Antitumor composition

Abstract

An object of the present invention is to provide a new combination of antitumor agents, which can significantly reduce tumors through synergistic antitumor effects while minimizing the increase of side effects. The present invention provides an antitumor composition, which comprises the following compound (B) or a salt thereof used in combination with the following antitumor agent (A): (A) at least one antitumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors; (B) a compound represented by the following formula (1) or a salt thereof: wherein R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms; R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

Description

Technical Field

[0001] The present disclosure relates to antitumor compositions, methods, etc. for preventing or treating tumors. The entire contents of all documents cited in this specification, including the following prior art documents (patent documents and non-patent documents), are incorporated herein by reference. Background Art

[0002] The number of deaths from malignant tumors (cancer) is on the rise. Therefore, better methods for treating malignant tumors are always desirable and such methods are always being developed.

[0003] [Citation List]

[0004] [Patent Document]

[0005] PTL 1: WO2012 / 046825 Summary of the invention

[0006] [Technical issues]

[0007] Generally, when an anti-tumor agent is administered alone in chemotherapy for tumors, especially malignant tumors, there are limitations in terms of side effects, etc., and sufficient anti-tumor effects cannot be exerted. Therefore, multi-drug combination therapy combining two or three or more drugs is performed in clinical settings. This combination therapy aims to reduce side effects or enhance anti-tumor effects by combining different anti-tumor agents, such as 1) reducing insensitive cell populations, 2) preventing or delaying the emergence of drug resistance, and 3) dispersing toxicity by combining drugs with different toxicity characteristics.

[0008] However, even if different antitumor agents are randomly combined for combination therapy, it is not always possible to obtain an effect of enhancing the antitumor effect. In addition, when multiple antitumor agents are used in combination, side effects tend to increase together with the antitumor effect. For this reason, there is a demand for a novel combination of antitumor agents that can significantly reduce tumors through synergistic antitumor effects while minimizing the increase in side effects.

[0009] [Technical solution]

[0010] The present inventors have found that the combined use of a specific antitumor agent and a specific compound can achieve an antitumor effect much higher than that achieved by using them alone, and have made further improvements.

[0011] The present disclosure covers subject matter such as described in the following items.

[0012] Item 1. An antitumor composition comprising the following compound (B) or a salt thereof used in combination with the following antitumor agent (A):

[0013] (A) at least one anti-tumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors;

[0014] (B) a compound represented by the following formula (1) or a salt thereof:

[0015]

[0016] in

[0017] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0018] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0019] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0020] Item 2. An antitumor composition comprising the following compound (B) or a salt thereof for administration to a person who will receive or has received the following antitumor agent (A):

[0021] (A) at least one anti-tumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors;

[0022] (B) a compound represented by the following formula (1) or a salt thereof:

[0023]

[0024] in

[0025] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0026] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0027] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0028] Item 3. An anti-tumor composition comprising:

[0029] The following antitumor agent (A), and

[0030] The following compound (B) or a salt thereof:

[0031] (A) at least one anti-tumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors;

[0032] (B) a compound represented by the following formula (1) or a salt thereof:

[0033]

[0034] in

[0035] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0036] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0037] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0038] Item 4. The composition according to any one of items 1 to 3, wherein compound (B) or a salt thereof is at least one compound or a salt thereof selected from the following:

[0039] N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0040] N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0041] N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0042] 2-Fluoro-N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0043] 2,3,4-trifluoro-N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}benzamide,

[0044] N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0045] N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0046] N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0047] 2-Fluoro-N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0048] N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0049] N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0050] N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0051] N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0052] N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0053] 2-Fluoro-N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0054] 2,3,4-trifluoro-N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}benzamide,

[0055] 2,3,4-trifluoro-N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}benzamide,

[0056] N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0057] 2-fluoro-N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide, and

[0058] 2,3,4-Trifluoro-N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}benzamide.

[0059] Item 5. A pharmaceutical kit comprising:

[0060] The following antitumor agent (A), and

[0061] An antitumor composition comprising the following compound (B) or a salt thereof:

[0062] (A) at least one anti-tumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors;

[0063] (B) a compound represented by the following formula (1) or a salt thereof:

[0064]

[0065] in

[0066] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0067] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0068] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0069] Item 6. A method for preventing or treating tumors, the method comprising administering to a subject the following anti-tumor agent (A) and the following compound (B) or a salt thereof:

[0070] (A) at least one anti-tumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors;

[0071] (B) a compound represented by the following formula (1) or a salt thereof:

[0072]

[0073] in

[0074] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0075] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0076] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0077] Item 7. The following compound (B) or a salt thereof, used in combination with the following anti-tumor agent (A) for preventing or treating tumors:

[0078] (A) at least one anti-tumor agent selected from alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors;

[0079] (B) a compound represented by the following formula (1) or a salt thereof:

[0080]

[0081] in

[0082] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0083] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0084] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0085] Item 8. The composition according to any one of Items 1 to 4, the kit according to Item 5, the method for prevention or treatment according to Item 6, or the compound or salt thereof according to Item 7, wherein the anti-tumor agent (A) is at least one selected from the following:

[0086] Compounds having 2 or 3 or more alkyl moieties capable of covalently bonding to DNA bases,

[0087] An anti-CD20 antibody or a fragment thereof that recognizes the CD20 antigen, an aptamer that recognizes the CD20 antigen, or a complex molecule comprising them (preferably an antibody-drug conjugate (ADC)),

[0088] DNA methyltransferase inhibitors,

[0089] Pyrimidine antimetabolites, purine antimetabolites, antifolates, Bcl-2 inhibitors, and tyrosine kinase inhibitors.

[0090] Item 9. The composition according to any one of Items 1 to 4, the kit according to Item 5, the method for prevention or treatment according to Item 6, or the compound or salt thereof according to Item 7, wherein the anti-tumor agent (A) is at least one selected from the following:

[0091] Nitrogen mustards (e.g., dichloromethane hydrochloride, cyclophosphamide, ifosfamide, melphalan, chlorambucil, and bendamustine), aziridines and epoxides (e.g., thiotepa, mitomycin C, and anhydrodulcitol), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, and ranimustine), hydrazine and triazene derivatives (e.g., procarbazine, dacarbazine, and temozolomide),

[0092] Rituximab, ibrutinib, antibodies that have been shown to be equivalent to rituximab or ibrutinib (biosimilars of rituximab or ibrutinib), ibritumomab tiuxetan,

[0093] Decitabine, azacitidine, and zebularine,

[0094] Cytarabine, 5-FU (5-fluorouracil), azathioprine, mercaptopurine, and methotrexate,

[0095] Venetoclax, and

[0096] Lapatinib, gefitinib, imatinib, ponatinib, erlotinib, ibrutinib, axitinib, lenvatinib, afatinib, and gilteritinib.

[0097] [Beneficial Effects]

[0098] The combined use of a specific antitumor agent and a specific compound can achieve an antitumor effect much higher than that achieved by using them alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] [ Figure 1 ] Figure 1 The results of the isobologram analysis shown show the combined effect of N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide hydrochloride (also referred to as "Compound A" in the figure description) and bendamustine on the human follicular lymphoma cell line DOHH-2.

[0100] [ Figure 2 ] Figure 2 The results of the isobologram analysis shown show the combined effect of Compound A and bendamustine on the human breast cancer cell line MDA-MB-231.

[0101] [ Figure 3 ] Figure 3 Shown are the results of an isobologram analysis showing the combined effect of Compound A and decitabine on the human acute myeloid leukemia (AML) cell line CMK-86.

[0102] [ Figure 4 ] Figure 4Shown are the results of an isobologram analysis showing the combined effect of Compound A and busulfan on the human acute myeloid leukemia (AML) cell line MV-4-11.

[0103] [ Figure 5 ] Figure 5 The results of the isobologram analysis are shown, showing the combined effect of Compound A and lomustine on the human chronic myeloid leukemia (CML) cell line K562.

[0104] [ Figure 6 ] Figure 6 The results of the isobologram analysis shown show the combined effect of Compound A and Thiotepa on the human lung cancer cell line A549.

[0105] [ Figure 7 ] Figure 7 The results of the isobologram analysis are shown, showing the combined effect of Compound A and lomustine on the human pancreatic cancer cell line BxPC-3.

[0106] [ Figure 8 ] Figure 8 Shown are the results of an isobologram analysis showing the combined effect of Compound A and melphalan on the human acute myeloid leukemia (AML) cell line KG-1.

[0107] [ Fig. 9 ] Fig. 9 The results of the isobologram analysis are shown, showing the combined effect of Compound A and lomustine on the human diffuse large B-cell lymphoma (DLBCL) cell line U2932.

[0108] [ Fig.10 ] Fig.10 Shown are the results of an isobologram analysis showing the combined effect of Compound A and melphalan on the human diffuse large B-cell lymphoma (DLBCL) cell line U2932.

[0109] [ Fig.11 ] Fig.11 Shown are the results of an isobologram analysis showing the combined effect of Compound A and melphalan on the human acute myeloid leukemia (AML) cell line MV-4-11.

[0110] [ Fig.12 ] Fig.12 The isobologram analysis results shown show the combined effect of Compound A and lomustine on the human gastric cancer cell line MKN45.

[0111] [ Fig.13 ] Fig.13 Shown are the results of an isobologram analysis showing the combined effect of Compound A and decitabine on the human acute myeloid leukemia (AML) cell line MV-4-11.

[0112] [ Fig.14 ] Fig.14 Shown are isobologram analysis results showing the combined effect of Compound A and decitabine on the human acute myeloid leukemia (AML) cell line OCI-AML2.

[0113] [ Fig.15 ] Fig.15 Shown are the results of an isobologram analysis showing the combined effect of Compound A and cytarabine on the human acute myeloid leukemia (AML) cell line MV-4-11.

[0114] [ Fig.16 ] Fig.16 The results of the isobologram analysis shown show the combined effect of Compound A and bendamustine on the human breast cancer cell line BT-474.

[0115] [ Fig.17 ] Fig.17 Shown are the results of an isobologram analysis showing the combined effect of Compound A and melphalan on the human multiple myeloma cell line RPMI 8226.

[0116] [ Fig.18 ] Fig.18 Shown are the results of an isobologram analysis showing the combined effect of Compound A and venetoclax on the human acute myeloid leukemia (AML) cell line KG-1.

[0117] [ Fig.19 ] Fig.19 Shown are the results of an isobologram analysis showing the combined effect of Compound A and ranimustine on the human chronic myeloid leukemia (CML) cell line K562.

[0118] [ Fig. 20 ] Fig. 20 The results of the isobologram analysis shown show the combined effect of Compound A and Thiotepa on the human ovarian cancer cell line SK-OV-3.

[0119] [ Fig.21 ] Fig.21 Shown are isobologram analysis results showing the combined effect of Compound A and ibrutinib on the human acute myeloid leukemia (AML) cell line MV-4-11.

[0120] [ Fig. 22 ] Fig. 22 Shown are the results of an isobologram analysis showing the combined effect of Compound A and decitabine on the human myelodysplastic syndrome cell line SKM-1.

[0121] [ Fig.23 ] Fig.23 The results of the isobologram analysis are shown, showing the combined effect of Compound A and erlotinib on the human lung cancer cell line A549.

[0122] [ Fig.24 ] Fig.24 The isobologram analysis results shown show the combined effect of Compound A and gefitinib on the human lung cancer cell line A549.

[0123] [ Fig.25 ] Fig.25 The isobologram analysis results shown show the combined effect of Compound A and gefitinib on the human lung cancer cell line HCC 827.

[0124] [ Fig.26 ] Fig.26 Shown are the results of an isobologram analysis showing the combined effect of Compound A and imatinib on the human chronic myeloid leukemia (CML) cell line K562.

[0125] [ Fig. 27 ] Fig. 27 The isobologram analysis results shown show the combined effect of Compound A and Axitinib on the human renal cancer cell line ACHN.

[0126] [ Fig.28 ] Fig.28 The results of the isobologram analysis shown show the combined effect of Compound A and lapatinib on the human breast cancer cell line MDA-MB-453.

[0127] [ Fig.29 ] Fig.29 Shown are the results of an isobologram analysis showing the combined effect of Compound A and Lenvatinib on the human hepatoma cell line Hep G2.

[0128] [ Fig.30 ] Fig.30 The results of the isobologram analysis shown show the combined effect of Compound A and lapatinib on the human breast cancer cell line SK-BR-3.

[0129] [ Fig.31 ] Fig.31 The isobologram analysis results shown show the combined effect of Compound A and afatinib on the human lung cancer cell line A549.

[0130] [ Fig.32 ] Fig.32 The results of the isobologram analysis shown show the combined effect of Compound A and gilteritinib on the human acute myeloid leukemia (AML) cell line MV-4-11.

[0131] [ Fig.33 ] Fig.33 The results of the isobologram analysis shown show the combined effect of Compound A and 5-FU on the human head and neck (pharyngeal) cancer cell line FaDu.

[0132] [ Fig.34 ] Fig.34 Shown are the results of an isobologram analysis showing the combined effect of Compound A and vincristine on the human diffuse large B-cell lymphoma cell line OCI-Ly18.

[0133] [ Fig.35 ] Fig.35 Shown are the results of an isobologram analysis showing the combined effect of Compound A and docetaxel on the human breast cancer cell line BT-474.

[0134] [ Fig.36 ] Fig.36 Results are shown for the combined effect of Compound A and cyclophosphamide on the human diffuse large B-cell lymphoma cell line OCI-Ly7.

[0135] [ Fig.37 ] Fig.37 Results are shown for the combined effect of Compound A and cyclophosphamide on the human diffuse large B-cell lymphoma cell line OCI-Ly7.

[0136] [ Fig.38 ] Fig.38 Results are shown for the combined effect of Compound A and cyclophosphamide on the human diffuse large B-cell lymphoma cell line OCI-Ly7.

[0137] [ Fig.39 ] Fig.39 Results are shown for the combined effect of Compound A and lapatinib on the human breast cancer cell line MDA-MB-453.

[0138] [ Fig.40 ] Fig.40 Results are shown for the combined effect of Compound A and lapatinib on the human breast cancer cell line MDA-MB-453.

[0139] [ Fig.41 ] Fig.41 Results are shown for the combined effect of Compound A and bendamustine on the human diffuse large B-cell lymphoma cell line OCI-Ly7.

[0140] [ Fig.42 ] Fig.42 Results are shown for the combined effect of Compound A and rituximab on the human diffuse large B-cell lymphoma cell line OCI-Ly7.

[0141] [ Fig.43 ] Fig.43 Results are shown for the combined effect of Compound A and decitabine on the human acute myeloid leukemia (AML) cell line KG-1.

[0142] [ Fig.44 ] Fig.44 Shown are the results of the combined effect of Compound A and cytarabine on the human acute myeloid leukemia (AML) cell line KG-1.

[0143] [ Fig.45 ] Fig.45 Shown are the results of the three-drug combination effect of Compound A, bendamustine and rituximab on the human diffuse large B-cell lymphoma cell line OCI-Ly7.

[0144] [ Fig.46 ] Fig.46 Shown are the results of the three-drug combination effect of Compound A, decitabine, and venetoclax on the human acute myeloid leukemia (AML) cell line KG-1. DETAILED DESCRIPTION

[0145] Each embodiment included in the present disclosure is described in more detail below. The present disclosure preferably includes anti-tumor compositions, methods for preventing or treating tumors, anti-tumor kits, etc.; however, the present disclosure is not limited thereto. The present disclosure includes all embodiments disclosed herein and recognized by those skilled in the art.

[0146] The antitumor composition included in the present disclosure comprises the following compound (B) or a salt thereof. Hereinafter, the antitumor composition comprising compound (B) or a salt thereof is also referred to as the "antitumor composition of the present disclosure".

[0147] (B) a compound represented by the following formula (1) or a salt thereof:

[0148]

[0149] in

[0150] R 1 represents a halogen atom, an aryl group, an aryloxy group or a lower alkyl group optionally substituted by one or more halogen atoms;

[0151] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0152] m represents an integer from 1 to 3, provided that when m represents 2 or 3, R 1 Same or different.

[0153] Specific examples of each group shown in formula (1) are as follows.

[0154] Examples of the lower alkoxy group include a straight chain or branched alkoxy group having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a tert-butoxy group, a pentyloxy group and a hexyloxy group.

[0155] Examples of the lower alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, 2,2-dimethylpropyl, 1-ethylpropyl, butyl, isobutyl, tert-butyl, isopentyl, pentyl and hexyl.

[0156] Examples of the lower alkyl group optionally substituted by one or more halogen atoms include, in addition to the above-mentioned lower alkyl groups, a straight or branched alkyl group having 1 to 6 carbon atoms which may be substituted by 1 to 3 halogen atoms, for example, a trifluoromethyl group, a trichloromethyl group, a chloromethyl group, a bromomethyl group, a fluoromethyl group, an iodomethyl group, a difluoromethyl group, a dibromomethyl group, a dichloromethyl group, a 2-chloroethyl group, a 2,2,2-trifluoroethyl group, a 2,2,2-trichloroethyl group, a 3-chloropropyl group, a 2,3-dichloropropyl group, a 4,4,4-trichlorobutyl group, a 4-fluorobutyl group, a 5-chloropentyl group, a 3-chloro-2-methylpropyl group, a 5-bromohexyl group and a 5,6-dibromohexyl group.

[0157] Examples of the halogen atom include fluorine, chlorine, bromine and iodine atoms.

[0158] Examples of aryl groups include C6-C 10 Aromatic groups such as phenyl, 1-naphthyl, 2-naphthyl, o-tolyl, m-tolyl and p-tolyl.

[0159] Examples of aryloxy groups include C6-C 10 Aryloxy groups such as phenoxy, 1-naphthyloxy and 2-naphthyloxy.

[0160] Preferred examples are compounds represented by formula (1) or salts thereof, wherein:

[0161] R 1 represents an aryl group, an aryloxy group or a lower alkyl group which is optionally substituted by one or more halogen atoms;

[0162] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0163] m represents an integer 1.

[0164] Another preferred example is a compound represented by formula (1) or a salt thereof, wherein:

[0165] R 1 represents a halogen atom;

[0166] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0167] m represents an integer from 1 to 3;

[0168] The premise is that when m represents 2 or 3, R 1 Same or different.

[0169] Still another preferred example is a compound represented by formula (1) or a salt thereof, wherein:

[0170] R 1 represents halogen or lower alkyl optionally substituted by one or more halogen atoms;

[0171] R 2 represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group; and

[0172] m represents the integer 2;

[0173] The premise is R 1 Same or different.

[0174] More specific preferred examples of the compound represented by formula (1) include:

[0175] N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0176] N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0177] N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0178] 2-Fluoro-N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0179] 2,3,4-trifluoro-N-{6-[2-fluoro-4-(methylamino)phenoxy]pyridin-3-yl}benzamide,

[0180] N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide, N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0181] N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0182] 2-Fluoro-N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0183] N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0184] N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0185] N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0186] N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}-4-phenoxybenzamide,

[0187] N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}biphenyl-4-carboxamide,

[0188] 2-Fluoro-N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0189] 2,3,4-trifluoro-N-{6-[2-methoxy-4-(methylamino)phenoxy]pyridin-3-yl}benzamide,

[0190] 2,3,4-trifluoro-N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}benzamide,

[0191] N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide,

[0192] 2-fluoro-N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide, and

[0193] 2,3,4-Trifluoro-N-{6-[4-(methylamino)phenoxy]pyridin-3-yl}benzamide.

[0194] Salts of these compounds can also be preferably used.

[0195] Examples of the salt of the compound represented by formula (1) include inorganic acid salts such as sulfates, nitrates, hydrochlorides, phosphates and hydrobromides, and organic acid salts such as acetates, sulfonates (e.g., p-toluenesulfonate, methanesulfonate and ethanesulfonate), oxalates, maleates, fumarates, malates, tartrates, citrates, succinates and benzoates.

[0196] The compound represented by formula (1) or a salt thereof can be produced by a known method or a method that can be easily conceived from a known method. For example, the method described in PTL 1 (WO2012 / 046825) can be used for production.

[0197] The anti-tumor composition of the present invention is used in combination with the following anti-tumor agent (A):

[0198] (A) at least one anti-tumor agent selected from the group consisting of alkylating agents, CD20 recognition molecules, DNA methylation inhibitors, pyrimidine antimetabolites, purine antimetabolites, antifolate agents, Bcl-2 inhibitors and tyrosine kinase inhibitors.

[0199] Alkylating agents are a class of cytotoxic anticancer drugs (cancer suppressants). They have multiple alkyl components that can covalently bind to DNA bases and prevent DNA replication by connecting two DNA chains (interchain crosslinking). DNA bases, especially guanine, are nucleophilic. Typically, DNA bases and alkyl groups are covalently bonded by nucleophilic substitution reactions. The alkylating agent is preferably an alkylating agent with 2 or 3 or more alkyl components. More specific examples include nitrogen mustards (e.g., dichloromethyldiethylamine hydrochloride, cyclophosphamide, ifosfamide, melphalan, chlorambucil and bendamustine), aziridines and epoxides (e.g., thiotepa, mitomycin C and dehydrated dactyl), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine and ranimustine), hydrazine and triazene derivatives (e.g., procarbazine, dacarbazine and temozolomide), etc.

[0200] The CD20 recognition molecules mentioned herein are molecules that recognize CD20 antigen (Bp35) and have anti-tumor effects. Examples include anti-CD20 antibodies or fragments thereof that recognize CD20 antigen, aptamers that recognize CD20 antigen, and complex molecules containing them. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. In addition, preferred antibodies are chimeric antibodies (rodents (e.g., mice, rats, or Chinese hamsters) and human chimeras), humanized antibodies, and fully humanized antibodies. Preferred examples of anti-CD20 antibodies include rituximab, imostat, antibodies that have been confirmed to be equivalent to rituximab or imostat (biosimilars of rituximab or imostat), etc. In addition, specific examples of the complex molecules mentioned herein preferably include antibody-drug conjugates (ADCs). The antibody-drug conjugate is preferably a conjugate in which the antibody is connected to an anticancer agent via a connector. Examples of such antibody-drug conjugates (ADCs) include ibritumomab tiuxetan, in which the antibody is linked to a compound containing a radioisotope (tiuxetan).

[0201] The DNA methylation inhibitor is preferably a DNA methyltransferase inhibitor. More specific examples include decitabine, azacitidine, zebularine, and the like.

[0202] The antimetabolite is preferably a pyrimidine antimetabolite, a purine antimetabolite or an antifolate. More specifically, examples of pyrimidine antimetabolites include cytarabine and 5-FU (5-fluorouracil), examples of purine antimetabolites include azathioprine and mercaptopurine, and examples of antifolates include methotrexate.

[0203] Bcl-2 (B-cell lymphoma 2) is known to be an oncogene that regulates apoptosis by regulating mitochondrial membrane permeability. Examples of Bcl-2 inhibitors include venetoclax.

[0204] Examples of tyrosine kinase inhibitors include lapatinib, gefitinib, imatinib, ponatinib, erlotinib, ibrutinib, axitinib, lenvatinib, afatinib, gilteritinib, and the like.

[0205] The antitumor composition of the present disclosure can be prepared by combining compound (B) or a salt thereof with, for example, a pharmaceutically acceptable carrier. The preparation can be performed by a known method or a method that can be easily conceived from a known method.

[0206] The antitumor composition of the present disclosure is preferably used in the form of a common pharmaceutical preparation. Such pharmaceutical preparations are obtained by formulating with commonly used excipients or additives such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and lubricants.

[0207] The form of the pharmaceutical preparation can be selected from various forms according to the therapeutic purpose. Typical examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (such as solutions and suspensions), and the like.

[0208] In order to form tablets, various known carriers can be used. Examples of the carrier include lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose and other additives, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone and other binders, dry starch, sodium alginate, agar powder, laminarin powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyoxyethylene sorbitan, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and other disintegrants, disintegration inhibitors such as sucrose, stearin, cocoa butter, hardened oil, quaternary ammonium base, sodium lauryl sulfate and other absorption promoters, glycerin, starch and other wetting agents, starch, lactose, kaolin, bentonite, colloidal silicic acid and other adsorbents, purified talc, stearate, boric acid powder, polyethylene glycol and other lubricants.

[0209] For example, in the case of preparing sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double-layer or multi-layer tablets, etc., such tablets can be coated with known coating materials as required.

[0210] In order to form pills, various known carriers can be used. Examples of the carrier include glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc and other additives, gum arabic powder, tragacanth powder, gelatin, ethanol and other binders, laminarin, agar and other disintegrators.

[0211] In order to form suppositories, various known carriers can be used. Examples of the carriers include polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.

[0212] In order to form an injection, the solution, emulsion or suspension is sterilized and preferably made isotonic with blood. A variety of known and widely used excipients can be used to prepare solutions, emulsions or suspensions. Examples of such excipients include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, fatty acid esters of polyoxyethylene sorbitan, etc. In this case, the pharmaceutical preparation may contain a sufficient amount of sodium chloride, glucose or glycerol to prepare an isotonic solution, and may contain common solubilizers, buffers, soothing agents, etc. In addition, if necessary, the pharmaceutical preparation may contain colorants, preservatives, flavorings, sweeteners and / or other drugs.

[0213] The amount of compound (B) or its salt contained in the antitumor composition of the present disclosure is not particularly limited, and can be appropriately selected from a wide range. For example, the amount of compound (B) or its salt in the composition is about 0.1 to 70% by weight, preferably about 0.1 to 30% by weight.

[0214] There is no restriction on the route of administration of the antitumor composition of the present disclosure. For example, the antitumor composition can be administered by a route suitable for the form of the preparation, the age and sex of the patient, the condition of the disease or other conditions. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. Injections are administered intravenously alone or mixed with common infusion solutions such as glucose solutions and amino acid solutions, or administered alone intramuscularly, intradermally, subcutaneously or intraperitoneally as needed. Suppositories are administered rectally.

[0215] The dosage of the antitumor composition of the present disclosure can be appropriately set according to the method of use, the age and sex of the patient, the severity of the disease, the dosage and administration time of the antitumor agent (A) and other conditions. Although not particularly limited, the dosage of the antitumor composition is, for example, 0.001 mg / kg body weight / day to 100 mg / kg body weight / day, preferably 0.001 mg / kg body weight / day to 50 mg / kg body weight / day, as a single dose or divided doses.

[0216] There is no particular limitation on the combination of the antitumor composition of the present invention and the antitumor agent (A). For example, more specifically, the antitumor composition of the present invention may be, for example, (i) an antitumor composition used in combination with an antitumor agent (A), (ii) an antitumor composition for administration to a person who will receive or has received an antitumor agent (A), or (iii) an antitumor composition further comprising an antitumor agent (A).

[0217] The antitumor composition (i) includes all embodiments in which it is used in combination with the antitumor agent (A). For example, it includes an embodiment in which the antitumor composition (i) is used for administration to a person who will receive or has received the antitumor agent (A). In other words, the antitumor composition (i) includes the antitumor composition (ii). In addition, for example, the antitumor composition (i) also includes an embodiment in which it is mixed with the antitumor agent (A) to prepare an antitumor composition that not only contains compound (B) or its salt, but also contains the antitumor agent (A). In other words, the antitumor composition (i) includes a composition for preparing the antitumor composition (iii). In addition, for example, the antitumor composition (i) also includes an embodiment in which it is used together with the antitumor agent (A) as a collection kit. In other words, the antitumor composition (i) includes an embodiment in which it is used together with the antitumor agent (A) to construct a kit (particularly an antitumor kit).

[0218] The antitumor composition (ii) is used for administration to a person who will receive or has received an antitumor agent (A). In this case, the person who will receive the antitumor agent (A) is a person who receives the antitumor agent (A) after or at the same time as the antitumor composition of the present disclosure is administered. In addition, in this case, the person who has received the antitumor agent (A) is a person who receives the antitumor agent (A) before the antitumor composition of the present disclosure is administered. In any of these cases, there is no particular restriction on the time interval between the administration of the antitumor composition of the present disclosure and the administration of the antitumor agent (A), as long as the antitumor agent (A) and the antitumor composition of the present disclosure are administered in combination to produce a synergistic effect for tumor treatment. For example, no time interval (i.e., simultaneous administration) may be provided, one administration may start during another administration, one administration may start immediately after another administration is completed, one administration may start a few minutes to tens of minutes after another administration is completed, or one administration may start, for example, about 1 to 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) hours after another administration is completed.

[0219] The antitumor composition (iii) is an antitumor composition comprising not only the compound (B) or its salt, but also the antitumor agent (A). The dosage form of the antitumor composition can be suitably set in consideration of the dosage form of the antitumor agent (A), etc. For example, it can be prepared by mixing the compound (B) or its salt with the antitumor agent (A) or during the preparation of the antitumor agent (A). Therefore, the dosage form of the antitumor composition (iii) is preferably the same as the dosage form of the antitumor agent (A) used for preparation.

[0220] The present disclosure also includes a drug kit comprising an antitumor agent (A) and an antitumor composition comprising a compound (B) or a salt thereof. The drug kit is also referred to as a "drug kit of the present disclosure". The drug kit of the present disclosure is preferably an antitumor drug kit (i.e., a drug kit for preventing or treating tumors). In addition, in addition to the antitumor agent (A) and the antitumor composition comprising a compound (B) or a salt thereof, the drug kit may suitably include a drug, a drug delivery device (e.g., a syringe), instructions for use (attached documents), etc., which are more preferably used to prevent or treat tumors.

[0221] The present disclosure also includes a method for preventing or treating tumors, the method comprising administering an anti-tumor agent (A) and a compound (B) or a salt thereof to a subject. The prevention or treatment method is also referred to as "the prevention or treatment method of the present disclosure". The prevention or treatment method of the present disclosure includes a method for preventing or treating tumors comprising separately administering an anti-tumor agent (A) and an anti-tumor composition comprising a compound (B) or a salt thereof, and a method for preventing or treating tumors comprising administering an anti-tumor composition (iii). In the former method (a method for preventing or treating tumors comprising separate administration), there is no particular restriction on the dosing schedule of the anti-tumor agent (A) and the anti-tumor composition comprising a compound (B) or a salt thereof, as long as the anti-tumor agent (A) and the anti-tumor composition of the present disclosure are administered in combination to produce a synergistic effect for tumor treatment. For example, no time interval may be provided (i.e., simultaneous administration), one administration may be started during another administration, one administration may be started immediately after another administration is completed, one administration may be started a few minutes to tens of minutes after another administration is completed, or one administration may be started, for example, about 1 to 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) hours after another administration is completed. One administration may be before or after another administration.

[0222] Furthermore, there is no particular limitation on the subject as long as the subject is in need of tumor treatment.

[0223] In the present disclosure, a tumor is a disease caused by abnormal cell proliferation, preferably a malignant tumor (cancer). More specific examples include breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ, and inflammatory breast cancer), prostate cancer (e.g., hormone-dependent prostate cancer and hormone-independent prostate cancer), pancreatic cancer (e.g., pancreatic ductal carcinoma), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, and adenosquamous carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and malignant mesothelioma), colon cancer (e.g., gastrointestinal stromal tumor), rectal cancer (e.g., gastrointestinal stromal tumor), colorectal cancer (e.g., familial colorectal cancer), and ovarian cancer (e.g., ovarian cancer). rectal cancer, hereditary nonpolyposis colorectal cancer, and gastrointestinal stromal tumors), small intestine cancer (e.g., gastrointestinal stromal tumors), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, and hypopharyngeal cancer), salivary gland cancer, brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, and anaplastic astrocytoma), schwannoma, liver cancer (e.g., primary liver cancer and extrahepatic bile duct cancer), kidney cancer (e.g., renal cell carcinoma and ureteral transitional cell carcinoma), biliary Tubal cancer, endometrial cancer, cervical cancer, uterine sarcoma, ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumors, ovarian germ cell tumors, and ovarian tumors of low malignant potential), bladder cancer, urethral cancer, skin cancer (e.g., intraocular (eye) melanoma and Merkel cell carcinoma), hemangioma, malignant lymphoma (e.g., non-Hodgkin lymphoma and Hodgkin lymphoma), malignant melanoma, thyroid cancer (e.g., medullary thyroid cancer), parathyroid cancer, nasal cancer, paranasal cancer, myeloma (e.g., multiple myeloma Tumors), bone tumors (such as osteosarcoma, Ewing's tumor and soft tissue sarcoma), myelodysplastic tumors, myeloproliferative tumors, angiofibroma, retinosarcoma, penile cancer, testicular tumors, pediatric solid cancers (such as Wilms' tumor and pediatric renal tumors), Kaposi's sarcoma, Kaposi's sarcoma caused by AIDS, maxillary sinus tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, leukemia (such as acute myeloid leukemia, chronic myeloid leukemia and acute lymphoblastic leukemia), etc. Among them, the types of cancers that are particularly preferred as the administration targets of the anti-tumor composition, pharmaceutical kit or prevention or treatment method of the present invention include malignant lymphoma, leukemia, myeloma, breast cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, etc.

[0224] In the present specification, the term "comprising" includes "consisting essentially of" and "consisting of.

[0225] The various features (properties, structures, functions, etc.) explained in the above-described embodiments may be combined in any manner to specify the subject matter covered in the present disclosure.

[0226] [Example]

[0227] The subject matter covered in the present disclosure is described in more detail below. However, the subject matter is not limited to the following embodiments.

[0228] According to the method described in the examples (especially Example 24) of WO2012 / 046825, N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide hydrochloride (white powder) was prepared. Hereinafter, N-{6-[2-methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide hydrochloride is also referred to as compound A. The obtained white powder of compound A was used as "compound A bulk powder" in the following studies.

[0229] [In vitro growth inhibition assay]

[0230] Example 1: Combination effect of compound A and bendamustine on human follicular lymphoma cell line DOHH-2

[0231] 1) Test substance

[0232] Compound A bulk powder was used. Bendamustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0233] 2) Cell culture

[0234] DOHH-2 cells were purchased from Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH (DSMZ). The cells were cultured in RPMI-1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum, penicillin and streptomycin at 37°C and 5% CO2. The cells were collected and resuspended by conventional methods and seeded on 96-well plates at a density of 1000 cells / well. The number of seeded cells was defined as the number of cells from which a linear cell growth curve could be obtained until the 6th day.

[0235] 3) Drug addition

[0236] After culturing the cells at 37°C and 5% CO2 for 24 hours, medium containing various concentrations of compound A or various concentrations of antitumor agents used in combination was added. After agitating the plate, the cells were cultured at 37°C and 5% CO2 for 120 hours.

[0237] 4) Cell growth inhibition assay

[0238] The cell growth inhibition test was performed using the CellTiter-Fluor cell viability assay (Promega). Specifically, the CellTiter-Fluor reagent was added to each well to be measured and then incubated. The fluorescence intensity was then measured using a fluorescence microplate reader at an excitation wavelength of 400 nm and a fluorescence wavelength of 505 nm.

[0239] 5) Calculation of cell viability

[0240] The cell-free blank value was subtracted from the measured value of each well, and the ratio relative to the drug-free control was calculated and defined as the cell viability. The IC 50 Values ​​were determined using the cell viability.

[0241] 6) Equi-effect diagram analysis

[0242] The combined effect of Compound A and the combined antitumor agent (bendamustine) was evaluated using the isobologram method. Specifically, the evaluation was performed as follows.

[0243] Determine the IC of compound A at each concentration of the antitumor agent 50 The fractional inhibitory concentration (FIC) of the antitumor agent was calculated by dividing the concentration of each antitumor agent by the IC 50 The FIC of compound A is determined by dividing the IC of compound A by 50 The value was divided by the IC of compound A alone 50 The IC values ​​of the antitumor agents at each concentration of compound A were determined. 50 The FIC of compound A was determined by dividing the concentration of each compound A by the IC of compound A alone. 50 The FIC of the anti-tumor drug is determined by dividing the IC 50 The value is divided by the IC of the antineoplastic agent alone 50 The total FIC value of each combination was defined as the CI value of each combination. The CI mean and 95% confidence interval were calculated. Table 1 shows the details of the calculation.

[0244] [Table 1]

[0245]

[0246] also, Figure 1The graph shown plots the FIC of each drug (Compound A and bendamustine). In the isobologram, blocks located to the upper right, almost on the line, or to the lower left of the straight line connecting the points where the FIC of each drug is 1 mean that antagonism, additive, or synergistic effects are exhibited, respectively. Figure 1 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and bendamustine exhibits a synergistic effect on the cell growth inhibition of DOHH-2.

[0247] Example 2: Combination effect of compound A and bendamustine on human breast cancer cell line MDA-MB-231

[0248] 1) Test substance

[0249] Compound A bulk powder was used. Bendamustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0250] 2) Cell growth inhibition assay

[0251] MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC).

[0252] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 2000 cells / well. Figure 2 The isoelectric plot analysis results are shown. Figure 2 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and bendamustine exhibits a synergistic effect on the cell growth inhibition of MDA-MB-231.

[0253] Example 3: Combination effect of compound A and decitabine on human acute myeloid leukemia (AML) cell line CMK-86 answer

[0254] 1) Test substance

[0255] Bulk powder of Compound A was used. Decitabine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0256] 2) Cell growth inhibition assay

[0257] CMK-86 cells were purchased from Health Science Research Resources Bank.

[0258] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 12,000 cells / well. Figure 3The isoelectric plot analysis results are shown. Figure 3 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and decitabine exhibits a synergistic effect on the cell growth inhibition of CMK-86.

[0259] Example 4: Combination effect of compound A and busulfan on human acute myeloid leukemia (AML) cell line MV-4-11

[0260] 1) Test substance

[0261] Bulk powder of Compound A was used. Busulfan used was purchased from Wako Pure Chemical Industries, Ltd.

[0262] 2) Cell growth inhibition assay

[0263] MV-4-11 cells were purchased from ATCC.

[0264] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Figure 4 The isoelectric plot analysis results are shown. Figure 4 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and busulfan exhibits a synergistic effect on the cell growth inhibition of MV-4-11.

[0265] Example 5: Combination effect of compound A and lomustine on human chronic myeloid leukemia (CML) cell line K562

[0266] 1) Test substance

[0267] Compound A bulk powder was used. Lomustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0268] 2) Cell growth inhibition assay

[0269] K562 cells were purchased from Health Science Research Resources Bank.

[0270] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1. Figure 5 The isoelectric plot analysis results are shown. Figure 5 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and lomustine exhibits a synergistic effect on the cell growth inhibition of K562.

[0271] Example 6: Combination effect of compound A and thiotepa on human lung cancer cell line A549

[0272] 1) Test substance

[0273] Compound A bulk powder was used. Thiotepa used was purchased from Sigma-Aldrich.

[0274] 2) Cell growth inhibition assay

[0275] A549 cells were purchased from Health Science Research Resources Bank.

[0276] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1. Figure 6 The isoelectric plot analysis results are shown. Figure 6 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and thiotepa exhibits a synergistic effect on the cell growth inhibition of A549.

[0277] Example 7: Combination effect of compound A and lomustine on human pancreatic cancer cell line BxPC-3

[0278] 1) Test substance

[0279] The lomustine used was purchased from Tokyo Chemical Industry Co., Ltd. Compound A bulk powder was used.

[0280] 2) Cell growth inhibition assay

[0281] BxPC-3 cells were purchased from ATCC.

[0282] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1. Figure 7 The isoelectric plot analysis results are shown. Figure 7 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and lomustine exhibits a synergistic effect on the cell growth inhibition of BxPC-3.

[0283] Example 8: Combination effect of compound A and melphalan on human acute myeloid leukemia (AML) cell line KG-1

[0284] 1) Test substance

[0285] Compound A bulk powder was used. Melphalan used was purchased from Sigma-Aldrich.

[0286] 2) Culture medium and cell passaging

[0287] KG-1 cells were purchased from Health Science Research Resources Bank.

[0288] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Figure 8 The isoelectric plot analysis results are shown. Figure 8 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and melphalan exhibits a synergistic effect on the cell growth inhibition of KG-1.

[0289] Example 9: Effect of Compound A and lomustine on human diffuse large B-cell lymphoma (DLBCL)

[0290] Combination effects in cell line U2932

[0291] 1) Test substance

[0292] Compound A bulk powder was used. Lomustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0293] 2) Cell growth inhibition assay

[0294] U2932 cells were purchased from DSMZ.

[0295] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 2000 cells / well. Fig. 9 The isoelectric plot analysis results are shown. Fig. 9 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and lomustine exhibits a synergistic effect on the cell growth inhibition of U2932.

[0296] Example 10: Combination of Compound A and Melphalan on Human Diffuse Large B-cell Lymphoma (DLBCL) Cell Line U2932 Synergistic Effect

[0297] 1) Test substance

[0298] Compound A bulk powder was used. Melphalan used was purchased from Sigma-Aldrich.

[0299] 2) Cell growth inhibition assay

[0300] U2932 cells were purchased from DSMZ.

[0301] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 2000 cells / well. Fig.10 The isoelectric plot analysis results are shown. Fig.10The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and melphalan exhibits a synergistic effect on the cell growth inhibition of U2932.

[0302] Example 11: Combination effect of compound A and melphalan on human acute myeloid leukemia (AML) cell line MV-4-11 answer

[0303] 1) Test substance

[0304] Compound A bulk powder was used. Melphalan used was purchased from Sigma-Aldrich.

[0305] 2) Cell growth inhibition assay

[0306] MV-4-11 cells were purchased from ATCC.

[0307] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.11 The isoelectric plot analysis results are shown. Fig.11 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and melphalan exhibits a synergistic effect on the cell growth inhibition of MV-4-11.

[0308] Example 12: Combination effect of compound A and lomustine on human gastric cancer cell line MKN45 1) Test substance

[0309] Compound A bulk powder was used. Lomustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0310] 2) Cell growth inhibition assay

[0311] MKN 45 cells were purchased from Health Science Research Resources Bank.

[0312] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1. Fig.12 The isoelectric plot analysis results are shown. Fig.12 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and lomustine exhibits a synergistic effect on the cell growth inhibition of MKN 45.

[0313] Example 13: Combination of Compound A and Decitabine on Human Acute Myeloid Leukemia (AML) Cell Line MV-4-11 effect

[0314] 1) Test substance

[0315] Bulk powder of Compound A was used. Decitabine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0316] 2) Cell growth inhibition assay

[0317] MV-4-11 cells were purchased from ATCC.

[0318] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.13 The isoelectric plot analysis results are shown. Fig.13 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and decitabine exhibits a synergistic effect on the cell growth inhibition of MV-4-11.

[0319] Example 14: Combination of Compound A and Decitabine on Human Acute Myeloid Leukemia (AML) Cell Line OCI-AML2 effect

[0320] 1) Test substance

[0321] Bulk powder of Compound A was used. Decitabine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0322] 2) Cell growth inhibition assay

[0323] OCI-AML2 cells were purchased from DSMZ.

[0324] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 4000 cells / well. Fig.14 The isoelectric plot analysis results are shown. Fig.14 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and decitabine exhibits a synergistic effect on the cell growth inhibition of OCI-AML2.

[0325] Example 15: Combination of Compound A and Cytarabine on Human Acute Myeloid Leukemia (AML) Cell Line MV-4-11 effect

[0326] 1) Test substance

[0327] Compound A bulk powder was used. Cytarabine used was purchased from Wako Pure Chemical Industries, Ltd.

[0328] 2) Cell growth inhibition assay

[0329] MV-4-11 cells were purchased from ATCC.

[0330] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.15 The isoelectric plot analysis results are shown. Fig.15 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and cytarabine exhibits a synergistic effect on the cell growth inhibition of MV-4-11.

[0331] Example 16: Combination effect of Compound A and bendamustine on human breast cancer cell line BT-474

[0332] 1) Test substance

[0333] Compound A bulk powder was used. Bendamustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0334] 2) Cell growth inhibition assay

[0335] BT-474 cells were purchased from ATCC.

[0336] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.16 The isoelectric plot analysis results are shown. Fig.16 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and bendamustine exhibits a synergistic effect on the cell growth inhibition of BT-474.

[0337] Example 17: Combination effect of compound A and melphalan on human multiple myeloma cell line RPMI 8226

[0338] 1) Test substance

[0339] Compound A bulk powder was used. Melphalan used was purchased from Sigma-Aldrich.

[0340] 2) Cell growth inhibition assay

[0341] RPMI 8226 cells were purchased from ATCC.

[0342] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 4000 cells / well. Fig.17 The isoelectric plot analysis results are shown. Fig.17The block shown in FIG. 1 is located at the lower left of the straight line, indicating that the combined use of compound A and melphalan exhibits a synergistic effect on the cell growth inhibition of RPMI 8226.

[0343] Example 18: Combination Effect of Compound A and Venetoclax on Human Acute Myeloid Leukemia (AML) Cell Line KG-1

[0344] 1) Test substance

[0345] Compound A bulk powder was used. Venetoclax used was purchased from LC Laboratories.

[0346] 2) Cell growth inhibition assay

[0347] KG-1 cells were purchased from Health Science Research Resources Bank.

[0348] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.18 The isoelectric plot analysis results are shown. Fig.18 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and venetoclax showed a synergistic effect on the cell growth inhibition of KG-1.

[0349] Example 19: Combination effect of compound A and ranimustine on human chronic myeloid leukemia (CML) cell line K562

[0350] 1) Test substance

[0351] Compound A bulk powder was used. Ranomustine used was purchased from Toronto Research Chemicals.

[0352] 2) Cell growth inhibition assay

[0353] K562 cells were purchased from Health Science Research Resources Bank.

[0354] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1. Fig.19 The isoelectric plot analysis results are shown. Fig.19 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and ranimustine exhibits a synergistic effect on the cell growth inhibition of K562.

[0355] Example 20: Combination effect of compound A and thiotepa on human ovarian cancer cell line SK-OV-3

[0356] 1) Test substance

[0357] Compound A bulk powder was used. Thiotepa used was purchased from Sigma-Aldrich.

[0358] 2) Cell growth inhibition assay

[0359] SK-OV-3 cells were purchased from ATCC.

[0360] Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1. Fig. 20 The isoelectric plot analysis results are shown. Fig. 20 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and thiotepa exhibits a synergistic effect on the cell growth inhibition of SK-OV-3 cells.

[0361] Example 21: Combination of Compound A and Ibrutinib on Human Acute Myeloid Leukemia (AML) Cell Line MV-4-11 effect

[0362] 1) Test substance

[0363] Compound A bulk powder was used. Ibrutinib used was purchased from MedChem Express.

[0364] 2) Cell growth inhibition assay

[0365] MV-4-11 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.21 The isoelectric plot analysis results are shown.

[0366] Fig.21 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and ibrutinib exhibits a synergistic effect on the cell growth inhibition of MV-4-11.

[0367] Example 22: Combination effect of compound A and decitabine on human myelodysplastic syndrome cell line SKM-1

[0368] 1) Test substance

[0369] Bulk powder of Compound A was used. Decitabine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0370] 2) Cell growth inhibition assay

[0371] SKM-1 cells were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank (JCRB). Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of inoculated cells was 8000 cells / well. Fig. 22 The isoelectric plot analysis results are shown.

[0372] Fig. 22 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and decitabine exhibits a synergistic effect on the cell growth inhibition of SKM-1.

[0373] Example 23: Combination effect of compound A and erlotinib on human lung cancer cell line A549

[0374] 1) Test substance

[0375] Compound A bulk powder was used. Erlotinib used was purchased from Wako Pure Chemical Industries, Ltd.

[0376] 2) Cell growth inhibition assay

[0377] A549 cells were purchased from Health Science Research Resources Bank. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 1000 cells / well. Fig.23 The isoelectric plot analysis results are shown.

[0378] Fig.23 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and erlotinib showed a synergistic effect on the cell growth inhibition of A549.

[0379] Example 24: Combination effect of compound A and gefitinib on human lung cancer cell line A549

[0380] 1) Test substance

[0381] Bulk powder of Compound A was used. Gefitinib used was purchased from Wako Pure Chemical Industries, Ltd.

[0382] 2) Cell growth inhibition assay

[0383] A549 cells were purchased from Health Science Research Resources Bank. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 1000 cells / well. Fig.24 The isoelectric plot analysis results are shown.

[0384] Fig.24 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and gefitinib exhibits a synergistic effect on the cell growth inhibition of A549.

[0385] Example 25: Combination effect of compound A and gefitinib on human lung cancer cell line HCC 827

[0386] 1) Test substance

[0387] Bulk powder of Compound A was used. Gefitinib used was purchased from Wako Pure Chemical Industries, Ltd.

[0388] 2) Cell growth inhibition assay

[0389] HCC 827 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 4000 cells / well. Fig.25 The isoelectric plot analysis results are shown.

[0390] Fig.25 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and gefitinib exhibits a synergistic effect on the cell growth inhibition of HCC 827.

[0391] Example 26: Combination effect of compound A and imatinib on human chronic myeloid leukemia (CML) cell line K562

[0392] 1) Test substance

[0393] Bulk powder of Compound A was used. Imatinib used was purchased from Wako Pure Chemical Industries, Ltd.

[0394] 2) Cell growth inhibition assay

[0395] K562 cells were purchased from Health Science Research Resources Bank. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 1000 cells / well. Fig.26 The isoelectric plot analysis results are shown.

[0396] Fig.26 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and imatinib exhibits a synergistic effect on the cell growth inhibition of K562.

[0397] Example 27: Combination effect of compound A and axitinib on human renal cancer cell line ACHN

[0398] 1) Test substance

[0399] Compound A bulk powder was used. Axitinib used was purchased from Selleck Biotech.

[0400] 2) Cell growth inhibition assay

[0401] ACHN cells were obtained from Otsuka Pharmaceutical TRC. Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig. 27 The isoelectric plot analysis results are shown.

[0402] Fig. 27 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and axitinib showed a synergistic effect on the cell growth inhibition of ACHN.

[0403] Example 28: Combination effect of compound A and lapatinib on human breast cancer cell line MDA-MB-453

[0404] 1) Test substance

[0405] Compound A bulk powder was used. Lapatinib used was purchased from LC Laboratories.

[0406] 2) Cell growth inhibition assay

[0407] MDA-MB-453 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.28 The isoelectric plot analysis results are shown.

[0408] Fig.28 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and lapatinib exhibits a synergistic effect on the cell growth inhibition of MDA-MB-453 cells.

[0409] Example 29: Combination effect of compound A and lenvatinib on human hepatoma cell line HepG2

[0410] 1) Test substance

[0411] Compound A bulk powder was used. Lenvatinib used was purchased from Funakoshi Co., Ltd.

[0412] 2) Cell growth inhibition assay

[0413] Hep G2 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 2000 cells / well. Fig.29 The isoelectric plot analysis results are shown.

[0414] Fig.29 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and lenvatinib exhibits a synergistic effect on the cell growth inhibition of Hep G2.

[0415] Example 30: Combination effect of compound A and lapatinib on human breast cancer cell line SK-BR-3

[0416] 1) Test substance

[0417] Compound A bulk powder was used. Lapatinib used was purchased from LC Laboratories.

[0418] 2) Cell growth inhibition assay

[0419] SK-BR-3 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 4000 cells / well. Fig.30 The isoelectric plot analysis results are shown.

[0420] Fig.30 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and lapatinib exhibits a synergistic effect on the cell growth inhibition of SK-BR-3 cells.

[0421] Example 31: Combination effect of compound A and afatinib on human lung cancer cell line A549

[0422] 1) Test substance

[0423] Bulk powder of compound A was used. Afatinib used was purchased from Tokyo Chemical Industry Co., Ltd.

[0424] 2) Cell growth inhibition assay

[0425] A549 cells were purchased from Health Science Research Resources Bank. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 1000 cells / well. Fig.31 The isoelectric plot analysis results are shown.

[0426] Fig.31 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and afatinib shows a synergistic effect on the cell growth inhibition of A549.

[0427] Example 32: Combination of Compound A and Gilteritinib on Human Acute Myeloid Leukemia (AML) Cell Line MV-4-11 effect

[0428] 1) Test substance

[0429] Compound A bulk powder was used. Gilteritinib used was purchased from Cayman Chemical Company.

[0430] 2) Cell growth inhibition assay

[0431] MV-4-11 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.32 The isoelectric plot analysis results are shown.

[0432] Fig.32 The block shown in is located at the lower left of the straight line, indicating that the combined use of compound A and gilteritinib exhibits a synergistic effect on the cell growth inhibition of MV-4-11.

[0433] Example 33: Combination effect of compound A and 5-FU on human head and neck (pharyngeal) cancer cell line FaDu

[0434] 1) Test substance

[0435] Compound A bulk powder was used. 5-FU used was purchased from Sigma-Aldrich.

[0436] 2) Cell growth inhibition assay

[0437] FaDu cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 1000 cells / well. Fig.33 The isoelectric plot analysis results are shown.

[0438] Fig.33 The block shown in is located at the lower left of the straight line, indicating that the combined use of Compound A and 5-FU exhibits a synergistic effect on the cell growth inhibition of FaDu.

[0439] Comparative Example 1: Combination of Compound A and Vincristine on Human Diffuse Large B-cell Lymphoma Cell Line OCI-Ly18 effect

[0440] 1) Test substance

[0441] Bulk powder of Compound A was used. Vincristine used was purchased from Wako Pure Chemical Industries, Ltd.

[0442] 2) Cell growth inhibition assay

[0443] OCI-Ly18 cells were purchased from DSMZ. Cell culture, drug addition, cell growth inhibition test, calculation of cell viability and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 16,000 cells / well. Fig.34 The isoelectric plot analysis results are shown. Fig.34 The plot shown in is almost on the straight line, indicating that the combined use of Compound A and vincristine only exhibits an additive effect on the cell growth inhibition of OCI-Ly18.

[0444] Comparative Example 2: Combination effect of compound A and docetaxel on human breast cancer cell line BT-474 1) Test substance

[0445] Compound A bulk powder was used. Docetaxel was purchased from Wako Pure Chemical Industries, Ltd.

[0446] 2) Cell growth inhibition assay

[0447] BT-474 cells were purchased from ATCC. Cell culture, drug addition, cell growth inhibition test, calculation of cell survival rate and isobologram analysis were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Fig.35 The isoelectric plot analysis results are shown.

[0448] Fig.35The plot shown in is almost on the straight line, indicating that the combination of Compound A and docetaxel only exhibits an additive effect on the cell growth inhibition of BT-474.

[0449] Example 34: Effect of Compound A and Bendamustine on the proliferation of human diffuse large B-cell lymphoma (DLBCL) cell line OCI- Combinatorial effects of Ly7

[0450] 1) Test substance

[0451] Compound A bulk powder was used. Bendamustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0452] 2) Cell growth inhibition assay

[0453] OCI-Ly7 cells were purchased from DSMZ. Cell culture, drug addition, cell growth inhibition test and calculation of cell viability were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Table 2 shows the results.

[0454] [Table 2]

[0455]

[0456]

[0457] 3) Statistical analysis

[0458] The combined index (CI) and 95% confidence interval were calculated using SAS software Release 9.3 (SAS Institute JAPAN).

[0459] The CI was 0.67, and the 95% confidence interval was 0.37 to 0.96.

[0460] A CI greater than 1, equal to 1 or less than 1 means that an antagonistic, additive or synergistic effect is exhibited, respectively. Therefore, it was found that the combination of Compound A and bendamustine showed a synergistic effect on the growth inhibition of OCI-Ly7 cells.

[0461] Example 35: Combination effect of Compound A and bendamustine on human mantle cell lymphoma cell line REC-1

[0462] 1) Test substance

[0463] Compound A bulk powder was used. Bendamustine used was purchased from Tokyo Chemical Industry Co., Ltd.

[0464] 2) Cell growth inhibition assay

[0465] REC-1 cells were purchased from DSMZ.

[0466] Cell culture, drug addition, cell growth inhibition test and calculation of cell viability were performed in the same manner as in Example 1, except that the number of seeded cells was 8000 cells / well. Using the results obtained, the combination index (CI) and 95% confidence interval were calculated in the same manner as in Example 34.

[0467] The CI was 0.65, and the 95% confidence interval was 0.34 to 0.96. Thus, it was found that the combined use of Compound A and bendamustine showed a synergistic effect on REC-1 cell growth inhibition.

[0468] [In vivo anti-tumor effect test]

[0469] Example 36: Combination of Compound A and Cyclophosphamide on Human Diffuse Large B-cell Lymphoma Cell Line OCI-Ly7 effect

[0470] 1) Test substance

[0471] Bulk powder of compound A was used. Cyclophosphamide (Acedaxing Injection (trademark) 100 mg) used was purchased from Shionogi & Co., Ltd.

[0472] 2) Preparation of test substances

[0473] Compound A was suspended in 1% hydroxypropyl methylcellulose solution to prepare a 10 mg / mL (100 mg / kg dosing solution) test substance suspension. The test substance suspension was diluted with 1% hydroxypropyl methylcellulose solution to prepare 3 mg / mL (30 mg / kg dosing solution) and 1 mg / mL (10 mg / kg dosing solution) test substance suspensions. In addition, 100 mg of commercially available injection carcinogen was dissolved in 20 mL of saline and a 5 mg / mL solution (50 mg / kg dosing solution) was prepared when used. This solution was used as cyclophosphamide.

[0474] 3) Cells

[0475] OCI-Ly7 was obtained from DSMZ. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 1.5×10 8 The cells were suspended in serum-free RPMI 1640 medium at a density of 10 cells / mL.

[0476] 4) Animals

[0477] Five-week-old female SCID mice (CB-17 / Icr-scid / scid Jcl, produced by CLEA Japan, Inc.) were used. The female SCID mice were maintained under specific pathogen-free (SPF) conditions with standard diet and drinking water throughout the study. The cell suspension was incubated at 3×10 7 The amount of cells / individual was inoculated into the subcutaneous space of the right axillary region of 6-week-old SCID mice. Using tumor volume as an index, grouping was performed by a stratified random sampling method using SAS software R9.3 (SAS Institute Japan).

[0478] 5) Dosing and Measurement

[0479] Administration started on the next day (day 2) of grouping. Compound A was orally administered once a day for 14 consecutive days, and cyclophosphamide was intraperitoneally administered once every 3 or 4 days for a total of 4 times. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the monotherapy group, as in the combination therapy group. All animals received oral administration for 14 consecutive days and intraperitoneal administration once every 3 or 4 days (4 times in total). Each group (N=8) was treated as follows.

[0480] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0481] (2) Compound A (10 mg / kg) treatment group: 10 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0482] (3) Compound A (30 mg / kg) treatment group: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0483] (4) Compound A (100 mg / kg) treatment group: 100 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0484] (5) Cyclophosphamide (50 mg / kg) treatment group: 50 mg / kg cyclophosphamide (administered intraperitoneally on days 2, 5, 9, and 12)

[0485] (6) Combination Group 1: 10 mg / kg Compound A (oral administration once a day for 14 consecutive days) + 50 mg / kg cyclophosphamide (intraperitoneal administration on days 2, 5, 9 and 12)

[0486] (7) Combination Group 2: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days) + 50 mg / kg cyclophosphamide (intraperitoneal administration on days 2, 5, 9 and 12)

[0487] (8) Combination Group 3: 100 mg / kg Compound A (oral administration once a day for 14 consecutive days) + 50 mg / kg cyclophosphamide (intraperitoneal administration on days 2, 5, 9 and 12)

[0488] Body weight and tumor diameter were measured over time using an electronic balance and electronic digital calipers. Tumor volume was calculated from tumor diameter using the following formula: (long diameter × [short diameter] 2 × 0.5). Using the calculated tumor volume values, the relative tumor volume (the ratio of the tumor volume on the day of measurement to the baseline tumor volume on day 1) was calculated. The time for the relative tumor volume of each animal to reach 1600% from the grouping day (day 1) was estimated using the data of the relative tumor volume reaching 1600% on the adjacent measurement day. Tumor growth delay (TGD) was calculated as the difference in the median time for the relative tumor volume of the treatment and control groups to reach 1600%. In addition, the tumor growth delay rate (%TGD) was calculated according to the following formula:

[0489] %TGD={(TC) / C}×100,

[0490] in:

[0491] T: median time for relative tumor volume to reach 1600% in the treatment group

[0492] C: Median time for relative tumor volume to reach 1600% in the control group

[0493] 6) Statistical analysis

[0494] The effect of Compound A monotherapy on tumor growth delay was assessed using a two-tailed Steel's test based on the time to 1600% relative tumor volume per animal between the control group (Group 1) and each Compound A monotherapy group (Groups 2, 3, and 4).

[0495] The effect of cyclophosphamide monotherapy on tumor growth delay was assessed using a two-tailed Wilcoxon rank sum test based on the time to 1600% relative tumor volume per animal between the control group (Group 1) and the cyclophosphamide monotherapy group (Group 5).

[0496] In order to study the effect of the combination therapy group of compound A and cyclophosphamide on tumor growth delay, a two-tailed Steel test was performed to compare each monotherapy group (groups 2 and 5) with the combination therapy group (group 6), to compare each monotherapy group (groups 3 and 5) with the combination therapy group (group 7), and to compare each monotherapy group (groups 4 and 5) with the combination therapy group (group 8), until the time before the relative tumor volume reached 1600%. When the combination therapy group was significant relative to each monotherapy group, it was determined that there was a combination effect.

[0497] When significant differences were observed in the combination therapy groups relative to each monotherapy group, two-way ANOVA (two-tailed, factors: group and dose) was performed to evaluate the interaction effects between the Compound A treatment groups (Groups 1 and 2) and the cyclophosphamide treatment groups (Groups 5 and 6), between the Compound A treatment groups (Groups 1 and 3) and the cyclophosphamide treatment groups (Groups 5 and 7), and between the Compound A treatment groups (Groups 1 and 4) and the cyclophosphamide treatment groups (Groups 5 and 8).

[0498] 7) Results

[0499] The time required for the relative tumor volume to reach 1600% as the evaluation endpoint was 12.7 days (median time) in the control group. In monotherapy, the tumor growth delay rate (%) at 10, 30 and 100 mg / kg compound A was 3.5%, 81.1% and 147.6%, respectively. The tumor growth delay rate (%) at 50 mg / kg cyclophosphamide was 83.9%. It was shown that the monotherapy of 30 or 100 mg / kg compound A or 50 mg / kg cyclophosphamide was effective.

[0500] In the combination therapy, the group of 10 mg / kg Compound A + 50 mg / kg cyclophosphamide, the group of 30 mg / kg Compound A + 50 mg / kg cyclophosphamide, and the group of 100 mg / kg Compound A + 50 mg / kg cyclophosphamide showed tumor growth delay rates (%) of >287.0%, >293.7%, and >293.7%, respectively. When Compound A was administered in combination with cyclophosphamide, a combination effect and a significant interaction were observed, and the combination effect was significantly synergistic. In the combination therapy, the tumor was reduced at 10 mg / kg and higher Compound A, and completely regressed at 30 mg / kg and higher Compound A. In addition, in the administration groups of 30 or 100 mg / kg Compound A in combination with cyclophosphamide, no tumor regrowth was observed during the observation period after administration (Day 16 to Day 51) (Tables 3 and Fig.36 , 37 and 38).

[0501] 8) Conclusion

[0502] These results demonstrate that 30 or 100 mg / kg Compound A monotherapy, 50 mg / kg cyclophosphamide monotherapy, and 10, 30, or 100 mg / kg Compound A in combination with 50 mg / kg cyclophosphamide have antitumor effects on OCI-Ly7 tumors. The combination therapy was well tolerated and showed significant synergistic effects.

[0503] [Table 3]

[0504]

[0505] a Steel test results for the control group and each Compound A monotherapy group for the evaluation endpoint (relative tumor volume 1600%)

[0506] b Results of the Wilcoxon rank sum test for the control group and the cyclophosphamide monotherapy group for the evaluation endpoint (relative tumor volume 1600%)

[0507] c,d,e Steel test results for each monotherapy group (10 mg / kg, 30 mg / kg or 100 mg / kg compound A or 50 mg / kg cyclophosphamide) and the corresponding combination therapy group (compound A + 50 mg / kg cyclophosphamide) for the evaluation endpoint (relative tumor volume 1600%) Since significant differences in tumor growth delay were observed between the combination therapy group and the corresponding monotherapy group (50 mg / kg cyclophosphamide and each dose of compound A), a two-way ANOVA was performed to evaluate the interaction effect by comparing the non-cyclophosphamide treatment group (control, compound A monotherapy) with the cyclophosphamide treatment group (cyclophosphamide monotherapy, combination therapy). Significant synergistic effects were observed in the interaction effect of the combination therapy group (p < 0.01).

[0508] Example 37: Combination effect of compound A and lapatinib on human breast cancer cell line MDA-MB-453

[0509] 1) Test substance

[0510] Compound A bulk powder was used. Lapatinib used was lapatinib di-p-toluenesulfonate purchased from LC Laboratories.

[0511] 2) Preparation of test substances

[0512] Compound A was suspended in a 1% hydroxypropyl methylcellulose solution to prepare a 20 mg / mL test substance suspension (suspension for preparing a single dosing solution and suspension for preparing a 100 mg / kg combined dosing solution). The 20 mg / mL test substance suspension was diluted with a 1% hydroxypropyl methylcellulose solution to prepare a 10 mg / mL (100 mg / kg dosing solution), 6 mg / mL (suspension for preparing a 30 mg / kg combined dosing solution) and 3 mg / mL (30 mg / kg dosing solution) test substance suspension. Lapatinib was suspended in a 1% hydroxypropyl methylcellulose solution to prepare a 20 mg / mL suspension for preparing a 100 mg / kg lapatinib combined dosing solution. The 20 mg / mL suspension was diluted with a 1% hydroxypropyl methylcellulose solution to prepare a 10 mg / mL suspension for a 100 mg / kg single dosing. The dosing solution for the combination group was prepared by mixing 6 mg / mL Compound A suspension with 20 mg / mL lapatinib suspension at 1:1 or mixing 20 mg / mL Compound A suspension with 20 mg / mL lapatinib suspension at 1:1.

[0513] 3) Cells

[0514] MDA-MB-453 was obtained from ATCC. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 1.0×10 8 The cells were suspended in serum-free RPMI 1640 medium at a density of 10 cells / mL.

[0515] 4) Animals

[0516] Five-week-old female NOD-SCID mice (NOD.CB17-Prkdcscid / J, produced by Charles River Japan Co., Ltd.) were used. The female NOD-SCID mice were raised in the same manner as in Example 36. The cell suspension was plated at 2×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old NOD-SCID mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0517] 5) Dosing and Measurement

[0518] The administration started on the next day after grouping (Day 2), and Compound A and lapatinib were orally administered once a day for 28 consecutive days. Each group (N=8) was treated as follows.

[0519] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution

[0520] (2) Compound A (30 mg / kg) treatment group: 30 mg / kg Compound A (oral administration once a day for 28 consecutive days)

[0521] (3) Compound A (100 mg / kg) treatment group: 100 mg / kg Compound A (oral administration once a day for 28 consecutive days)

[0522] (4) Lapatinib (100 mg / kg) treatment group: 100 mg / kg lapatinib (orally administered once a day for 28 consecutive days)

[0523] (5) Combination group 1: 30 mg / kg compound A (orally administered once a day for 28 consecutive days) + 100 mg / kg lapatinib (orally administered once a day for 28 consecutive days)

[0524] (6) Combination group 2: 100 mg / kg compound A (administered orally once a day for 28 consecutive days) + 100 mg / kg lapatinib (administered orally once a day for 28 consecutive days)

[0525] Measurement, calculation of tumor growth delay and statistical analysis were performed in the same manner as in Example 36.

[0526] 6) Results

[0527] The time required for the relative tumor volume to reach 200% as the endpoint of the assessment was 22.8 days (median time) in the control group. In monotherapy, the tumor growth delay rate (%) was 72.4% and >334.2% at 30 and 100 mg / kg compound A, respectively. The tumor growth delay rate (%) was 50.0% at 100 mg / kg lapatinib. It was shown that the monotherapy of 30 or 100 mg / kg compound A or 100 mg / kg lapatinib was effective.

[0528] In the combination therapy, the group of 30 mg / kg compound A + 100 mg / kg lapatinib and the group of 100 mg / kg compound A + 100 mg / kg lapatinib both showed a tumor growth delay rate (%) of >334.2%. When 30 mg / kg compound A was administered in combination with 100 mg / kg lapatinib, a combination effect and a significant interaction were observed, and the combination effect was significantly synergistic. In the combination therapy, the tumor completely regressed at two doses of compound A. In addition, in the group of 100 mg / kg compound A + 100 mg / kg lapatinib, no tumor regrowth was observed until 2 months after the completion of the combined administration (Table 4 and Table 5). Fig.39 and 40 ).

[0529] 7) Conclusion

[0530] These results demonstrate that 30 or 100 mg / kg Compound A monotherapy, 100 mg / kg Lapatinib monotherapy, and 30 or 100 mg / kg Compound A combined with 100 mg / kg Lapatinib have antitumor effects on MDA-MB-453 tumors. The combination therapy was well tolerated and showed significant synergistic effects.

[0531] [Table 4]

[0532]

[0533] a Steel test results for the control group and each Compound A monotherapy group for the evaluation endpoint (relative tumor volume 200%)

[0534] b Results of Wilcoxon rank sum test for the control group and lapatinib monotherapy group for the evaluation endpoint (relative tumor volume 200%)

[0535] c,d Steel test results for each monotherapy group (30 mg / kg or 100 mg / kg compound A or 100 mg / kg lapatinib) and the corresponding combination therapy group (compound A+100 mg / kg lapatinib) for the evaluation endpoint (relative tumor volume 200%) Since significant differences in tumor growth delay were observed between the combination therapy group of 30 mg / kg compound A+100 mg / kg lapatinib and the corresponding monotherapy group, a two-way ANOVA was performed to evaluate the interaction effect by comparing the non-lapatinib treatment group (control, compound A monotherapy) with the lapatinib treatment group (lapatinib monotherapy, combination therapy). Significant synergistic effects were observed in the interaction effect of the combination therapy group (p<0.01).

[0536] Example 38: Combination of Compound A and Bendamustine on Human Diffuse Large B-cell Lymphoma Cell Line OCI-Ly7 Synergistic Effect

[0537] 1) Test substance

[0538] Bulk powder of Compound A was used. Bendamustine used was bendamustine hydrochloride hydrate purchased from Tokyo Chemical Industry Co., Ltd.

[0539] 2) Preparation of test substances

[0540] Compound A was suspended in a 1% hydroxypropylmethylcellulose solution to prepare a 3 mg / mL (30 mg / kg dosing solution) test substance suspension. Bendamustine was dissolved in saline and a 1.5 mg / mL solution (15 mg / kg dosing solution) was prepared when used.

[0541] 3) Cells

[0542] OCI-Ly7 was obtained from DSMZ. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 1.5×10 8 The cells were suspended in serum-free RPMI 1640 medium at a density of 10 cells / mL.

[0543] 4) Animals

[0544] Five-week-old female SCID mice (CB-17 / Icr-scid / scid Jcl, produced by CLEA Japan, Inc.) were used. The female SCID mice were raised in the same manner as in Example 36. The cell suspension was incubated at 3×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old SCID mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0545] 5) Dosing and Measurement

[0546] Dosing started on the next day of grouping (Day 2). Compound A was orally administered once a day for 14 consecutive days, and bendamustine was intraperitoneally administered for a total of 5 times on Days 2, 3, 4, 5, and 6. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the single-dose group, as in the combination group. All animals received oral administration for 14 consecutive days and intraperitoneal administration on Days 2, 3, 4, 5, and 6 (a total of 5 times). Each group (N=6) was treated as follows.

[0547] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0548] (2) Compound A (30 mg / kg) treatment group: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0549] (3) Bendamustine (15 mg / kg) treatment group: 15 mg / kg bendamustine (administered intraperitoneally on days 2, 3, 4, 5, and 6)

[0550] (4) Combination group: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days) + 15 mg / kg Bendamustine (intraperitoneal administration on days 2, 3, 4, 5 and 6)

[0551] Measurement, calculation of tumor growth delay and statistical analysis were performed in the same manner as in Example 36.

[0552] 6) Results

[0553] The time required for the relative tumor volume to reach 1600%, which is the evaluation endpoint, was 15.5 days (median time) in the control group. In monotherapy, the tumor growth delay rate (%) at 30 mg / kg Compound A was 54.2%. The tumor growth delay rate (%) at 15 mg / kg Bendamustine was 56.5%. It was shown that the monotherapy of 30 mg / kg Compound A or 15 mg / kg Bendamustine was effective.

[0554] In the combination therapy, the group of 30 mg / kg Compound A + 15 mg / kg Bendamustine showed a tumor growth delay rate (%) of >222.6%. When Compound A was administered in combination with Bendamustine, a combination effect and a significant interaction were observed, and the combination effect was significantly synergistic. In addition, by the combination therapy, the tumor completely regressed (Table 5 and Fig.41 ).

[0555] 7) Conclusion

[0556] These results demonstrate that 30 mg / kg Compound A monotherapy, 15 mg / kg Bendamustine monotherapy, and 30 mg / kg Compound A and 15 mg / kg Bendamustine combination therapy have antitumor effects on OCI-Ly7 tumors. The combination therapy was well tolerated and showed significant synergistic effects.

[0557] [Table 5]

[0558]

[0559] a Results of Wilcoxon rank sum test for the control group and each Compound A monotherapy group and the control group and bendamustine monotherapy group for the evaluation endpoint (relative tumor volume 1600%)

[0560] bSteel test results for each monotherapy group (30 mg / kg Compound A or 15 mg / kg bendamustine) and combination therapy group (30 mg / kg Compound A + 15 mg / kg bendamustine) for the evaluation endpoint (relative tumor volume 1600%) Since significant differences in tumor growth delay were observed between the combination therapy group and the corresponding monotherapy group, a two-way ANOVA was performed to evaluate the interaction effect by comparing the non-bendamustine treatment group (control, Compound A monotherapy) with the bendamustine treatment group (bendamustine monotherapy, combination therapy). A significant synergistic effect was observed in the interaction effect of the combination therapy group (p<0.01).

[0561] Example 39: Combination of Compound A and Rituximab on Human Diffuse Large B-cell Lymphoma Cell Line OCI-Ly7 Synergistic Effect

[0562] 1) Test substance

[0563] Bulk powder of compound A was used. Rituximab (Rituxan (trademark) 100 mg / 10 mL) was purchased from Genentech, Inc.

[0564] 2) Preparation of test substances

[0565] Compound A was suspended in a 1% hydroxypropylmethylcellulose solution to prepare a 3 mg / mL (30 mg / kg dosing solution) test substance suspension. Rituximab was diluted with saline and a 1 mg / mL solution (10 mg / kg dosing solution) was prepared when used.

[0566] 3) Cells

[0567] OCI-Ly7 was obtained from DSMZ. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 1.5×10 8 The cells were suspended in serum-free RPMI 1640 medium at a density of 10 cells / mL.

[0568] 4) Animals

[0569] Five-week-old female nude mice (BALB / c Slc-nu / nu, produced by Japan SLC, Inc.) were used. The female nude mice were raised in the same manner as in Example 36. The cell suspension was incubated at 3×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old nude mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0570] 5) Dosing and Measurement

[0571] Dosing started on the next day (day 2) of grouping. Compound A was orally administered once a day for 21 consecutive days, and rituximab was intraperitoneally administered for a total of 3 times on the 2nd, 9th and 16th days. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the single-dose group, as in the combination group. All animals received oral administration for 21 consecutive days and intraperitoneal administration on the 2nd, 9th and 16th days (3 times in total). Each group (N=6) was treated as follows.

[0572] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0573] (2) Compound A (30 mg / kg) treatment group: 30 mg / kg Compound A (oral administration once a day for 21 consecutive days)

[0574] (3) Rituximab treatment group: 10 mg / kg rituximab (intraperitoneal administration on days 2, 9, and 16)

[0575] (4) Combination group: 30 mg / kg compound A (oral administration once a day for 21 consecutive days) + 10 mg / kg rituximab (intraperitoneal administration on days 2, 9, and 16)

[0576] Measurement, calculation of tumor growth delay and statistical analysis were performed in the same manner as in Example 36.

[0577] 6) Results

[0578] The time required for the relative tumor volume to reach 600%, which was the endpoint of the evaluation, was 16.3 days (median time) in the control group. In monotherapy, the tumor growth delay rate (%) at 30 mg / kg compound A was 73.9%. The tumor growth delay rate (%) at 10 mg / kg rituximab was 74.2%. It was shown that the monotherapy of 30 mg / kg compound A or 10 mg / kg rituximab was effective.

[0579] In the combination therapy, the group of 30 mg / kg Compound A + 10 mg / kg Rituximab showed a tumor growth delay rate (%) of >421.5%. When Compound A was administered in combination with Rituximab, a combination effect and a significant interaction were observed, and the combination effect was significantly synergistic. In addition, by the combination therapy, the tumor completely regressed (Table 6 and Fig.42 ).

[0580] 7) Conclusion

[0581] These results demonstrate that 30 mg / kg Compound A monotherapy, 10 mg / kg Rituximab monotherapy, and 30 mg / kg Compound A combined with 10 mg / kg Rituximab have anti-tumor effects on OCI-Ly7 tumors. The combination therapy was well tolerated and showed significant synergistic effects.

[0582] [Table 6]

[0583]

[0584] a Results of Wilcoxon rank sum test for the control group and each Compound A monotherapy group and the control group and rituximab monotherapy group for the evaluation endpoint (relative tumor volume 600%)

[0585] b Steel test results for each monotherapy group (30 mg / kg Compound A or 10 mg / kg Rituximab) and combination therapy group (30 mg / kg Compound A + 10 mg / kg Rituximab) for the evaluation endpoint (relative tumor volume 600%) Since significant differences in tumor growth delay were observed between the combination therapy group and the corresponding monotherapy group, a two-way ANOVA was performed to evaluate the interaction effect by comparing the non-rituximab treatment group (control, Compound A monotherapy) with the rituximab treatment group (rituximab monotherapy, combination therapy). A significant synergistic effect was observed in the interaction effect of the combination therapy group (p<0.01).

[0586] Example 40: Combination effect of compound A and decitabine on human acute myeloid leukemia (AML) cell line KG-1

[0587] 1) Test substance

[0588] Bulk powder of compound A was used. Decitabine (5-aza-2′-deoxycytidine) used was purchased from Tokyo Chemical Industry Co., Ltd.

[0589] 2) Preparation of test substances

[0590] Compound A was suspended in a 1% hydroxypropylmethylcellulose solution to prepare a 3 mg / mL dosing solution of 30 mg / kg Compound A. Decitabine was diluted with saline to prepare a 0.1 mg / mL dosing solution of 1 mg / kg decitabine.

[0591] 3) Cells

[0592] KG-1 was obtained from the Health Science Research Resources Bank. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 2.5×10 8 The cells were suspended in serum-free RPMI1640 medium at a density of 10 cells / mL.

[0593] 4) Animals

[0594] Five-week-old female SCID mice (CB-17 / Icr-scid / scid Jcl, produced by CLEA Japan, Inc.) were used. The female SCID mice were raised in the same manner as in Example 36. The cell suspension was incubated at 5×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old SCID mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0595] 5) Dosing and Measurement

[0596] Administration started on the next day of grouping (day 2), and tumor diameter and body weight were finally measured and evaluated on the next day (day 23) after 21 days of administration. Each group (N=8) was treated as follows. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the single administration group, as in the combination group.

[0597] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0598] (2) Compound A treatment group: 30 mg / kg Compound A (oral administration once a day for 21 consecutive days)

[0599] (3) Decitabine treatment group: 1 mg / kg decitabine (intraperitoneal administration on days 2, 5, 9, 12, 16 and 19)

[0600] (4) Combination group: 30 mg / kg Compound A (oral administration once a day for 21 consecutive days) + 1 mg / kg decitabine (intraperitoneal administration on days 2, 5, 9, 12, 16 and 19)

[0601] Body weight and tumor diameter were measured over time using an electronic balance and electronic digital calipers. Tumor volume was calculated from tumor diameter using the following formula: (long diameter × [short diameter] 2× 0.5). Antitumor activity was expressed as the ratio of relative tumor volume of each treatment group to that of the control group (T / C%). T / C% on day 23 was calculated for each group by the following formula: relative tumor volume of each group on day 23 [(tumor volume of each group on day 23 / tumor volume of each group on day 1) × 100] / average relative tumor volume of the control group on day 23 [(tumor volume of the control group on day 23 / tumor volume of the control group on day 1) × 100] × 100.

[0602] 6) Statistical analysis

[0603] The effect of the combination of Compound A and decitabine (Group 4) on relative tumor volume (Day 23) was evaluated by Dunnett's test (two-tailed) for Compound A alone (Group 2) and decitabine alone (Group 3). Since significant differences were observed in Group 4 for each monotherapy group, a two-way ANOVA (two-tailed) was performed to evaluate the interaction effect between the Compound A treatment groups (Groups 1 and 2) and the decitabine treatment groups (Groups 3 and 4).

[0604] 7) Results

[0605] Compound A and decitabine showed antitumor effects on KG-1 tumors. Combination therapy of decitabine and compound A significantly inhibited tumor growth more effectively than monotherapy of each drug (Table 7 and Fig.43 ).

[0606] 8) Conclusion

[0607] The combination therapy using Compound A and decitabine was well tolerated and showed significant synergistic effects in a mouse model bearing KG-1 tumors. This result supports the conduct of a clinical trial using the combination chemotherapy of Compound A and decitabine.

[0608] Example 41: Combination effect of compound A and cytarabine on human acute myeloid leukemia (AML) cell line KG-1

[0609] 1) Test substance

[0610] Bulk powder of compound A was used. Cytarabine (Cylocide (registered trademark) injection, 60 mg) used was purchased from Nippon Shinyaku Co., Ltd.

[0611] 2) Preparation of test substances

[0612] Compound A was suspended in a 1% hydroxypropylmethylcellulose solution to prepare a 3 mg / mL dosing solution of 30 mg / kg Compound A. Cytarabine was diluted with saline to prepare a 5 mg / mL dosing solution of 50 mg / kg Cytarabine.

[0613] 3) Cells

[0614] KG-1 was obtained from the Health Science Research Resources Bank. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 2.5×10 8 The cells were suspended in serum-free RPMI1640 medium at a density of 10 cells / mL.

[0615] 4) Animals

[0616] Five-week-old female SCID mice (CB-17 / Icr-scid / scid Jcl, produced by CLEA Japan, Inc.) were used. The female SCID mice were raised in the same manner as in Example 36. The cell suspension was incubated at 5×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old SCID mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0617] 5) Dosing and Measurement

[0618] Administration started on the next day (day 2) of grouping. The next day (day 16) after the 14-day administration was completed, the tumor diameter and body weight were finally measured and evaluated. Each group (N=6) was treated as follows. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the single administration group, as in the combination group.

[0619] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0620] (2) Compound A treatment group: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0621] (3) Cytarabine treatment group: 50 mg / kg cytarabine (intraperitoneal administration on days 2, 3, 4, 5, 6, 9, 10, 11, 12 and 13)

[0622] (4) Combination group: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days) + 50 mg / kg cytarabine (intraperitoneal administration on days 2, 3, 4, 5, 6, 9, 10, 11, 12 and 13)

[0623] Body weight and tumor diameter were measured over time using an electronic balance and electronic digital calipers. Tumor volume was calculated from tumor diameter using the following formula: (long diameter × [short diameter] 2× 0.5). Antitumor activity was expressed as the ratio of relative tumor volume of each treatment group to that of the control group (T / C%). T / C% on day 16 was calculated for each group by the following formula: relative tumor volume of each group on day 16 [(tumor volume of each group on day 16 / tumor volume of each group on day 1) × 100] / average relative tumor volume of the control group on day 16 [(tumor volume of the control group on day 16 / tumor volume of the control group on day 1) × 100] × 100.

[0624] 6) Statistical analysis

[0625] The effect of the combination of Compound A and cytarabine (Group 4) on relative tumor volume (Day 16) was evaluated by Dunnett's test (two-tailed) for Compound A alone (Group 2) and cytarabine alone (Group 3). Since significant differences were observed in Group 4 for each monotherapy group, a two-way ANOVA (two-tailed) was performed to evaluate the interaction effect between the Compound A treatment groups (Groups 1 and 2) and the cytarabine treatment groups (Groups 3 and 4).

[0626] 7) Results

[0627] Compound A and cytarabine showed antitumor effects on KG-1 tumors. The combination therapy of cytarabine and compound A was more effective than the monotherapy of each drug in significantly inhibiting tumor growth (Table 7 and Fig.44 ).

[0628] 8) Conclusion

[0629] The combination therapy using Compound A and cytarabine was well tolerated and showed significant synergistic effects in a mouse model bearing KG-1 tumors. This result supports the conduct of a clinical trial using the combination chemotherapy of Compound A and cytarabine.

[0630] [Table 7]

[0631]

[0632] *Synergistic combination effect

[0633] Example 42: Effect of Compound A, Bendamustine and Rituximab on Human Diffuse Large B-cell Lymphoma Cell Lines Triple-drug combination effects of OCI-Ly7

[0634] 1) Test substance

[0635] The bulk powder of compound A was used. The bendamustine used was bendamustine hydrochloride hydrate purchased from Tokyo Chemical Industry Co., Ltd. The rituximab used (Rituxan (registered trademark) 100 mg / 10 mL) was purchased from Genentech, Inc.

[0636] 2) Preparation of test substances

[0637] Compound A was suspended in a 1% hydroxypropylmethylcellulose solution to prepare a 3 mg / mL (30 mg / kg dosing solution) test substance suspension. Bendamustine was dissolved in saline and a 1 mg / mL solution was prepared when used (10 mg / kg dosing solution). Rituximab was diluted with saline and a 1 mg / mL solution was prepared when used (10 mg / kg dosing solution).

[0638] 3) Cells

[0639] OCI-Ly7 was obtained from DSMZ. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 1.5×10 8 The cells were suspended in serum-free RPMI 1640 medium at a density of 10 cells / mL.

[0640] 4) Animals

[0641] Five-week-old female SCID mice (CB-17 / Icr-scid / scid Jcl, produced by CLEA Japan, Inc.) were used. The female SCID mice were raised in the same manner as in Example 36. The cell suspension was incubated at 3×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old SCID mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0642] 5) Dosing and Measurement

[0643] Dosing started on the next day of grouping (Day 2). Compound A was orally administered once a day for 14 consecutive days. Bendamustine was administered intraperitoneally for a total of 5 times on Days 2, 3, 4, 5 and 6. Rituximab was administered intraperitoneally for a total of two times on Days 2 and 9. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the single-dose group, as in the combination group. All animals received oral administration for 14 consecutive days and intraperitoneal administration on Days 2, 3, 4, 5, 6 and 9. Each group (N=6) was treated as follows.

[0644] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0645] (2) Compound A (30 mg / kg) treatment group: 30 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0646] (3) Bendamustine (10 mg / kg) + Rituximab (10 mg / kg) treatment group: 10 mg / kg Bendamustine (intraperitoneal administration on days 2, 3, 4, 5, and 6) + 10 mg / kg Rituximab (intraperitoneal administration on days 2 and 9)

[0647] (4) Three-drug combination group: 30 mg / kg Compound A (administered orally once a day for 14 consecutive days) + 10 mg / kg Bendamustine (administered intraperitoneally on days 2, 3, 4, 5, and 6) + 10 mg / kg Rituximab (administered intraperitoneally on days 2 and 9)

[0648] Measurement, calculation of tumor growth delay and statistical analysis were performed in the same manner as in Example 36.

[0649] 6) Results

[0650] The time required for the relative tumor volume to reach 800%, which was the endpoint of the evaluation, was 9.9 days (median time) in the control group. The compound A (30 mg / kg) treatment group showed a tumor growth delay rate (%) of 83.8%. The bendamustine (10 mg / kg) + rituximab (10 mg / kg) treatment group showed a tumor growth delay rate (%) of 144.4%. It was shown that 30 mg / kg of compound A and 10 mg / kg of bendamustine + 10 mg / kg of rituximab were effective.

[0651] In the three-drug combination therapy, the group administered with 30 mg / kg Compound A + 10 mg / kg Bendamustine + 10 mg / kg Rituximab showed a tumor growth delay rate (%) of >829.3%. When Compound A was administered in combination with Bendamustine and Rituximab, a three-drug combination effect and a significant interaction were observed, and the three-drug combination effect was significantly synergistic. In addition, the tumor completely regressed by the three-drug combination therapy (Table 8 and Table 9). Fig.45 ).

[0652] 7) Conclusion

[0653] It has been shown that 30 mg / kg of Compound A monotherapy, 10 mg / kg bendamustine + 10 mg / kg rituximab combination therapy, and 30 mg / kg Compound A, 10 mg / kg bendamustine, and 10 mg / kg rituximab triple combination therapy have significant anti-tumor activity in a mouse model bearing OCI-Ly7 tumors. The triple combination therapy was well tolerated and showed significant synergistic effects.

[0654] [Table 8]

[0655]

[0656] a Wilcoxon rank sum test results for the control group and the compound A treatment group and the control group and the bendamustine + rituximab treatment group for the evaluation endpoint (relative tumor volume 800%)

[0657] b Steel test results for the evaluation endpoint (relative tumor volume 800%) of the compound A (30 mg / kg) treatment group, the bendamustine (10 mg / kg) + rituximab (10 mg / kg) treatment group, and the triple drug treatment group (30 mg / kg compound A + 10 mg / kg bendamustine + 10 mg / kg rituximab)

[0658] Since significant differences in tumor growth delay were observed between the three-drug treatment group, the corresponding Compound A (30 mg / kg) treatment group, and the bendamustine (10 mg / kg) + rituximab (10 mg / kg) treatment group, a two-way ANOVA was performed to evaluate the interaction effect by comparing the non-bendamustine + rituximab treatment group (control, Compound A treatment group) with the bendamustine + rituximab treatment group (bendamustine + rituximab treatment group, three-drug treatment group). A significant synergistic effect was observed in the interaction effect of the three-drug treatment group (p < 0.01).

[0659] Example 43: Effects of Compound A, Decitabine and Venetoclax on the Human Acute Myeloid Leukemia (AML) Cell Line KG-1 The three-drug combination effect

[0660] 1) Test substance

[0661] Compound A bulk powder was used. Venetoclax was purchased from LC Laboratories. Decitabine (5-aza-2′-deoxycytidine) was purchased from Tokyo Chemical Industry Co., Ltd.

[0662] 2) Preparation of test substances

[0663] Compound A and venetoclax were suspended in a 1% hydroxypropylmethylcellulose solution to prepare a 5 mg / mL dosing solution of 50 mg / kg Compound A. Decitabine was diluted with saline to prepare a 0.1 mg / mL dosing solution of 1 mg / kg decitabine.

[0664] 3) Cells

[0665] KG-1 was obtained from the Health Science Research Resources Bank. The cells were cultured in RPMI 1640 medium supplemented with 10% inactivated (treated at 56°C for 30 minutes) fetal bovine serum at 37°C and 5% CO2. The collected cells were cultured at 2.5×10 8 The cells were suspended in serum-free RPMI1640 medium at a density of 10 cells / mL.

[0666] 4) Animals

[0667] Five-week-old female SCID mice (CB-17 / Icr-scid / scid Jcl, produced by CLEA Japan, Inc.) were used. The female SCID mice were raised in the same manner as in Example 36. The cell suspension was incubated at 5×10 7 The cells were inoculated in an amount of 10 cells / individual into the subcutaneous space of the right axillary region of 6-week-old SCID mice, and the mice were grouped in the same manner as in Example 36 using tumor volume as a grouping index.

[0668] 5) Dosing and Measurement

[0669] Administration started on the next day of grouping (Day 2), and tumor diameter and body weight were finally measured and evaluated on the next day (Day 16) after the 14-day administration was completed. Each group (N=6) was treated as follows. In order to match the combination group, the solvent of each drug was administered to the control group and the solvent of the combination drug was administered to the single administration group, as in the combination group.

[0670] (1) Control (solvent control) group: 1% hydroxypropyl methylcellulose solution, saline

[0671] (2) Compound A treatment group: 50 mg / kg Compound A (oral administration once a day for 14 consecutive days)

[0672] (3) Venetoclax + decitabine treatment group: 50 mg / kg venetoclax (orally administered once a day for 14 consecutive days) + 1 mg / kg decitabine (intraperitoneally administered on days 2, 5, 9, and 12)

[0673] (4) Three-drug combination group: 50 mg / kg Compound A (oral administration once a day for 14 consecutive days) + 50 mg / kg venetoclax (oral administration once a day for 14 consecutive days) + 1 mg / kg decitabine (intraperitoneal administration on days 2, 5, 9 and 12)

[0674] Body weight and tumor diameter were measured over time using an electronic balance and electronic digital calipers. Tumor volume was calculated from tumor diameter using the following formula: (long diameter × [short diameter] 2× 0.5). Antitumor activity was expressed as the ratio of relative tumor volume of each treatment group to that of the control group (T / C%). T / C% on day 16 was calculated for each group by the following formula: relative tumor volume of each group on day 16 [(tumor volume of each group on day 16 / tumor volume of each group on day 1) × 100] / average relative tumor volume of the control group on day 16 [(tumor volume of the control group on day 16 / tumor volume of the control group on day 1) × 100] × 100.

[0675] 6) Statistical analysis

[0676] The effect of Compound A and the three-drug combination of venetoclax and decitabine (Group 4) on relative tumor volume (Day 16) was evaluated by Dunnett's test (two-tailed) for Compound A alone (Group 2) and venetoclax + decitabine (Group 3). Since significant differences were observed in Group 4 for Groups 2 and 3, a two-way ANOVA (two-tailed) was performed to evaluate the interaction effect between the control and Compound A treatment groups (Groups 1 and 2) and the venetoclax + decitabine treatment groups and the three-drug combination groups (Groups 3 and 4).

[0677] 6) Results

[0678] Compound A and venetoclax + decitabine showed antitumor effects on KG-1 tumors. The three-drug combination therapy of venetoclax, decitabine and compound A significantly inhibited tumor growth more effectively than single administration of compound A and administration of venetoclax and decitabine (Table 9 and Fig.46 ).

[0679] 7) Conclusion

[0680] The three-drug combination therapy using Compound A, venetoclax, and decitabine was well tolerated and showed significant synergistic effects in a mouse model bearing KG-1 tumors. This result supports the conduct of a clinical trial using combination chemotherapy using Compound A, venetoclax, and decitabine.

[0681] [Table 9]

[0682]

[0683] *Synergistic combination effect

Claims

1. Use of an antitumor composition in the preparation of an antitumor drug, the antitumor composition comprising the following antitumor agent (A) and the following compound (B) or a salt thereof: (A) at least one antineoplastic agent selected from bendamustine, decitabine, busulfan, lomustine, thiotepa, melphalan, cytarabine, venetoclax, ranimustine, ibrutinib, erlotinib, gefitinib, imatinib, axitinib, lapatinib, lenvatinib, afatinib, gilteritinib, 5-fluorouracil, cyclophosphamide and rituximab; (B) N-{6-[2-Methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide.

2. The use according to claim 1, wherein the antitumor drug is used to administer compound (B) or a salt thereof to a person who will receive or has received an antitumor agent (A), and wherein one of compound (B) or a salt thereof and the antitumor agent (A) is administered a few minutes to several tens of minutes or 1 to 12 hours after the completion of administration of the other.

3. An antitumor composition comprising: The following antitumor agent (A), and The following compound (B) or a salt thereof: (A) at least one antineoplastic agent selected from bendamustine, decitabine, busulfan, lomustine, thiotepa, melphalan, cytarabine, venetoclax, ranimustine, ibrutinib, erlotinib, gefitinib, imatinib, axitinib, lapatinib, lenvatinib, afatinib, gilteritinib, 5-fluorouracil, cyclophosphamide and rituximab; (B) N-{6-[2-Methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide.

4. A medicine kit comprising: The following antitumor agent (A), and An antitumor composition comprising the following compound (B) or a salt thereof: (A) at least one antineoplastic agent selected from bendamustine, decitabine, busulfan, lomustine, thiotepa, melphalan, cytarabine, venetoclax, ranimustine, ibrutinib, erlotinib, gefitinib, imatinib, axitinib, lapatinib, lenvatinib, afatinib, gilteritinib, 5-fluorouracil, cyclophosphamide and rituximab; (B) N-{6-[2-Methyl-4-(methylamino)phenoxy]pyridin-3-yl}-4-(trifluoromethyl)benzamide.

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