WO2016194390A1 - Pharmaceutical composition for use in the treatment of cancer - Google Patents

Abstract

Provided are a method and a pharmaceutical composition for treating cancer using a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof. Also provided are combination use of the compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof and an anti-CTLA-4 antibody, and combination use of the compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof, an anti-CTLA-4 antibody and an anti-PD-1 antibody.

Description

[Title established by the ISA under Rule 37.2] PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OF CANCER

The present application relates to a method and a pharmaceutical composition for treating cancer.

Background

Vascular adhesion protein-1 (referred to as VAP-1 hereafter) is an amine oxidase that is abundant in human plasma also called as semicarbazide sensitive amine oxidase, SSAO. VAP-1 expression in vascular endothelium and vascular smooth muscle is significantly increased in inflammatory lesions. Physiological roles of VAP-1 have not been elucidated although VAP-1 gene was cloned in 1998. VAP-1 is reported as a membrane protein that is regulated by inflammatory cytokines and controls rolling and migration of lymphocytes and NK cells by acting as an adhesion molecule. It is also suggested that a physiological substrate for SSAO is methylamine; and that VAP-1 produces, through its amine oxidase activity, hydrogen peroxides and aldehydes, which are known to play a significant role for its adhesion activity.

Thiazole, benzene, and thiophene derivatives having VAP-1 inhibitory activity are disclosed in WO2009/096609 and WO2009/145360. Those compounds, however, have not been known to have anti-tumor effects.

[PTL 1] WO2009/096609
[PTL 2] WO2009/145360
[PTL 3] US2011/0015240A
[PTL 4] US2011/0059957A
[PTL 5] JPS61-239891A
[PTL 6] US4,888,283B
[PTL 7] WO1993/23023
[PTL 8] WO2002/02090
[PTL 9] WO2002/02541
[PTL 10] US2002/0173521A
[PTL 11] WO2002/38152
[PTL 12] WO2002/38153
[PTL 13] WO2004/087138
[PTL 14] WO2004/067521
[PTL 15] WO2006/011631
[PTL 16] WO2006/028269
[PTL 17] WO2008/066145

[NPL 1] Diabetologia, 42 (1999) 233-237
[NPL 2] Diabetes Medicine, 16 (1999) 514-521

Summary

The inventors have found that compounds having VAP-1 inhibitory activity disclosed in WO2009/096609 and WO2009/145360 reduced tumor volume in tumor model mice, and further showed synergistic anti-tumor effect in combination with an anti-CTLA-4 antibody, or with an anti-CTLA-4 antibody and an anti-PD-1 antibody.

In an aspect, the present application provides a pharmaceutical composition for treating cancer, which comprises a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof.

In another aspect, the present application provides a method of treating cancer, comprising administering a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof to a mammal in need thereof.

In another aspect, the present application provides use of a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof for the manufacture of a medicament for treating cancer.

In another aspect, the present application provides a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof for use in the treatment of cancer.

In another aspect, the present application provides a pharmaceutical composition for treating cancer comprising an anti-CTLA-4 antibody, which is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof.

In another aspect, the present application provides a pharmaceutical composition for treating cancer comprising an anti-PD-1 antibody, which is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof and an anti-CTLA-4 antibody.

The compound having VAP-1 inhibitory activity shows anti-tumor effects and further synergistic effects in combination with an anti-CTLA-4 antibody, or with an anti-CTLA-4 antibody and an anti-PD-1 antibody. Provided by the present disclosure is useful for treating cancer.

Fig. 1A shows the decrease of tumor volume in tumor model mice by administration of 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide (hereafter, "Compound A"). The label "VAP-1" in this figure and the following figures indicates a compound having VAP-1 inhibitory activity, i.e., a VAP-1 inhibitor. Such a VAP-1 inhibitor includes, e.g. Compound A as stated above, Compound B as defined later and the like. Specifically, the label VAP-1 in Fig. 1A indicates the result of Compound A. Fig. 1B shows tumor antigen-specific IFN-γ production by CD8⁺ cells obtained from tumor mass or draining lymph node. TIL: Tumor-infiltrating lymphocytes; dLN: Draining lymph node. Fig. 1C shows the direct effects of Compound A on cancer cells. Fig. 2 shows the combination effects of Compound A and an anti-CTLA-4 antibody on tumor volume. Fig. 3 shows the reduction of soluble VAP-1 (sVAP-1) by Compound A in serum of tumor model mice. Fig. 4 shows the effects of Compound A in a different mouse tumor model. Fig. 5 shows the effects of Compound A in NOG mouse model and CD8 depletion model. Fig. 6 shows the results of FACS (Fluorescence Activated Cell Sorting) analysis of tumor-infiltrating lymphocytes. Fig. 7 shows the change in the population of tumor-infiltrating lymphocytes induced by Compound A. Fig. 8 shows the change of activated T cells in tumor-infiltrating lymphocytes induced by Compound A. Fig. 9 shows the effects of Compound A on T cell population in draining lymph node. Fig. 10 shows the change in myeloid-derived suppressor cells in tumor induced by Compound A. Fig. 11 shows the effects of Compound A on dendritic cell activation in tumor. Fig. 12 shows the decrease of tumor volume in tumor model mice by Compound B (4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate). Fig. 13 shows the combination effects of Compound A and an anti-PD-1 antibody on tumor volume. Fig. 14 shows the increase of PD-1 positive cells by Compound A. Fig. 15 shows the effects on tumor volume of Compound A, doublet combination of an anti-CTLA-4 antibody and an anti-PD-1 antibody, or triplet combination of Compound A, an anti-CTLA-4 antibody, and an anti-PD-1 antibody.

The present application provides a pharmaceutical composition for treating cancer, which comprises a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof. In one embodiment, the compound is a compound represented by the formula (I):

X-Y-A-B-D-E (I)

wherein
X is

wherein
A¹ is a residue derived from benzene or a heterocycle containing at least one nitrogen or sulfur atom;
B¹ is hydrogen, hydroxy, halogen, lower alkyl, lower cycloalkyl, lower haloalkyl, lower alkoxy, acyl, acylamino, optionally substituted carbamoyl, (lower alkyl)sulfonylamino, or (lower alkyl)carbonyloxy, with the proviso that, when A¹ is a residue derived from thiazole, B¹ is not acylamino;
B² is hydrogen or a group containing at least one nitrogen atom, with the proviso that, when A¹ is a residue derived from thiazole, B² is not acylamino; or
alternatively, B¹ and B² may together form a cyclic structure;
A² is a divalent residue derived from optionally substituted thiazole;
R¹ is acyl;
Y is represented by the following formula:
J-L-M (II)
wherein
J is a bond, lower alkylene, lower alkenylene, lower alkynylene, -(CH₂)_n-O-, -(CH₂)_n-NH-, -(CH₂)_n-CO-, or -(CH₂)_n-SO₂-, wherein n is an integer of from 0 to 6;
L is a bond, -O-, -NH-, -CO-, or -SO₂-; and
M is a bond, lower alkylene, lower alkenylene, or lower alkynylene, with the proviso that, when J is -(CH₂)_n-O-, L is not -O-, -NH-, or -SO₂-; when J is -(CH₂)_n-NH-, L is not -O- or -NH-; when J is -(CH₂)_n-CO-, L is not -CO-; and when J is -(CH₂)_n-SO₂-, L is not -O- or -SO₂-;
A is a divalent residue derived from optionally substituted benzene or optionally substituted thiophene;
B is -(CH₂)_m-CO-, -(CH₂)_m-O-CO-, -(CH₂)_m-S-CO-, or -(CH₂)_m-NR²-CO-, wherein R² is hydrogen, lower alkyl, or acyl, and m is an integer of from 0 to 6;
D is -NR³-, wherein R³ is hydrogen, lower alkyl, alkoxycarbonyl, or acyl; and
E is optionally substituted amino;
or a pharmaceutically acceptable salt thereof.

The terms as used herein are explained in details below.

The term "halogen" includes fluorine, bromine, chlorine, and iodine.

The term "lower" as used for a functional group means that the group has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, unless otherwise specified.

The term "lower alkyl" includes straight chain or branched chain alkyl having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl, or hexyl. Preferred is C₁-C₄ alkyl.

The term "lower haloalkyl" includes lower alkyl substituted with halogen. When two or more halogen substituents are present, the substituents may be the same or different. Examples of the lower haloalkyl include trifluoromethyl, trichloromethyl, and pentafluoroethyl.

The term "lower alkylene" includes straight chain or branched chain alkylene having 1 to 6 carbon atoms, such as methylene, ethylene, trimethylene, propylene, ethylidene, or propylidene. Preferred is C₁-C₄ alkylene.

The term "lower alkenylene" includes straight chain or branched chain alkenylene having 2 to 6 carbon atoms. Examples of such lower alkenylene include vinylene, 1-propenylene, 1-methyl-l-propenylene, 2-methyl-1-propenylene, 2-propenylene, 2-butenylene, 1-butenylene, 3-butenylene, 2-pentenylene, 1-pentenylene, 3-pentenylene, 4-pentenylene, 1,3-butadienylene, 1,3-pentadienylene, 2-penten-4-ynylene, 2-hexenylene, 1-hexenylene, 5-hexenylene, 3-hexenylene, 4-hexenylene, 3,3-dimethyl-1-propenylene, 2-ethyl-1-propenylene, 1,3,5-hexatrienylene, 1,3-hexadienylene, and 1,4-hexadienylene. Preferred is C₂-C₄ alkenylene.

The term "lower alkenylene" may be in an E- or Z-form. When the compound of the formula (I) has a lower alkenylene moiety, the compound may be a geometric isomer having E- or Z-configuration in the lower alkenylene moiety.

The term "lower alkynylene" includes straight chain or branched chain alkynylene having 2 to 6 carbon atoms and 1 to 3 triple bonds. Examples of such lower alkynylene include ethynylene, 1-propynylene, 1-methyl-1-propynylene, 2-methyl-1-propynylene, 2-propynylene, 2-butynylene, 1-butynylene, 3-butynylene, 2-pentynylene, 1-pentynylene, 3-pentynylene, 4-pentynylene, 2-pentyn-4-ynylene, 2-hexynylene, 1-hexynylene, 5-hexynylene, 3-hexynylene, 4-hexynylene, 3,3-diethyl-1-propynylene, and 2-ethyl-1-propynylene. Preferred is C₂-C₄ alkynylene.

The term "aryl" includes C₆-C₁₀ aryl such as phenyl or naphthyl. The aryl may be optionally substituted at any one or more positions. Examples of substituents on the aryl include methyl, ethyl, hydroxy, methoxy, amino, acetyl, and halogen. When two or more substituents are present, the substituents may be the same or different.

The term "aralkyl" includes aralkyl whose aryl moiety has 6 to 10 carbon atoms (i.e., the aryl moiety is C₆-C₁₀ aryl as defined herein about the term "aryl") and whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"). Examples of such aralkyl include benzyl, phenethyl, 1-naphthylmethyl, 2-naphthylmethyl, 3-phenylpropyl, 4-phenylbutyl, and 5-phenylpentyl.

The term "lower cycloalkyl" includes cycloalkyl having 3 to 6 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term "heterocycle" includes "aromatic heterocycle" and "non-aromatic heterocycle". The aromatic heterocycle includes 5- to 10-membered aromatic heterocycle containing 1 to 3 hetero atoms selected from nitrogen, oxygen, and sulfur atoms. Examples of such aromatic heterocycle include thiophene, furan, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, pyridine, pyridazine, pyrimidine, and pyrazine. The non-aromatic heterocycle includes 5- to 10-membered non-aromatic heterocycle containing 1 to 3 hetero atoms selected from nitrogen, oxygen, and sulfur atoms. Examples of such non-aromatic heterocycle include pyrrolidine, imidazoline, pyrazolidine, pyrazoline, piperidine, piperazine, homopiperazine, triethylenediamine, morpholine, thiomorpholine, dioxolane, oxazolidine, thiazolidine, triazolidine, and 2,5-diazabicyclo[2.2.1]heptane.

The term "acyl" includes (lower alkyl)carbonyl, (lower cycloalkyl)carbonyl, and arylcarbonyl.

The term "(lower alkyl)carbonyl" includes alkylcarbonyl whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"), such as acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, or hexanoyl.

The term "(lower cycloalkyl)carbonyl" includes cycloalkylcarbonyl whose cycloalkyl moiety has 3 to 6 carbon atoms (i.e., the cycloalkyl moiety is C₃-C₆ cycloalkyl as defined herein about the term "lower cycloalkyl"), such as cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, or cyclohexylcarbonyl.

The term "arylcarbonyl" includes arylcarbonyl whose aryl moiety has 6 to 10 carbon atoms (i.e., the aryl moiety is C₆-C₁₀ aryl as defined herein about the term "aryl"), such as benzoyl or naphthoyl.

The term "acylamino" includes acylamino whose acyl moiety is the acyl as defined herein. Examples of such acylamino include acetylamino, propionylamino, butyrylamino, isobutyrylamino, valerylamino, isovalerylamino, pivaloylamino, hexanoylamino, cyclopropylcarbonylamino, cyclobutylcarbonylamino, cyclopentylcarbonylamino, cyclohexylcarbonylamino, benzoylamino, and naphthoylamino.

The term "lower alkoxy" includes straight chain or branched chain alkoxy having 1 to 6 carbon atoms. Examples of such lower alkoxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, tert-pentoxy, and hexoxy. Preferred is C₁-C₄ alkoxy. The lower alkoxy may be optionally substituted with one or more substituents. Examples of the substituents include halogen, acyl, and aminocarbonyl (the terms "halogen" and "acyl" are as defined herein). When two or more substituents are present, the substituents may be the same or different.

The term "alkoxy" includes optionally substituted alkyloxy, lower cycloalkyloxy, and aralkyloxy.

The term "alkyloxy" in the term "optionally substituted alkyloxy" includes alkyloxy whose alkyl moiety has 1 to 10 carbon atoms. Examples of such alkyloxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, tert-pentyloxy, hexyloxy, and decyloxy. Examples of substituents on the "optionally substituted alkyloxy" include halogen, acyl, and aminocarbonyl (the terms "halogen" and "acyl" are as defined herein). The alkyloxy may be substituted at any or more positions. When two or more substituents are present, the substituents may be the same or different.

The term "lower cycloalkyloxy" includes cycloalkyloxy whose cycloalkyl moiety has 3 to 6 carbon atoms (i.e., the cycloalkyl moiety is C₃-C₆ cycloalkyl as defined herein about the term "lower cycloalkyl"), such as cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, or cyclohexyloxy.

The term "aralkyloxy" includes aralkyloxy whose aryl moiety has 6 to 10 carbon atoms (i.e., the aryl moiety is C₆-C₁₀ aryl as defined herein about the term "aryl") and whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"). Examples of such aralkyloxy include benzyloxy, phenethyloxy, 1-naphthylmethyloxy, 2-naphthylmethyloxy, 3-phenylpropyloxy, 4-phenylbutyloxy, and 5-phenylpentyloxy.

The term "alkoxycarbonyl" includes alkyloxycarbonyl, (lower cycloalkyloxy)carbonyl, and aralkyloxycarbonyl.

The term "alkyloxycarbonyl" includes alkyloxycarbonyl whose alkyl moiety has 1 to 10 carbon atoms, such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, tert-pentyloxycarbonyl, hexyloxycarbonyl, or decyloxycarbonyl. The term "(lower alkoxy)carbonyl" is alkyloxycarbonyl whose alkyl moiety has 1 to 6 carbon atoms.

The term "(lower cycloalkyloxy)carbonyl" includes cycloalkyloxycarbonyl whose cycloalkyl moiety has 3 to 6 carbon atoms (i.e., the cycloalkyl moiety is C₃-C₆ cycloalkyl as defined herein about the term "lower cycloalkyl"), such as cyclopropyloxycarbonyl, cyclobutyloxycarbonyl, cyclopentyloxycarbonyl, or cyclohexyloxycarbonyl.

The term "aralkyloxycarbonyl" includes aralkyloxycarbonyl whose aryl moiety has 6 to 10 carbon atoms (i.e., the aryl moiety is C₆-C₁₀ aryl as defined herein about the term "aryl") and whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"). Examples of such aralkyloxycarbonyl include benzyloxycarbonyl, phenethyloxycarbonyl, 1-naphthylmethyloxycarbonyl, 2-naphthylmethyloxycarbonyl, 3-phenylpropyloxycarbonyl, 4-phenylbutyloxycarbonyl, and 5-phenylpentyloxycarbonyl.

The term "optionally substituted carbamoyl" includes unsubstituted carbamoyl, and carbamoyl substituted with one or two substituents. The optionally substituted carbamoyl is represented by the formula -CONR⁴R⁵.

R⁴ and R⁵ may be the same or different, and are independently selected from hydrogen; and lower alkyl, acyl, in particular (lower alkyl)carbonyl, alkoxycarbonyl, in particular (lower alkoxy)carbonyl, aryl, aralkyl, lower cycloalkyl, sulfuryl, sulfinyl, phosphoryl, and heterocyclyl, each of which is optionally substituted with one or more substituents such as hydroxy. The terms "lower alkyl", "acyl", "(lower alkyl)carbonyl", "alkoxycarbonyl", "(lower alkoxy)carbonyl", "aryl", "aralkyl", and "lower cycloalkyl" are as defined herein. The term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein.

The term "(lower alkyl)sulfonylamino" includes alkylsulfonylamino and di(alkylsulfonyl)amino whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"). Examples of such (lower alkyl)sulfonylamino include methylsulfonylamino, ethylsulfonylamino, propylsulfonylamino, isopropylsulfonylamino, butylsulfonylamino, isobutylsulfonylamino, sec-butylsulfonylamino, tert-butylsulfonylamino, pentylsulfonylamino, tert-pentylsulfonylamino, and hexylsulfonylamino; and di(methylsulfonyl)amino, di(ethylsulfonyl)amino, and (methylsulfonyl)(ethylsulfonyl)amino).

The term "(lower alkyl)carbonyloxy" includes alkylcarbonyloxy whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"), such as methylcarbonyloxy, ethylcarbonyloxy, propylcarbonyloxy, isopropylcarbonyloxy, butylcarbonyloxy, isobutylcarbonyloxy, sec-butylcarbonyloxy, tert-butylcarbonyloxy, pentylcarbonyloxy, tert-pentylcarbonyloxy, or hexylcarbonyloxy.

The term "(lower alkyl)amino" includes alkylamino and dialkylamino whose alkyl moiety has 1 to 6 carbon atoms (i.e., the alkyl moiety is C₁-C₆ alkyl as defined herein about the term "lower alkyl"). Examples of such (lower alkyl)amino include methylamino, ethylamino, propylamine, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, tert-pentylamino, and hexylamino; and dimethylamino, diethylamino, dipropylamino, ethylmethylamino, methylpropylamino, and ethylpropylamino.

In the formula (I), A¹ is a residue derived from benzene or a heterocycle containing at least one nitrogen or sulfur atom, such as a residue derived from benzene, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, or thiophene. A¹ binds to Y at any position on the ring.

Preferably, A¹ is a residue derived from benzene, pyridine, pyridazine, pyrimidine, pyrazine, imidazole, thiazole, or thiophene.

In the formula (I), A² is a divalent residue derived from optionally substituted thiazole.

Examples of the "divalent residue derived from optionally substituted thiazole", i.e., A², includes

The thiazole may be optionally substituted at any one or more positions. Examples of substituents on the "optionally substituted thiazole" include the followings:
(1) halogen, such as fluoro, chloro, and bromo;
(2) alkoxycarbonyl as defined herein, such as ethoxycarbonyl;
(3) optionally substituted aryl, wherein the term "aryl" is as defined herein and may be substituted at any one or more positions with, for example, -SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein), such as phenyl and 4-(methylsulfonyl)phenyl;
(4) a group of the formula: -CONR_aR_b, wherein R_a is hydrogen, lower alkyl, aryl, or aralkyl and R_b is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein), such as N-methylaminocarbonyl, N-phenylaminocarbonyl, N,N-dimethylaminocarbonyl, and N-benzylaminocarbonyl);
(5) a group of the formula: -CONH-(CH₂)_k-aryl, wherein k is an integer of from 0 to 6, and the term "aryl" is as defined herein and may be optionally substituted at any one or more positions with one to five substituents independently selected from the group consisting of -NO₂, -SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein), -CF₃ and -O-aryl (the term "aryl" is as defined herein);
(6) a group of the formula: -CONH-(CH₂)_s-heterocyclyl, wherein s is an integer of from 0 to 6, and the term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein (e.g., pyridine);
(7) a group of the formula: -CO-heterocyclyl, wherein the term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein (e.g., pyrrolidine, piperidine, piperazine, and thiomorpholine), and may be optionally substituted at any one or more positions with one to five substituents independently selected from the group consisting of -CO-(lower alkyl) (the term "lower alkyl" is as defined herein), -CO-O-(lower alkyl) (the term "lower alkyl" is as defined herein), -SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein), oxo (i.e., =O), and -CONR_cR_d wherein R_c is hydrogen, lower alkyl, aryl, or aralkyl, and R_d is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein);
(8) a group of the formula: -(CH₂)_t-aryl, wherein t is an integer of from 1 to 6, and the term "aryl" is as defined herein and may be optionally substituted at any one or more positions by one to five substituents independently selected from the group consisting of -S-(lower alkyl) (the term "lower alkyl" is as defined herein); -SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein); -SO₂-NR_VR_W, wherein R_V is hydrogen, lower alkyl, aryl, or aralkyl and R_W is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein); -CO₂-(lower alkyl) (the term "lower alkyl" is as defined herein); -NHCO-O-(lower alkyl) (the term "lower alkyl" is as defined herein); and -CONR_eR_f, wherein R_e is hydrogen, lower alkyl, aryl, or aralkyl and R_f is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein);
(9) a group of the formula: -(CH₂)_o-heterocyclyl, wherein o is an integer of from 0 to 6, and the term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein (e.g., pyrrolidine, piperidine, piperazine, morpholine, and thiomorpholine), and may be optionally substituted at any one or more positions by one to five substituents; the substituent(s) being independently selected from the group consisting of oxo (i.e., =O); -CO-(lower alkyl) (the term "lower alkyl" is as defined herein); -CO-O-(lower alkyl) (the term "lower alkyl" is as defined herein); -SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein); -CO-(heterocyclyl), wherein the term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein, such as pyrrolidine, piperazine, or morpholine, and may be optionally substituted at any one or more positions by one to five substituents independently selected from the group consisting of lower alkyl and halogen such as fluoro, chloro, or bromo (the term "lower alkyl" is as defined herein); and -CONR_gR_h, wherein R_g is hydrogen, lower alkyl, aryl, or aralkyl, and R_h is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein);
(10) a group of the formula: -(CH₂)_p-NR_iR_j, wherein p is an integer of from 0 to 6; R_i is hydrogen, acyl, lower alkyl, aryl, or aralkyl; and R_i is hydrogen, acyl, lower alkyl, aryl, or aralkyl, wherein the terms "acyl", "lower alkyl", "aryl", and "aralkyl" are as defined herein, and the "lower alkyl" may be optionally substituted at any one or more positions by one to five substituents independently selected from the formula: -CONR_kR_l, wherein R_k is hydrogen, lower alkyl, aryl, or aralkyl and R_l is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein);
(11) a group of the formula: -CON(H or lower alkyl)-(CHR_m)_q-T, wherein q is an integer of from 0 to 6; the term "lower alkyl" is as defined herein; R_m is hydrogen, aralkyl as defined herein or alkyl as defined herein (in particular, lower alkyl), wherein the aralkyl and alkyl may be optionally substituted at any one or more positions by one to three substituents independently selected from the group consisting of -OH and -CONH₂; and T is hydrogen; -CONR_nR_o, wherein R_n is hydrogen, lower alkyl, aryl, or aralkyl and R_o is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein); -NH-CO-R_p, wherein R_p is lower alkyl or aralkyl (the terms "lower alkyl" and "aralkyl" are as defined herein); -NH-SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein); -SO₂-(lower alkyl) (the term "lower alkyl" is as defined herein); -heterocyclyl, wherein the term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein (e.g., pyridine, pyrrolidine, or morpholine), and may be optionally substituted at any one or more positions by one to three substituents (e.g., oxo, i.e., =O)); or -CO-(heterocyclyl) (the term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein, such as piperidine or morpholine); and
(12) a group of the formula: -(CH₂)_r-CO-NR_tR_u, wherein r is an integer of from 1 to 6, R_t is hydrogen, lower alkyl, aryl, or aralkyl and R_u is hydrogen, lower alkyl, aryl, or aralkyl (the terms "lower alkyl", "aryl", and "aralkyl" are as defined herein).

In the formula (I), A is a divalent residue derived from optionally substituted benzene or optionally substituted thiophene.

The benzene and thiophene may be optionally substituted at any one or more positions. Examples of substituents on the "optionally substituted benzene" and the "optionally substituted thiophene" include halogen (e.g., fluoro, chloro, and bromo), lower alkyl (e.g., methyl and ethyl), lower alkoxy (e.g., methoxy), acyl (e.g., acetyl), and haloalkyl (e.g., trifluoromethyl). When two or more substituents are present, the substituents may be the same or different.

The benzene or thiophene is substituted with (i.e., binds to) Y and B at any two positions. The benzene may be ortho-, meta- or para-substituted. The thiophene may be substituted at any two positions of positions 2, 3, 4, and 5.

The benzene is preferably meta- or para-substituted with Y and B as shown below:

The thiophen is preferably substituted with Y and B at positions 2 and 5 (or 5 and 2), or positions 2 and 4 (or 5 and 3) as shown below:

In the formula (I), A¹ is substituted with B¹ and B² at any two positions.

In the formula (I) , B¹ is hydrogen, hydroxy, halogen, lower alkyl, lower cycloalkyl, lower haloalkyl, lower alkoxy, acyl, acylamino, optionally substituted carbamoyl, (lower alkyl)sulfonylamino, or (lower alkyl)carbonyloxy, with the proviso that, when A¹ is a residue derived from thiazole, B¹ is not acylamino. The terms "halogen", "lower alkyl", "lower cycloalkyl", "lower haloalkyl", "lower alkoxy", "acyl", "acylamino", "optionally substituted carbamoyl", "(lower alkyl)sulfonylamino", and "(lower alkyl)carbonyloxy" are as defined herein.

Examples of B¹ include hydrogen; hydroxy; halogen, such as fluoro, chloro, or bromo; lower alkyl, such as methyl, ethyl, or isopropyl; lower cycloalkyl, such as cyclopropyl; lower haloalkyl, such as trifluoromethyl; lower alkoxy, such as methoxy, ethoxy, trifluoromethoxy, or aminocarbonylmethoxy; acyl, such as acetyl or ethylcarbonyl; acylamino, such as acetylamino or ethylcarbonylamino; optionally substituted aminocarbonyl (carbamoyl), such as aminocarbonyl, N-methylaminocarbonyl, or N,N-dimethylaminocarbonyl; (lower alkyl)sulfonylamino, such as methylsulfonylamino, ethylsulfonylamino, cyclopropylsulfonylamino, or bis(methylsulfonyl)amino; (lower alkyl)carbonyloxy, such as methylcarbonyloxy.

B¹ is preferably hydrogen, methyl, trifluoromethyl, acetylamino, aminocarbonyl (carbamoyl), aminocarbonylmethoxy, methylsulfonylamino, ethylsulfonylamino, or methylcarbonyloxy.

In the formula (I), B² is hydrogen or a group containing at least one nitrogen atom, with the proviso that, when A¹is a residue derived from thiazole, B² is not acylamino. The term "group containing at least one nitrogen atom" may be acylamino; (lower alkyl)amino optionally substituted with amino or acetylamino; optionally substituted heterocyclyl containing at least one nitrogen atom; or methyl substituted with (lower alkyl)amino or optionally substituted heterocyclyl containing at least one nitrogen atom. The terms "acylamino" and "(lower alkyl)amino" are as defined herein. The term "heterocyclyl containing at least one nitrogen atom" of the term "optionally substituted heterocyclyl containing at least one nitrogen atom" is a residue derived from a heterocycle that contains at least one nitrogen atom as defined herein.

Examples of substituents on the "optionally substituted heterocyclyl containing at least one nitrogen atom" include lower alkyl (e.g., methyl), (lower alkyl)carbonyl (e.g., acetyl), and hydroxy. When two or more substituents are present, the substituents may be the same or different.

Specific examples of B² include acylamino, such as acetylamino or ethylcarbonylamino; (lower alkyl)amino optionally substituted with amino or acetylamino, such as methylamino, ethylamino, dimethylamino, diethylamino, dipropylamino, ethylmethylamino, methylpropylamino, ethylpropylamino, ethyl(aminoethyl)amino, or ethyl(acetylaminoethyl)amino; heterocyclyl that is derived from a heterocycle containing at least one nitrogen atom, such as pyrrolidine, piperidine, piperazine, homopiperadine, morpholine, triethylenediamine, pyrazole, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, imidazole, oxazole, isoxazole, thiazole, isothiazole, or 2,5-diazabicyclo[2.2.1]heptane, wherein the heterocyclyl may be optionally substituted, for example, with methyl or acetyl; methyl substituted with (lower alkyl)amino or optionally substituted heterocyclyl containing at least one nitrogen atom, such as dimethylaminomethyl, pyrrolidylmethyl, piperidylmethyl, piperazinylmethyl, or morpholinomethyl.

B² is preferably hydrogen, dimethylamino, diethylamino, dipropylamino, ethylmethylamino, methylpropylamino, ethylpropylamino, acetylamino, morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, piperazinylmethyl, 2,5-diazabicyclo[2.2.1]hept-2-yl, or 5-acetyl-2,5-diazabicyclo[2.2.1]hept-2-yl.

Alternatively, B¹ and B² may together form a cyclic structure. The term "cyclic structure" may be 5- to 7-membered heterocyclic structure having at least one nitrogen atom as a ring-constituting atom, such as morpholine or piperazine. The "cyclic structure" may be optionally substituted, for example, with oxo, lower alkyl (e.g., methyl), lower haloalkyl (e.g., trifluoromethyl), or lower acyl (e.g., acetyl). When two or more substituents are present, the substituents may be the same or different.

In the formula (I), Y is a group of the formula (II): J-L-M, wherein J is a bond, lower alkylene, lower alkenylene, lower alkynylene, -(CH₂)_n-O-, -(CH₂)_n-NH-, -(CH₂)_n-CO-, or -(CH₂)_n-SO₂-; L is a bond, -O-, -NH-, -CO-, or -SO₂-; and M is a bond, lower alkylene, lower alkenylene, or lower alkynylene; with the proviso that, when J is -(CH₂)_n-O-, L is not -O-, -NH-, or -SO₂-; when J is -(CH₂)_n-NH-, L is not -O- or -NH-; when J is -(CH₂)_n-CO-, L is not -CO-; and when J is -(CH₂)_n-SO₂-, L is not -O- or -SO₂-, wherein n is an integer of from 0 to 6. The terms "lower alkylene", "lower alkenylene", and "lower alkynylene" recited for J or M are as defined herein.

Examples of the group of the formula (II) include -(CH₂)_n-, -(CH₂)_n-NH-(CH₂)_n'-, -(CH₂)_n-O-(CH₂)_n'-, -(CH₂)_n-CO-O-(CH₂)_n'-, -(CH₂)_n-O-CO-(CH₂)_n'-, -(CH₂)_n-CO-NH-(CH₂)_n'-, -(CH₂)_n-NH-CO-(CH₂)_n'-, -(CH₂)_n-SO₂-NH-(CH₂)_n'-, -(CH₂)_n-NH-SO₂-(CH₂)_n'-, vinyl, and ethynylene, wherein n and n' are independently an integer of from 0 to 6, preferably, an integer of from 0 to 3. The group of formula (II) is preferably -(CH₂)_n-, -(CH₂)_n-NH-(CH₂)_n'-, -(CH₂)_n-O-(CH₂)_n'-, -(CH₂)_n-CO-O-(CH₂)_n'-, -(CH₂)_n-CO-NH-(CH₂)_n'-, or ethynylene, and more preferably, -(CH₂)_n- or ethynylene. Specific examples of the group of formula (II) include -(CH₂)₂-, -CH₂-O-, -CH₂-NH-, -CO-O-, -CO-NH-, and ethynylene.

Y is preferably -(CH₂)₂- or ethynylene.

In the formula (I), B is -(CH₂)_m-CO-, -(CH₂)_m-O-CO-, -(CH₂)_m-S-CO-, or -(CH₂)_m-NR²-CO-, wherein m is an integer of from 0 to 6, and R² is hydrogen, lower alkyl, or acyl. The terms "lower alkyl" and "acyl" are as defined herein. Preferably, m is an integer of from 0 to 3.

Specific examples of B include -CO- (i.e., -(CH₂)_m-CO- wherein m is 0), -CH₂-CO-, -(CH₂)₂-CO-, -O-CO-, -CH₂-O-CO-, -(CH₂)₂-O-CO-, -(CH₂)₃-O-CO-, -CH₂-NH-CO-, -(CH₂)₂-NH-CO-, -(CH₂)₃-NH-CO-, -S-CO-, -CH₂-S-CO-, and -(CH₂)₂-S-CO-.

In the formula (I), D is -NR³-, wherein R³ is hydrogen, lower alkyl, acyl, or (lower alkoxy)carbonyl. The terms "lower alkyl", "acyl", and "(lower alkoxy)carbonyl" are as defined herein. D may be -NH- or -N(CH₃)-, preferably -NH-.

In the formula (I), E is optionally substituted amino, including unsubstituted amino, and amino substituted by one or two substituents. The term "optionally substituted amino" is represented by the formula: -NR⁶R⁷.

In the formula, R⁶ and R⁷ may be the same or different, and independently selected from hydrogen, optionally substituted lower alkyl, acyl, in particular (lower alkyl)carbonyl, alkoxycarbonyl, in particular (lower alkoxy)carbonyl, aryl, aralkyl, lower cycloalkyl, sulfuryl, sulfinyl, phosphoryl, and heterocyclyl. The terms "lower alkyl", "acyl", "(lower alkyl)carbonyl", "alkoxycarbonyl", "(lower alkoxy)carbonyl", "aryl", "aralkyl", and "lower cycloalkyl" are as defined herein. The term "heterocyclyl" is a residue derived from the "heterocycle" as defined herein.

Specific examples of each of R⁶ and R⁷ include hydrogen, lower alkyl (e.g., methyl or ethyl), acetyl, butanoyl, decanoyl, 3-hydroxypropanoyl, 6-hydroxyhexanoyl, ethoxycarbonyl, butoxycarbonyl, decyloxycarbonyl, and 2-hydroxyethoxycarbonyl.

E is preferably -NH₂.

Alternatively, E may be an amino that is protected with R⁶ and/or R⁷ by methods such as those described in "Protective Groups in Organic Synthesis 3rd Edition" (John Wiley and Sons, 1999) (i.e., R⁶ and/or R⁷ may be an amino protecting group).

The -B-D-E moiety of the formula (I) preferably has -CO-, -O-CO-, -CH₂-CO-, -(CH₂)₂-CO-, -CH₂-O-CO-, -(CH₂)₂-O-CO-, -(CH₂)₃-O-CO-, -CH₂-NH-CO-, -(CH₂)₂-NH-CO- or -(CH₂)₃-NH-CO- as B; -NH- as D; and -NH₂ as E. The -B-D-E moiety is preferably -CO-NH-NH₂, -O-CO-NH-NH₂, -CH₂-CO-NH-NH₂, -(CH₂)₂-CO-NH-NH₂, -CH₂-O-CO-NH-NH₂, -(CH₂)₂-O-CO-NH-NH₂, -(CH₂)₃-O-CO-NH-NH₂, -CH₂-NH-CO-NH-NH₂, -(CH₂)₂-NH-CO-NH-NH₂, or -(CH₂)₃-NH-CO-NH-NH₂.

When the compound of the formula (I) has an asymmetric carbon atom in its structure, the present disclosure encompasses all enantiomers and diastereomers of the compound.

In one embodiment, the compound of the formula (I) is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide of the following formula:

In another embodiment, the compound of the formula (I) is (4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate of the following formula:

The pharmaceutically acceptable salt as used herein includes salts with inorganic or organic base, and acid addition salts. Examples of salts with inorganic or organic base include alkali metal salts (e.g., sodium salt and potassium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), ammonium salts, and amine salts (e.g., triethylamine salt and N-benzyl-N-methylamine salt). The acid addition salts include salts with mineral acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid), and salts with organic acids (e.g., tartaric acid, acetic acid, trifluoroacetic acid, citric acid, malic acid, lactic acid, fumaric acid, maleic acid, benzoic acid, glycol acid, gluconic acid, succinic acid, and arylsulfonic acid such as p-toluenesulfonic acid).

The compound of the formula (I) may be prepared by methods as described in WO 2009/096609 or WO 2009/145360.

The compound having VAP-1 inhibitory activity is useful for the treatment of cancer. As used herein, the term "treatment of cancer" or "treating cancer" includes therapeutic and prophylactic treatment of cancer, such as prophylaxis of symptoms, and cure, amelioration, reduction, prevention of exacerbation, or any other control of symptoms.

The cancer includes leukemia, lymphoma, blastoma, carcinoma (malignant tumor derived from epithelial cells) and sarcoma (malignant tumor arising from connective tissue derived from non-epithelial cells). Examples of the cancer include large intestine cancer (including colon cancer and rectal cancer), melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, lung cancer (e.g., non-small-cell lung cancer), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or orbital malignant melanoma, uterine cancer, ovarian cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute/chronic leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia and chronic lymphoblastic leukemia, pediatric solid tumor, lymphocytic lymphoma, bladder cancer, ureteral cancer, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, cancer caused by exposure to asbestos or other environmental factor, and a combination thereof. The pharmaceutical composition of the present application is particularly useful for the treatment of carcinoma (malignant tumor derived from epithelial cells), or large intestine cancer (including colon cancer and rectal cancer), melanoma (e.g., metastatic malignant melanoma), kidney cancer, prostate cancer, breast cancer, and lung cancer (e.g., non-small-cell lung cancer).

The compound having VAP-1 inhibitory activity may be used in combination with a different agent or therapy that is effective for the treatment of cancer. In one embodiment, the compound having VAP-1 inhibitory activity is used in combination with an anti-CTLA-4 antibody. In a further embodiment, the compound having VAP-1 inhibitory activity is used in combination with an anti-CTLA-4 antibody and an anti-PD-1 antibody.

The antibody includes monoclonal and polyclonal antibodies and antibody fragments. The antibody may be a chimeric, humanized, or human antibody. The antibody fragments include heavy and light chain variable regions (VH and VL), F(ab')2, Fab', Fab, Fv, Fd, single-domain FV (sdFv), single-chain FV (scFV), and complexes thereof.

The term "CTLA-4" refers to Cytotoxic T-Lymphocyte Antigen 4, i.e., a member of the immunoglobulin superfamily, which is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. In addition, as used herein, the term "anti-CTLA-4 antibody" refers to an antibody that selectively binds to a CTLA-4 polypeptide. An exemplary anti-CTLA-4 antibody includes, but not limited to, e.g. ipilimumab and tremelimumab.

The term "PD-1" refers to programmed cell death protein 1, or a protein originally isolated from a T-cell hybridoma undergoing T-cell receptor activation-induced cell death. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. In addition, the term "anti-PD-1 antibody" refers to an antibody that selectively binds to a PD-1 polypeptide. An exemplary anti-PD-1 antibody includes, but not limited to, e.g. nivolumab and pembrolizumab.

The subject according to the present application is a mammal such as human, mouse, rat, swine, dog, cat, horse, or bovine. Preferably, the subject is human.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier together with the compound having VAP-1 inhibitory activity as an active ingredient. The pharmaceutically acceptable carrier may be any carrier generally used in pharmaceutical compositions unless it has undesired physicochemical properties such as solubility or reactivity for the compound having VAP-1 inhibitory activity, or it is not suitable for the route of administration.

The amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may vary depending on the type of formulation, and it is typically 0.00001 to 10.0 % by weight, 0.001 to 5 % by weight, or 0.001 to 1 % by weight.

Dosage forms, dosage amounts, and frequency of administration are appropriately determined according to diseases to be treated, condition of the subject, and target sites. Dosage forms for oral administration includes solid dosage forms (such as capsule, tablet, and powder) or liquid dosage forms (such as solution and suspension). Dosage forms for parenteral administration includes aseptic solutions or suspensions for injection or infusion. The oral solid formulation may contain a conventional vehicle. The oral liquid formulation may contain one or more additives such as aromatics, colorants, preservatives, stabilizers, solubilizers, or suspending agents. The parenteral formulation may be aseptic, aqueous or nonaqueous solutions or suspensions and may contain additives such as preservatives, stabilizers, buffer agents, solubilizers, and suspending agents. Where necessary, the formulation may further contain isotonic agents.

The pharmaceutical composition may be administered by any route. The route of administration includes systemic administration (such as oral administration or administration by injection) and local administration (such as ocular instillation, intraocular administration, or transdermal administration). The pharmaceutical composition may also be administered by transmucosal administration such as intranasal, buccal, intravaginal, intrarectal, or sublingual administration.

The compound of the formula (I) may be administered about 0.03 ng/kg body weight/day to about 300 mg/kg body weight/day, preferably about 0.003 μg/kg body weight/day to about 10 mg/kg body weight/day. Dosage regimens such as frequency of administration per day and intervals of administration are appropriately adjusted. For example, a daily dose may be administered by a single dose or several divided doses (such as two, three, or four doses) or continuously. The administration of the compound may be daily administration, or once every few days, once a week, once every few weeks, once a month, or once every few months.

The anti-CTLA-4 antibody and the anti-PD-1 antibody may be administered at about 0.0001 to 100 mg/kg body weight, preferably 0.01 to 10 mg/kg body weight, more preferably 0.3 to 10 mg/kg body weight/day, respectively. For example, the antibody may be administered at about 0.3 mg/kg body weight, about 1 mg/kg body weight, about 2 mg/kg body weight, about 3mg/kg body weight, about 5 mg/kg body weight, about 9 mg/kg body weight, or about 10 mg/kg body weight. The dosage amount of the antibody may be administered by a single dose or several divided doses (such as two, three, or four doses) or continuously. The administration of the antibody may be daily administration, or once every few days, once a week, once every few weeks, once a month, or once every few months. For example, the antibody may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferably, the anti-CTLA-4 antibody and the anti-PD-1 antibody are administered intravenously.

The term "about" as used herein means that the recited value may vary by plus or minus 30 %, preferably plus or minus 20 %, more preferably plus or minus 10%.

The compound having VAP-1 inhibitory activity and an agent used in combination may be contained in separate formulations, respectively, or in a single formulation. The compound having VAP-1 inhibitory activity and the combined agent may be provided in a kit. For example, the kit comprises a pharmaceutical composition comprising the compound having VAP-1 inhibitory activity and a pharmaceutical composition comprising an anti-CTLA-4 antibody, and optionally a pharmaceutical composition comprising an anti-PD-1 antibody. The pharmaceutical composition and kit of the present application may be distributed with package inserts that indicate dosage and administration of the compound having VAP-1 inhibitory activity, or dosage and administration of the compound having VAP-1 inhibitory activity for use in combination with the combined agent, and where appropriate, packages and/or written instructions.

The compound having VAP-1 inhibitory activity and the combined agent may be administered concurrently or separately. The term "concurrently" means that the compound having VAP-1 inhibitory activity and the combined agent are administered in the same administration schedule, and the compound and the agent may be contained in separate formulations, respectively, or in a single formulation. The term "separately" means that the compound having VAP-1 inhibitory activity and the combined agent are administered in different administration schedules, both of which may be individually determined.

For example, the present invention provides the followings:
1. A pharmaceutical composition for treating cancer, which comprises a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof.
2. The pharmaceutical composition of item 1, wherein the compound is represented by the formula (I):

X-Y-A-B-D-E (I)

wherein
X is

wherein
A¹ is a residue derived from benzene or a heterocycle containing at least one nitrogen or sulfur atom;
B¹ is hydrogen, hydroxy, halogen, lower alkyl, lower cycloalkyl, lower haloalkyl, lower alkoxy, acyl, acylamino, optionally substituted carbamoyl, (lower alkyl)sulfonylamino, or (lower alkyl)carbonyloxy, with the proviso that, when A¹ is a residue derived from thiazole, B¹ is not acylamino;
B² is hydrogen or a group containing at least one nitrogen atom, with the proviso that, when A¹ is a residue derived from thiazole, B² is not acylamino; or
alternatively, B¹ and B² may together form a cyclic structure;
A² is a divalent residue derived from optionally substituted thiazole;
R¹ is acyl;
Y is represented by the following formula:
J-L-M (II)
wherein
J is a bond, lower alkylene, lower alkenylene, lower alkynylene, -(CH₂)_n-O-, -(CH₂)_n-NH-, -(CH₂)_n-CO-, or -(CH₂)_n-SO₂-, wherein n is an integer of from 0 to 6;
L is a bond, -O-, -NH-, -CO-, or -SO₂-; and
M is a bond, lower alkylene, lower alkenylene, or lower alkynylene;
with the proviso that, when J is -(CH₂)_n-O-, L is not -O-, -NH-, or -SO₂-; when J is -(CH₂)_n-NH-, L is not -O- or -NH-; when J is -(CH₂)_n-CO-, L is not -CO-; and when J is -(CH₂)_n-SO₂-, L is not -O- or -SO₂-;
A is a divalent residue derived from optionally substituted benzene or optionally substituted thiophene;
B is -(CH₂)_m-CO-, -(CH₂)_m-O-CO-, -(CH₂)_m-S-CO-, or -(CH₂)_m-NR²-CO-, wherein R² is hydrogen, lower alkyl, or acyl, and m is an integer of from 0 to 6;
D is -NR³-, wherein R³ is hydrogen, lower alkyl, alkoxycarbonyl, or acyl; and
E is optionally substituted amino.
3. The pharmaceutical composition of item 2, wherein A¹ is a residue derived from benzene, pyridine, pyridazine, pyrimidine, pyrazine, imidazole, thiazole, or thiophene.
4. The pharmaceutical composition of any one of item 2 or 3, wherein B¹ is hydrogen, methyl, trifluoromethyl, acetylamino, aminocarbonyl (carbamoyl), aminocarbonylmethoxy, methylsulfonylamino, ethylsulfonylamino, or methylcarbonyloxy.
5. The pharmaceutical composition of any one of items 2-4, wherein B² is hydrogen, dimethylamino, diethylamino, dipropylamino, ethylmethylamino, methylpropylamino, ethylpropylamino, acetylamino, morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, piperazinylmethyl, 2,5-diazabicyclo[2.2.1]hept-2-yl, or 5-acetyl-2,5-diazabicyclo[2.2.1]hept-2-yl.
6. The pharmaceutical composition of any one of items 2-5, wherein A² is

7. The pharmaceutical composition of any one of items 2-6, wherein Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3, or ethynylene.
8. The pharmaceutical composition of any one of items 2-7, wherein -Y-A-B- is

9. The pharmaceutical composition of any one of items 2-8, wherein -B-D-E- is -CO-NH-NH₂, -O-CO-NH-NH₂, -CH₂-CO-NH-NH₂, -(CH₂)₂-CO-NH-NH₂, -CH₂-O-CO-NH-NH₂, -(CH₂)₂-O-CO-NH-NH₂, -(CH₂)₃-O-CO-NH-NH₂, -CH₂-NH-CO-NH-NH₂, -(CH₂)₂-NH-CO-NH-NH₂, or -(CH₂)₃-NH-CO-NH-NH₂.
10. The pharmaceutical composition of any one of items 2-9, wherein A¹ is a residue derived from pyridine.
11. The pharmaceutical composition of any one of items 2-10, wherein B¹ is hydrogen.
12. The pharmaceutical composition of any one of items 2-11, wherein B² is morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, or piperazinylmethyl.
13. The pharmaceutical composition of any one of items 2-12, wherein Y is -(CH₂)_n- and n is an integer of from 0 to 3.
14. The pharmaceutical composition of any one of items 2-13, wherein -Y-A-B- is

15. The pharmaceutical composition of any one of items 2-14, wherein B is -(CH₂)_m-CO- and m is an integer of from 0 to 3.
16. The pharmaceutical composition of any one of items 2-14, wherein B is -(CH₂)_m-O-CO- and m is an integer of from 0 to 3.
17. The pharmaceutical composition of any one of items 2-16, wherein D is -NH-.
18. The pharmaceutical composition of any one of items 2-17, wherein E is -NH₂.
19. The pharmaceutical composition of any one of items 2-18, wherein
X is

wherein A¹ is a residue derived from benzene, pyridine, pyridazine, pyrimidine, pyrazine, imidazole, thiazole, or thiophene;
B¹ is hydrogen, methyl, trifluoromethyl, acetylamino, aminocarbonyl (carbamoyl), aminocarbonylmethoxy, methylsulfonylamino, ethylsulfonylamino, or methylcarbonyloxy;
B² is hydrogen, dimethylamino, diethylamino, dipropylamino, ethylmethylamino, methylpropylamino, ethylpropylamino, acetylamino, morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, piperazinylmethyl, 2,5-diazabicyclo[2.2.1]hept-2-yl, or 5-acetyl-2,5-diazabicyclo[2.2.1]hept-2-yl;
A² is

R¹ is acyl;
Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3, or ethynylene;
-Y-A-B- is

and
-B-D-E- is -CO-NH-NH₂, -O-CO-NH-NH₂, -CH₂-CO-NH-NH₂, -(CH₂)₂-CO-NH-NH₂, -CH₂-O-CO-NH-NH₂, -(CH₂)₂-O-CO-NH-NH₂, -(CH₂)₃-O-CO-NH-NH₂, -CH₂-NH-CO-NH-NH₂, -(CH₂)₂-NH-CO-NH-NH₂, or -(CH₂)₃-NH-CO-NH-NH₂.
20. The pharmaceutical composition of any one of items 2-18, wherein
X is

wherein A¹ is a residue derived from pyridine;
B¹ is hydrogen;
B² is morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, or piperazinylmethyl;
Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3;
-Y-A-B- is

B is -(CH₂)_m-CO-, wherein m is an integer of from 0 to 3;
D is -NH-; and
E is -NH₂.
21. The pharmaceutical composition of any one of items 2-18, wherein the compound is represented by the formula (I):

X-Y-A-B-D-E (I)

wherein
X is R¹-NH-A²-
wherein A² is

R¹ is C₁-C₆ alkylcarbonyl;
Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3;
-Y-A-B- is

B is -(CH₂)_m-O-CO-, wherein m is an integer of from 0 to 3;
D is -NH-; and
E is -NH₂.
22. The pharmaceutical composition of any one of items 2-20, wherein the compound is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide.
23. The pharmaceutical composition of any one of items 2-19 and 21, wherein the compound is 4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate.
24. The pharmaceutical composition of any one of items 1-23, wherein the pharmaceutical composition is for use in combination with an anti-CTLA-4 antibody.
25. The pharmaceutical composition of item 24, wherein the anti-CTLA-4 antibody is ipilimumab or tremelimumab.
26. The pharmaceutical composition of any one of items 1-25, wherein the pharmaceutical composition is for use in combination with an anti-PD-1 antibody.
27. The pharmaceutical composition of item 26, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.
28. The pharmaceutical composition of any one of items 1-27, wherein the cancer is large intestine cancer, melanoma, kidney cancer, prostate cancer, breast cancer, or lung cancer.
29. A pharmaceutical composition for treating cancer comprising an anti-CTLA-4 antibody, which is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof.
30. The pharmaceutical composition of item 29, wherein the compound is the compound recited in any one of items 2-23.
31. The pharmaceutical composition of item 30, wherein the compound is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide.
32. The pharmaceutical composition of item 30, wherein the compound is 4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate.
33. The pharmaceutical composition of any one of items 29-32, wherein the pharmaceutical composition is for use in combination with an anti-PD-1 antibody.
34. A pharmaceutical composition for treating cancer comprising an anti-PD-1 antibody, which is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof and an anti-CTLA-4 antibody.
35. The pharmaceutical composition of item 34, wherein the compound is the compound recited in any one of items 2-23.
36. The pharmaceutical composition of item 35, wherein the compound is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide.
37. The pharmaceutical composition of item 35, wherein the compound is 4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate.
38. A method of treating cancer, comprising administering a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof to a mammal in need thereof.
39. The method of item 38, further comprising administering an anti-CTLA-4 antibody to the mammal.
40. The method of item 38 or 39, further comprising administering an anti-PD-1 antibody to the mammal.
41. Use of a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof for the manufacture of a medicament for treating cancer.
42. The use of item 41, wherein the medicament is for use in combination with an anti-CTLA-4 antibody.
43. The use of item 41 or 42, wherein the medicament is for use in combination with an anti-PD-1 antibody.
44. A compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof for use in the treatment of cancer.
45. The compound of item 44, wherein the compound is for use in combination with an anti-CTLA-4 antibody.
46. The compound of item 44 or 45, wherein the compound is for use in combination with an anti-PD-1 antibody.
47. Use of an anti-CTLA-4 antibody for the manufacture of a medicament for treating cancer, wherein the medicament is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof.
48. An anti-CTLA-4 antibody for use in the treatment of cancer, wherein the anti-CTLA-4 antibody is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof.
49. Use of an anti-PD-1 antibody for the manufacture of a medicament for treating cancer, wherein the medicament is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof and an anti-CTLA-4 antibody.
50. An anti-PD-1 antibody for use in the treatment of cancer, wherein the anti-PD-1 antibody is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof and an anti-CTLA-4 antibody.

The present application will be described in detail with reference to the following examples, which, however, are not intended to limit the scope of the present application.

1. Methods
(1) Administration of a VAP-1 inhibitor
Colon carcinoma MC38 cells (5 × 10⁵ cells/100 μl) were subcutaneously transplanted into lateral abdominal sites of C57/B6j mice (female, 6 weeks; CLEA Japan, Inc.). Five days after transplantation, development of tumor was confirmed and tumor volume was calculated according to the following formula: (maximum tumor diameter) x (minimum tumor diameter) x (minimum tumor diameter)/2. The mice were divided into two groups showing no significant difference. To one group, Compound A (2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide) (10 mg/kg in 100 μl of saline, pH 6.0) was intraperitoneally administered daily, once per day, for two weeks. To another group, which is a control group, 100 μl of the solvent (saline, pH 6.0) was intraperitoneally administered. The tumor volume was determined once every two or three days. After the two-week administration, effects of the compound were analyzed.

NOG mice (Central Institute for Experimental Animals) and BALB/c mice (female, 6 weeks; CLEA Japan, Inc.) that received MC38 cells and colon carcinoma CT26 cells, respectively, were also examined in the same manner as the C57/B6j mice.

To MC38-transplanted C57/B6j mice, Compound B (4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate) in 100 μl of 0.5% methylcellulose solution (100 mg/kg or 500 mg/kg) was orally administered by a probe daily, once per day. Tumor volume was determined in the same manner as the C57/B6j mice that received Compound A.

(2) Combination use of a VAP-1 inhibitor and anti-CTLA-4 or anti-PD-1 antibody
An anti-CTLA-4 antibody (InVivoMAb anti m CTLA4, Clone: 9D9, Isotype: Mouse IgG2b, BioXcell, catalog number BE0164) or an isotype control thereof (InVivoMAb Mouse IgG2b Isotype control; Unknown Specificity, Clone: MPC-11, BioXcell, catalog number BE0086), or an anti-PD-1 antibody (InVivoMAb anti m PD-1, Clone: RMP1-14, Isotype: Rat IgG2a, BioXcell, catalog number BE0146) or an isotype control thereof (InVivoMAb Polyclonal Rat IgG, Clone: Rat IgG, catalog number BE0094) (100 mg/kg in 100 μl saline, pH 6.0) was intraperitoneally administered three times in total, on the date of first administration of Compound A (day 1), three days after the first administration (day 4), and six days after the first administration (day 7). Tumor volume was determined as described above. Four-group comparison was performed between the control group, the Compound A group, the combination group of Compound A and the anti-CTLA-4 antibody, and the combination group of Compound A and the anti-PD-1 antibody.

(3) Triplet combination of a VAP-1 inhibitor, anti-CTLA-4 antibody and anti-PD-1 antibody
As described in Section 1(2) above, the anti-CTLA-4 antibody and the anti-PD-1 antibody or isotype controls thereof (100 mg/kg in 100 μl saline, pH 6.0, respectively) were intraperitoneally administered on the date of first administration of Compound A (day 1), three days after the first administration (day 4), and six days after the first administration (day 7) and tumor volume was determined as described above. Four-group comparison was performed between the control group, the Compound A group, the doublet combination group of the anti-CTLA-4 antibody and the anti-PD-1 antibody, and the triplet combination group of Compound A, the anti-CTLA-4 antibody, and the anti-PD-1 antibody.

(4) CD8 depletion model
To the mice of Section 1(2) above, an anti-CD8 antibody (InVivoMAb anti m CD8a, Clone: 53.6.72, Isotype: RatIgG2a, BioXcell, catalog number BE0004-1) or an isotype control thereof (InVivoMAb Polyclonal Rat IgG, Clone: Rat IgG, catalog number BE0094) (100 mg/kg in 100 μl saline, pH 6.0) was further administered three times in total, on the date of first administration of Compound A (day 1), three days after the first administration (day 4), and six days after the first administration (day 7). Tumor volume was determined as described above. Four-group comparison was performed between the control group, the Compound A group, the anti-CD8 antibody group, and the combination group of Compound A and the anti-CD8 antibody.

(5) Induction of tumor antigen-specific CD8 cells
CD8 positive cells were sorted out from tumor mass or draining lymph node by CD8a (Ly-2) microbeads, mouse (Miltenyi Biotec, 130-049-401). As antigen presenting cells, 32 Gy-irradiated spleen cells of a healthy mouse were used. The CD8 positive cells and the antigen presenting cells were mixed and MC38-specific tumor antigen, Gp70, (1 mg/ml) or β-gal as a control (1 mg/ml) was added to the mixture. The cells were cultured for 5 days to induce tumor-specific CD8 cells. The cells were then collected, and Gp70 (1 mg/ml) or β-gal (1 mg/ml) was again added to the collected cells. After one day of culture, the supernatant of the cells was collected. The amount of IFN-γ in the supernatant was determined by IFN-γ ELISA Set (Mouse IFN-γ ELISA, SetBD OptEIA^TM, catalog number 555138).

(6) FACS
Single-cell suspension was prepared from tumor mass and treated with 2.4G2 blocking antibody (Anti-mouse CD16/CD32, Clone: 2.4G2, Bay bioscience, catalog number 40-0161). The single-cell suspension was stained with each of the following antibodies. For intranuclear staining of Foxp3, FOXP3 Fix/Perm Buffer Set (Biolegend, catalog number 421403) was used.

CD4 (PE/Dazzle 594, RatIgG2a, RM4-5, Biolegend 100566)
Foxp3 (PC5.5, RatIgG2a, FJK-16s, eBioscience 35-5773-82)
CD25 (PC7, RatIgG2b, PC61, BD 552880)
CTLA-4 (APC, HamIgG, UC10-4B9, Biolegend 106310)
CD8 (Alexa700, RatIgG2a, 53-6.7 Biolegend 100730)
PD-1 (APCCY7, RatIgG1 29F.1A12, Biolegend 135224)
CD3 (BrilliantViolet421, HamIgG, 145-2c-11, Biolegend 100336)
CD45 (V500, RatIgG2b, 30-F11, BD 561487)

(7) Cell proliferation assay
MC38 cells were seeded onto 96-well flat-bottom plates at 5 x 10³ cells/well. Direct cytotoxicity was determined with serially-diluted Compound A from 100 μg/ml, which was 10 times higher than the blood concentration of Compound A in mice intraperitoneally administered at 10 mg/kg in Section 1(1) above (10 μg/ml). Compound A was added to RPMI medium at 100 μg/ml, and RPMI medium containing 50, 25, 12.5, 6.25, 3.125, or 1.56 μg/ml, or at 782, 391, 196, or 98 ng/ml was prepared by two-fold serial dilutions. RPMI medium without Compound A was used as a control. The medium was added to the plate containing MC38 cells, and the cells were cultured for three days. The assays were performed in triplicate. Proliferation of cancer cells were measured by Premix WST-1 Cell Proliferation Assay System (Takara Bio Inc., product code MK400).

(8) Statistical analysis
Data was analyzed by t test to compare two independent groups. In the test, P<0.05 was considered statistically significant.

2. Results
(1) Anti-tumor effects of Compound A
Tumor volume in MC38-transplanted C57/B6j mice was determined as described in Section 1(1). As shown in Fig. 1A, Compound A significantly reduced tumor volume compared to the control.

CD8⁺ cells were obtained from tumor mass or draining lymph node, and IFN-γ production by the CD8⁺ cells in response to tumor antigen Gp70 was determined. As shown in Fig. 1B, Compound A, which is labeled as "VAP-1" in the figure, increased tumor antigen-specific IFN-γ production.

Subsequently, it was determined if Compound A directly affected proliferation of tumor cells. As shown in Fig. 1C, Compound A suppressed proliferation of MC38 cells at 50 μg/ml or more while there was no clear suppression up to 25 μg/ml. Thus, Compound A did not affect proliferation of the tumor cell line at the concentration used in vivo experiments in Section 1(1).

(2) Synergistic anti-tumor effects of Compound A and anti-CTLA-4 antibody
Combination effects of Compound A and an anti-CTLA-4 antibody on tumor volume were determined as described in Section 1(2). As shown in Fig. 2, the combination of Compound A and the anti-CTLA-4 antibody ("combination") significantly reduced tumor volume compared to the control ("control") and the anti-CTLA-4 antibody ("CTLA4"). Also, the reduction of tumor volume observed with the combination was greater than that observed with Compound A although the difference was not statistically significant.

(3) Reduction of sVAP-1 activity by Compound A
Peripheral blood was collected from the MC38-transplanted model mice described in Section 2(1) after the two-week administration of Compound A. Serum sVAP-1 activity was measured according to the following protocol. As shown in Fig. 3, serum sVAP-1 activity was significantly decreased in the MC38-transplanted model mice compared to normal mice, and further decreased by administration of Compound A.

Measurement of VAP-1/SSAO activity
(a) Frozen plasma was mixed with water, quickly thawed, and centrifuged at 1,000 x g for 3 min at 4°C.
(b) In a 2 ml polypropylene tube, 50 μl of plasma sample and 100 μl of reaction solution (final concentration: 50 mM phosphate buffer, pH 7.4 and 0.5 mM pargyline) were mixed to prepare a reaction mixture.
(c) The reaction mixture was pre-incubated on a shaker for 20 min at room temperature.
(d) To the reaction mixture, 50 μl of substrate solution (final concentration: 10 μM ¹⁴C-benzylamine) was added, and the resulting reaction mixture was mixed gently.
(e) The reaction mixture was incubated on a shaker 24 h at 37°C.
(f) To the reaction mixture, 200 μl of citric acid (2 M) was added, and the resulting reaction mixture was mixed to terminate the enzyme reaction.
(g) The reaction mixture was mixed with 1 ml of a toluene/ethyl acetate mixture (1:1, v/v), and centrifuged at 1,700 x g for 5 min at room temperature.
(h) To prepare a sample for radioactivity measurement, 750 μl of supernatant was collected and mixed with 3 ml of scintillation cocktail (Ultima Gold).
(i) Radioactivity of the sample was measured for 5 min by a liquid scintillation counter.
(j) The amount of radioactivity of the supernatant was calculated from that of the measured sample, and divided by the specific radioactivity of ¹⁴C-benzylamine to calculate the amount of ⁴C-benzaldehyde produced (pmol). The amount of ⁴C-benzaldehyde was divided by the volume of the measured sample (ml) and reaction time (min) to calculate VAP-1/SSAO activity (pmol/mL/min).
(k) The VAP-1/SSAO activity (pmol/mL/min) was divided by the concentration of total protein in the sample measured by Bradford assay to calculate VAP-1/SSAO activity per mg total protein (pmol/mg protein/min).

(4) Effects of Compound A on different mouse tumor model
Effects of Compound A on CT26-transplanted BALB/c mice were also examined as described in Section 2(1) for the MC38-transplanted model mice. As shown in Fig. 4, Compound A significantly reduced tumor volume compared to the control in both mouse models.

(5) Effects of Compound A on NOG mouse and CD8 depletion models
Effects of Compound A were examined in immunodeficient NOG mouse and CD8 depletion models, the results of which are shown in Fig.5 at the left and right sides, respectively. As shown in Fig. 5, tumor growth in MC38-transplanted NOG mice ("control" at left side) was faster than that in MC38-transplanted C57B6j mice ("control" at right side). In MC38-transplanted NOG mice, the tumor growth was not affected by administration of Compound A. In the CD8 depletion model mice, i.e. MC38-transplanted C57B6j mice that received the anti-CD8 antibody to have CD8⁺ cells depleted, anti-tumor effect of Compound A was not observed ("VAP-1+dCD8"). Also, as shown in Fig. 5 at right side, tumor volume in mice that received the anti-CD8 antibody ("dCD8" or "VAP-1+dCD8") was increased compared to the control ("control"). These results suggest that tumor growth in MC38-transplanted mice was suppressed to some extent by immune response, in particular by CD8-mediated immune response, and the suppressive effect is enhanced by theVAP-1 inhibitor.

(6) FACS analysis of tumor-infiltrating lymphocytes
From tumor mass of MC38-transplanted C57B6j mice, single cell suspension was prepared and analyzed by FACS. CD3⁺/CD45⁺ cells were gated and analyzed as tumor-infiltrating lymphocytes (TIL). The CD3⁺/CD45⁺ cells were divided into CD4⁺ or CD8⁺ cells, and then CD4⁺CD25⁺FoxP3⁺ cells were analyzed as regulatory T cells (Treg) (Fig. 6, upper). In CD8⁺ cells, expression of T cell activation markers, PD-1 and CTLA-4, was determined (Fig. 6, lower).

(7) Increase of tumor-infiltrating CD8⁺ cells by Compound A
From the results of FACS analysis of Section 2(6), the absolute numbers of CD3⁺, CD4⁺, or CD8⁺ cells and Treg as well as the relative ratio of each type of cells were determined in the tumor-infiltrating lymphocytes of MC38-transplanted C57B6j mice. As shown in Fig. 7, Compound A increased the absolute number and ratio of CD8⁺ cells in the tumor-infiltrating lymphocytes, and decreased the ratio of Treg in CD4⁺ cells.

(8) Induction of T cell activation by Compound A
From the results of FACS analysis of Section 2(6), the absolute number and relative ratio of activated T cells were determined in the tumor-infiltrating lymphocytes of MC38-transplanted C57B6j mice. As shown in Fig. 8, Compound A increased the absolute number of cells positive for the T cell activation marker, PD-1 or CTLA-4.

(9) Effects of Compound A on T cell population in draining lymph node
In addition to the tumor-infiltrating lymphocytes, T cell population including the number of activated T cells in draining lymph node was analyzed in MC38-transplanted C57B6j mice. As shown in Fig. 9, Compound A did not change the T cell population in draining lymph node.

(10) Effects of Compound A on myeloid-derived suppressor cells
Effects of Compound A on tumor myeloid-derived suppressor cells (MDSCs) were examined in MC38-transplanted C57B6j mice. As shown in Fig. 10, Compound A decreased infiltration of granulocytic MDSCs (G-MDSCs) (CD11b⁺Gr-1^high), which were potent immune-suppressive cells. This result suggests that the anti-tumor effect of the VAP-1 inhibitor is mediated, at least in part, by the change of tumor-infiltrating MDSCs.

(11) Effects of Compound A on dendritic cell activation in tumor
Dendritic cell (DC) activation in tumor was examined in MC38-transplanted C57B6j mice. As shown in Fig. 11, the number of CD80⁺, CD83⁺, PD-L1⁺, or CD86⁺ cells was not changed by the administration of Compound A. These results demonstrate that the VAP-1 inhibitor does not affect DC activation.

(12) Anti-tumor effect of Compound B
As described in Section 2(1) above, anti-tumor effect of Compound B orally administered to MC38-transplanted C57B6j mice (100 mg/kg or 500 mg/kg, in 100 μl saline, pH 6.0) was determined. As shown in Fig. 12, Compound B decreased tumor volume in a dose-dependent manner although the difference was not statistically significant.

(13) Anti-tumor effect of combination of Compound A and anti-PD-1 antibody
As described in Section 2(2) above, effects of combination of Compound A and an anti-PD-1 antibody on tumor volume was determined. As shown in Fig. 13, Compound A, the anti-PD-1 antibody, and combination of Compound A and the anti-PD-1 antibody (see "VAP-1", "PD-1", and "combination" in Fig. 13, respectively) decreased tumor volume compared to the control (see "control" in Fig. 13) although there is no statistical significance between the combination group and the control group.

(14) Increase of PD-1 positive cells by Compound A
From the results of FACS analysis of Section 2(6), the ratio of PD-1⁺ cells in tumor-infiltrating lymphocytes was determined in MC38-transplanted C57B6j mice that received Compound A, the anti-CTLA-4 antibody, or combination of Compound A and the anti-CTLA-4 antibody. As shown in Fig. 14 at left side, PD-1 expression in CD8⁺ cells of the combination group was relatively higher than that of the control group, the Compound A group, or the anti-CTLA-4 antibody group. These results suggest that the combination of Compound A and the anti-CTLA-4 antibody produces immunological environment that facilitates activation of tumor-infiltrating CD8⁺ cells compared to the compound or antibody alone. Also, as shown in Fig. 14 at right side, the combination increased the ratio of cells positive for a Treg marker, Foxp3 or PD-1, suggesting improvement of immunosuppressive environment.

(15) Triplet combination of Compound A, anti-CTLA-4 antibody, and anti-PD-1 antibody
According to the method in Section 1(3) above, effects of triplet combination of Compound A, an anti-CTLA-4 antibody, and an anti-PD-1 antibody on tumor volume was examined. As shown in Fig. 15, tumor volume was significantly decreased either with Compound A alone, with doublet combination of the anti-CTLA-4 antibody and the anti-PD-1 antibody, or with triplet combination of Compound A, the anti-CTLA-4 antibody and the anti-PD-1 antibody. Notably, tumor was disappeared in 2 of 5 mice in the triplet combination group but not in the doublet combination group (data not shown).

Claims (30)

1. A pharmaceutical composition for treating cancer, which comprises a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof.
2. The pharmaceutical composition of claim 1, wherein the compound is represented by the formula (I):
   
   X-Y-A-B-D-E (I)
   
   wherein
   X is
   

   

   wherein
   A¹ is a residue derived from benzene or a heterocycle containing at least one nitrogen or sulfur atom;
   B¹ is hydrogen, hydroxy, halogen, lower alkyl, lower cycloalkyl, lower haloalkyl, lower alkoxy, acyl, acylamino, optionally substituted carbamoyl, (lower alkyl)sulfonylamino, or (lower alkyl)carbonyloxy, with the proviso that, when A¹ is a residue derived from thiazole, B¹ is not acylamino;
   B² is hydrogen or a group containing at least one nitrogen atom, with the proviso that, when A¹ is a residue derived from thiazole, B² is not acylamino; or
   alternatively, B¹ and B² may together form a cyclic structure;
   A² is a divalent residue derived from optionally substituted thiazole;
   R¹ is acyl;
   Y is represented by the following formula:
   J-L-M (II)
   wherein
   J is a bond, lower alkylene, lower alkenylene, lower alkynylene, -(CH₂)_n-O-, -(CH₂)_n-NH-, -(CH₂)_n-CO-, or -(CH₂)_n-SO₂-, wherein n is an integer of from 0 to 6;
   L is a bond, -O-, -NH-, -CO-, or -SO₂-; and
   M is a bond, lower alkylene, lower alkenylene, or lower alkynylene;
   with the proviso that, when J is -(CH₂)_n-O-, L is not -O-, -NH-, or -SO₂-; when J is -(CH₂)_n-NH-, L is not -O- or -NH-; when J is -(CH₂)_n-CO-, L is not -CO-; and when J is -(CH₂)_n-SO₂-, L is not -O- or -SO₂-;
   A is a divalent residue derived from optionally substituted benzene or optionally substituted thiophene;
   B is -(CH₂)_m-CO-, -(CH₂)_m-O-CO-, -(CH₂)_m-S-CO-, or -(CH₂)_m-NR²-CO-, wherein R² is hydrogen, lower alkyl, or acyl, and m is an integer of from 0 to 6;
   D is -NR³-, wherein R³ is hydrogen, lower alkyl, alkoxycarbonyl, or acyl; and
   E is optionally substituted amino.
3. The pharmaceutical composition of claim 2, wherein
   X is
   

   

   wherein A¹ is a residue derived from benzene, pyridine, pyridazine, pyrimidine, pyrazine, imidazole, thiazole, or thiophene;
   B¹ is hydrogen, methyl, trifluoromethyl, acetylamino, aminocarbonyl (carbamoyl), aminocarbonylmethoxy, methylsulfonylamino, ethylsulfonylamino, or methylcarbonyloxy;
   B² is hydrogen, dimethylamino, diethylamino, dipropylamino, ethylmethylamino, methylpropylamino, ethylpropylamino, acetylamino, morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, piperazinylmethyl, 2,5-diazabicyclo[2.2.1]hept-2-yl, or 5-acetyl-2,5-diazabicyclo[2.2.1]hept-2-yl;
   A² is
   

   

   R¹ is acyl;
   Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3, or ethynylene;
   -Y-A-B- is
   

   

   and
   -B-D-E- is -CO-NH-NH₂, -O-CO-NH-NH₂, -CH₂-CO-NH-NH₂, -(CH₂)₂-CO-NH-NH₂, -CH₂-O-CO-NH-NH₂, -(CH₂)₂-O-CO-NH-NH₂, -(CH₂)₃-O-CO-NH-NH₂, -CH₂-NH-CO-NH-NH₂, -(CH₂)₂-NH-CO-NH-NH₂, or -(CH₂)₃-NH-CO-NH-NH₂.
4. The pharmaceutical composition of claim 2, wherein
   X is
   

   

   wherein A¹ is a residue derived from pyridine;
   B¹ is hydrogen;
   B² is morpholino, piperidino, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-acetylpiperidinyl, morpholinomethyl, or piperazinylmethyl;
   Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3;
   -Y-A-B- is
   

   

   B is -(CH₂)_m-CO-, wherein m is an integer of from 0 to 3;
   D is -NH-; and
   E is -NH₂.
5. The pharmaceutical composition of claim 2, wherein
   X is R¹-NH-A²-
   wherein A² is
   

   

   R¹ is C₁-C₆ alkylcarbonyl;
   Y is -(CH₂)_n-, wherein n is an integer of from 0 to 3;
   -Y-A-B- is
   

   

   B is -(CH₂)_m-O-CO-, wherein m is an integer of from 0 to 3;
   D is -NH-; and
   E is -NH₂.
6. The pharmaceutical composition of any one of claims 2-4, wherein the compound is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide.
7. The pharmaceutical composition of any one of claims 2, 3 and 5, wherein the compound is 4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate.
8. The pharmaceutical composition of any one of claims 1-7, wherein the pharmaceutical composition is for use in combination with an anti-CTLA-4 antibody.
9. The pharmaceutical composition of claim 8, wherein the anti-CTLA-4 antibody is ipilimumab or tremelimumab.
10. The pharmaceutical composition of any one of claims 1-9, wherein the pharmaceutical composition is for use in combination with an anti-PD-1 antibody.
11. The pharmaceutical composition of claim 10, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.
12. The pharmaceutical composition of any one of claims 1-11, wherein the cancer is large intestine cancer, melanoma, kidney cancer, prostate cancer, breast cancer, or lung cancer.
13. A pharmaceutical composition for treating cancer comprising an anti-CTLA-4 antibody, which is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof.
14. The pharmaceutical composition of claim 13, wherein the compound is the compound recited in any one of claims 2-7.
15. The pharmaceutical composition of claim 14, wherein the compound is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide.
16. The pharmaceutical composition of claim 14, wherein the compound is 4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate.
17. The pharmaceutical composition of any one of claims 13-16, wherein the pharmaceutical composition is for use in combination with an anti-PD-1 antibody.
18. A pharmaceutical composition for treating cancer comprising an anti-PD-1 antibody, which is for use in combination with a compound having VAP-1 inhibitory activity or a pharmaceutically acceptable salt thereof and an anti-CTLA-4 antibody.
19. The pharmaceutical composition of claim 18, wherein the compound is the compound recited in any one of claims 2-7.
20. The pharmaceutical composition of claim 19, wherein the compound is 2-(4-{2-[5-(4-acetylpiperazin-1-yl)pyridin-2-yl]ethyl}phenyl)acetohydrazide.
21. The pharmaceutical composition of claim 19, wherein the compound is 4-{2-[2-(acetylamino)-1,3-thiazol-4-yl]ethyl}benzyl hydrazinecarboxylate.
22. A method of treating cancer, comprising administering a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof to a mammal in need thereof.
23. The method of claim 22, further comprising administering an anti-CTLA-4 antibody to the mammal.
24. The method of claim 22 or 23, further comprising administering an anti-PD-1 antibody to the mammal.
25. Use of a compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof for the manufacture of a medicament for treating cancer.
26. The use of claim 25, wherein the medicament is for use in combination with an anti-CTLA-4 antibody.
27. The use of claim 25 or 26, wherein the medicament is for use in combination with an anti-PD-1 antibody.
28. A compound having VAP-1 inhibitory activity or a pharmaceutically salt thereof for use in the treatment of cancer.
29. The compound of claim 28, wherein the compound is for use in combination with an anti-CTLA-4 antibody.
30. The compound of claim 28 or 29, wherein the compound is for use in combination with an anti-PD-1 antibody.
    

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