractions collected based on UV signal at 236 nm) to give (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid; dihydrochloride (Example compound 17) as a pale pink solid (8 mg, 0.022 mmol, 26% yield). HPLC purity: 95%; MS (ESI+) m / z = 359 [M+H] + .

[0271] 1H NMR (600 MHz, DMSO-d6) δ 14.90 (s, 1H), 8.93 (s, 3H), 7.75 (d, J = 9.2 Hz, 1H), 7.15 (dd, J = 9.3, 2.4 Hz, 1H), 6.91 (d, J = 2.3 Hz, 1H), 4.65 (t, J = 7.6 Hz, 1H), 3.84 - 3.71 (m, 13H).

[0272] Example 18 (S)-2-amino-4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid dihydrochloride (Example compound 18) Example 18 was carried out according to Scheme 7 below. [ka]

[0273] (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester (compound 18-1) To a solution of (2S)-2-(tert-butoxycarbonylamino)-5-[2-(methylamino)-5-nitro-anilino]-5-oxo-pentanoic acid methyl ester (Scheme 1) (50 g, 98.2 mmol) in MeOH (150 mL) was added hydrochloric acid (12.0 mol / L, 73.6 mL, 884 mmol) and the mixture was stirred at 70 °C for 30 min. The reaction mixture was dissolved in water (500 mL) and washed with EtOAc (2 × 150 mL). The aqueous phase was mixed with EtOAc (300 mL) and solid K2CO3 was added until pH 9-10. The aqueous phase was extracted with EtOAc (2 × 100 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure to give methyl (2S)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoate as a reddish solid (26.0 g, 82.7 mmol, 84% yield). The crude product was used in the next step without further purification. HPLC purity: 93%; MS (ESI+) m / z = 293 [M+H] + .

[0274] To a solution of (2S)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid methyl ester (26 g, 82.7 mmol) in THF (100 mL) were added triethylamine (13.8 mL, 99.3 mmol) and di-tert-butyl dicarbonate (20.9 mL, 91 mmol) and the mixture was stirred at room temperature overnight. The crude product was concentrated under reduced pressure, dissolved in EtOAc (400 mL) and washed with 0.5 N HCl (400 mL). The two phases were separated and the aqueous phase was extracted with EtOAc (2× 200 mL). The organic phase was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was dissolved in a small amount of THF and Et2O was added. The mixture was cooled to 0° C. and left for 30 min. The solid was filtered and washed with cold Et2O. The solid was dried under reduced pressure to give (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester as an off-white solid (27 g, 68.8 mmol, 83% yield). HPLC purity: 100%; MS (ESI+) m / z = 393 [M+H] + .

[0275] (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 18-2) (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid methyl ester (1.95 g, 4.97 mmol) was dissolved in MeOH (15 mL) and placed in a H reactor. 10% Pd / C (264 mg) was added and the reactor was sealed. Nitrogen purging was performed. Hydrogen gas was charged to the reactor at 3 bar and the reaction was stirred at 35° C. for 2 h. Hydrogen gas was evacuated with a nitrogen purge. The catalyst was removed by filtration through Celite. The solvent was evaporated to give the crude product (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester as an off-white (slightly pink) solid (1.8 g, quantitative), which was used in the next step without further purification. HPLC purity: 95%; MS (ESI+) m / z 363 [M+H] + .

[0276] (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 18-3) 2-Chloroacetaldehyde (1.30 g, 1.05 mL, 8.28 mmol, 50%, 10 equiv.) was added to a solution of (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (300 mg, 0.83 mmol), TFA (0.369 mL, 4.97 mmol, 6 equiv.) and MeOH (10 mL). The reaction mixture was stirred at 0° C. for 15 min, then NaBH3CN (104 mg, 1.66 mmol, 2 equiv.) was added over 5 min and the mixture was stirred at 0° C. for 20 min. Water (10 mL), NaHCO3 (10 mL) and EtOAc (10 mL) were added and the layers were separated. The aqueous phase was extracted with EtOAc (3×10 mL). The combined organic phase was washed with brine (2×20 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified on a silica column and the product was eluted with 80% EtOAc in p-ether. Fractions were pooled and concentrated to give (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (207 mg, 0.425 mmol, 51% yield). HPLC purity: 93%; MS (ESI+) m / z 487 [M+H] + .

[0277] (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonyl-amino)butanoic acid (compound 18-4) A mixture of (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (207 mg, 0.425 mmol) and LiOH·H2O (54 mg, 1.27 mmol) was suspended in 1,4-dioxane (12 mL) and water (4 mL). The reaction mixture was heated at 50 °C for 20 min. The solution was allowed to cool to room temperature and water (20 mL) was added. The mixture was then brought to pH 5 with 1 M HCl and the product was extracted with EtOAc (3 × 70 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous MgSO4, filtered and concentrated on a rotary evaporator to give (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonyl-amino)butanoic acid (188 mg, 0.397 mmol, 94% yield). HPLC purity: 95%; MS (ESI+) m / z 473 [M+H] + .

[0278] (S)-2-Amino-4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid dihydrochloride (Example compound 18) (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonyl-amino)butanoic acid (188 mg, 0.397 mmol) was dissolved in dichloromethane (3 mL) and 4M HCl in dioxane (1.0 mL) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours and then diethyl ether (15 mL) was added. Upon addition of diethyl ether, a precipitate formed which was isolated by filtration and dried in vacuum to give (S)-2-amino-4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid dihydrochloride (Example compound 18) (118 mg, 0.288 mmol, 73% yield). HPLC purity: 95%; MS (ESI+) m / z 373 [M+H] + .

[0279] 1 H NMR (600 MHz, CD3OD) δ 7.71 (d, J = 9.3 Hz, 1H), 7.18 (dd, J = 9.3, 2.4 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 4.23 (dd, J = 7.3, 5.5 Hz, 1H), 3.99 (s, 3H), 3.89 (t, J = 6.8 Hz, 4H), 3.76 (t, J = 6.7 Hz, 4H), 3.54 - 3.44 (m, 1H), 3.44 - 3.35 (m, 1H), 2.61 - 2.49 (m, 1H), 2.49 - 2.37 (m, 1H).

[0280] Example 19 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid; dihydrochloride (Example compound 19) [ka]

[0281] To a solution of (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid ethyl ester; dihydrochloride (Example compound 3) (15.3 mg, 0.0264 mmol) in THF:MeOH (2:1, 1 mL) was added an aqueous solution of lithium hydroxide monohydrate (0.5 M, 0.0555 mL). The mixture was stirred at room temperature overnight. The sample was filtered and purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 2% B to 31% B in 15 min, fractions collected based on UV signal at 235 nm). HCl in ethanol (2.5 M, 1 mL) was added to the combined fractions and the sample was immediately lyophilized to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanoic acid; dihydrochloride (Example compound 19) as a white solid (5.8 mg, 0.0106 mmol, 40% yield). HPLC purity: 99%; MS (ESI+) m / z = 472 [M+H] + .

[0282] 1H NMR (600 MHz, DMSO-d6) δ 14.72 (s, 1H), 12.91 (s, 1H), 8.86 (d, J = 7.7 Hz, 1H), 8.55 (d, J = 5.2 Hz, 3H), 7.73 (d, J = 9.2 Hz, 1H), 7.13 (dd, J = 9.4, 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 4.19 (dd, J = 7.7, 5.1 Hz, 2H), 3.86 (s, 3H), 3.81 (t, J = 6.8 Hz, 4H), 3.76 (t, J = 6.9 Hz, 4H), 3.31 (h, J = 9.4 Hz, 2H), 2.33 (ddd, J = 13.1, 9.4, 6.8 Hz, 1H), 2.23 (ddt, J = 13.9, 9.5, 6.9 Hz, 1H), 2.19 - 2.10 (m, 1H), 0.94 (dd, J = 6.9, 3.6Hz, 6H).

[0283] Example 20 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 20) [ka]

[0284] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 1) (15 mg, 0.0255 mmol) was dissolved in HCl (4 M in dioxane, 0.319 mL) and stirred at room temperature for 48 h. The crude product was purified by preparative chromatography (acidic buffer; flow rate 25 mL / min, gradient from 9% B to 42% B in 15 min. Fractions were collected based on the UV signal at 235 nm). (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 20) was obtained as a white solid (7.3 mg, 0.013 mmol, 51% yield). HPLC purity: 99%; MS (ESI+) m / z = 486 [M+H] + .

[0285] 1 H NMR (600 MHz, DMSO-d6) δ 14.64 (s, 1H), 12.92 (s, 1H), 9.00 (d, J = 7.4 Hz, 1H), 8.49 (d, J = 5.6 Hz, 3H), 7.73 (d, J = 9.2 Hz, 1H), 7.13 (d, J = 9.0 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 4.26 (ddd, J = 10.0, 7.3, 5.1 Hz, 1H), 4.05 - 3.99 (m, 1H), 3.86 (s, 3H), 3.82 (t, J = 6.9 Hz, 4H), 3.76 (d, J = 6.3 Hz, 4H), 3.30 (s, 2H), 2.32 (p, J = 7.2 Hz, 1H), 2.26 - 2.19 (m, 1H), 1.76 - 1.69 (m, 1H), 1.57 (pt, J = 9.4, 5.0 Hz, 2H), 0.89 (dd, J = 24.6, 6.6 Hz, 6H).

[0286] Example 21 (2S)-2-[[(2R)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 21) [ka]

[0287] Example 21 was carried out according to Scheme 8 below. [ka]

[0288] (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid methyl ester (compound 21-1) To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoic acid (500 mg, 0.00176 mol), (2R)-2-amino-4-methyl-pentanoic acid methyl ester; hydrochloride (0.417 g, 0.00229 mol), 1-methylimidazole (0.436 mL, 0.00547 mol) in MeCN (8 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphoric acid (0.545 g, 0.00194 mol) at room temperature. The mixture was stirred at room temperature for 1 h. EtOAc (10 mL) was added followed by H2O (10 mL). The two phases were separated, the aqueous phase was washed with EtOAc (5 mL) and the combined organic phase was dried over MgSO4 and evaporated under reduced pressure. The crude product was adsorbed onto diatomaceous earth and purified on a Buchi Pure C-810 Flash system (20 g column, EtOAc:petroleum ether 0:100 to 50:50) to give (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid methyl ester as a white solid (480 mg, 0.00117 mol, 66% yield). HPLC purity: 95%; MS (ESI+) m / z 411 [M+H] + .

[0289] (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid (compound 21-2) To a solution of (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid methyl ester (200 mg, 0.000487 mol) in THF (10 mL) was added a solution of lithium hydroxide monohydrate (0.0215 g, 0.000512 mol) in water (0.5 mL). The mixture was stirred at room temperature for 1 h, and EtOAc (10 mL), 1N HCl (0.5 mL) and H2O (10 mL) were added. The two phases were separated and the aqueous phase was further extracted with EtOAc (10 mL). The combined organic phase was washed with brine (10 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoic acid was obtained as a colorless sticky oil (0.185 g, 0.000467 mol, 96% yield). HPLC purity: 94%; MS (ESI+) m / z 341 [M-tBu] + .

[0290] (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (compound 21-3) To (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methylpentanoic acid (95.0 mg, 0.240 mmol) in DMF (3.0 mL) was added HATU (91.1 mg, 0.240 mmol) and DIEA (0.0820 mL, 0.479 mmol) at room temperature, followed by (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 38) (125 mg, 0.264 mmol). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with H2O and extracted with DCM. The organic phase was dried (Na2SO4), concentrated in vacuo, and purified by preparative chromatography. The pure fractions were lyophilized to give (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]butanoic acid ethyl ester as a white solid (0.080 g, 0.103 mmol, 43% yield). HPLC purity: 97%; MS (ESI+) m / z = 779 [M+H] + .

[0291] (2S)-2-[[(2R)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 21) To (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[[(2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]butanoic acid ethyl ester (80.0 mg, 0.103 mmol) in EtOH (1 mL) was added HCl in dioxane (4.00 mol / L, 0.128 mL, 0.513 mmol) and the reaction mixture was stirred overnight. After completion of the reaction, the solvent was evaporated and the crude product was purified by preparative chromatography. Pure fractions were collected and lyophilized to give (2S)-2-[[(2R)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride salt (Example compound 21) as a white solid (6 mg, 0.0079 mmol, 8% yield). HPLC purity: 99%; MS (ESI+) m / z = 679 [M+H] + .

[0292] 1H NMR (600 MHz, DMSO-d6) δ 14.15 (s, 1H), 8.80 - 8.64 (m, 2H), 8.06 (s, 3H), 7.38 - 7.22 (m, 2H), 7.22 - 7.08 (m, 2H), 6.95- 6.81 (m, 1H), 4.51 - 4.27 (m, 2H), 4.14 - 3.96 (m, 3H), 3.89 - 3.67 (m, 12H), 3.13 (s, 1H), 3.14 (m, 2H), 3.07 (dd, J = 14.3,4.9 Hz, 1H), 2.87 (dd, J = 14.2, 8.3 Hz, 1H), 2.35 - 2.25 (m, 1H), 2.21 - 2.07 (m, 1H), 1.76 - 1.60 (m, 1H), 1.58 -1.39 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H).

[0293] Example 22 (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride (Example compound 22) [ka]

[0294] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 equiv., 68 mg, 0.355 mmol) was dissolved in 4-methylmorpholine (1.15 equiv., 0.039 mL, 0.355 mmol), ethyl cyanoglyoxylate-2-oxime (1.00 equiv., 44 mg, 0.308 mmol), (2S)-2-amino-3-(4-fluorophenyl)-2-propanediol (1.00 equiv., 44 mg, 0.308 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 equiv., 68 mg, 0.355 mmol) in DMF (2 mL). To a mixture of (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 20) (1.00 equiv., 150 mg, 0.308 mmol) was added (1.15 equiv., 74.9 mg, 0.355 mmol) and (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 20) (1.00 equiv., 150 mg, 0.308 mmol). The reaction mixture was stirred at room temperature for 3 h. Water was added and the mixture was extracted with DCM. The organic phase was washed with 10% K2HPO4 and 10% NaCl, followed by an acidic wash with 25 M HCl and 10% NaCl, passed through a phase separation cartridge and evaporated. The crude product was purified by preparative chromatography to give (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride salt (Example compound 22) as a white solid (4 mg, 0.0053 mmol, 2% yield). HPLC purity: 99%; MS (ESI+) m / z = 679 [M+H] + .

[0295] 1H NMR (600 MHz, DMSO-d6) δ 14.34 (br s, 1H), 8.72 (d, J = 7.7 Hz, 2H), 8.34 (s, 3H), 7.68 (s, 1H), 7.25 (dd, J = 8.3, 5.5 Hz, 2H), 7.01 (t, J = 8.7 Hz, 2H), 6.90 (d, J = 2.4 Hz, 1H), 4.43 (dq, J = 15.5, 7.7 Hz, 2H), 4.05 - 3.96 (m, 2H), 3.95 (q, J = 6.5, 5.8 Hz, 1H), 3.84 - 3.69 (m, 11H), 3.13 - 2.98 (m, 4H), 2.93 (dd, J = 13.9, 9.0 Hz, 1H), 2.23 (dq, J = 13.4, 6.8, 6.1 Hz, 1H), 2.10 (dd, J = 15.9, 8.1 Hz, 1H), 1.69 (dp, J = 13.5, 6.7 Hz, 1H), 1.47 (t, J = 7.4 Hz, 2H), 1.08 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 19.1, 6.5 Hz, 6H).

[0296] Example 23 (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-4-methyl-pentanoic acid butyl ester dihydrochloride (Example compound 23) [ka]

[0297] 4-Methylmorpholine (1.15 equiv., 0.039 mL, 0.355 mmol) was dissolved in (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 20) (1.00 equiv., 150 mg, 0.308 mmol) in DMF (2 mL). A mixture of 1.15 equiv., (2S)-2-amino-4-methyl-pentanoic acid ethyl ester (1.15 equiv., 56 mg, 0.355 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 equiv., 68 mg, 0.355 mmol) and ethyl cyanoglyoxylate-2-oxime (1.00 equiv., 44 mg, 0.308 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. Water was added and the mixture was extracted with DCM. The organic phase was washed with 10% K2HPO4 and 10% NaCl followed by an acidic wash with 0.25 M HCl and 10% NaCl, passed through a phase separation cartridge and evaporated. The resulting crude product was purified by preparative chromatography to give (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 23) as a solid (5.1 mg, 0.00728 mmol, 2% yield). HPLC purity: 95%; MS (ESI+) m / z = 627 [M+H] + .

[0298] 1H NMR (600 MHz, DMSO-d6) δ 14.41 (br s, 1H), 8.76 (s, 1H), 8.54 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 123.3 Hz, 3H), 7.70 (s, 1H), 6.90 (d, J = 2.3 Hz, 1H), 6.57 (s, 1H), 4.39 (dt, J = 13.0, 6.5 Hz, 1H), 4.27 (ddd, J = 10.6, 7.5, 4.9 Hz, 1H), 4.10 - 4.00 (m, 2H), 3.97 (d, J = 7.5 Hz, 1H), 3.91 - 3.71 (m, 11H), 3.1 (m, 2H), 2.25 (dt, J = 13.5, 7.2 Hz, 1H), 2.13 (s, 1H), 1.73 (dt, J = 13.4, 6.3 Hz, 1H), 1.64 (dd, J = 13.9, 7.0 Hz, 1H), 1.53 (tddd, J = 22.7, 14.1, 9.6, 5.1 Hz, 4H), 1.15 (t, J = 7.1 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.78 (d, J = 6.5 Hz, 3H).

[0299] Example 24 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 2-morpholinoethyl ester dihydrochloride (Example compound 24) [ka]

[0300] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (1.00 equiv, 50 mg, 0.0758 mmol) was suspended in THF (2 mL) and thionyl chloride (5.53 equiv, 31 uL, 0.420 mmol) was added. The reaction was stirred at room temperature for 30 minutes and then 4-(2-hydroxyethyl)morpholine (5.00 equiv, 0.046 mL, 0.379 mmol) was added. The mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 2-morpholinoethyl ester; dihydrochloride (Example compound 24) as a solid (6 mg, 0.0089 mmol, 12% yield). HPLC purity: 99%; MS (ESI+) m / z = 599 [M+H] + .

[0301] 1 H NMR (600 MHz, DMSO-d6) δ 14.79 (s, 1H), 11.42 (s, 1H), 9.36 (d, J = 7.3 Hz, 1H), 8.55 (s, 2H), 7.72 (s, 1H), 7.12 (s, 1H), 6.93 (s, 1H), 4.50 (s, 1H), 4.44 (td, J = 13.5, 11.9, 5.2 Hz, 2H), 4.11 (s, 1H), 3.96 - 3.74 (m, 15H), 3.56 (s, 2H), 3.15 (s, 6H), 2.32 (q, J = 7.6 Hz, 2H), 1.74 (dq, J = 13.3, 6.6, 6.1 Hz, 1H), 1.63 (dddd, J = 18.6, 13.8, 9.1, 4.6 Hz, 2H), 0.91 (dd, J = 20.5, 6.5 Hz, 6H).

[0302] Example 25 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 2-isopropoxyethyl ester dihydrochloride (Example compound 25) [ka]

[0303] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (1.00 equiv., 50 mg, 0.0758 mmol) was added in portions to a solution of ethylene glycol monoisopropyl ether (5.00 equiv., 0.042 mL, 0.379 mmol) and thionyl chloride (5.53 equiv., 31 uL, 0.420 mmol) in MeCN (2 mL) at -5°C. The reaction was allowed to warm to room temperature and then heated at 30°C for 3 hours. The solvent was removed in vacuo as an azeotrope with toluene and then dried using a high vacuum pump. The resulting crude product was purified by preparative chromatography to give a solid of (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 2-isopropoxyethyl ester; dihydrochloride (Example compound 25) (6.2 mg, 0.0096 mmol, 12% yield). HPLC purity: 95%; MS (ESI+) m / z = 572 [M+H] + .

[0304] 1H NMR (600 MHz, DMSO-d6) δ 14.77 (s, 1H), 9.25 (d, J = 7.0 Hz, 1H), 8.56 (s, 2H), 7.72 (s, 1H), 7.12 (s, 1H), 6.93 (dd, J = 10.1, 2.4 Hz, 1H), 6.55 (s, 1H), 4.37 - 4.28 (m, 1H), 4.23 (dq, J = 8.1, 4.4, 3.9 Hz, 1H), 4.09 (ddt, J = 13.8, 10.7, 5.5 Hz, 2H), 3.90 - 3.74 (m, 11H), 3.54 (td, J = 8.6, 7.0, 3.6 Hz, 3H), 2.36 - 2.24 (m, 2H), 1.77 (dd, J = 13.9, 6.7 Hz, 1H), 1.60 (ddt, J = 28.1, 13.4, 8.4 Hz, 2H), 1.05 (dd, J = 18.5, 6.1 Hz, 6H), 0.91 (dd, J = 25.5, 6.5 Hz, 6H). One CH2 signal is obscured by the H2O signal.

[0305] Example 26 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid isopropyl ester dihydrochloride (Example compound 26) [ka]

[0306] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (1.00 equiv., 50 mg, 0.0758 mmol) was added in portions to a solution of isopropanol (200 equiv., 1.2 mL, 15.2 mmol) and thionyl chloride (5.53 equiv., 31 uL, 0.420 mmol) at -5°C. The reaction was allowed to warm to room temperature and stirred for 3 hours. The solvent was evaporated in vacuo as an azeotrope with toluene and then dried using a high vacuum pump. The resulting crude product was purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid isopropyl ester; dihydrochloride (Example compound 26) as an off-white solid (32 mg, 0.053 mmol, 69% yield). HPLC purity: 98%; MS (ESI+) m / z = 528 [M+H] + .

[0307] 1H NMR (600 MHz, DMSO-d6) δ 14.79 (s, 1H), 9.20 (d, J = 7.0 Hz, 1H), 8.55 (s, 3H), 7.74 (d, J = 9.2 Hz, 1H), 7.15 (d, J = 9.2 Hz, 1H), 6.93 (s, 1H), 4.90 (p, J = 6.3 Hz, 1H), 4.26 (dt, J = 11.0, 5.7 Hz, 1H), 4.08 (s, 1H), 3.90 (s, 3H), 3.80 (dt, J = 33.7, 6.9 Hz, 8H), 3.37 - 3.32 (m, 2H), 2.31 (dh, J = 22.0, 7.3 Hz, 2H), 1.75 (p, J = 6.7 Hz, 1H), 1.62 (ddd, J = 15.0, 10.2, 5.1 Hz, 1H), 1.53 (ddd, J = 13.9, 9.2, 4.9 Hz, 1H), 1.18 (dd, J = 11.4, 6.3 Hz, 6H), 0.90 (dd, J = 27.2, 6.5 Hz, 6H).

[0308] Example 27 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid methyl ester; dihydrochloride (Example compound 27) [ka]

[0309] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example compound 20) (1.00 equiv, 35 mg, 0.06260 mmol) was dissolved in 0.4 mL of MeOH (137 equiv). Thionyl chloride (5.72 equiv, 30 uL, 0.411 mmol) was added dropwise to the solution at room temperature. The reaction was stirred at room temperature for 4.5 hours. The crude reaction mixture was purified by preparative chromatography to give off-white (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid methyl ester; dihydrochloride salt (Example compound 27) (14 mg, 0.0244 mmol, 33% yield). HPLC purity: 97%; MS (ESI+) m / z = 500 [M+H] + .

[0310] 1 H NMR (600 MHz, DMSO-d6) δ 14.86 (s, 1H), 9.32 (d, J = 7.1 Hz, 1H), 8.61 (s, 3H), 7.74 (d, J = 9.2 Hz, 1H), 7.15 (d, J = 9.2 Hz, 1H), 6.94 (s, 1H), 4.33 (dt, J = 11.1, 5.5 Hz, 1H), 4.10 (s, 1H), 3.91 (s, 3H), 3.80 (dt, J = 33.0, 6.9 Hz, 8H), 3.65 (s, 3H), 3.35 (h, J = 10.1, 8.5 Hz, 2H), 2.33 (ddq, J = 30.2, 15.1, 7.5 Hz, 2H), 1.75 (p, J = 6.7 Hz, 1H), 1.65 (td, J = 12.2, 10.3, 5.1 Hz, 1H), 1.54 (ddd, J = 14.2, 9.6, 4.7 Hz, 1H), 0.90 (dd, J = 23.3, 6.5 Hz, 6H).

[0311] Example 28 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 3-(dimethylamino)propyl ester; dihydrochloride (Example compound 28) [ka]

[0312] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 1) (1.00 equiv., 50 mg, 0.0851 mmol) was dissolved in THF (1 mL). HCl (4M in EtOAc) (10.0 equiv., 0.21 mL, 0.851 mmol) was added, followed by 3-dimethylamino-1-propanol (10.0 equiv., 0.10 mL, 0.851 mmol). The reaction was stirred at 80° C. for 4 h. The mixture was filtered and purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid 3-(dimethylamino)propyl ester; dihydrochloride salt (Example compound 28) as an off-white solid (2 mg, 0.0031 mmol, 3% yield). HPLC purity: 95%; MS (ESI+) m / z = 571 [M+H] + .

[0313] 1H NMR (600 MHz, DMSO-d6) δ 15.9 (br s, 1H), 10.7 (br s, 1H), 9.43 (d, J = 7.1 Hz, 1H), 8.55 (d, J = 73.7 Hz, 3H), 7.79 (m, 1H), 6.93 (s, 1H), 4.34 (s, 1H), 4.18 - 4.12 (m, 2H), 3.76 (s, 11H), 3.09 (s, 1H), 2.73 (d, J = 20.6 Hz, 6H), 2.34 (s, 1H), 2.01 (q, J = 7.8, 7.3 Hz, 2H), 1.81 - 1.52 (m, 4H), 1.23 (s, 4H), 0.97 - 0.89 (m, 6H).

[0314] Example 29 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (2-methoxy-1-methyl-ethyl) ester; dihydrochloride (Example compound 29) [ka]

[0315] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 20) (1.0 eq, 36 mg, 0.0644 mmol) was dissolved in 1-methoxy-2-propanol (55 eq, 0.40 mL, 4.07 mmol) and thionyl chloride (5.6 eq, 30 uL, 0.411 mmol) was added dropwise at room temperature. The reaction was stirred at room temperature for 1 hour and then heated at 40° C. for 21 hours. The crude reaction mixture was purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (2-methoxy-1-methyl-ethyl) ester; dihydrochloride salt (Example compound 29) (2.7 mg, 0.0043 mmol, 6% yield) as an off-white solid. HPLC purity: 99%; MS (ESI+) m / z = 558 [M+H] + .

[0316] 1H NMR (600 MHz, DMSO-d6) δ 14.88 (s, 1H), 9.30 (p, J = 6.1 Hz, 1H), 8.60 (s, 3H), 7.75 (d, J = 9.2 Hz, 1H), 7.15 (d, J = 9.1 Hz, 1H), 6.94 (s, 1H), 4.96 (dtd, J = 16.3, 6.5, 4.0 Hz, 1H), 4.28 (qd, J = 11.2, 10.4, 6.1 Hz, 1H), 4.13 - 4.05 (m, 1H), 3.91 (s, 3H), 3.83 (t, J = 6.8 Hz, 4H), 3.78 (t, J = 6.5 Hz, 4H), 3.40 - 3.33 (m, 4H), 3.22 (d, J = 13.7 Hz, 3H), 2.34 (dd, J = 18.1, 9.2 Hz, 2H), 1.77 (td, J = 9.9, 9.3, 4.6 Hz, 1H), 1.62 (ddd, J = 15.1, 12.3, 7.7 Hz, 1H), 1.53 (ddt, J = 13.8, 9.6, 4.8 Hz, 1H), 1.14 (dd, J = 11.9, 6.5 Hz, 3H), 0.90 (dd, J = 26.5, 6.6 Hz, 6H).

[0317] Example 30 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,N,4-trimethyl-pentanamide; dihydrochloride (Example compound 30) [ka]

[0318] 3-(3-Dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride (1.15 equiv., 59 mg, 0.308 mmol) was added to a mixture of (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 20) (1.00 equiv., 150 mg, 0.268 mmol), dimethylamine hydrochloride (1.15 equiv., 25 mg, 0.308 mmol), (2E)-ethyl cyano(hydroxyimino)acetate (1.00 equiv., 38 mg, 0.268 mmol) and N-methylmorpholine (2.00 equiv., 0.059 mL, 0.536 mmol) in DMF (1 mL). The reaction was stirred at room temperature for 3 hours. Water and DCM were added and the mixture was extracted twice with 10% K2HPO4 and 10% NaCl, followed by an acidic wash with 25M HCl and 10% NaCl. The organic phase was concentrated and the resulting crude product was purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,N,4-trimethyl-pentanamide; dihydrochloride salt (Example compound 30) as a solid (13 mg, 0.022 mmol, 8% yield). HPLC purity: 95%; MS (ESI+) m / z = 513 [M+H] + .

[0319] 1H NMR (600 MHz, DMSO-d6) δ 14.63 (s, 1H), 8.87 (s, 1H), 8.51 (s, 3H), 7.73 (s, 1H), 7.14 (s, 1H), 6.91 (d, J = 2.4 Hz, 1H), 4.72 (ddd, J = 11.0, 7.5, 3.6 Hz, 1H), 4.00 (s, 1H), 3.89 (s, 3H), 3.82 (d, J = 6.7 Hz, 4H), 3.77 (t, J = 6.4 Hz, 4H), 3.3 (m, 2H), 3.04 (s, 3H), 2.84 (s, 3H), 2.39 - 2.30 (m, 1H), 2.19 (dq, J = 14.3, 7.3 Hz, 1H), 1.75 - 1.67 (m, 1H), 1.50 (ddd, J = 14.7, 10.8, 4.2 Hz, 1H), 1.39 (ddd, J = 13.7, 9.8, 3.6 Hz, 1H), 0.92 (t, J = 6.1 Hz, 6H).

[0320] Example 31 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,4-dimethyl-pentanamide; dihydrochloride (Example compound 31) [ka]

[0321] Example 31 was carried out according to Scheme 9 below. [ka]

[0322] N-[(1S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-1-[[(1S)-3-methyl-1-(methylcarbamoyl)butyl]carbamoyl]propyl]carbamic acid tert-butyl ester; dihydrochloride (compound 31-1) (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (step 4 of the synthetic pathway for example compound 1) (50 mg, 0.0814 mmol) in methylamine (40% in MeOH) (63 mg, 0.814 mmol) was placed in a sealed tube and stirred at 80° C. for 20 min under microwave irradiation. The solvent was removed under reduced pressure and the crude product was purified by preparative chromatography to give N-[(1S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-1-[[(1S)-3-methyl-1-(methylcarbamoyl)butyl]carbamoyl]propyl]carbamic acid tert-butyl ester; dihydrochloride (35 mg, 0.0586 mmol, 64% yield) as a white solid. HPLC purity: 99%; MS (ESI+) m / z = 599 [M+H] + .

[0323] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,4-dimethyl-pentanamide; dihydrochloride (Example compound 31) N-[(1S)-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-1-[[(1S)-3-methyl-1-(methylcarbamoyl)butyl]carbamoyl]propyl]carbamic acid tert-butyl ester (35 mg, 0.0586 mmol) was dissolved in MeCN (1 mL). Hydrochloric acid (4 M in dioxane) (0.15 mL, 0.586 mmol) was added and the mixture was stirred at room temperature for 3 h. The reaction mixture was purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,4-dimethyl-pentanamide; dihydrochloride salt (Example compound 31) as an off-white solid (14 mg, 0.024 mmol, 40% yield). HPLC purity: 95%; MS (ESI+) m / z = 499 [M+H] + .

[0324] 1 H NMR (600 MHz, DMSO-d6) δ 14.62 (s, 1H), 8.79 (s, 1H), 8.48 (s, 3H), 8.18 (q, J = 4.6 Hz, 1H), 7.74 (s, 1H), 7.15 (s, 1H), 6.91 (d, J = 2.4 Hz, 1H), 4.29 (ddd, J = 10.0, 7.6, 5.0 Hz, 1H), 3.98 (s, 1H), 3.90 (s, 3H), 3.82 (s, 4H), 3.77 (t, J = 6.4 Hz, 4H), 3.3 (m, 2H), 2.59 (d, J = 4.6 Hz, 3H), 2.33 (s, 1H), 2.21 (dq, J = 14.0, 7.2 Hz, 1H), 1.66 (dt, J = 13.3, 6.5 Hz, 1H), 1.52 (ddd, J = 14.7, 10.0, 5.1 Hz, 1H), 1.43 (ddd, J = 13.8, 9.0, 5.1 Hz, 1H), 0.90 (dd, J = 15.6, 6.6 Hz, 6H).

[0325] Example 32 (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 32) [ka]

[0326] Example 32 was carried out according to Scheme 10 below. [ka]

[0327] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[tert-butoxycarbonyl(methyl)amino]butanoyl]amino]-4-methyl-pentanoic acid (compound 32-1) To a solution of (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (step 4 of the synthetic route of Example compound 1) (300 mg, 0.488 mmol) in dry THF (3 mL) was added sodium hydride (50%; 59 mg, 1.46 mmol). The mixture was stirred at room temperature until gas evolution ceased. 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (0.12 mL, 1.03 mmol) was added to the reaction mixture followed by methyl iodide (0.27 mL, 4.39 mmol). The reaction was stirred at room temperature overnight. The reaction was carefully quenched with water and purified by preparative chromatography to give (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[tert-butoxycarbonyl(methyl)amino]butanoyl]amino]-4-methyl-pentanoic acid as a white solid (105 mg, 0.175 mmol, 36% yield). HPLC purity: 99%; MS (ESI+) m / z = 600 [M+H] + .

[0328] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 32) (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[tert-butoxycarbonyl(methyl)amino]butanoyl]amino]-4-methyl-pentanoic acid (42 mg, 0.0699 mmol) was added portionwise to a solution of ethanol (2 mL) and thionyl chloride (31 uL, 0.420 mmol) at -5°C. The reaction was stirred at room temperature for 5 minutes and then refluxed for 2 hours. The reaction was allowed to cool to room temperature and the solvent was evaporated in vacuo as an azeotrope with toluene and then dried using a high vacuum pump. The crude product was purified by preparative chromatography to give (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride salt (Example compound 32) as an off-white solid (25 mg, 0.041 mmol, 59% yield). HPLC purity: 99%; MS (ESI+) m / z = 528 [M+H] + .

[0329] 1H NMR (600 MHz, DMSO-d6) δ 14.84 (s, 1H), 9.43 (d, J = 7.0 Hz, 3H), 7.73 (d, J = 9.2 Hz, 1H), 7.14 (d, J = 9.4 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 4.35 (ddd, J = 11.0, 7.0, 4.7 Hz, 1H), 4.12 (p, J = 7.0 Hz, 2H), 4.04 (dd, J = 8.0, 4.8 Hz, 1H), 3.90 (s, 3H), 3.83 (t, J = 6.8 Hz, 4H), 3.77 (t, J = 6.5 Hz, 4H), 3.30 (t, J = 8.4 Hz, 2H), 2.55 (s, 3H), 2.47 (dd, J = 8.5, 4.9 Hz, 1H), 2.28 (dq, J = 14.8, 7.9 Hz, 1H), 1.69 (dddd, J = 39.0, 0.91 (dd, J = 26.6, 6.5 Hz, 6H).

[0330] Example 33 (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 33) [ka]

[0331] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-[tert-butoxycarbonyl(methyl)amino]butanoyl]amino]-4-methyl-pentanoic acid (compound 32-1) (12 mg, 0.0200 mmol) was dissolved in ethanol (1 mL). HCl (4 M in dioxane) (50 uL, 0.200 mmol) was added and the reaction was stirred at room temperature for 3 h. The reaction mixture was filtered and purified by preparative chromatography to give (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride salt (Example compound 33) as an off-white solid (4 mg, 0.0069 mmol, 34% yield). HPLC purity: 97%; MS (ESI+) m / z = 500 [M+H] + .

[0332] 1 H NMR (600 MHz, DMSO-d6) δ 14.43 (s, 1H), 13.03 (s, 1H), 9.13 (s, 2H), 7.63 (s, 1H), 7.16 - 6.98 (m, 1H), 6.92 (d, J = 2.4 Hz, 1H), 4.34 (q, J = 7.5 Hz, 1H), 3.95 (dd, J = 7.8, 4.7 Hz, 1H), 3.87 - 3.72 (m, 11H), 3.14 (s, 3H), 2.57 (s, 3H), 2.44 - 2.36 (m, 1H), 2.21 (ddt, J = 19.1, 13.7, 5.8 Hz, 1H), 1.70 (dp, J = 13.2, 6.6 Hz, 1H), 1.62 (t, J = 7.4 Hz, 2H), 0.92 (dd, J = 28.5, 6.5 Hz, 6H).

[0333] Example 34 (2S)-2-[[(2S)-2-acetamido-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; hydrochloride (Example compound 34) [ka]

[0334] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; hydrochloride (Example compound 2) (15 mg, 0.0233 mmol) was dissolved in acetonitrile (1 mL). N,N-diisopropylethylamine (0.00553 mL, 0.0317 mmol) was added, followed by acetyl chloride (0.00227 mL, 0.0318 mmol) and the reaction was stirred at room temperature for 3 hours. The reaction mixture was purified by preparative chromatography to give (2S)-2-[[(2S)-2-acetamido-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; hydrochloride salt (Example compound 34) as an off-white solid (7 mg, 0.0108 mmol, 41% yield). HPLC purity: 99%; MS (ESI+) m / z = 608 [M+H] + .

[0335] 1H NMR (600 MHz, DMSO-d6) δ 14.20 (s, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.18 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.29 - 7.19 (m, 2H), 7.14 - 7.04 (m, 3H), 6.86 (d, J = 2.4 Hz, 1H), 4.40 (dtd, J = 20.2, 8.2, 6.1 Hz, 2H), 4.00 (qd, J = 7.2, 2.2 Hz, 2H), 3.84 - 3.74 (m, 11H), 3.05 (q, J = 9.3, 8.4 Hz, 2H), 3.00 - 2.88 (m, 2H), 2.09 (ddd, J = 13.9, 8.2, 4.3 Hz, 1H), 2.04 - 1.94 (m, 1H), 1.76 (s, 3H), 1.06 (t, J = 7.1 Hz, 3H).

[0336] Example 35 (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; hydrochloride (Example compound 35) [ka]

[0337] (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 4) (11 mg, 0.0182 mmol) was dissolved in acetonitrile (1 mL). N,N-Diisopropylethylamine (0.00381 mL, 0.0219 mmol) was added, followed by acetyl chloride (0.00156 mL, 0.0219 mmol) and the reaction was stirred at room temperature for 3 hours. The reaction mixture was purified by preparative chromatography to give (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; hydrochloride salt (Example compound 35) as a white solid (8 mg, 0.0138 mmol, 76% yield). HPLC purity: 99%; MS (ESI+) m / z = 542 [M+H] + .

[0338] 1H NMR (600 MHz, DMSO-d6) δ 14.16 (s, 1H), 8.56 (d, J = 7.7 Hz, 1H), 8.41 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 9.2 Hz, 1H), 7.12 (dd, J = 9.2, 2.4 Hz, 1H), 6.87 (d, J = 2.3 Hz, 1H), 4.83 (td, J = 8.0, 6.5 Hz, 1H), 4.26 (ddd, J = 9.8, 7.6, 5.2 Hz, 1H), 4.00 (qd, J = 7.2, 1.2 Hz, 2H), 3.91 (s, 3H), 3.81 (dd, J = 8.9, 6.4 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.29 (dd, J = 15.0, 7.8 Hz, 2H), 1.80 (s, 3H), 1.61 - 1.49 (m, 2H), 1.46 (ddd, J = 13.7, 8.9, 5.1 Hz, 1H), 1.11 (t, J = 7.1 Hz, 3H), 0.83 (dd, J = 25.2, 6.4 Hz, 6H).

[0339] Example 36 (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; hydrochloride (Example compound 36) [ka]

[0340] (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride (Example compound 5) (15 mg, 0.0228 mmol) was dissolved in acetonitrile (1 mL). N,N-diisopropylethylamine (0.00476 mL, 0.0273 mmol) was added, followed by acetyl chloride (0.00195 mL, 0.0273 mmol) and the reaction was stirred at room temperature for 3 hours. The reaction mixture was purified by preparative chromatography to give (2S)-2-[[(2S)-2-acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; hydrochloride salt (Example compound 36) as a white solid (10.6 mg, 0.0168 mmol, 71% yield). HPLC purity: 97%; MS (ESI+) m / z = 594 [M+H] + .

[0341] 1 H NMR (600 MHz, DMSO-d6) δ 14.18 (s, 1H), 8.62 (d, J = 7.6 Hz, 1H), 8.34 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.24 - 7.18 (m, 2H), 7.12 (dd, J = 9.2, 2.4 Hz, 1H), 7.10 - 7.03 (m, 2H), 6.86 (d, J = 2.4 Hz, 1H), 4.83 (td, J = 8.0, 6.5 Hz, 1H), 4.45 (td, J = 8.0, 6.1 Hz, 1H), 3.97 (qd, J = 7.1, 2.1 Hz, 2H), 3.88 (s, 3H), 3.85 - 3.79 (m, 4H), 3.75 (t, J = 7.1 Hz, 4H), 3.25 (dd, J = 15.0, 7.9 Hz, 2H), 3.01 - 2.89 (m, 2H), 1.79 (s, 3H), 1.04 (t, J = 7.1 Hz, 3H).

[0342] Example 37 (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid; dihydrochloride (Example compound 37) [ka]

[0343] To a solution of (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 7) (6 mg, 0.0102 mmol) in THF:MeOH (2:1, 1 mL) was added an aqueous solution of lithium hydroxide monohydrate (0.5 M, 0.0238 mL). The mixture was stirred overnight at room temperature, then filtered and purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid; dihydrochloride (Example compound 37) as a white solid (1 mg, 0.00177 mmol, 16% yield). HPLC purity: 99%; MS (ESI+) m / z = 486 [M+H] + .

[0344] 1H NMR (600 MHz, DMSO-d6) δ 14.50 (s, 1H), 13.04 (s, 1H), 9.14 (d, J = 8.0 Hz, 1H), 8.38 (d, J = 5.4 Hz, 3H), 7.73 (d, J = 9.2 Hz, 1H), 7.13 (dd, J = 9.3, 2.4 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 4.37 (td, J = 8.8, 4.0 Hz, 1H), 3.91 (s, 3H), 3.86 (q, J = 6.6 Hz, 1H), 3.81 (t, J = 6.8 Hz, 4H), 3.76 (t, J = 6.6 Hz, 4H), 3.32 - 3.22 (m, 2H), 2.36 (t, J = 8.8 Hz, 1H), 2.12 (ddt, J = 14.0, 9.2, 4.7 Hz, 1H), 1.69 (dq, J = 13.3, 6.6 Hz, 1H), 1.59 (dt, J = 13.9, 7.1 Hz, 1H), 1.51 (dt, J = 14.2, 7.4 Hz, 1H), 0.90 (dd, J = 22.4, 6.5 Hz, 6H).

[0345] Example 38 (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride (Example compound 38) [ka]

[0346] To a solution of (2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid ethyl ester (synthesized according to Scheme 6 using (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid ethyl ester shown in Scheme 1 as the starting material) (559 mg, 1.06 mmol, 95% purity) in EtOH (5 mL) was added hydrochloric acid in dioxane (4.00 mol / L, 2.65 mL, 10.6 mmol) and the reaction mixture was stirred at 25° C. for 3 h. The solvent was evaporated under reduced pressure to give the crude product, which was purified by preparative chromatography to give (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester; dihydrochloride salt (Example compound 38) as a white solid (416 mg, 0.877 mmol, 83% yield). HPLC purity: 99%; MS (ESI+) m / z = 401 [M+H] + .

[0347] 1 H NMR (600 MHz, DMSO-d6) δ 14.81 (s, 1H), 8.86 (s, 3H), 7.85 - 7.68 (m, 1H), 7.15 (d, J = 9.6 Hz, 1H), 6.92 (d, J = 5.0 Hz, 1H), 4.27 (s, 1H), 4.21 (t, J = 6.5 Hz, 2H), 3.93 (t, J = 4.4 Hz, 3H), 3.84 (q, J = 6.0 Hz, 4H), 3.78 (t, J = 5.8 Hz, 4H), 3.36 (dq, J = 16.9, 6.1 Hz, 2H), 2.44 - 2.31 (m, 2H), 1.25 (q, J = 6.3 Hz, 3H).

[0348] Example 39 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid; dihydrochloride (Example compound 39) [ka]

[0349] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester; dihydrochloride (Example compound 2) (10.2 mg, 0.015 mmol) was dissolved in HCl (4 M in dioxane, 0.0758 mL) and stirred at room temperature for 48 hours. The reaction mixture was purified by preparative chromatography to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid; dihydrochloride butanoate (Example compound 39) as a white solid (2.3 mg, 0.00348 mmol, 25% yield). HPLC purity: 99%;MS (ESI+) m / z = 538 [M+H] + .

[0350] 1H NMR (600 MHz, DMSO-d6) δ 14.74 (s, 1H), 13.10 (s, 1H), 9.23 (s, 1H), 8.54 (s, 3H), 7.73 (d, J = 9.3 Hz, 1H), 7.36 (ddd, J = 8.1, 5.6, 2.1 Hz, 2H), 7.16 - 7.07 (m, 3H), 6.91 (d, J = 2.3 Hz, 1H), 4.47 (td, J = 8.4, 4.8 Hz, 1H), 4.04 (q, J = 5.9 Hz, 1H), 3.87 (d, J = 1.5 Hz, 3H), 3.82 (d, J = 6.9 Hz, 4H), 3.76 (d, J = 6.2 Hz, 4H), 3.32 (s, 2H), 3.10 (dd, J = 14.1, 4.7 Hz, 1H), 2.98 (dd, J = 14.1, 8.9 Hz, 1H), 2.38 - 2.22 (m, 2H).

[0351] Example 40 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 40) [ka]

[0352] (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 13-11) (90.0 mg, 0.000133 mol) was dissolved in 1,4-dioxane (5.0 mL), then water (1.0 mL) and LiOH·H2O (3 equiv., 16 mg) were added. The mixture was stirred at 50 °C for 2 h. Water (10 mL) and DCM (10 mL) were added and the pH was adjusted to 5-6 with HCl (aq). The phases of the mixture were separated and the organic phase was washed with water and dried over MgSO4. The solvent was removed in vacuum. The residue was dissolved again in DCM (2 mL), then HCl (4M in 1,4-dioxane, 1.0 mL) was added and the solution was stirred at room temperature for 30 min. The mixture was evaporated to dryness to give (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride salt (Example compound 40) as a solid (70 mg, 0.113 mmol, 90% yield). HPLC purity: 89%; MS (ESI+) m / z = 548 [M+H] + .

[0353] Example 41 (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoic acid (Example compound 41) [ka]

[0354] Example 41 was carried out according to Scheme 11 below. [ka]

[0355] (2S)-5-(2-anilino-5-nitro-anilino)-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid methyl ester (compound 41-1) Boc-Glu-OMe (1.63 g, 0.00624 mol) was dissolved in dry DMF (15 mL). DIEA (2.97 mL, 0.0170 mol) was added followed by 4-nitro-N1-phenyl-benzene-1,2-diamine (1.30 g, 0.00567 mol; 13-4) and COMU (2.91 g, 0.00681 mol). The reaction was stirred at room temperature overnight. Most of the DMF was evaporated in vacuo, then toluene (10 mL) was added and the organic phase was washed with water (3× 100 mL). The organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (41-1) as a dark brown oily crude product (5.61 g). HPLC purity: 48%; (ESI+) m / z 373 [M+H-BOC] + This material was used in the next step without further purification.

[0356] (2S)-2-Amino-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid methyl ester (compound 41-2) To a solution of crude (2S)-5-(2-anilino-5-nitro-anilino)-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid methyl ester (5.61 g, 48% purity; 41-1) in EtOH (100 mL) was added 12 M HCl (15 mL) and the mixture was stirred at 70 °C for 45 min. The reaction mixture was diluted with water (100 mL), carefully neutralized to pH 7 with 4 M NaOH (ca. 45 mL) and extracted with EtOAc (3 x 100 mL). The organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (41-2) as a dark red-brown solid (2.04 g). HPLC purity: 60%; MS (ESI+) m / z 355 [M+H] + This material was used in the next step without further purification.

[0357] (2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid methyl ester (compound 41-3) To a solution of crude (2S)-2-amino-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid methyl ester (2.04 g) in THF (25 mL) was added TEA (2.38 mL) and di-tert-butyl dicarbonate (1.49 g) and the mixture was stirred at room temperature for 1 h. The solvent was evaporated and the residue was dissolved in EtOAc. The organic phase was washed with 5% citric acid, dried over MgSO4, filtered and the solvent was evaporated. The crude product was purified by flash chromatography on silica eluting with a gradient of 20-80% EtOAc in petroleum ether to give the title compound (41-3) (1.13 g, 0.00249 mmol, 44% yield). HPLC purity: 95%; MS (ESI+) m / z 455 [M+H] + .

[0358] (2S)-4-(5-amino-1-phenyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 41-4) To a solution of (2S)-2-(tert-butoxycarbonylamino)-4-(5-nitro-1-phenyl-benzimidazol-2-yl)butanoic acid methyl ester (0.330 g, 0.000581 mol, 80% purity) in MeOH (20 mL) was added Pd / C (10.0%, 0.0734 g, 0.000069 mol) and evacuated. A balloon of hydrogen gas was attached and the mixture was stirred at room temperature for 4 h. The Pd / C was removed by filtration and the solvent was evaporated to dryness to give (2S)-4-(5-amino-1-phenyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (190 mg, 0.000636 mol; 41-4). HPLC purity: 90%; MS (ESI+) m / z 425 [M+H] + .

[0359] (2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 41-5) NaBH3CN (120 mg) was added to a mixture of (2S)-4-(5-amino-1-phenyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (90%, 300 mg, 0.636 mmol, 90% purity), 2-chloroacetaldehyde (50%, 0.24 mL) and TFA (0.19 mL) in EtOH (20 mL). The mixture was stirred at room temperature for 3 h. Three excess equivalents of NaBH3CN, 2-chloroacetaldehyde and TFA were added and the reaction was stirred at room temperature for 1 h. Toluene and saturated NaHCO3 solution were added and the organic phase was separated and washed with water. The solvent was evaporated and the light brown solid residue was purified by flash chromatography on silica eluting with 50-100% EtOAc in petroleum ether to give (2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (190 mg, 0.000311 mol, 49% yield). HPLC purity: 90%; MS (ESI+) m / z = 550 [M+H] + .

[0360] (2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoic acid; dihydrochloride (Example compound 41) (2S)-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (190 mg, 0.000311 mol) was dissolved in 1,4-dioxane (5.00 mL), then water (1.5 mL) and LiOH·H2O (44 mg) were added. The mixture was stirred at 50 °C for 20 min, then water (10 mL) and DCM (10 mL) were added and the pH was adjusted to 6-7 with 1 M hydrochloric acid. The two phases were separated and the organic phase was washed with water and dried over MgSO4. HCl (4 M in 1,4-dioxane, 0.50 mL, 0.004 mol) was added to the organic phase and the clear solution was stirred at room temperature for 1 h. The precipitate was removed from the solvent and washed with diethyl ether. It was then redissolved in a small amount of water and immediately lyophilized to give a solid of (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoic acid; dihydrochloride (Example Compound 41) (138 mg, 0.000242 mol, 78% yield). HPLC purity: 89%; MS (ESI+) m / z = 435 [M+H] + .

[0361] Example 42 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid; 2,2,2-trifluoroacetic acid (Example compound 42) [ka]

[0362] (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid ethyl ester; dihydrochloride (compound 6) (12 mg, 0.02 mmol) was dissolved in a mixture of THF (1 mL), MeOH (0.5 mL) and 0.5 M LiOH (aq, 0.13 mL, 0.065 mmol). The reaction mixture was stirred at room temperature overnight. The organic solvent was evaporated and the residue was purified by preparative HPLC to give a solid of (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid; 2,2,2-trifluoroacetic acid (Example compound 42) (2.6 mg, 0.00454 mmol, 21% yield). HPLC purity: 95%; MS (ESI+) m / z = 458 [M+H] + .

[0363] Example 43 (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid; dihydrochloride (Example compound 43) [ka]

[0364] To (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester; dihydrochloride salt (Example compound 10) (12 mg, 0.02 mmol) was added 1 M hydrochloric acid (1 mL) and the reaction was stirred at room temperature overnight. Concentrated HCl was added (10 drops). After 4 hours, an additional 0.3 mL of concentrated HCl was added and the mixture was stirred overnight. The mixture was transferred to a round bottom flask and water was added to the mixture before lyophilization. (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid; dihydrochloride (Example compound 43) was obtained as a solid (5 mg, 0.00917 mmol, 44% yield). HPLC purity: 95%; MS (ESI+) m / z = 472 [M+H] + .

[0365] 1 H NMR (600 MHz, DMSO-d6) δ 14.78 (s, 1H), 9.40 (d, J = 7.9 Hz, 1H), 8.38 (s, 3H), 7.76 (d, J = 9.2 Hz, 1H), 7.16 (dd, J = 9.2, 2.4 Hz, 1H), 6.94 (d, J = 2.3 Hz, 1H), 4.93 (td, J = 7.9, 5.7 Hz, 1H), 3.96 (s, 3H), 3.80 (dt, J = 33.0, 6.8 Hz, 9H), 3.64 (dd, J = 15.5, 5.9 Hz, 1H), 3.60 - 3.56 (m, 2H), 1.67 - 1.60 (m, 1H), 1.50 (t, J = 7.2 Hz, 2H), 0.85 (t, J = 7.2 Hz, 6H).

[0366] Example 44 (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 44) [ka]

[0367] To (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 4) (10 mg, 0.017 mmol) was added 1 M hydrochloric acid (1 mL) and the reaction was stirred at room temperature overnight. Concentrated HCl was added (10 drops) and the reaction was stirred overnight. The mixture was transferred to a round bottom flask, diluted with water, and lyophilized to give (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 44) ​​as a solid (5 mg, 0.00917 mmol, 53% yield). HPLC purity: 95%; MS (ESI+) m / z = 472 [M+H] + .

[0368] 1 H NMR (600 MHz, DMSO-d6) δ 14.61 (s, 1H), 9.11 (d, J = 7.7 Hz, 1H), 8.73 (s, 3H), 7.75 (d, J = 9.3 Hz, 1H), 7.16 (d, J = 9.4 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 4.51 - 4.44 (m, 1H), 4.27 (q, J = 7.6 Hz, 1H), 3.94 (s, 3H), 3.87 - 3.75 (m, 9H), 3.66 (d, J = 6.6 Hz, 2H), 1.76 - 1.68 (m, 1H), 1.55 (dt, J = 8.8, 4.6 Hz, 2H), 0.88 (dd, J = 19.5, 6.5 Hz, 6H).

[0369] Example 45 (2R)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester di-2,2,2-trifluoroacetic acid (Example compound 45) [ka]

[0370] Example 45 was carried out according to Scheme 12 below. [ka]

[0371] Reagents and conditions: a) 1.2 eq. SOCl2, ethanol, 0°C to reflux, b) HATU, TEA, DMF, c) 10% palladium on carbon (wet with approximately 55% water, EtOH, 5% AcOH, d) 2-chloroacetaldehyde (15 eq.), NaBH3CN (10 eq.), TFA (12 eq.), EtOH, e) 4M HCl in dioxane:EtOH:DCM.

[0372] R-Leucine ethyl ester hydrochloride (compound 45-1) A 25 mL flask was charged with (2R)-2-amino-4-methyl-pentanoic acid (1.00 g, 0.00762 mol) and absolute ethanol (6.00 mL). The reaction was cooled on an ice bath and thionyl chloride (0.667 mL, 0.00915 mol) was added dropwise to give a slurry. After addition, the reaction was stirred at reflux for 2 h 30 min. The solvent was evaporated and the residue was suspended in Et2O (16 mL) and stirred for 1 h. The product was filtered to give the title compound (45-1) (1.36 g, 0.00693 mol, 91% yield). HPLC purity: 99%; MS (ESI+) m / z = 160 [M+H] + .

[0373] ((S)-2-((tert-butoxycarbonyl)amino)-4-(1-methyl-5-nitro-1H-benzo[d]imidazol-2-yl)butanoyl)-D-leucine ethyl ester (compound 45-2) To a mixture of (2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (Intermediate 2) (0.500 g, 0.00132 mol), R-leucine ethyl ester hydrochloride (0.259 g, 0.00132 mol) and TEA (0.553 mL, 0.00396 mol) in DMF (4.60 mL) was added HATU (0.502 g, 0.00132 mol). The reaction was stirred for 2 h and then water (9 mL) was added. The product was an oil. The liquid was removed with a pipette. To the damp semi-solid was added EtOH (5 mL). The semi-solid was heated to 80° C. to dissolve. Water (5 mL) was added until signs of precipitation were observed. Heating was stopped. The product was an oil. The solvent was evaporated, the residue was dissolved in toluene and washed with water. The organic phase was dried over MgSO4, filtered and the solvent was evaporated to give the title compound (45-2) (0.575 g, 0.00111 mol, 84% yield). HPLC purity: 97%; MS (ESI+) m / z = 520 [M+H] + This material was used in the next step without further purification.

[0374] (2R)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 45-3) A mixture of (2R)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (575 mg, 0.00111 mol) and 10% palladium on carbon (wet with about 55% water (0.262 g, 0.00111 mol)) was stirred overnight in EtOH (25.0 mL) containing AcOH (1.30 mL). The solid material was filtered through Celite to give the title compound (542 mg, 1.11 mmol, 100% yield). HPLC purity: 98%; MS (ESI+) m / z = 490 [M+H] + This material was used in the next step without further purification.

[0375] (2R)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 45-4) At room temperature, NaBH3CN (0.348 g, 0.00554 mol) was added to a mixture of (2R)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (0.542 g, 0.00111 mol; 45-3), 2-chloroacetaldehyde (50%, 0.701 mL, 0.00554 mol) and TFA (0.493 mL, 0.00664 mol) in EtOH (30 mL). After stirring for 20 min, NaBH3CN (0.348 g, 0.00554 mol) and 2-chloroacetaldehyde (0.542 g, 0.00111 mol; 45-3) were added to a mixture of (2R)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (0.542 g, 0.00111 mol; 45-3), 2-chloroacetaldehyde (50%, 0.701 mL, 0.00554 mol) and TFA (0.493 mL, 0.00664 mol). Additional chloroacetaldehyde (5.0%, 0.701 mL, 0.00554 mol) was added. The reaction was stirred for an additional 20 min, then 2-chloroacetaldehyde (50%, 0.701 mL, 0.00554 mol) was added. The reaction was stirred for an additional 2 h, after which toluene was added and the organic phase was washed with sodium bicarbonate and brine. The organic phase was dried over MgSO4, filtered, and evaporated under reduced pressure. The compound was purified by flash chromatography on spherical silica gel, eluting with 2% EtOH in DCM to give the title compound 45-4 (209 mg, 0.340 mmol, 31% yield). HPLC purity: 95%; MS (ESI+) m / z = 614 [M+H] + .

[0376] (2R)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester di-2,2,2-trifluoroacetic acid (Example compound 45) 4M HCl in dioxane (1.40 mL) was added to (2R)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (0.209 g, 0.000340 mol) in a mixture of DCM (1.40 mL) and EtOH (0.860 mL). The reaction was stirred for 5 h and then the solvent was evaporated to give a crude product of 90% purity. The crude product was dissolved in MeCN containing 30% water and purified by preparative HPLC on an ACE column, eluting with 10-60% MeCN / water (0.1% TFA) to give the title compound (Example compound 45) (104 mg, 0,140 mmol, 49% yield). HPLC purity: 99%;MS (ESI+) m / z = 514 [M+H] + .

[0377] 1 H NMR (600 MHz, DMSO-d6 δ 9.18 - 8.99 (m, 1H), 8.41 (s, 3H), 7.70 - 7.59 (m, 1H), 7.13 - 7.02 (m, 1H), 6.94 (d, J = 2.3 Hz, 1H), 4.33 (q, J = 7.8 Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 4.07 - 4.01 (m, 1H), 3.85 - 3.71 (m, 11H), 3.17 - 3.07 (m, 2H), 2.25 (q, J = 7.4 Hz, 2H), 1.65 - 1.52 (m, 3H), 1.21 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H).

[0378] Example 46 (2S)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 46) [ka]

[0379] Example 46 was carried out according to Scheme 13 below. [ka]

[0380] (2R)-2-(tert-butoxycarbonylamino)-5-[2-(methylamino)-5-nitro-anilino]-5-oxo-pentanoic acid methyl ester (compound 46-1) To a solution of N-tert-butoxycarbonyl-D-glutamic acid α-methyl ester (1.00 equiv., 2.00 g, 7.65 mmol), 1-methylimidazole (1.00 equiv., 0.61 mL, 7.65 mmol) and N1-methyl-4-nitro-benzene-1,2-diamine (1.34 g, 8.037 mmol) in MeCN (20 mL) was added chloro(dimethylamino)-N,N-dimethylmethaniminium hexafluorophosphate (TCFH) (1.10 equiv., 2.36 g, 8.42 mmol) slowly. The mixture was stirred at room temperature for 1 h, and then water (200 mL) was added. The organic phase was passed through a phase separation cartridge, evaporated and dried in vacuum to give a solid: (2R)-2-(tert-butoxycarbonylamino)-5-[2-(methylamino)-5-nitro-anilino]-5-oxo-pentanoic acid methyl ester (3.1 g, 7.55 mmol, 99% yield). HPLC purity: 99%; MS (ESI+) m / z = 843 [2M+Na] + .

[0381] (2R)-2-Amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (compound 46-2) (2R)-2-(tert-butoxycarbonylamino)-5-[2-(methylamino)-5-nitro-anilino]-5-oxo-pentanoic acid methyl ester (1.00 equiv., 3.10 g, 7.55 mmol) was added to hydrochloric acid (4 M) (18.0 equiv., 34 mL, 136 mmol). After the addition, the mixture was heated and stirred at 100° C. for 2 h without a stopper. The reaction was allowed to cool to room temperature and a small amount of brown solid was filtered off. The filtrate was cooled in an ice bath and 10 M NaOH was carefully added until the pH was 2. Then 4 M NaOH solution was added until the pH was 5.2. The resulting solid was filtered, washed with water and Et2O, and dried in vacuum to give a solid of (2R)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (2.3 g, 7.52 mmol, >99% yield). HPLC purity: 91%; MS (ESI+) m / z = 279 [M+H] + .

[0382] (2R)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (compound 46-3) To a solution of (2R)-2-amino-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (1.00 equiv., 2.30 g, 7.52 mmol) in water (10 mL) was added aqueous NaOH (4 M) (1.10 equiv., 2.1 mL, 8.27 mmol). To the solution was added di-tert-butyl dicarbonate (1.40 equiv., 2.4 mL, 10.5 mmol) and the mixture was stirred at room temperature for 2 h. A precipitate was obtained. Further 4 M NaOH (0.5 equiv.) was added and the reaction was stirred at room temperature for 3 h. The pH was adjusted to 9.2 and the reaction mixture was washed with Et2O (3× 300 mL). The resulting organic phase was discarded and the aqueous phase was evaporated to remove Et2O residues. 4 M HCl was added dropwise to the aqueous phase. The resulting solid was collected, washed with water, and dried to give a solid of (2R)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (1.85 g, 4.59 mmol, 61% yield). HPLC purity: 94%; MS (ESI+) m / z = 379 [M+H] + .

[0383] (2R)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid (compound 46-4) (2R)-2-(tert-butoxycarbonylamino)-4-(1-methyl-5-nitro-benzimidazol-2-yl)butanoic acid (1.00 equiv., 300 mg, 0.769 mmol) was dissolved in ethanol (5 mL) and placed in a H reactor. Pd / C 10% (0.0500 equiv., 8.2 mg, 0.0385 mmol) was added and the reactor was sealed. A nitrogen purge was performed. Hydrogen gas was charged to the reactor at 3 bar and the reaction was stirred at room temperature for 2 h. The hydrogen gas was vented with a nitrogen purge. The catalyst was removed by filtration through Celite. The solvent was evaporated to give (2R)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid (235 mg, 0.690 mmol, 94% yield). HPLC purity: 95%; MS (ESI+) m / z = 349 [M+H] + .

[0384] (2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid (compound 46-5) (2R)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid (1.00 equiv., 253 mg, 0.690 mmol) was suspended in ethanol (2 mL). Sodium cyanoborohydride (3.00 equiv., 130 mg, 2.07 mmol) was added, followed by trifluoroacetic acid (1.50 equiv., 0.079 mL, 1.03 mmol) and then chloroacetaldehyde (50%, 3.00 equiv., 0.26 mL, 2.07 mmol) was added dropwise. The reaction mixture was stirred for 30 min. Two excess equivalents of NaBH3CN, chloroacetaldehyde and TFA were added and the reaction was stirred at room temperature for 2 h. The pH was adjusted to 3.6 by dropwise addition of 4M NaOH. The ethanol was evaporated, water was added and the aqueous phase was extracted with EtOAc. The organic phase was evaporated to isolate the crude product (2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid as a solid (303 mg, 0.557 mmol, 81% yield). HPLC purity: 87%; MS (ESI+) m / z = 473 [M+H] + .

[0385] (2S)-2-[[(2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 46-6) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 equiv., 41 mg, 0.211 mmol) was added to a mixture of (2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid (1.00 equiv., 100 mg, 0.184 mmol), (2S)-2-amino-4-methyl-pentanoic acid ethyl ester (1.15 equiv., 34 mg, 0.211 mmol), ethyl cyanoglyoxylate-2-oxime (1.00 equiv., 26 mg, 0.184 mmol) and 4-methylmorpholine (1.15 equiv., 23 uL, 0.211 mmol) in MeCN (3 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered and purified by preparative chromatography. The pure fractions were lyophilized to give (2S)-2-[[(2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (17 mg, 0.0275 mmol, 15% yield). HPLC purity: 99%; MS (ESI+) m / z = 614 [M+H] + .

[0386] (2S)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 46) (2S)-2-[[(2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (1.00 equiv, 17.1 mg, 0.0275 mmol) was dissolved in MeCN (2 mL) and hydrochloric acid (4 M in dioxane) (5.00 equiv, 35 uL, 0.139 mmol) was added. The reaction was stirred at room temperature for 1 h. The mixture was filtered and purified by preparative chromatography, and the pure fractions were pooled and lyophilized to give (2S)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride salt (Example compound 46) as a solid (14.3 mg, 0.0234 mmol, 84% yield). HPLC purity: 96%; MS (ESI+) m / z = 514 [M+H] + .

[0387] 1 H NMR (600 MHz, DMSO-d6) δ 14.81 (s, 1H), 9.23 (d, J = 7.6 Hz, 1H), 8.59 (s, 3H), 7.75 (d, J = 9.1 Hz, 1H), 7.15 (d, J = 9.2 Hz, 1H), 6.94 (d, J = 2.3 Hz, 1H), 4.30 - 4.24 (m, 1H), 4.11 (q, J = 7.1 Hz, 2H), 4.04 (s, 1H), 3.89 (s, 3H), 3.83 (t, J = 6.7 Hz, 4H), 3.77 (t, J = 6.6 Hz, 4H), 3.2-3-3 (m, 2H), 2.32 (dp, J = 29.8, 7.6 Hz, 2H), 1.69 - 1.57 (m, 2H), 1.53 (ddd, J = 13.8, 8.5, 5.6 Hz, 1H), 1.20 (t, J = 7.1 Hz, 3H), 0.87 (dd, J = 21.4, 6.3 Hz, 6H).

[0388] Example 47 (2R)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 47) [ka]

[0389] Example 47 was carried out according to Scheme 13, but using D-leucine ethyl ester hydrochloride (1.15 equiv., 41 mg, 0.211 mmol) as starting material in the peptide coupling with compound 46-5 to give (2R)-2-[[(2R)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; 2,2,2-trifluoroacetic acid, isolated as a solid (27 mg, 0.0375 mmol, 20% yield). HPLC purity: 99%; MS (ESI+) m / z = 614 [M+H] + .

[0390] (2R)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 47) was obtained as a solid (6 mg, 0.0097 mmol, 25% yield). HPLC purity: 95%; MS (ESI+) m / z = 514 [M+H] + .

[0391] 1H NMR (600 MHz, DMSO-d6) δ 14.79 (s, 1H), 9.25 (d, J = 7.0 Hz, 1H), 8.69 - 8.47 (m, 3H), 7.75 (d, J = 9.1 Hz, 1H), 7.15 (d, J = 9.2 Hz, 1H), 6.93 (s, 1H), 4.31 (dt, J = 11.3, 5.7 Hz, 1H), 4.16 - 4.04 (m, 3H), 3.90 (s, 3H), 3.83 (d, J = 6.7 Hz, 4H), 3.78 (d, J = 6.4 Hz, 4H), 3.38 - 3.33 (m, 2H), 2.32 (dp, J = 23.6, 7.6, 7.0 Hz, 2H), 1.75 (h, J = 6.9 Hz, 1H), 1.64 (td, J = 12.3, 10.8, 5.1 Hz, 1H), 1.55 (ddd, J = 14.0, 9.4, 4.8 Hz, 1H), 1.18 (t, J = 7.2 Hz, 3H), 0.90 (dd, J = 25.1, 6.5 Hz, 6H).

[0392] Example 48 (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester di-2,2,2-trifluoroacetic acid (Example compound 48) [ka]

[0393] Example 48 was carried out according to Scheme 14 below. [ka]

[0394] Reagents and conditions: a) MeNH2, MeOH, 0 °C, b) 20% ammonium sulfide in water, MeOH, heat, c) HATU, DIPEA, DMF, room temperature, d) n-PrOH, 100 °C, e) LiOH·H2O, THF, MeOH, H2O, room temperature, f) HATU, DIPEA, DMF, room temperature, g) Pd / C10%, H2, EtOH, h) chloroacetaldehyde solution (~55 wt% in water), NaBH3CN, TFA, EtOH, i) TFA, DCM, 0 °C to room temperature, then 12 M HCl.

[0395] N-Methyl-3,5-dinitro-pyridin-2-amine (Compound 48-1) To a suspension of 2-chloro-3,5-dinitropyridine (4.0 g, 0.0196 mol) in MeOH (40 mL) was added dropwise a mixture of MeNH2 (4.32 mL, 40% by volume, 0.059 mol) and MeOH (16 mL) in water at 0-5 °C. The resulting yellow precipitate was collected by filtration, washed with MeOH and water, and dried in vacuum overnight to give N-methyl-3,5-dinitro-pyridin-2-amine (3.00 g, 0.0151 mol, 77% yield). HPLC purity: 95%; MS (ESI+) m / z=199 [M+H] + .

[0396] N2-Methyl-5-nitro-pyridine-2,3-diamine (compound 48-2) N-Methyl-3,5-dinitro-pyridin-2-amine (2.80 g, 14.1 mmol) was suspended in methanol (60 mL) and a 20% aqueous solution of ammonium sulfide (24.1 mL) was added. The temperature was raised to 75° C. and the mixture was stirred for 2.5 h. The reaction was then stirred at room temperature overnight. The mixture was placed in an ice bath to give a precipitate. The solid was collected by filtration and dried overnight in vacuum to give N2-Methyl-5-nitro-pyridine-2,3-diamine as a red-brown solid (3.39 g, more than theoretical yield). This material was used in the next step without further purification. HPLC purity: 90%; MS (ESI+) m / z=169 [M+H] + .

[0397] (2S)-2-(tert-butoxycarbonylamino)-5-[[2-(methylamino)-5-nitro-3-pyridyl]amino]-5-oxo-pentanoic acid benzyl ester (compound 48-3) To a solution of (4S)-5-benzyloxy-4-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (1.00 g, 2.97 mmol) in dimethylformamide (7.50 mL) was added HATU (1.36 g, 3.57 mmol) and DIEA (1.04 mL, 5.45 mmol), the reaction was stirred for 2 minutes, and then N2-methyl-5-nitro-pyridine-2,3-diamine (compound 48-2) (0.500 g, 2.97 mmol) was added. The reaction was stirred at room temperature for 22 hours. EtOAc was added and the organic phase was washed with brine (3x), dried over magnesium sulfate, decanted and concentrated to give (2S)-2-(tert-butoxycarbonylamino)-5-[[2-(methylamino)-5-nitro-3-pyridyl]amino]-5-oxo-pentanoic acid benzyl ester (2.74 g, more than theoretical yield). This material was used in the next step without further purification. HPLC purity: 80%; MS (ESI+) m / z=488 [M+H] + .

[0398] (2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoic acid benzyl ester (compound 48-4) (2S)-2-(tert-butoxycarbonylamino)-5-[[2-(methylamino)-5-nitro-3-pyridyl]amino]-5-oxo-pentanoic acid benzyl ester (1.07 g, 2.19 mmol) in 1-propanol (20.0 mL) was heated at 100° C. for 6 days (a slow reaction compared to when carried out on a small scale). 1-propanol was evaporated and EtOAc was added. The organic phase was washed with brine, dried over magnesium sulfate and concentrated. The crude material was purified by flash chromatography (SiO2 12 g, petroleum ether / EtOAc 0-100%) to give (2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoic acid benzyl ester (280 mg, 27% yield). HPLC purity: 90%;MS (ESI+) m / z= 470 [M+H] + .

[0399] (2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoic acid (compound 48-5) To a solution of (2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoic acid benzyl ester (0.280 g, 0.596 mmol) in tetrahydrofuran (1.20 mL) / methanol (1.20 mL) containing water (0.2 mL) was added LiOH·H2O (0.0425 g, 1.01 mmol). The reaction was stirred at room temperature for 2 h. EtOAc and 5% citric acid were added and the two phases were separated. The organic phase was dried over magnesium sulfate, filtered and the solvent was evaporated to give (2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoic acid (0.240 g). This material was used in the next step without further purification. HPLC purity: 95%;MS (ESI+) m / z= 380 [M+H] + .

[0400] (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 48-6) To a solution of (2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoic acid (0.240 g, 0.633 mmol) in N,N-dimethylformamide (1.50 mL) was added (2S)-2-amino-4-methyl-pentanoic acid ethyl ester hydrochloride (0.124 g, 0.633 mmol) and HATU (0.289 g, 0.759 mmol), followed by N,N-diisopropylethylamine (0.204 g, 1.58 mmol). The reaction was then stirred overnight at room temperature. EtOAc was added and the organic phase was washed with brine (3x), dried over MgSO4, filtered and concentrated. The crude material was purified by flash chromatography (SiO2, petroleum ether / EtOAc 0-100%) to give (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (0.340 g). HPLC purity: 90%; MS (ESI+) m / z= 521 [M+H] + .

[0401] (2S)-2-[[(2S)-4-(6-amino-3-methyl-imidazo[4,5-b]pyridin-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 48-7) A mixture of (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-6-nitro-imidazo[4,5-b]pyridin-2-yl)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (0.340 g, 0.653 mmol) and Pd / C 10% (0.0695 g, 0.653 mmol) in ethanol (20.0 mL) was stirred overnight under a hydrogen gas atmosphere (atmospheric pressure). The catalyst was filtered off and the solvent was evaporated to give the title compound (2S)-2-[[(2S)-4-(6-amino-3-methyl-imidazo[4,5-b]pyridin-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester as a clear, sticky oil (0.273 g, 85% yield). This material was used in the next step without further purification. HPLC purity: 90%; MS (ESI+) m / z= 491 [M+H] + .

[0402] (2S)-2-[[(2S)-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 48-8) To (2S)-2-[[(2S)-4-(6-amino-3-methyl-imidazo[4,5-b]pyridin-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (170 mg, 0.346 mmol) was added chloroacetaldehyde (0.22 mL, 50% aqueous solution) and TFA (0.12 mL) at room temperature. Sodium cyanoborohydride (109 mg, 1.73 mmol) was then added and the reaction was stirred at room temperature for 30 min, at which point 50% conversion was observed. Again, the same amount of all the above reagents was added and the reaction was stirred for an additional 30 min. The volatiles were evaporated in vacuo. The crude product was dissolved in ethyl acetate, washed with saturated aqueous sodium carbonate, brine, dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash chromatography (12 g silica column, 70-90% MeOH in DCM) to give the title compound (2S)-2-[[(2S)-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester as a colorless thick oil (75 mg, 35% yield). HPLC purity: 95%; MS (ESI+) m / z= 615 [M+H] + .

[0403] (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; di-2,2,2-trifluoroacetic acid (Example compound 48) To a solution of (2S)-2-[[(2S)-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 48-8) (35 mg, 0.057 mmol) in DCM (2 mL) was added TFA (0.1 mL) at 5° C. The reaction was stirred at 5° C. for 30 min and allowed to warm to room temperature. An additional 0.2 mL of TFA was then added and the reaction was stirred at room temperature for 3 h. The crude material was purified by reverse phase acidic preparative HPLC (10-90% acetonitrile, 0.1% TFA in water). The pure fractions were concentrated to give (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; di-2,2,2-trifluoroacetic acid (Example Compound 48) as a solid (11.3 mg, 39% yield). HPLC purity: 95%; MS (ESI+) m / z= 515 [M+H] + .

[0404] 1 H NMR (400 MHz, DMSO-d6) δ 9.04 (d, J = 7.2 Hz, 1H), 8.57 - 8.26 (m, 3H), 8.10 (d, J = 2.6 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 4.38 - 4.30 (m, 1H), 4.12 - 4.08 (m, 2H), 4.05 - 4.02 (m, 1H), 3.81 - 3.73 (m, 11H), 3.13 (t, J = 8.0 Hz, 2H), 2.39 - 2.22 (m, 2H), 1.77 - 1.67 (m, 1H), 1.65 - 1.52 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H).

[0405] Example 49 (2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid; dihydrochloride (Example compound 49) [ka]

[0406] Example 49 was carried out according to Scheme 15 below. [ka]

[0407] Reagents and conditions: a) 2-bromoethanol-d4, KI, CaCO3, 1,4-dioxane / water, 90 °C, 17 h, b) mesyl chloride, triethylamine, room temperature, 1 h, c) LiCl, DMF, 60 °C, 2 h, d) LiOH·H2O, 1,4-dioxane, 50 °C, 20 min, e) HCl in 1,4-dioxane, DCM, room temperature, 1 h.

[0408] (2S)-4-[5-[bis(1,1,2,2-tetradeuterio-2-hydroxy-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 49-1) To a solution of (2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 18-2) (50 mg, 0.138 mmol) in 1,4-dioxane (1.00 mL) / water (0.500 mL) in a sealed reaction tube, CaCO3 (21.5 mg, 0.214 mmol) and a catalytic amount of KI (5.1 mg, 0.0306 mmol) were added, followed by 2-bromoethanol-d4 (39.5 mg, 0.306 mmol). The reaction was heated at 90 °C for 17 h, after which the mixture was diluted with acetonitrile and purified by preparative HPLC on an XBridge column, eluted with 10–80% MeCN in water containing 50 mM NH4HCO3. The title compound (49-1) was obtained as a white solid (29 mg) with ~19% mono-alkylated by-product and 2% over-alkylated by-product. MS (ESI+) m / z=459 [M+H] + .

[0409] (2S)-4-[5-[bis(1,1,2,2-tetradeuterio-2-methanesulfonyloxy-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 49-2) (2S)-4-[5-[bis(1,1,2,2-tetradeuterio-2-hydroxy-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (29 mg, 0.06 mmol) was dissolved in dry dichloromethane (1.00 mL) and cooled to 0° C. Then, triethylamine (20 mg, 0.20 mmol) and mesyl chloride (0.0096 mL, 0.124 mmol) were added, and the mixture was stirred at room temperature until the reaction was complete. Water was added, the DCM phase was washed, and dried to give the title compound (49-2) as a crude product (31 mg) as a brown oil, which was used in the next step without further purification. MS (ESI+) m / z=615 [M+H] + .

[0410] (2S)-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (compound 49-3) (2S)-4-[5-[bis(1,1,2,2-tetradeuterio-2-methanesulfonyloxy-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (21 mg, 0.035 mmol) was dissolved in dry dimethylformamide (0.50 mL), then LiCl (30 mg, 0.70 mmol) was added and the mixture was stirred at 60 °C until the reaction was complete. The mixture was purified by flash chromatography on silica using 50-100% EtOAc in petroleum ether to give the product as an off-white oil (14 mg, 92% yield). HPLC purity: 90%; MS (ESI+) m / z=495 [M+H] + .

[0411] (2S)-2-Amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid dihydrochloride (Example Compound 49) (2S)-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoic acid methyl ester (13 mg, 0.026 mmol) was dissolved in 1,4-dioxane (1.0 mL), then water (0.3 mL) and LiOH·H2O (26 mg, 0.063 mmol) were added. The mixture was stirred at 50 °C for 20 min, water (2 mL) and dichloromethane (1 mL) were added, and the pH was adjusted to 5-6 with hydrochloric acid. The two phases were separated, and the organic phase was washed with water and dried. Excess HCl (4 mol / L) in 1,4-dioxane was added to the dried organic phase, and the clear solution was stirred at room temperature for 30 min. The mixture was concentrated to dryness. A solid of (2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid dihydrochloride (Example Compound 49) was obtained (6 mg, 55% yield). HPLC purity: 97%; MS (ESI+) m / z=381 [M+H] + .

[0412] Example 50 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester; dihydrochloride (Example compound 50) [ka]

[0413] Example 50 was carried out according to Scheme 16 below. [ka]

[0414] Reagents and conditions: a) 2-bromoethanol-d4, KI, CaCO3, 1,4-dioxane / water, 90 °C, 17 h, b) mesyl chloride, triethylamine, room temperature, 1 h, c) LiCl, DMF, 60 °C, 2 h, d) LiOH·H2O, 1,4-dioxane, 50 °C, 20 min, e) HCl in 1,4-dioxane, DCM, room temperature, 1 h.

[0415] (2S)-2-[[(2S)-4-[5-[bis(1,1,2,2-tetradeuterio-2-hydroxy-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 50-1) The title compound (50-1) was prepared from (2S)-2-[[(2S)-4-(5-amino-1-methyl-benzimidazol-2-yl)-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (prepared as described in step 2 for the synthesis of example compound 1) by the procedure described for the synthesis of compound 49-1 (39 mg, 65% yield). HPLC purity: 80%; MS (ESI+) m / z=586 [M+H] + .

[0416] (2S)-2-[[(2S)-4-[5-[bis(1,1,2,2-tetradeuterio-2-methylsulfonyloxy-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 50-2) The title compound (50-2) was prepared from compound 50-1 by the procedure described in the synthesis of compound 49-2 (26 mg, 71% yield). MS (ESI+) m / z=742 [M+H] + .

[0417] (2S)-2-[[(2S)-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(tert-butoxycarbonylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (compound 50-3) The title compound (50-3) was prepared from compound 50-2 by the procedure described in the synthesis of compound 49-3 (20 mg, 92% yield). HPLC purity: 90%; MS (ESI+) m / z=622 [M+H] + .

[0418] (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example compound 50) The title compound (Example compound 50) was prepared from compound 50-3 by the procedure described in step 5 of the synthesis of Example compound 1, with slight changes in conditions (solvent: DCM, reaction time: 1 h) (9 mg, 4% yield). HPLC purity: 95%; MS (ESI+) m / z=522 [M+H] + .

[0419] Example 51 (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid; dihydrochloride (Example compound 51) [ka]

[0420] Example 51 was made according to Scheme 16. This compound was prepared from compound 50-3 by the procedure described in the final step in the synthesis of example compound 49 (8 mg, 68% yield). HPLC purity: 95%; MS (ESI+) m / z=494 [M+H] + .

[0421] Biological Examples: Biological Example 1 In vitro cytotoxicity study using MM.1S cell viability assay Methods: MM.1S cells were cultured in RPMI medium 1640 (IX) supplemented with 10 μg / μL penicillin / streptomycin, 10% heat-inactivated FBS, 0.1 mM sodium pyruvate, 1× MEM NEAA (ThermoFisher) and 1 mM HEPES. Cells were grown to log phase and then exposed to test compounds.

[0422] Test compounds (Example compounds 1-36, 38-41, and 45-48) and comparison compounds (bendamustine, busulfan, melphalan, chlorambucil, and 4-hydroperoxycyclophosphamide) were dissolved in 100% DMSO and placed in a 384 assay plate. Further DMSO was added to achieve the same final DMSO concentration (0.2%) in all wells. 25 μL of cell suspension at 100000 cells / mL was added to each well. Cells were cultured at 37°C in a CO2 incubator for 72 hours. After the plates were brought to room temperature for 30 minutes, 20 μL of CellTiter-Glo® 2.0 (Promega) reagent was added to all wells. The plates were then allowed to stabilize for 20 minutes before recording luminescence (emission filter 700 nm) on an Envision plate reader. Plate reader data were normalized to the negative control (no treatment) and positive control (cells treated with 400 μM chlorpromazine for 72 h) and fitted to a four-parameter logistic regression curve to determine the IC 50 was calculated.

[0423] Results: Test compounds were evaluated for their ability to inhibit MM.1S cell proliferation and cause cytotoxicity in vitro. A dose response could be extracted from the experiment and is shown in Table 1. The cytotoxicity of the test compounds towards MM.1S cells was favorable. Example compounds 1-16, 21-32, 34-36, 38 and 45-48 were found to be more cytotoxic in this assay than bendamustine, busulfan, melphalan, chlorambucil and 4-hydroperoxycyclophosphamide.

[0424] [Table 1]

[0425] Biological Example 2 In vitro cytotoxicity studies with blood cell lines, human peripheral blood mononuclear cells and normal fibroblasts Methods: The cell lines used in the experiments are listed in Table 2. All cells were cultured in RPMI medium 1640 (IX). All media were supplemented with 10 μg / μL penicillin / streptomycin, 10% heat-inactivated FBS, 0.1 mM sodium pyruvate, 1× MEM NEAA (ThermoFisher) and 1 mM HEPES. For DERL-2 cells, FBS was 20% and 40 ng / mL IL-2 was added. hPBMCs were thawed, conditioned overnight and then stimulated with 10 μg / mL PHA-M and 40 ng / mL IL-2 for 4 hours before exposure to test compounds. All cells except hPBMCs were grown to log phase before exposure to test compounds.

[0426] [Table 2]

[0427] Test compounds were dissolved in 100% DMSO and placed in a 384 assay plate. Further DMSO was added to achieve the same final DMSO concentration (0.2%) in all wells. 25 μL of cell suspension at 100,000 cells / mL was added to each well. Cells were cultured for 72 hours at 37°C in a CO2 incubator. After the plates were brought to room temperature for 30 minutes, 20 μL of CellTiter-Glo® 2.0 (Promega) reagent was added to all wells. The plates were then allowed to stabilize for 20 minutes before recording luminescence (emission filter 700 nm) on an Envision plate reader. Plate reader data were normalized to the negative control (no treatment) and positive control (cells treated with 400 μM chlorpromazine for 72 hours) and fitted to a four-parameter logistic regression curve to obtain IC 50 was calculated.

[0428] Results: Test compounds were evaluated for their ability to inhibit cell proliferation in vitro using several cell lines of hematological origin. Cytotoxicity was also evaluated in non-malignant human peripheral blood mononuclear cells (hPBMC) and fibroblasts (BJ). Table 3 shows the dose response extracted from the experiments for all compounds tested. The table summarizes the results as cytotoxicity IC 50 Shown as μM ± standard deviation (number of experiments). nd means the value was not determined.

[0429] [Table 3-1]

[0430] [Table 3-2]

[0431] It can be seen that the compounds of the present invention are consistently more cytotoxic than the two prior art compounds.

[0432] Biological Example 3 Example 18 (after treatment with Example 1), Cellular retention / efflux study of bendamustine and melphalan Method: 2×10 6 MM.1S cells at 1000 cells / mL were seeded at 250 μL / well in a 96-well plate. Cells were incubated in the presence of test compounds for 5, 15 and 60 minutes, after which the cells were pelleted by centrifugation at 4° C. for 5 minutes. Cell media was immediately collected in a second plate and stored at −80° C. until analysis. Pelleted cells were washed once with ice-cold PBS and then stored at −80° C. until analysis.

[0433] Cells were lysed and proteins in the medium were precipitated with acetonitrile:dimethylformamide (9:1) containing an internal standard. Samples were centrifuged and the supernatants were analyzed by LC-MS / MS (ACQUITY UPLC - Xevo TQ-S micro) to determine compound concentrations.

[0434] Example compound 1, bendamustine and melphalan were used as test compounds.

[0435] Results: The results are shown in Figure 1. Figure 1 (A) shows the intracellular and extracellular concentrations of a metabolic compound (Example compound 18) formed from bendamustine and Example compound 1. It can be seen that Example compound 18 is rapidly formed in cells after treatment with Example compound 1, and is at a low level in the extracellular medium. This result indicates that Example compound 1 is hydrolyzed in cells, and the metabolite of Example compound 1 is retained in the cells. The intracellular concentration of bendamustine is low, and the level decreases over time. The extracellular concentration of bendamustine is extremely high, and decreases over time.

[0436] Figure 1(B) shows the intracellular and extracellular concentrations of melphalan during the experiment. After MM.1S cells were treated in vitro with melphalan, the intracellular concentration of melphalan was low. The extracellular concentration of melphalan was high and remained constant throughout the experiment.

[0437] Biological Example 4: In vivo cytotoxicity of test compounds in a chicken embryo xenograft model Method: 3×10 6 SU-DHL-4 tumor cells were inoculated into fertilized White Leghorn eggs on day 9 (post-fertilization). 100 μL of either Example Compound 1 (8.4 μM (0.008 mg / kg), 33.5 μM (0.033 mg / kg), 167.5 μM (0.164 mg / kg)), bendamustine (8.4 μM (0.005 mg / kg), 33.5 μM (0.02 mg / kg), 167.5 μM (0.1 mg / kg)) or vehicle was injected into live eggs on days 11, 13, 15 and 17. On day 18, tumors were removed and weighed.

[0438] Results: Example compound 1 showed a dose-dependent effect on tumor weight (Figure 2). For example compound 1, tumor reduction compared to vehicle was 38%, 70% and 82% at 8.4μM, 33.5μM and 167.5μM, respectively. For bendamustine, tumor reduction compared to vehicle was only evident at 33.5μM and 167.5μM, with a reduction of 30% and 76%, respectively. Thus, example compound 1 shows potent tumor cytotoxicity in this model.

[0439] Biological Example 5 Cellular Retention / Efflux Studies of PDC Compounds and Metabolites Methods: As described in Biological Example 3.

[0440] Example compounds 1, 2, 3, 4, 6, 7 and 10 were examined. All compounds tested formed intracellular metabolites. The compounds used to treat the cells were metabolized to an ester hydrolysis compound (referred to as metabolite A) and an amide hydrolysis compound (referred to as metabolite B). The structures of the example compounds and the structures of the two metabolites of each compound are shown in Table 4. Metabolite B formed under the conditions tested was the compound with the structure of Example 17 and 18.

[0441] [ka]

[0442] [Table 4-1]

[0443] [Table 4-2]

[0444] The intracellular concentration of the metabolites slowly decreased over time. In the extracellular medium, the metabolites were barely detectable or undetectable. This result indicates that the example compounds were hydrolyzed in the cells and the metabolites of the example compounds were retained in the cells.

[0445] Biological Example 6: Examination of direct DNA damage by test compounds Methods: DNA was prepared from MM.1S cells by QIAGEN Genome Chip 20 / G according to the protocol described by Furda et al., Methods Mol Biol, 2012. 1 μg DNA was treated with 0.25 μM compound for 30 min at 37°C. DMSO was used as a control. 20 ng of treated DNA was PCR amplified using Phusion Hot Start II High Fidelity PCR Master Mix (Thermo Scientific) and either primers for HPRT or primers for mitochondrial DNA (mtDNA) to obtain fragments of 10.4 kb or 8.9 kb, respectively. PCR reactions were separated on a 1% agarose gel and visualized with a ChemiDoc™ MP Imaging System (BioRad). Examples compounds 1, 2 and 38, as well as compounds 18 and 20, metabolites of compound 1, were investigated. They were compared to bendamustine, melphalan and DMSO controls. The results for compounds 1, 2, 18, and 20 using HPRT primers are shown in Figure 4(A), and the results for compounds 18 and 38 using HPRT and mtDNA primers are shown in Figure 4(C).

[0446] Results: From Figure 4, it can be seen that melphalan and bendamustine have no or very little DNA-damaging effect on both nuclear and mitochondrial DNA at the concentrations tested. Example compounds 1 and 2 have strong DNA-damaging effect on both nuclear and mitochondrial DNA, as shown by the fact that no PCR bands could be detected on the gel. Example compounds 18 and 38 effectively damage both nuclear and mitochondrial DNA, and only weak PCR bands were detected. Example compound 20 also has DNA-damaging effect, but to a lesser extent than Example compounds 1, 2 and 18, and more than bendamustine or melphalan.

[0447] Biological Example 7: Examination of DNA fragmentation by test compounds Method: 1×10 6 MM.1S cells (CRL-2974, ATCC) were treated with 0.006 μM, 0.06 μM, 0.6 μM, or 6 μM of Example Compound 1 or bendamustine for 24 hours. After incubation, cells were washed with PBS (10010-015, Gibco), fixed with flow cytometry fixation buffer (FC004, R&D Systems) for 10 minutes, and stored overnight at -20°C in 70% ethanol. Deoxythymidine analog (BrdUTP) was added to label DNA breakage sites. The next day, cells were washed and stained with APO-BrdU™ TUNEL Assay Kit according to the manufacturer's protocol, followed by analysis on a BD FACSCanto™ II. The percentage of TUNEL-positive cells indicates the percentage of cells with fragmented DNA.

[0448] Results: The dose-response effect of Example Compound 1 and bendamustine on DNA damage in 24 hours of incubation is shown in Figure 5 (A). All data are expressed as mean ± SD (n = 2). 24 hours of exposure to Example Compound 1 induced DNA damage in a dose-dependent manner, while no obvious effect of bendamustine on DNA damage was observed in 24 hours of incubation. Representative FACS histograms of Alexa fluor 488 intensity after 24 hours of treatment with 0.6 μM of test compound or bendamustine (n = 2) are shown in Figure 5 (B). The top two are for Example Compound 1, the middle two are for bendamustine, and the bottom two are for DMSO control. Notably, 24 hours of treatment with 0.6 μM of Example Compound 1, but not bendamustine, resulted in DNA breaks in more than 90% of cells tested. Example Compound 1 is more potent in causing DNA damage.

Claims

1. Formula (I) 【Chemical Formula 1】 (wherein X is C １－６ alkylene; W １ , W ２ , W ３ and W ４ are each CH, or one of W １ , W ２ , W ３ and W ４ is N and the others are CH; R １ is C １－４ alkyl which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen, and is selected from the group consisting of halogen; R ２ is phenyl which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen, and C １－６ alkyl which may be substituted with 1, 2 or 3 substituents independently selected from H and halogen, and is selected from the group consisting of; R ３ is a group represented by formula (II) 【Chemical Formula 2】 or formula (III) 【Chemical Formula 3】 , Here, R ４ is N(R ｃ )(R ｄ ) and the group represented by formula (IV) 【Chemical Formula 4】 and is selected from the group consisting of; R ４ when is a group represented by formula (IV), R ５ is R ｂ , and R ４ when is N(R ｃ )(R ｄ ), R ５ is R ｂ and formula (V) [Chemical Formula 5] selected from the group consisting of groups represented by; Each R ａ is independently selected from the group consisting of H, C １－６ alkyl, -CH ２ -phenyl or -CH ２ -heterocyclyl, wherein the heterocyclyl is a 3- to 12-membered heterocyclyl having 1, 2, 3, and 4 heteroatoms selected from N, O, and S, and the C １－６ alkyl may be substituted with 1, 2, or 3 substituents independently selected from the group consisting of -OH, -OC １－６ alkyl, -NH ２ , -NH C(=NH)NH ２ , -C(O)OH, -C(O)NH ２ , -SH, -SCH ３ and halogen, and the phenyl or the heterocyclyl may be substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, -NH ２ , -OH, -OC １－６ alkyl and -NO ２ ; R ｂ is -OH, -N(R ｅ )(R ｆ ), and halogen, -OH, -CN, -N(R ｅ )(R ｆ ), -C ６－１０ aryl or -OC １－６ alkyl which may be substituted with one or more substituents selected from the group consisting of 3- to 12-membered heterocycles having 1, 2, or 3 halogens and having 1, 2, or 3 O, N, or S atoms, and the alkyl may be interrupted by 1, 2, or 3 O, N, or S atoms; R ｃ and R ｄ are each independently H, -C １－６ alkyl, C(O)C １－６ alkyl and -CH ２Independently selected from the group consisting of -phenyl, wherein said alkyl or said phenyl may be substituted with 1, 2 or 3 substituents selected from among halogens; R ｅ and R ｆ are each, independently of one another, H and -C １－６ alkyl, independently selected from the group consisting of, said alkyl may be substituted with 1, 2 or 3 substituents selected from among halogens, or R ｅ and R ｆ together with the nitrogen atom to which they are attached, form a 4-, 5- or 6-membered heterocyclic ring which may be substituted with 1, 2 or 3 substituents selected from among halogens), or a pharmaceutically acceptable salt, ester, amide or carbamate thereof, or a salt of such an ester, amide or carbamate.

2. The compound according to claim 1, having a structure represented by formula (Ia) 【Chemical Formula 6】 For example, a structure represented by formula (Ib) 【Chemical Formula 7】 The compound according to claim 1, having a structure represented by the formula:

3. R １ is H, the compound according to claim 1.

4. R ２ is phenyl which may be substituted with 1, 2 or 3 halogens, and C which may be substituted with 1, 2 or 3 halogens １－６ alkyl, and is selected from the group consisting of, for example, C which may be substituted with 1, 2 or 3 halogens １－４ alkyl, for example methyl, the compound according to claim 1.

5. X is -CH ２ - or -CH ２ -CH ２ -, the compound according to claim 1.

6. R ３ is of formula (IIa) 【Chemical Formula 8】 or a group represented by formula (IIIa) [Chemical Formula 9] The compound according to claim 1, which is a group represented by

7. R ４ is N(R ｃ )(R ｄ ) and a group represented by formula (IVa) [Chemical Formula 10] The compound according to claim 1, which is selected from the group consisting of

8. R ４ When is a group represented by formula (IV) or formula (IVa), R ５ is R ｂ and when R ４ is N(R ｃ )(R ｄ ), R ５ is R ｂ and a group represented by formula (Va) [Chemical Formula 11] The compound according to claim 1, which is selected from the group consisting of

9. R ４ is NH ２ The compound according to claim 1, which is

10. Each R ａ is independently selected from the group consisting of C １－６ alkyl and -CH ２ -phenyl, and the C １－６ alkyl may be substituted with -OH, -NH ２ , -NHC(=NH)NH ２ , -C(O)OH, -C(O)NH ２ , -SH, -SCH ３ or halogen (e.g., F or Cl), and the phenyl may be substituted with halogen (e.g., F or Cl), -NH ２ , -OH, -O-C １－６ alkyl and -NO ２The compound according to claim 1, which may be substituted with 1, 2 or 3 substituents independently selected from the group consisting of.

11. R ａ is -C １－６ alkyl and -CH ２ -phenyl, wherein the phenyl may be substituted with 1, 2 or 3 halogens, the compound according to claim 10.

12. R ４ When it is a group represented by formula (IV) or formula (IVa), R ５ is -OC １－６ alkyl, and when R ４ is NH ２ , R ５ is -OC １－６ alkyl or a group represented by formula (V) or (Va), the compound according to claim 1.

13. R ｂ When it is -OC １－６ alkyl, R ｂ is selected from the group consisting of methoxy, ethoxy and isopropoxy, the compound according to claim 12.

14. The compound is (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example Compound 1); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example Compound 2); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methyl-butanic acid ethyl ester (Example Compound 3); Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoate (Example Compound 4); Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoate (Example Compound 5); Ethyl (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoate (Example Compound 6); Ethyl (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoate (Example Compound 7); Ethyl (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoate (Example Compound 8); Ethyl (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoate (Example Compound 9); Ethyl (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoate (Example Compound 10); Ethyl (2S)-2-[[(2S)-2-amino-3-(4-fluorophenyl)propanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoate (Example Compound 11); (2S)-2-[[(2S)-2-Amino-3-methyl-butanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid ethyl ester (Example Compound 12); (2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example Compound 13); (2S)-2-[[(2S)-2-Amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid methyl ester (Example Compound 14); (2S)-2-[[(2S)-2-Amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid isopropyl ester (Example Compound 15); (2S)-2-[[(2S)-2-[[(2S)-2-Amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example Compound 16); (2S)-2-[[(2R)-2-[[(2S)-2-Amino-3-(4-fluorophenyl)propanoyl]amino]-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example Compound 21); (2S)-2-[[(2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example Compound 22); Ethyl (2S)-2-[[(2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoate (Example Compound 23); 2-Morpholinoethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 24); 2-Isopropoxyethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 25); Isopropyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 26); Methyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 27); 3-(Dimethylamino)propyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 28); (2-Methoxy-1-methyl-ethyl) (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 29); (2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,N,4-trimethyl-pentanamide (Example Compound 30); (2S)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-N,4-dimethyl-pentanamide (Example Compound 31); (2S)-2-[[(2S)-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example Compound 32); (2S)-2-[[(2S)-2-Acetamido-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example Compound 34); (2S)-2-[[(2S)-2-Acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example Compound 35); (2S)-2-[[(2S)-2-Acetamido-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-(4-fluorophenyl)propanoic acid ethyl ester (Example Compound 36); (2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid ethyl ester (Example Compound 38); (2R)-2-[[(2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid ethyl ester (Example Compound 45); Ethyl (2S)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 46); Ethyl (2R)-2-[[(2R)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 47); Ethyl (2S)-2-[[(2S)-2-amino-4-[6-[bis(2-chloroethyl)amino]-3-methyl-imidazo[4,5-b]pyridin-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 48); and Ethyl (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoate (Example Compound 50) or a compound according to claim 1, selected from the group consisting of a pharmaceutically acceptable salt, amide or carbamate thereof, including salts of such amides or carbamates.

15. The compound is (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-methylbutanoic acid (Example Compound 19); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methylpentanoic acid (Example Compound 20); (2S)-2-[[(2S)-4-[5-[Bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]-2-(methylamino)butanoyl]amino]-4-methyl-pentanoic acid (Example Compound 33); (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid (Example Compound 37); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-3-(4-fluorophenyl)propanoic acid (Example Compound 39); (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example Compound 40); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-3-methyl-butanoic acid (Example Compound 42); (2S)-2-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid (Example Compound 43); (2S)-2-[[(2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoyl]amino]-4-methyl-pentanoic acid (Example Compound 44) and (2S)-2-[[(2S)-2-amino-4-[5-[bis(2-chloro-1,1,2,2-tetra-deuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoyl]amino]-4-methyl-pentanoic acid (Example Compound 51) The compound according to claim 1, selected from the group consisting of or a salt thereof.

16. A pharmaceutical composition comprising the compound according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, further comprising another therapeutic agent, such as a steroid, a checkpoint inhibitor, a nuclear transport inhibitor, an anti-apoptosis inhibitor, adoptive cell therapy, a bispecific T cell engager (BiTE), an immunomodulatory imide drug (IMiD), a proteasome inhibitor (PI), a histone deacetylase (HDAC) inhibitor, a peptide drug conjugate (PDC), an alkylating agent or a DNA intercalator.

18. The pharmaceutical composition according to claim 16, for use in the prevention or treatment of cancer.

19. The pharmaceutical composition according to claim 17, for use in the prevention or treatment of cancer.

20. The pharmaceutical composition according to claim 18, wherein the cancer is selected from multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma, for example, multiple myeloma, acute myeloid leukemia or lymphoma.

21. The pharmaceutical composition according to claim 19, wherein the cancer is selected from multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma, for example, multiple myeloma, acute myeloid leukemia or lymphoma.

22. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is administered together with (for example, simultaneously, sequentially or separately) one or more other therapeutic agents, such as a steroid, a checkpoint inhibitor, a nuclear transport inhibitor, an anti-apoptosis inhibitor, adoptive cell therapy, a bispecific T cell engager (BiTE), an immunomodulatory imide drug (IMiD), a proteasome inhibitor (PI), a histone deacetylase (HDAC) inhibitor, a peptide drug conjugate (PDC), an alkylating agent or a DNA intercalator.

23. The pharmaceutical composition according to claim 21, wherein the pharmaceutical composition is administered (e.g., simultaneously, sequentially, or separately) with one or more other therapeutic agents, such as steroids, checkpoint inhibitors, nuclear transport inhibitors, anti-apoptosis inhibitors, adoptive cell therapy, bispecific T cell engagers (BiTE), immunomodulatory imide drugs (IMiD), proteasome inhibitors (PI), histone deacetylase (HDAC) inhibitors, peptide drug conjugates (PDC), alkylating agents, or DNA intercalators.

24. Use of a compound according to any one of claims 1 to 15 in the manufacture of a medicament for preventing or treating cancer, wherein, for example, the cancer is any one of multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia, and lymphoma, such as multiple myeloma, acute myeloid leukemia, or lymphoma.

25. Formula (I) 【Chemical Formula 12】 (wherein, X is C １－６ alkylene; W １ 、W ２ 、W ３ and W ４ are each CH, or one of W １ 、W ２ 、W ３ and W ４ is N and the others are CH; R １ is C １－４ alkyl which may be substituted with 1, 2, or 3 substituents independently selected from H and halogen, and is selected from the group consisting of halogen; R ２ is selected from the group consisting of phenyl which may be substituted with 1, 2, or 3 substituents independently selected from H and halogen, and C １－６ alkyl which may be substituted with 1, 2, or 3 substituents independently selected from H and halogen; R ３ is of formula (VIa) 【Chemical Formula 13】 (wherein R ｃ and R ｄ are each independently selected from the group consisting of H, -C １－６ alkyl, C(O)C １－６ alkyl and -CH ２ -phenyl, and the alkyl or the phenyl may be substituted with 1, 2 or 3 substituents selected from among halogens; R ｇ is selected from the group consisting of H and -C １－６ alkyl, and the alkyl may be substituted with 1, 2 or 3 substituents selected from among halogens)) is a group represented by) A compound represented by or a salt or solvate thereof.

26. Formula (Ia) 【Chemical Formula 14】 having a structure represented by, or a structure represented by formula (Ib) 【Chemical Formula 15】 or R １ is H, R ２ is selected from the group consisting of phenyl which may be substituted with 1, 2 or 3 halogens, and C1-6 alkyl which may be substituted with 1, 2 or 3 halogens, for example, C1-4 alkyl which may be substituted with 1, 2 or 3 halogens, for example methyl, or X is -CH2- or -CH2-CH2-; R ｃ and R ｄ are each H; R ｇ is H, the compound according to claim 25.

27. The compound is (2S)-2-amino-3-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]propanoic acid (Example Compound 17); (S)-2-amino-4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid (Example Compound 18); Ethyl (2S)-2-amino-4-[5-[bis(2-chloroethyl)amino]-1-methyl-benzimidazol-2-yl]butanoate (Example Compound 38); (2S)-2-Amino-4-[5-[bis(2-chloroethyl)amino]-1-phenyl-benzimidazol-2-yl]butanoic acid (Example Compound 41); and (2S)-2-Amino-4-[5-[bis(2-chloro-1,1,2,2-tetradeuterio-ethyl)amino]-1-methyl-benzimidazol-2-yl]butanoic acid (Example Compound 49); or a salt thereof The compound according to claim 25, selected from the group consisting of: **Claim 28** For the treatment of cancer selected from the group consisting of multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma, such as multiple myeloma, acute myeloid leukemia or lymphoma, of formula (I) **Chemical Formula 16** (In the formula, X is C １－６ alkylene; W １ , W ２ , W ３ and W ４ are each CH, or one of W １ , W ２ , W ３ and W ４ is N and the others are CH; R １ is C １－４ alkyl, optionally substituted with 1, 2 or 3 substituents independently selected from H, halogen, and selected from the group consisting of halogen; R ２ is selected from the group consisting of phenyl, optionally substituted with 1, 2 or 3 substituents independently selected from H, halogen, and C １－６ alkyl, optionally substituted with 1, 2 or 3 substituents independently selected from H, halogen; R ３ is of formula (VI) **Chemical Formula 17** (wherein, R ｃ and R ｄ are each independently selected from the group consisting of H, -C １－６ alkyl, C(O)C １－６ alkyl and -CH ２ -phenyl, and the alkyl or the phenyl may be substituted with 1, 2 or 3 substituents selected from among halogens; R ｇ is selected from the group consisting of H and -C １－６ alkyl, and the alkyl may be substituted with 1, 2 or 3 substituents selected from among halogens)), a group represented by) A pharmaceutical composition containing a compound represented by or a salt or solvate thereof.

Images