Anticancer composition

Abstract

The present invention provides a novel cancer therapeutic means, which is a combination of a reversible BTK inhibitor and a BCL-2 inhibitor. Specifically, provided is an anticancer agent composition obtained by combining, for instance, BTK inhibitor (I-A) and venetoclax.

Description

[Technical Field]

[0001] The present invention relates to an anticancer composition. More specifically, the present invention relates to an anticancer composition comprising a combination of a reversible BTK inhibitor and a BCL-2 inhibitor. [Background technology]

[0002] Bruton's tyrosine kinase (BTK) is a member of the Tec family of non-receptor tyrosine kinases and is an important signaling enzyme expressed in all hematopoietic cell types except T lymphocytes and natural killer cells. BTK is a key regulator of B cell survival, differentiation, proliferation, and activation, and plays a crucial role in B cell signaling (Non-Patent Documents 1 and 2). The B cell receptor (BCR) on the cell surface transmits signals into the cell via BTK, which is located downstream of BCR. Therefore, aberrant activation of B cell signaling pathways is thought to promote the proliferation and survival of cancer cells, such as B cell lymphoma and chronic lymphocytic leukemia (Non-Patent Document 3). BTK is also known to play an important role in numerous other cell signaling pathways and is thought to be involved in allergic diseases, autoimmune diseases, and inflammatory diseases (Non-Patent Document 1). For example, BTK plays an important role in signal transduction of the high-affinity IgE receptor (FcεRI) in mast cells, and mast cells lacking BTK are known to exhibit reduced degranulation and reduced production of proinflammatory cytokines (Non-Patent Document 4). Experiments with BTK-deficient mice suggest that BTK is involved in systemic lupus erythematosus (SLE) (Non-Patent Document 5). Furthermore, BTK mutant mice are resistant to the development of collagen-induced arthritis (Non-Patent Document 6). Ibrutinib, an irreversible BTK inhibitor, is an anticancer drug used to treat B-cell tumors. Recently, it has been shown that the C481S mutation in BTK induces resistance to ibrutinib during ibrutinib treatment (Non-Patent Document 7). Furthermore, it has recently been reported that the p65BTK isoform is expressed downstream of RAS signaling in solid tumors other than hematological cancers and has a profound effect on the proliferation of solid tumors such as colon cancer cells (Non-Patent Document 8). Therefore, compounds with BTK inhibitory activity are expected to be useful in the treatment of diseases in which BTK signaling is involved, such as cancer, B-cell lymphoma, and chronic lymphocytic leukemia, as well as solid cancers in which p65BTK is expressed.Furthermore, they are also useful for treating allergic diseases, autoimmune diseases, inflammatory diseases, etc. Furthermore, there is a need for reversible BTK inhibitors that are effective against cancers with BTK mutations that are resistant to irreversible BTK inhibitors such as ibrutinib. In particular, oxoisoquinoline derivatives and triazine derivatives with reversible BTK inhibitory activity have been reported (see Patent Documents 1 and 2).

[0003] BCL-2 (B-cell lymphoma 2) is a member of the BCL-2 family of proteins that regulate cell death. It primarily functions on the mitochondrial membrane, negatively regulating apoptosis by positively and negatively regulating the permeability of the outer mitochondrial membrane. BCL-2 was discovered through a translocation frequently observed in follicular lymphoma. It has since been reported to be activated in lymphoid B-cell tumors, such as diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), as well as multiple myeloma and T-cell tumors. Therefore, molecules that inhibit BCL-2 are useful for treating these cancers (Non-Patent Document 9). Several BCL-2 inhibitors have been reported to selectively bind to BCL-2, inhibiting its activity and potently inducing apoptosis, either alone or in combination with other therapeutic agents, demonstrating antitumor effects (Non-Patent Document 10). As mentioned above, BTK inhibitors are already being used clinically for anti-cancer treatment, but there have been reports of patients for whom the inhibitors are ineffective, and satisfactory therapeutic effects have not always been achieved (Non-Patent Document 11).

[0004] In recent years, it has been reported that combining the BTK inhibitor ibrutinib with the BCL-2 inhibitor venetoclax in hematological tumors enhances the antitumor effect, and combination therapy of a BTK inhibitor and a Bcl-2 inhibitor is gaining increasing attention as a new cancer treatment (Patent Document 3, Non-Patent Document 12). However, there has been no disclosure whatsoever about the combination of a reversible BTK inhibitor and a BCL-2 inhibitor according to the present invention, nor has there been any report on the anticancer effect of the combination of a reversible BTK inhibitor and a BCL-2 inhibitor according to the present invention. [Primary Technology Documents] [Chartered documents]

[0005]

Patent Document 1

Patent document 2

Patent document 3

Non-licensed literature

[0006] [Non-licensed document 1] Satterthwaite,ABand Witte,ON,Immunol.Rev.,2000,175,120-127 [Non-licensed document 2] Kurosaki,T.,Curr.Opin.Immunol.,2000,12,276-281 [Non-licensed document 3] Davis,RE,et al.,Nature,2010,463,88-92

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0007] The present invention aims to provide an anticancer composition (combination drug) that effectively induces cell death in cancer cells by combining a reversible BTK inhibitor with a BCL-2 inhibitor. [Means for solving the problem]

[0008] The present invention relates to an anticancer composition that combines a reversible BTK inhibitor with a BCL-2 inhibitor. More specifically, the present invention relates to the following: (1) A pharmaceutical composition for cancer treatment comprising a reversible BTK inhibitor and a BCL-2 inhibitor. (2) The pharmaceutical composition according to (1), wherein the reversible BTK inhibitor is an oxoisoquinoline derivative represented by the following formula (I) or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 1 represents a lower alkyl group which may have a substituent, and Q represents a structure selected from the following structures (a), (b), and (c): [ka] R 2 and R 3 each independently represents a hydrogen atom, an optionally substituted lower alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted heterocyclic group. (3) The reversible BTK inhibitor is a compound in which Q is structure (a) and R 1 is a hydroxymethyl group, or a pharmaceutically acceptable salt thereof. (4) The pharmaceutical composition according to (1), wherein the reversible BTK inhibitor is an oxoisoquinoline derivative represented by the following formula (Ia) or a pharmaceutically acceptable salt thereof: [ka] (In the formula, R 3a represents a tetrahydropyridyl group which may have a substituent.

[0009] (5) The pharmaceutical composition according to (4), wherein the reversible BTK inhibitor is an oxoisoquinoline derivative having the structure of compound (IA) or a pharmaceutically acceptable salt thereof. Formula (IA): 2-(3-{2-amino-6-[1-(oxetan-3-yl)-1,2,3,6-tetrahydropyridin-4-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one [ka] (IA) (6) The pharmaceutical composition according to (1), wherein the reversible BTK inhibitor is a triazine derivative represented by the following formula (II) or a pharmaceutically acceptable salt thereof: [ka] (In the formula, Z 1 represents a lower alkyl group which may have a substituent; Z 2represents a hydrogen atom or a lower alkyl group which may have a substituent; A represents a nitrogen atom or CZ 3 represents Z 3 represents a hydrogen atom, a cyano group, an acyl group which may have a substituent, a sulfonyl group which may have a substituent, or a carbamoyl group which may have a substituent; Z 4 represents a lower alkyl group which may have a substituent, or a cycloalkyl group which may have a substituent. (7) The reversible BTK inhibitor is Z 1 is a hydroxymethyl group or a pharmaceutically acceptable salt thereof. (8) The pharmaceutical composition according to (6), wherein the reversible BTK inhibitor is a triazine derivative having the structure of the following formula (II-A) or a pharmaceutically acceptable salt thereof: Formula (II-A): 2-(3-{4-amino-6-[(1-methyl-1H-pyrazol-4-yl)amino]-1,3,5-triazin-2-yl}-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one [ka] (II-A)

[0010] (9) The pharmaceutical composition according to (1) to (8), wherein the BCL-2 inhibitor is venetoclax, navitoclax, obatoclax, obatoclax mesylate, sabutoclax, APG-1252, AZD-0466, APG-2575, ABBV-167, S-65487 or S-55746. (10) The pharmaceutical composition described in (9), wherein the BCL-2 inhibitor is venetoclax. (11) The pharmaceutical composition according to (9), wherein the BCL-2 inhibitor is navitoclax. (12) The pharmaceutical composition according to (9), wherein the BCL-2 inhibitor is S-55746. (13) The pharmaceutical composition described in (5), wherein the BCL-2 inhibitor is venetoclax. (14) The pharmaceutical composition according to (5), wherein the BCL-2 inhibitor is navitoclax. (15) The pharmaceutical composition according to (5), wherein the BCL-2 inhibitor is S-55746. (16) The pharmaceutical composition described in (8), wherein the BCL-2 inhibitor is venetoclax. (17) The pharmaceutical composition according to (8), wherein the BCL-2 inhibitor is navitoclax. (18) The pharmaceutical composition according to (8), wherein the BCL-2 inhibitor is S-55746. (19) Use of compound (I) and a BCL-2 inhibitor for producing the pharmaceutical composition according to (1) above. (20) A method for treating cancer, characterized by using a combination of the agents according to any one of (1) to (18). [Effects of the Invention]

[0011] The combination of a reversible BTK inhibitor and a BCL-2 inhibitor can induce cell death more efficiently than the use of either the BTK inhibitor or the BCL-2 inhibitor alone. In particular, the BTK inhibitor can suppress cell proliferation by controlling abnormal growth signals in cancer cells, while the BCL-2 inhibitor can effectively induce cell death by restoring the suppressed apoptosis-inducing ability of cancer cells. Therefore, the combination of this BTK inhibitor and BCL-2 inhibitor is expected to have a synergistic anti-cancer effect and is useful as a pharmaceutical composition for the prevention or treatment of cancer, etc. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the cell growth inhibition rate showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) against a human DLBCL cell line (OCI-Ly10). [Figure 2]FIG. 2 is a diagram of BLISS scores showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) on a human DLBCL cell line (OCI-Ly10). [Figure 3] FIG. 3 is an isobologram (50% inhibition) showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) on a human DLBCL cell line (OCI-Ly10). [Figure 4] FIG. 4 is an Fa-CI plot showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) on a human DLBCL cell line (OCI-Ly10). [Figure 5] FIG. 5 is a graph showing the cell growth inhibition rate showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) on BTK-C481S mutant OCI-Ly10 cells. [Figure 6] FIG. 6 is a graph of BLISS scores showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) on BTK-C481S mutant OCI-Ly10 cells. [Figure 7] Figure 7 is an isobologram (50% inhibition) showing the interaction of the combination of compound (IA) and a BCL-2 inhibitor (venetoclax) on BTK-C481S mutant OCI-Ly10 cells. [Figure 8] Figure 8 is a Fa-CI plot showing the combined interaction of compound (IA) and a BCL-2 inhibitor (venetoclax) on BTK-C481S mutant OCI-Ly10 cells. BEST MODE FOR CARRYING OUT THE INVENTION

[0013] (1) Reversible BTK inhibitor One embodiment of the reversible BTK inhibitor is an oxoisoquinoline derivative represented by the following formula (I) or a pharmaceutically acceptable salt thereof, as described in International Publication No. 2018 / 097234 (Patent Document 1). [ka] (In the formula, R 1 represents a lower alkyl group which may have a substituent, and Q represents a structure selected from the following structures (a), (b), and (c): [ka] R 2 and R 3 each independently represents a hydrogen atom, an optionally substituted lower alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted heterocyclic group.

[0014] In formula (I) of the present specification, the lower alkyl group moiety of the lower alkyl group which may have a substituent may be any of a linear or branched alkyl group having 1 to 3 carbon atoms, and specific examples thereof include a methyl group, an ethyl group, an isopropyl group, etc. The cycloalkyl group moiety of the cycloalkyl group which may have a substituent may be any cyclic alkyl group having 3 to 6 carbon atoms, and specific examples thereof include a cyclopropyl group, a cyclobutyl group, and a cyclohexyl group. The aryl group moiety of the aryl group which may have a substituent may be either a monocyclic or bicyclic aryl group having 6 to 14 carbon atoms, and the bicyclic aryl group may be partially hydrogenated. Specific examples include a phenyl group, a naphthyl group, a tetrahydronaphthyl group, and an indenyl group.

[0015] The heteroaryl group moiety of the heteroaryl group, which may have a substituent, includes monocyclic aromatic heterocyclic groups and heteroaromatic fused ring groups. Examples of monocyclic aromatic heterocyclic groups include 5- or 6-membered monocyclic aromatic heterocyclic groups containing at least one heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom. Specific examples include pyrrolyl, imidazolyl, pyrazolyl, thienyl, thiazolyl, furanyl, pyridyl, pyrimidyl, and pyridazyl. Examples of heteroaromatic fused ring groups include bicyclic fused heterocyclic groups containing at least one heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom, each of which has 3 to 8 fused rings. Specific examples include tetrahydroisoquinolyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, and isoquinolyl.

[0016] The heterocyclic group moiety of the heterocyclic group which may have a substituent is a 4- to 6-membered monocyclic saturated heterocyclic group containing at least one heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom, and may have a partial unsaturated bond within the ring. Specific examples include a dihydrothiopyranyl group, a 1,1-dioxo-dihydrothiopyranyl group, and a tetrahydropyridyl group, and particularly preferably a tetrahydropyridyl group. Unless otherwise specified, the substituents in the "optionally substituted" lower alkyl group, optionally substituted cycloalkyl group, optionally substituted aryl group, optionally substituted heteroaryl group, and optionally substituted heterocyclic group may be one or more of any type of substituent at any chemically possible position, and when there are two or more substituents, the respective substituents may be the same or different.

[0017] Examples of the substituent of the optionally substituted lower alkyl group include a halogen atom, a C1-C4 alkoxy group, an amino group optionally substituted with one or two C1-C4 alkyl groups, a nitro group, a cyano group, a hydroxy group, a carbamoyl group optionally substituted with one or two C1-C4 alkyl groups, a carboxyl group, a formyl group, an acetyl group, a mesyl group, a benzoyl group, a C1-C6 acylamino group, and a C1-C6 acyloxy group. An example of the optionally substituted lower alkyl group is a hydroxymethyl group. Examples of the "optionally substituted" substituents for the optionally substituted cycloalkyl group, optionally substituted aryl group, optionally substituted heteroaryl group, and optionally substituted heterocyclic group include a halogen atom, an oxygen atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, an amino group optionally substituted with one or two C1-C4 alkyl groups, a nitro group, a cyano group, a hydroxy group, a carbamoyl group optionally substituted with one or two C1-C4 alkyl groups, a sulfonyl group optionally substituted with a C1-C4 alkyl group, a carboxyl group, a formyl group, an acetyl group, a mesyl group, a benzoyl group, an oxetanyl group, a C1-C6 acylamino group, and a C1-C6 acyloxy group. Compound (I) may have isomers depending on, for example, the type of substituent. In this specification, the chemical structure of only one form of the isomer may be described, but the present invention also includes all isomers (geometric isomers, optical isomers, tautomers, etc.) that can occur structurally, and also includes single isomers or mixtures thereof.

[0018] Pharmaceutically acceptable salts of Compound (I) include inorganic acid salts with hydrochloric acid, sulfuric acid, carbonic acid, phosphoric acid, etc., and organic acid salts with fumaric acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, etc. Other examples include alkali metal salts with sodium, potassium, etc., alkaline earth metal salts with magnesium, calcium, etc., organic amine salts with triethylamine, ethanolamine, etc., basic amino acid salts with lysine, arginine, ornithine, etc., as well as ammonium salts. Compound (I) and pharmaceutically acceptable salts thereof can be produced, for example, by the method described in Patent Document 1. In the production method described in Patent Document 1, if the defined groups are changed under the conditions of the method or if the method is unsuitable for carrying out, the compound can be easily produced by applying methods commonly used in organic synthetic chemistry, such as protection and deprotection of functional groups [T.W. Greene, Protective Groups in Organic Synthesis 3rd Edition, John Wiley & Sons, Inc., 1999]. Furthermore, the order of reaction steps such as the introduction of substituents can be changed as necessary.

[0019] The compounds of formula (I) above are preferably those in which Q is of structure (a), R 1 is a hydroxymethyl group, and more preferably compound (IA): 2-(3-{2-amino-6-[1-(oxetan-3-yl)-1,2,3,6-tetrahydropyridin-4-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one. [ka] (IA) Here, compound (IA) is the compound of Example 23 of Patent Document 1.

[0020] Another embodiment of the reversible BTK inhibitor is a triazine derivative represented by the following formula (II) or a pharmaceutically acceptable salt thereof, which is described in WO 2015 / 012149 (Patent Document 2). [ka] (In the formula, Z 1 represents a lower alkyl group which may have a substituent; Z 2 represents a hydrogen atom or a lower alkyl group which may have a substituent; A represents a nitrogen atom or CZ 3 represents Z 3represents a hydrogen atom, a cyano group, an acyl group which may have a substituent, a sulfonyl group which may have a substituent, or a carbamoyl group which may have a substituent; Z 4 represents a lower alkyl group which may have a substituent, or a cycloalkyl group which may have a substituent. In compound (II), the lower alkyl group moiety of the optionally substituted lower alkyl group may be any of a linear, branched, or cyclic alkyl group having 1 to 3 carbon atoms, and specific examples thereof include a methyl group, an isopropyl group, etc. The cycloalkyl group moiety of the cycloalkyl group which may have a substituent may be any cyclic alkyl group having 3 to 6 carbon atoms, and specific examples thereof include a cyclopropyl group and a cyclobutyl group.

[0021] The acyl group portion of the acyl group which may have a substituent may be a straight-chain, branched-chain, or cyclic alkyl group or an aryl group bonded to a carbonyl group, and examples thereof include a formyl group, an acetyl group, a propionyl group, an octanoyl group, a dodecanoyl group, a pivaloyl group, a cyclopropylcarbonyl group, and a benzoyl group. Examples of the sulfonyl group which may have a substituent include a methylsulfonyl group and an ethylsulfonyl group. Examples of the optionally substituted carbamoyl group include a methylcarbamoyl group, an ethylcarbamoyl group, and a dimethylcarbamoyl group.

[0022] Unless otherwise specified, the substituents in the "optionally substituted" of an optionally substituted lower alkyl group, an optionally substituted cycloalkyl group, an optionally substituted acyl group, an optionally substituted sulfonyl group, and an optionally substituted carbamoyl group may be one or more of any type of substituent at any chemically possible position, and when there are two or more substituents, the respective substituents may be the same or different, and examples include a halogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted amino group, a nitro group, a cyano group, a hydroxy group, a substituted or unsubstituted alkylamino group, a substituted or unsubstituted carbamoyl group, a carboxyl group, a formyl group, an acetyl group, a mesyl group, a benzoyl group, a substituted or unsubstituted acylamino group, and a substituted or unsubstituted acyloxy group.

[0023] Compound (II) may have isomers depending on, for example, the type of substituent. In this specification, the chemical structure of only one form of such isomer may be described, but the present invention also includes all isomers (geometric isomers, optical isomers, tautomers, etc.) that can occur structurally, and also includes single isomers or mixtures thereof. Pharmaceutically acceptable salts of Compound (II) include inorganic acid salts with hydrochloric acid, sulfuric acid, carbonic acid, phosphoric acid, etc., and organic acid salts with fumaric acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, etc. Other examples include alkali metal salts with sodium, potassium, etc., alkaline earth metal salts with magnesium, calcium, etc., organic amine salts with lower alkylamines, lower alcoholamines, etc., basic amino acid salts with lysine, arginine, ornithine, etc., as well as ammonium salts. Compound (II) and pharmaceutically acceptable salts thereof can be produced, for example, by the method described in Patent Document 2. In the production method described in Patent Document 2, if the defined groups change under the conditions of the method or if the method is unsuitable for carrying out, the compound can be easily produced by applying methods commonly used in organic synthetic chemistry, such as protection and deprotection of functional groups [T.W. Greene, Protective Groups in Organic Synthesis 3rd Edition, John Wiley & Sons, Inc., 1999]. Furthermore, the order of reaction steps such as the introduction of substituents can be changed as necessary.

[0024] The compound of the above formula (II) is preferably a compound in which A is a nitrogen atom, Z 1 is a hydroxymethyl group, and more preferably, compound (II-A): 2-(3-{4-amino-6-[(1-methyl-1H-pyrazol-4-yl)amino]-1,3,5-triazin-2-yl}-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one. [ka] (II-A) Here, compound (II-A) is the compound of Example 1 of Patent Document 2.

[0025] (2) BCL-2 inhibitors In the present invention, a BCL-2 inhibitor refers to a drug that has the effect of inhibiting the physiological function of BCL-2 in cells, and includes drugs that inhibit the physiological function of BCL-2, such as low molecular weight compounds, polypeptides, proteins, nucleic acids (such as siRNA, miRNA, and aptamers), and other high molecular weight compounds. BCL-2 is a member of the BCL-2 family of cells that regulates cell death and negatively regulates apoptosis. BCL-2 is activated in lymphoid B-cell tumors such as follicular lymphoma (DLBCL) and CLL, as well as in multiple myeloma and T-cell tumors. BCL-2 inhibitors induce apoptosis in these cancer cells, demonstrating antitumor effects. Therefore, even greater antitumor effects can be expected when used alone or in combination with other drugs. Examples of BCL-2 inhibitors include venetoclax, navitoclax, obatoclax, obatoclax mesylate, sabutoclax, APG-1252, AZD-0466, APG-2575, ABBV-167, S-65487, S-55746, and the like.

[0026] (3) Anticancer composition The anticancer composition of the present invention is a combination drug comprising a reversible BTK inhibitor and a BCL-2 inhibitor, and also encompasses a kit comprising the reversible BTK inhibitor and the BCL-2 inhibitor. The two inhibitors can be effectively used together as a mixture or separately for the treatment of tumors, particularly solid tumors such as breast cancer, colon cancer, and lung cancer, as well as blood cancers such as leukemia, lymphoma, and myeloma. The anti-cancer composition according to the present invention can be prepared and used in the form of a conventional pharmaceutical preparation (pharmaceutical composition) suitable for oral administration, parenteral administration, or topical administration. Formulations for oral administration include solid formulations such as tablets, granules, powders, and capsules, as well as liquid formulations such as syrups. These formulations can be prepared by conventional methods. Solid formulations can be prepared using conventional pharmaceutical carriers such as lactose, starches such as corn starch, crystalline cellulose such as microcrystalline cellulose, hydroxypropyl cellulose, calcium carboxymethylcellulose, talc, magnesium stearate, etc. Capsules can be prepared by encapsulating the granules or powders prepared in this manner. Syrups can be prepared by dissolving or suspending the compound of the present invention or a pharmaceutically acceptable salt thereof in an aqueous solution containing sucrose, carboxymethylcellulose, etc. Preparations for parenteral administration include injections such as drip infusions. Injection preparations can also be prepared by conventional methods and can be appropriately incorporated into isotonic agents (e.g., mannitol, sodium chloride, glucose, sorbitol, glycerol, xylitol, fructose, maltose, mannose), stabilizers (e.g., sodium sulfite, albumin), and preservatives (e.g., benzyl alcohol, methyl p-hydroxybenzoate).

[0027] In the present invention, the reversible BTK inhibitor and the BCL-2 inhibitor may be administered simultaneously, either as a mixture or as separate formulations, or as separate formulations in any order, sequentially, or at an appropriate time interval. The dosage of the anticancer composition of the present invention can vary depending on the reversible BTK inhibitor and BCL-2 inhibitor to be combined, the method of administration (oral, parenteral, or topical), the type of disease to which it is applied, the severity of the disease, the age and weight of the patient, etc., but the dosage of each inhibitor is typically in the range of 1 mg to 1,000 mg per day for adults, which can be administered orally or parenterally in a single dose, or in two or three divided doses. In particular, the anticancer composition of the present invention, which comprises a combination of compound (IA) as a reversible BTK inhibitor and venetoclax as a BCL-2 inhibitor, can be suitably used for treating tumors. [Example]

[0028] Test Example 1: Cell proliferation inhibition test of single agents (cell culture) Human lymphoma cell line OCI-Ly10 cells (obtained from University Health Network) were cultured in a 5% CO incubator using IMDM medium (Iscove's Modified Dulbecco's Medium, Thermo Fisher Scientific Inc.) containing 20% ​​fetal bovine serum (GE Healthcare) and 1% penicillin-streptomycin (Nacalai). Human lymphoma cell line U2932 cells (obtained from DSMZ) were cultured in a 5% CO2 incubator using RPMI medium (Roswell Park Memorial Institute medium, Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (GE Healthcare) and 1% penicillin-streptomycin (Nacalai).

[0029] (Cytostatic test) OCI-Ly10 or U2932 cells (20,000 cells / well) were seeded in a 96-well plate (cell plate), and test compounds diluted in medium were added to a final concentration of 0.009 nM to 30,000 nM (final DMSO concentration 0.3%). After 96 hours of incubation, Alamar Blue reagent (Thermo Fisher Scientific Inc.) was added. After 3 hours, fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The IC50 inhibitory activity was calculated as 100% for wells without compound or cells and 0% for wells without compound or cells. 50 The values ​​were calculated and the results are shown in Table 1. [Table 1] Example 1: Evaluation of the Effect of Combination Use of BTK Inhibitor and BCL-2 Inhibitor Using BLISS Score

[0030] The BLISS score is one of the criteria used to evaluate the synergistic effects of drug combinations (Borisy et al., Proc. Natl. Acad. Sci. USA, 100(13):7977-7982 (2003); Griner et al., Proc. Natl. Acad. Sci. USA, 111(6):2349-54 (2014)). We used this BLISS score to analyze the combined effect of a BTK inhibitor and a BCL-2 inhibitor on the growth inhibition of OCI-Ly10 cells. The BTK inhibitor (compound (IA)) (7 doses ranging from 0.03 nM to 30 nM plus DMSO control) and the BCL-2 inhibitor (venetoclax) (7 doses ranging from 0.1 nM to 90 nM plus DMSO control) were added to OCI-Ly10 cell plates in an 8x8 matrix (final DMSO concentration 0.6%). After 96 hours of culture, Alamar Blue reagent was added, and fluorescence was measured 3 hours later at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell growth inhibition and viability (100 - % inhibition) were calculated by setting wells without compound and cells as 100% and wells without compound and cells as 0%.

[0031] (Assessed by BLISS score) The theoretical BLISS independence (BLISS in ) can be calculated using the following formula when drug A and drug B are considered to act as single agents (i.e., the cell proliferation rate when the concentration of either compound is 0). G is the cell survival rate. BLISS in =G(A)×G(B) The BLISS score for each well is the theoretical BLISS score. in The BLISS score can be calculated by subtracting the actual measured cell proliferation rate from the BLISS score. If the BLISS score is positive, it is considered to have a synergistic effect. BLISS score = 100 × (BLISS in -G) The results of the cell proliferation inhibition rate in this test are shown in Figure 1, and the BLISS score is shown in Figure 2. In this study, as shown in Figures 1 and 2, the BLISS score for the combined use of the BTK inhibitor (compound (IA)) and the BCL-2 inhibitor (venetoclax) was positive, and the results of Example 1 indicate that the combined use of the BTK inhibitor and the BCL-2 inhibitor has a strong synergistic effect.

[0032] Example 2 Evaluation of the combined drug effect of a BTK inhibitor (compound (IA)) and a BCL-2 inhibitor (venetoclax) using isobologram method The isobologram method is a method for evaluating whether the combined effects of two drugs are additive, synergistic, or antagonistic (Chou Cancer Res. 70(2):440-6(2010)). Using this isobologram method, we analyzed the combined effect of a BTK inhibitor and a BCL-2 inhibitor on the growth inhibition of OCI-Ly10 cells. Based on the results of single-agent cell growth inhibition tests on OCI-Ly10 cells, the IC value of BTK inhibitors or BCL-2 inhibitors alone was 50 IC values ​​based on the concentration corresponding to 50 The two drugs were mixed in eight ratios: 1:0, 1:1, 1:5, 1:10, 10:1, 5:1, 3:1, and 0:1, and the final concentration of the mixture was IC 50 The IC values ​​were added to the OCI-Ly10 cell plate at concentrations ranging from 0.001 to 30 times the original concentration (final DMSO concentration: 0.3%). After 96 hours of culture, Alamar Blue reagent was added, and after 3 hours, the fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The IC values ​​for each mixture ratio were calculated. 50 The value was calculated. Single-agent IC 50 The concentrations of the BTK inhibitor and BCL-2 inhibitor required to achieve 50% growth inhibition were calculated from the values ​​and the mixing ratio of the two drugs. The results are shown in Figure 3, where the vertical axis represents the concentration of the BCL-2 inhibitor and the horizontal axis represents the concentration of the BTK inhibitor.

[0033] (evaluation by isobologram) The dose at which drug A and drug B exert a certain effect independently is D. A , D B When the dose-response curves of both drugs are parallel, the dose at which the combined use of both drugs produces the same effect as when they are administered alone is represented by line D. A D B If it is above, it is additive, line D A D B If it is at the bottom left of A D B When the response is in the upper right corner of the graph, it is judged to be an antagonistic response. In this test, as shown in Figure 3, the concentration of each mixture ratio required to exhibit 50% growth inhibition was the IC 50 Since the results of Example 2 fall to the lower left of the line connecting the values, the results of Example 2 indicate that the combined use of the BTK inhibitor of the present invention and the BCL-2 inhibitor has a strong synergistic effect.

[0034] Example 3 Evaluation of the combined drug effect of a BTK inhibitor (compound (IA)) and a BCL-2 inhibitor (venetoclax) by median-effect analysis Median-effect analysis is a theory proposed by Chou and Talalay, and is a method for evaluating whether the combined effect of two drugs used in combination is additive, synergistic, or antagonistic (Chou Cancer Res., 70(2):440-6(2010)). When the drug concentration is D, the 50% inhibitory concentration (median-effect dose) is Dm, the fraction of suppressed cells is Fa (fraction affected), the fraction of unsuppressed cells is Fu (fraction unaffected), and m is a coefficient, D=Dm(Fa / Fu) 1 / m The following relation holds: The combination index (CI), which is a quantitative indicator of the combined effect, is calculated by dividing the concentration of drug A by the concentration of drug A when the two drugs have exclusive effects. A+B ) A , x% inhibitory concentration of A alone is (Dx) A, the concentration of drug B when used in combination (D A+B ) B , x% inhibitory concentration of B alone is (Dx) B Then, CI=(D A+B ) A / (Dx) A +(D A+B ) B / (Dx) B is given by As described above, CI is expressed as a function of Fa, and a correlation diagram called an Fa-CI plot is obtained by plotting Fa on the horizontal axis and CI on the vertical axis. A CI of less than 1 indicates synergism, 1 indicates additive action, and a CI of greater than 1 indicates antagonism. Using the isobologram experimental data from Example 2, the Fa-CI of a BTK inhibitor and a BCL-2 inhibitor in OCI-Ly10 cells was plotted (Figure 4). As shown in Figure 4, at all mixing ratios, the CI was less than 1 when Fa was 0.5 (50% inhibition) or higher, indicating a strong synergistic effect when a BTK inhibitor and a BCL-2 inhibitor were used together.

[0035] Example 4 Dose-response study of BTK inhibitors in the presence of BCL-2 inhibitors IC of BTK inhibitors in the presence of various concentrations of BCL-2 inhibitors 50 The change in values ​​was examined. Based on the results of the BCL-2 inhibitor alone, three doses of the BCL-2 inhibitor were selected for each cancer cell line. The DMSO-added group was used as a control, and the BTK inhibitor was added to the cell plate at concentrations of 0.009 nM to 30,000 nM (final DMSO concentration, 0.4%). The IC2000 values ​​in the presence of each concentration of the BCL-2 inhibitor were measured in the same manner as in Test Example 1. 50 The value was calculated. IC in the presence of various concentrations of BCL-2 inhibitors 50 The values ​​are shown in Tables 2 to 9. In this study, as shown in Tables 2 to 9, the IC value of the BTK inhibitor was 1.0 in both OCI-Ly10 and U2932 lymphoma cells. 50 The values ​​decreased in a BCL-2 inhibitor concentration-dependent manner.

[0036] [Table 2] [Table 3]

[0037] [Table 4] [Table 5]

[0038] [Table 6] [Table 7]

[0039] [Table 8] [Table 9] The results of Example 4 demonstrate that the combined use of a BTK inhibitor and a BCL-2 inhibitor according to the present invention has a combined effect not only on specific cancer cells but also on other cancer cells.

[0040] Test Example 2: Cell proliferation inhibitory effect of a single agent on BTK-C481S mutant lymphoma cells (Generation of BTK-C481S mutant OCI-Ly10 cells) BTK-C481S mutant OCI-Ly10 cells were generated by point mutation knock-in using the CRISPR-Cas9 system. Cas9 protein (Thermo Fisher Scientific Inc.), guide RNA prepared with the GeneArt™ Precision gRNA Synthesis Kit (Thermo Fisher Scientific Inc.), and a single-stranded synthetic oligonucleotide to replace cysteine ​​residue 481 of BTK with serine residue were introduced into OCI-Ly10 cells by electroporation. The resulting modified OCI-Ly10 cells were cultured in the presence of ibrutinib, and the proliferating cells were designated BTK-C481S mutant OCI-Ly10 cells. The mutation introduction was confirmed by genomic DNA and mRNA sequence analysis.

[0041] (Cytostatic test) In the same manner as in Test Example 1, the IC 50 The value was calculated. The results are shown in Table 10. [Table 10]

[0042] Example 5 Evaluation of the Effect of a Combination of a BTK Inhibitor (Compound (IA)) and a BCL-2 Inhibitor (Venetoclax) on BTK-C481S Mutant Lymphoma Cells Using the BLISS Score The BLISS score was calculated in the same manner as in Example 1. The results of the cell proliferation inhibition rate in this test are shown in FIG. 5, and the BLISS score is shown in FIG. Similar to BTK wild-type OCI-Ly10 cells, the BLISS score for the combined use of a BTK inhibitor and a BCL-2 inhibitor in BTK-C481 mutant OCI-Ly10 cells was positive, indicating that the combined use of a BTK inhibitor and a BCL-2 inhibitor according to the present invention has a strong synergistic effect.

[0043] Example 6: Evaluation of the drug combination effect of a BTK inhibitor (compound (IA)) and a BCL-2 inhibitor (venetoclax) on BTK-C481S mutant lymphoma cells using the isobologram method In the same manner as in Example 2, an isobologram was plotted. The results are shown in Figure 7. In the BTK-C481 mutant OCI-Ly10 cells, as in the BTK wild-type OCI-Ly10 cells, the concentration of each mixture ratio required to exhibit 50% growth inhibition was found to be greater than the IC of the single agent. 50 Since the results of Example 6 fall to the lower left of the line connecting the values, the results of Example 6 indicate that the combined use of the BTK inhibitor and BCL-2 inhibitor according to the present invention has a strong synergistic effect.

[0044] Example 7: Evaluation of the drug combination effect of the BTK inhibitor compound (IA) and the BCL-2 inhibitor (venetoclax) on BTK-C481S mutant lymphoma cells by median-effect analysis The CI value was calculated in the same manner as in Example 3. The results are shown in Figure 8. At all mixing ratios, the CI was less than 1 at Fa = 0.5 (50% inhibition) or higher, indicating that the combined use of the BTK inhibitor of the present invention and a BCL-2 inhibitor had a strong synergistic effect in BTK-C481 mutant OCI-Ly10 lymphoma cells, just as it did in BTK wild-type OCI-Ly10 cells.

[0045] Example 8 Dose-response test of BTK inhibitors in the presence of BCL-2 inhibitors using BTK-C481S mutant lymphoma cells IC of BTK inhibitors in the presence of various concentrations of BCL-2 inhibitors using BTK-C481S mutant lymphoma cells 50 The change in values ​​was examined. Based on the results of the BCL-2 inhibitor alone, three doses of the BCL-2 inhibitor were selected for use with BTK-C481 mutant OCI-Ly10 cells. The BTK inhibitor was added to the cell plate at concentrations ranging from 0.009 nM to 30,000 nM (final DMSO concentration, 0.4%), with the DMSO group as a control. The IC2000 / 10 ... 50 The value was calculated. IC in the presence of various concentrations of BCL-2 inhibitors 50 The values ​​are shown in Tables 11 to 14. In this study, as shown in Tables 11 to 14, the IC value of the BTK inhibitor was significantly higher in the BTK-C481 mutant OCI-Ly10 cells. 50 The values ​​decreased in a BCL-2 inhibitor concentration-dependent manner.

[0046] [Table 11] [Table 12]

[0047] [Table 13] [Table 14] The results of Example 8 demonstrate that the combined use of a BTK inhibitor according to the present invention and a BCL-2 inhibitor has a combined effect not only on BTK wild-type OCI-Ly10 cells but also on BTK-C481 mutant OCI-Ly10 cells. [Industrial Applicability]

[0048] The present invention provides an anticancer agent composition that induces cell death more efficiently with higher selectivity and specificity against a broad range of cancer cells than when a BTK inhibitor or a BCL-2 inhibitor is used alone.

Claims

1. An oxoisoquinoline derivative represented by the following formula (I): 【Chemistry 1】 [In the formula, R １ represents a lower alkyl group which may have a substituent, and Q represents a structure selected from the following structures (a), (b), and (c): 【Chemistry 2】 R ２ and R 3 each independently represents a hydrogen atom, an optionally substituted lower alkyl group, an optionally substituted cycloalkyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted heterocyclic group. or a pharmaceutically acceptable salt thereof, and a BCL-2 inhibitor selected from venetoclax, navitoclax, obatoclax mesylate, and S-55746.

2. The reversible BTK inhibitor is selected from the group consisting of: Q having structure (a) and R １ The combination drug according to claim 1, which is an oxoisoquinoline derivative or a pharmaceutically acceptable salt thereof, wherein is a hydroxymethyl group.

3. The reversible BTK inhibitor is an oxoisoquinoline derivative represented by the following formula (Ia): 【Transformation 3】 (In the formula, R ３ａ represents a tetrahydropyridyl group which may have a substituent. or a pharmaceutically acceptable salt thereof.

4. The reversible BTK inhibitor is represented by the following formula (IA): 【Chemistry 4】 Formula (IA): 2-(3-{2-amino-6-[1-(oxetan-3-yl)-1,2,3,6-tetrahydropyridin-4-yl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one 4. The combination drug according to claim 3, which is an oxoisoquinoline derivative having the structure: or a pharmaceutically acceptable salt thereof.

5. A triazine derivative represented by the following formula (II): 【Transformation 5】 (In the formula, Z １ represents a lower alkyl group which may have a substituent; Z ２ represents a hydrogen atom or a lower alkyl group which may have a substituent; A represents a nitrogen atom or C-Z ３ represents Z ３ represents a hydrogen atom, a cyano group, an acyl group which may have a substituent, a sulfonyl group which may have a substituent, or a carbamoyl group which may have a substituent; Z ４ represents an optionally substituted lower alkyl group or an optionally substituted cycloalkyl group. or a pharmaceutically acceptable salt thereof, and a BCL-2 inhibitor selected from venetoclax, navitoclax, obatoclax mesylate, and S-55746.

6. The reversible BTK inhibitor is Z １ The combination drug according to claim 5, which is a triazine derivative or a pharmaceutically acceptable salt thereof, wherein is a hydroxymethyl group.

7. The reversible BTK inhibitor is a triazine derivative having the structure of the following formula (II-A): 【Transformation 6】 [Formula (II-A): 2-(3-{4-amino-6-[(1-methyl-1H-pyrazol-4-yl)amino]-1,3,5-triazin-2-yl}-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one] or a pharmaceutically acceptable salt thereof.

8. The combination pharmaceutical of claim 4, wherein the BCL-2 inhibitor is venetoclax.

9. The pharmaceutical combination of claim 4, wherein the BCL-2 inhibitor is navitoclax.

10. The pharmaceutical combination of claim 7, wherein the BCL-2 inhibitor is venetoclax.

11. The pharmaceutical combination of claim 7, wherein the BCL-2 inhibitor is navitoclax.

12. The combination pharmaceutical of claim 1, wherein the reversible BTK inhibitor and the BCL-2 inhibitor are contained in the same composition.

13. The combination pharmaceutical of any one of claims 1 to 11, wherein the reversible BTK inhibitor and the BCL-2 inhibitor are contained in separate compositions.

14. Use of the reversible BTK inhibitor and the BCL-2 inhibitor for the manufacture of the pharmaceutical combination of any one of claims 1 to 13.

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