Cancer treatment agent containing CLK inhibitor as active ingredient

Abstract

A useful drug combination for preventing or treating cancer or a precancerous lesion was discovered. The present invention provides the same as a medicinal drug.　A concomitant drug of the present invention is obtained by combining: a first component which is a CLK inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by formula (I), a salt thereof, or a hydrate thereof; and a second component comprising at least one antitumor agent.

Description

Cancer treatment containing a CLK inhibitor as an active ingredient

[0001] The present invention provides a compound according to the present invention for preventing or treating cancer or precancerous lesions, comprising a compound of the following formula (I): The present invention relates to a pharmaceutical composition comprising, as an active ingredient, 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine (hereinafter also referred to as "Compound A") represented by the formula:

[0002] Abnormal regulation of alternative splicing has been reported in various diseases, including neurodegenerative diseases, amyotrophic lateral sclerosis, and cancer. In particular, cancer-specific splice variants produced by aberrant alternative splicing have been shown to play important roles in cancer survival and invasion. Furthermore, recent studies have revealed that spliceosome components such as SF3B1, SRSF2, and U2AF1 are frequently mutated in myelodysplastic syndromes. These findings suggest that regulation of alternative splicing plays an important role in cancer.

[0003] The CLK family kinases are a type of dual-specificity protein kinase that possess both serine / threonine kinase activity and tyrosine kinase activity, and there are four kinases, CLK1 to CLK4. CLK phosphorylates SR proteins such as SRSF1, thereby controlling their localization and regulating the splicing regulatory mechanism mediated by the SR proteins. It has been shown that inhibiting CLK kinase activity can regulate alternative splicing, thereby inhibiting signals essential for cancer survival and inhibiting cancer cell proliferation. It has also been shown that CLK2 acts as an oncogene in breast cancer. Therefore, inhibition of CLK kinase activity is considered to be a promising cancer treatment.

[0004] Patent Document 1 discloses various compounds, including Compound A, that have inhibitory activity against cdc2-like kinase (hereinafter also referred to as "CLK").

[0005] International Publication No. 2017 / 188374

[0006] One of the objectives of the present invention is to find a useful combination of drugs for preventing or treating cancer or precancerous lesions, and to provide them as a pharmaceutical composition for simultaneous or sequential administration.

[0007] The present inventors have discovered a compound of the following formula (I): The present inventors have found that the use of a sirtuin activating factor (CLK) inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine or a derivative thereof represented by the following formula (I) in combination with an antitumor agent has a significantly superior antitumor effect compared to the use of the CLK inhibitor alone, and may therefore be useful in the prevention and / or treatment of cancer or precancerous lesions. Based on this finding, the present inventors have conducted extensive research and have completed the present invention. That is, the present invention is as follows. [1] A compound represented by the following formula (I): [2] The combination drug according to [1], wherein the first component and the second component are administered simultaneously or sequentially. [3] The combination drug according to [1] or [2], wherein the second component is an antitumor agent selected from a hormone therapy agent, a chemotherapy agent, an immunotherapy agent, and a molecular-targeted drug. [4] The combination drug according to any one of [1] to [3], wherein the antitumor agent of the second component is selected from the group consisting of antimetabolites, CDK inhibitors, anticancer antibiotics, proteasome inhibitors, topoisomerase I inhibitors, ALK inhibitors, alkylating agents, PI3K inhibitors, corticosteroids, plant-derived anticancer agents, mTOR inhibitors, BCL-2 inhibitors, Akt inhibitors, HER2 inhibitors, EGF receptor inhibitors, PARP inhibitors, CSF inhibitors, FLT3 inhibitors, Aurora inhibitors, MEK (MEK1 / 2) inhibitors, Raf inhibitors, PLK inhibitors, NEDD8 inhibitors, UAE inhibitors, differentiation inducers, cytotoxic agents, IDH1 / 2 inhibitors, VEGFR inhibitors, and anti-PD-1 antibodies. [5] The antitumor agent of the second component is 5-fluorouracil, abemaciclib, or actinomycin D. D), all-trans retinoic acid (all-transretinoic-acid), AZD6738, BAY1895344, bortezomib (bortezomib), camptothecin (camptothecin), ceritinib (ceritinib), crizotinib (crizotinib), cyclophosphamide (cyclophosphamide), cytarabine (cytarabine), dactolisib (dactolisib), dasatinib (dasatiniib), decitabine (decitabine), dexamethasone (dexamethasone), dinaciclib (dinaciclib),Docetaxel, doxorubicin, duvelisib, entinostat, epirubicin, etoposide, everolimus, gemcitabine, ibrutinib ib), imatinib, ipatasertib, tucatinib, KU-60019, lapatinib, masitinib, mercaptopurine, methotrexate, mitomycin C C), mitoxantrone, MK-1775, momelotinib, navitoclax, niraparib, olaparib, palbociclib, pexidartinib, PHA-793887, quizal Quizartinib, ruxolitinib, SCH900776, SN-38, topotecan, tozasertib, trametinib, UMI-77, VE-822, vemurafenib, venetoclax, Vinblastine, vincristine, volasertib, cisplatin, melphalan, oxaliplatin, EPZ-6438, JQ1, MLN-4924, MLN-7243, MPI-0479605, NMS- P715, carboplatin, paclitaxel, azacitidine, hydroxyurea, ivosidenib, enasidenib, gilteritinib, sorafenib,The combination drug according to any one of [1] to [4], wherein the antitumor agent of the second component is at least one selected from the group consisting of lenvatinib, regorafenib, midostaurin, and rucapalib. [6] The combination drug according to any one of [1] to [4], wherein the antitumor agent of the second component is an anti-PD-1 antibody. [7] The combination drug according to any one of [1] to [6], wherein the CLK-related disease is selected from the group consisting of colorectal cancer, lung cancer, mesothelioma, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, breast cancer, ovarian cancer, testicular tumor, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, gestational choriocarcinoma, brain tumor, retinoblastoma, skin cancer, sarcoma, malignant bone tumor, bladder cancer, acute myeloid leukemia, myelodysplastic syndrome, and other blood cancer. [8] The combination drug according to any one of [1] to [7], which is used for the prevention or treatment of myelodysplastic syndrome, acute myeloid leukemia, or ovarian cancer. [9] A compound represented by the following formula (I): a sirtuin-activating factor (CLK) inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[11] A pharmaceutical composition for administering to a patient in need thereof, simultaneously or sequentially, in combination with a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[12] A method for treating cancer or a precancerous lesion, comprising simultaneously or sequentially administering to a patient in need of treatment an effective amount of a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[13] A method for treating cancer or a precancerous lesion, comprising administering an effective amount of a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[14] Use of a CLK inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[15] Use of an antitumor agent for the manufacture of a therapeutic agent for cancer or precancerous lesions, which is administered in combination with a CLK inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[16] A combination drug for preventing or treating myelodysplastic syndrome, acute myeloid leukemia, or precancerous lesions thereof, comprising a first component that is a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I):

[17] A combination drug for preventing or treating myelodysplastic syndrome, acute myeloid leukemia, or precancerous lesions thereof, comprising a first component that is a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine represented by the following formula (I): and a second component comprising at least one antitumor agent selected from the group consisting of niraparib, docetaxel, carboplatin, and paclitaxel.

[0008] The combinations of the present invention are useful in the prevention and / or treatment of cancer.

[0009] IC of each compound (single agent) in HL-60 ５０ IC of each compound (single agent) in HL-60 ５０ The IC of each compound (single agent) in NCI-H661 ５０IC of each compound (single agent) in NCI-H661 ５０ The IC of each compound (single agent) in MV-4-11 ５０ FIG. 1 is a diagram illustrating the change in tumor volume after drug administration in a subcutaneous transplantation model of MC38 cells. FIG. 2 is a diagram illustrating the change in body weight after drug administration in a subcutaneous transplantation model of MC38 cells. FIG. 3 is a diagram illustrating the change in tumor volume after drug administration in a subcutaneous transplantation model of MV-4-11 cells. FIG. 4 is a diagram illustrating the change in body weight after drug administration in a subcutaneous transplantation model of MV-4-11 cells. FIG. 5 is a diagram illustrating the change in tumor volume after drug administration in a subcutaneous transplantation model of MV-4-11 cells. FIG. 6 is a diagram illustrating the change in body weight after drug administration in a subcutaneous transplantation model of MV-4-11 cells. FIG. 7 is a diagram illustrating the change in tumor volume after drug administration in a subcutaneous transplantation model of OVCAR-8 cells. FIG. 8 is a diagram illustrating the change in body weight after drug administration in a subcutaneous transplantation model of OVCAR-8 cells.

[0010] The following provides a detailed description of combinations within the scope of the present invention, particularly combination agents comprising a sirtuin-modifying inhibitor comprising Compound A or a derivative thereof in combination with an antitumor agent, pharmaceutical compositions comprising a sirtuin-modifying inhibitor comprising Compound A or a derivative thereof for use in combination with the antitumor agent, commercial packages comprising a combination pharmaceutical composition of a sirtuin-modifying inhibitor comprising Compound A or a derivative thereof in combination with an antitumor agent, and methods for preventing and / or treating cancer or precancerous lesions, comprising administering the combination agent, pharmaceutical composition, or combination of pharmaceutical compositions to a patient in need thereof.

[0011] (1) Compound A Within the scope of the present invention, “Compound A” refers to a compound having the following formula (I):

[0023] Furthermore, within the scope of the present invention, the derivative of Compound A includes salts and hydrates of Compound A, as well as optical isomers, tautomers, solvates, and the like thereof. Furthermore, the derivative of Compound A may be in the form of a crystal or a clathrate, and may be in the form of a single crystal, a mixed crystal form, a co-crystal, or the like. The salt of Compound A is preferably a pharmacologically acceptable salt, and examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Suitable examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, and ammonium salts. Suitable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Suitable examples of salts with basic amino acids include salts with arginine, lysine, and ornithine. Suitable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid. In addition, details of the method for producing Compound A or a derivative thereof are disclosed in International Publication No. 2017 / 188374, the contents of which are incorporated herein by reference.

[0012] (2) Antitumor Agent The "antineoplastic agent" used in the combination of the pharmaceutical composition of the present invention may be at least one antitumor agent selected from hormonal therapy agents, chemotherapeutic agents, immunotherapy agents, and molecular targeted agents including agents that inhibit the action of cell growth factors and their receptors, and may be, for example, a combination of at least two or at least three antitumor agents selected from the above.

[0013] Examples of hormone therapy agents include fosfestrol, diethylstilbestrol, chlorotrianisene, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone, mepartricin, raloxifene, ormeloxifene, levormeloxifene, antiestrogens (e.g., tamoxifen citrate, toremifene citrate), birth control pills, mepitiostane, testololactone, aminoglutethimide, LH-RH agonists (e.g., goserelin acetate, buserelin, leuprorelin acetate), droloxifene, epitiostanol, ethinyl sulfonate, Luestradiol, aromatase inhibitors (e.g., fadrozole hydrochloride, anastrozole, letrozole, exemestane, vorozole, formestane), antiandrogens (e.g., flutamide, bicalutamide, nilutamide, enzalutamide), 5α-reductase inhibitors (e.g., finasteride, epristeride, dutasteride), corticosteroid drugs (e.g., dexamethasone, prednisolone, betamethasone, triamcinolone), androgen synthesis inhibitors (e.g., abiraterone), retinoids and agents that slow the metabolism of retinoids (e.g., liarozole), thyroid hormones, and DDS preparations thereof can be used.

[0014] Examples of chemotherapeutic agents that can be used include alkylating agents, antimetabolites, anticancer antibiotics, and plant-derived anticancer agents.

[0015] Examples of alkylating agents include nitrogen mustard, nitrogen mustard-N-oxide hydrochloride, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, carboquone, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine, estramustine sodium phosphate, triethylenemelamine, carmustine, lomustine, streptomycin, and benzophenone. Putozocin, pipobroman, etoglucide, carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin, altretamine, ambamustine, dibrospidium hydrochloride, fotemustine, prednimustine, pumitepa, bendamustine, temozolomide, treosulfan, trofosfamide, zinostatin stimalamer, adozelesin, systostin, bizelesin, and DDS formulations thereof may be used.

[0016] Examples of the antimetabolite that can be used include mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, pemetrexed, enocitabine, cytarabine, cytarabine ocfosfate, ancitabine hydrochloride, 5-FU drugs (e.g., fluorouracil, tegafur, UFT, doxifluridine, carmofur, galocitabine, emitefur, capecitabine), aminopterin, nelzarabine, leucovorin calcium, thioguanine, butosin, calcium folinate, calcium levofolinate, cladribine, fludarabine, gemcitabine, pentostatin, piritrexim, idoxuridine, mitoguazone, tiazofurin, ambamustine, bendamustine, hydroxyurea, and DDS preparations thereof.

[0017] Examples of anticancer antibiotics that can be used include actinomycin D, actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, peplomycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, neocarzinostatin, mithramycin, sarkomycin, carzinophilin, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride, and DDS preparations thereof (e.g., doxorubicin-encapsulated PEG liposomes).

[0018] Examples of plant-derived anticancer agents that can be used include etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, cabazitaxel, vinorelbine, and DDS preparations thereof.

[0019] Examples of immunotherapeutic agents that can be used include picibanil, krestin, sizofiran, lentinan, ubenimex, interferon, interleukin, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum, levamisole, polysaccharide K, procodazole, anti-CTLA4 antibodies (e.g., ipilimumab, tremelimumab), anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab), and anti-PD-L1 antibodies.

[0020] Examples of molecular targeted drugs include drugs that inhibit the action of cell growth factors and their receptors, BCL-2 inhibitors, PARP inhibitors, etc. Here, the cell growth factor may be any substance that promotes cell proliferation, and typically includes factors that are peptides with a molecular weight of 20,000 or less and exert their effect at low concentrations by binding to a receptor. Specifically, (i) EGF (epidermal growth factor) or a substance having substantially the same activity as EGF, for example, TGFα; (ii) insulin or a substance having substantially the same activity as insulin, IGF (insulin-like growth factor; IGF-1, IGF-2); (iii) FGF (fibroblast growth factor) or a substance having substantially the same activity as FGF, for example, acidic FGF, basic FGF, KGF (epidermal growth factor), FGF-10; (iv) other cell growth factors, for example, CSF (colony stimulating factor), EPO (erythropoietin), IL-2 (interleukin-2), NGF (nerve growth factor), PDGF (platelet-derived growth factor), TGFβ (transforming growth factor β), HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), heregulin, and angiopoietin can be used.

[0021] Furthermore, the receptor for the cell growth factor may be any receptor that has the ability to bind to the above-mentioned cell growth factor. Specifically, an EGF receptor, a heregulin receptor, for example, HER3, an insulin receptor, an IGF-1 receptor, an IGF-2 receptor, an FGF-1 receptor or an FGF-2 receptor, a VEGF receptor, an angiopoietin receptor, for example, Tie2, a PDGF receptor, etc., can be used.

[0022] Therefore, drugs that inhibit the action of cell growth factors and their receptors include EGF inhibitors, TGFα inhibitors, heregulin inhibitors, insulin inhibitors, IGF inhibitors, FGF inhibitors, KGF inhibitors, CSF inhibitors, EPO inhibitors, IL-2 inhibitors, NGF inhibitors, PDGF inhibitors, TGFβ inhibitors, HGF inhibitors, VEGF inhibitors, angiopoietin inhibitors, EGF receptor inhibitors, HER2 inhibitors, HER4 inhibitors, insulin receptor inhibitors, IGF-1 receptor inhibitors, IGF-2 receptor inhibitors, FGF-1 receptor inhibitors, FG Examples of agents that can be used include F-2 receptor inhibitors, FGF receptor-3 inhibitors, FGF receptor-4 inhibitors, VEGF receptor inhibitors (VEGFR inhibitors), Tie-2 inhibitors, PDGF receptor inhibitors, Abl inhibitors, Raf inhibitors, FLT3 inhibitors, c-Kit inhibitors, Src inhibitors, PKC inhibitors, Smo inhibitors, ALK inhibitors, ROR1 inhibitors, Trk inhibitors, Ret inhibitors, mTOR inhibitors, Aurora inhibitors, PLK inhibitors, MEK (MEK1 / 2) inhibitors, MET inhibitors, CDK inhibitors, Akt inhibitors, ERK inhibitors, and PI3K inhibitors. More specifically, anti-VEGF antibodies (e.g., bevacizumab, ramucurumab, aflibercept), anti-HER2 antibodies (e.g., trastuzumab, pertuzumab), anti-EGFR antibodies (e.g., cetuximab, panitumumab, matuzumab, nimotuzumab), anti-HGF antibodies, imatinib, erlotinib, ib), gefitinib, sorafenib, sunitinib, dasatinib, lapatinib, vatalanib, ibrutinib, bosutinib, cabozantinib, crizotinib, alectinib, vismodegib, axitinib, motesanib,Nilotinib, 6-[4-(4-ethylpiperazin-1-ylmethyl)phenyl]-N-[1(R)-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE-788), vandetanib, temsirolimus, everolimus, enzastaurin, tozasertib, 2-[N-[3-[4-[5-[N-(3-fluorophenyl)carbamoylmethyl]-1H-pyrazol-3-ylamino]quinazolin-7-yloxy]propyl]-N-ethylamino]ethyl phosphate Ester (AZD-1152), 4-[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-ylamino]benzoic acid, N-[2-methoxy-5-[(E)-2-(2,4,6-trimethoxyphenyl)vinylsulfonylmethyl]phenyl]glycine sodium salt (ON-1910Na), volasertib, selumetinib, trametinib, N-[2(R),3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide (PD-0325901), bosutinib, regorafenib, afatinib Afatinib, Idelalisib, Ceritinib, Dabrafenib, anti-CD47 antibodies (e.g., Magrolimab), anti-CD33 / CD2 antibodies (e.g., BiTE), anti-CD123 / CD3 antibodies (e.g., Flotetuzumab), folate receptor ADCs (e.g., Mirvetuximab soravtansine soravtansine), menin-MLL binding inhibitors (e.g., diftmenib, revumenib, DS-1594b, JNJ-75276617), VEGFR inhibitors (e.g., cediranib), and the like can be used.

[0023] In one embodiment of the present invention, the antitumor agent is selected from the group consisting of antimetabolites, CDK inhibitors, anticancer antibiotics, proteasome inhibitors, topoisomerase I inhibitors, ALK inhibitors, alkylating agents, PI3K inhibitors, corticosteroid drugs, plant-derived anticancer agents, mTOR inhibitors, BCL-2 inhibitors, Akt inhibitors, HER2 inhibitors, EGF receptor inhibitors, PARP inhibitors, CSF inhibitors, FLT3 inhibitors, Aurora inhibitors, MEK (MEK1 / 2) inhibitors, Raf inhibitors, PLK inhibitors, NEDD8 inhibitors, UAE inhibitors, differentiation inducers, cytotoxic agents, IDH1 / 2 inhibitors, VEGFR inhibitors, and anti-PD-1 antibodies.

[0024] In a preferred embodiment of the present invention, the antitumor agent is specifically 5-fluorouracil, abemaciclib, actinomycin D D), all-trans retinoic acid (all-transretinoic acid), AZD6738, BAY1895344, bortezomib (bortezomib), camptothecin (camptothecin), ceritinib (ceritinib), crizotinib (crizotinib), cyclophosphamide (cyclophosphamide), cytarabine (cytarabine), dactolisib (dactolisib), dasatinib (dasatiniib), decitabine (decitabine), dexamethasone (dexamethasone), dinaciclib (dinaciclib), docetaxel (docetaxel), doxorubicin (d oxorubicin, duvelisib, entinostat, epirubicin, etoposide, everolimus, gemcitabine, ibrutinib, imatinib, ipatasertib, tucatinib, KU-60019, lapatinib, masitinib, mercaptopurine, methotrexate, mitomycin C C), mitoxantrone, MK-1775, momelotinib, navitoclax, niraparib, olaparib, palbociclib, pexidartinib, PHA-793887, quizartinib, ruxolitinib, SCH900776, SN-38, topotecan, tozasertib, trametinib, UMI-77,VE-822, vemurafenib, venetoclax, vinblastine, vincristine, volasertib, cisplatin, melphalan, oxaliplatin, EPZ-6438, JQ1, MLN-4924, MLN-7243, MPI-0479605, NMS-P715, carboplatin, It may be at least one selected from the group consisting of paclitaxel, azacitidine, hydroxyurea, ivosidenib, enasidenib, gilteritinib, sorafenib, lenvatinib, regorafenib, midostaurin, and rucaparib.

[0025] In specific embodiments of the present invention, the antitumor agent may be at least one antitumor agent selected from the group consisting of quizartinib, midostaurin, cytarabine, venetoclax, and azacitidine, and in specific embodiments of the present invention, the antitumor agent may be at least one antitumor agent selected from the group consisting of niraparib, docetaxel, carboplatin, and paclitaxel.

[0026] In a preferred embodiment of the present invention, the antitumor agent may be an anti-PD-1 antibody.

[0027] (3) Indications The pharmaceutical composition of the present invention, by combining a sirtuin activating protein (CLK) inhibitor comprising Compound A or a derivative thereof with an antitumor agent, exhibits selective and excellent inhibitory activity against CLK, and is also excellent in terms of efficacy, pharmacokinetics (e.g., absorbability, distribution, metabolism, excretion), solubility (e.g., water solubility), interaction with other pharmaceuticals (e.g., inhibitory activity against drug-metabolizing enzymes), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous system toxicity), and stability (e.g., chemical stability, stability against enzymes), and is therefore useful as a pharmaceutical.

[0028] Therefore, the pharmaceutical composition of the present invention can be used as a medicine for inhibiting excessive (abnormal) sirtuin activation in mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans).

[0029] Furthermore, the pharmaceutical composition of the present invention can be used as a medicament for preventing and / or treating cancer or precancerous lesions, including CLK-related diseases, which are diseases that may be affected by CLK. Furthermore, examples of the cancer or precancerous lesion include colorectal cancer (e.g., colon cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestinal cancer, breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, castration therapy-resistant prostate cancer), cancer), liver cancer (e.g. hepatocellular carcinoma, primary liver carcinoma, extrahepatic bile duct carcinoma), thyroid cancer (e.g. medullary thyroid carcinoma), kidney cancer (e.g. renal cell carcinoma, e.g. clear cell renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g. cervical cancer, uterine carcinoma, uterine sarcoma), gestational choriocarcinoma, brain tumors (e.g. medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g. basal cell carcinoma, malignant melanoma), sarcomas (e.g. rhabdomyosarcoma, smooth muscle tumor, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, acute myeloid leukemia, myelodysplastic syndrome, and other blood cancers or precancerous lesions. Here, the term "precancerous lesion" refers, in a narrow sense, to lesions formed by proliferation of cells in an intermediate stage of carcinogenesis, but in a broad sense, includes, in addition to such lesions, lesions that are likely to become cancerous (a state in which cancer is likely to occur) and lesions that are not themselves linked to cancer but serve as markers of a state in which cancer is likely to occur. The pharmaceutical composition of the present invention can be used as a pharmaceutical for preventing or treating these cancers or precancerous lesions, as a cancer growth inhibitor, a cancer metastasis suppressor, or an apoptosis promoter.

[0030] In particular, the pharmaceutical composition of the present invention can be used as a medicament for treating myelodysplastic syndrome, acute myeloid leukemia or ovarian cancer.

[0031] In a specific embodiment of the present invention, the pharmaceutical composition of the present invention may be a combination drug for preventing or treating myelodysplastic syndrome, acute myeloid leukemia, or a precancerous lesion thereof, comprising a first component which is a CLK inhibitor consisting of compound A, a salt thereof, or a hydrate thereof, in combination with a second component which is at least one antitumor agent selected from the group consisting of quizartinib, midostaurin, cytarabine, venetoclax, and azacitidine. In a specific embodiment of the present invention, the pharmaceutical composition of the present invention may be a combination drug for preventing or treating ovarian cancer or a precancerous lesion thereof, comprising a first component which is a CLK inhibitor consisting of compound A, a salt thereof, or a hydrate thereof, in combination with a second component which is at least one antitumor agent selected from the group consisting of niraparib, docetaxel, carboplatin, and paclitaxel.

[0032] (4) Dosage Form The pharmaceutical composition of the present invention may be any composition that allows the CLK inhibitor comprising Compound A or a derivative thereof to be combined with an anti-tumor agent upon administration. Thus, the pharmaceutical composition of the present invention may be a single formulation (e.g., a combination formulation) obtained by simultaneously formulating the CLK inhibitor comprising Compound A or a derivative thereof and at least one anti-tumor agent, or a combination of at least two formulations obtained by separately formulating the CLK inhibitor comprising Compound A or a derivative thereof and at least one anti-tumor agent.

[0033] Therefore, the administration form is not particularly limited, and examples thereof include: (i) administration of a composition containing the sirtuin activating factor (CLK) inhibitor comprising compound A or a derivative thereof and at least one anti-tumor agent, i.e., administration as a single formulation; (ii) simultaneous administration via the same administration route of at least two formulations obtained by separately formulating the sirtuin activating factor (CLK) inhibitor comprising compound A or a derivative thereof and at least one anti-tumor agent; and (iii) administration via the same administration route with a time lag between the two formulations obtained by separately formulating the sirtuin activating factor (CLK) inhibitor comprising compound A or a derivative thereof and at least one anti-tumor agent (e.g., administration via the same administration route of at least two formulations obtained by separately formulating the sirtuin activating factor (CLK) inhibitor comprising compound A or a derivative thereof and at least one anti-tumor agent). (iv) simultaneous administration of at least two formulations obtained by separately formulating the CLK inhibitor comprising Compound A or a derivative thereof and at least one anti-tumor agent via different administration routes; (v) staggered administration of at least two formulations obtained by separately formulating the CLK inhibitor comprising Compound A or a derivative thereof and at least one anti-tumor agent via different administration routes (e.g., administration of the CLK inhibitor comprising Compound A or a derivative thereof and the anti-tumor agent in the order of Compound A or a derivative thereof and the anti-tumor agent, or administration in the reverse order). When the pharmaceutical composition of the present invention is a combination of at least two formulations obtained by separately formulating the CLK inhibitor comprising Compound A or a derivative thereof and at least one anti-tumor agent, these formulations can be administered simultaneously or sequentially. Sequential administration herein includes administration of at least two formulations with a time lag.

[0034] The pharmaceutical compositions of the present invention can be used as medicines, and can be used as a single formulation containing, as active ingredients, a CLK inhibitor comprising Compound A or a derivative thereof and at least one antitumor agent, or as at least two formulations obtained by separately formulating the two or more compounds. Each of these formulations can be used in the form of a solid, semi-solid, or liquid pharmaceutical preparation (e.g., tablet, pellet, troche, capsule, suppository, cream, ointment, aerosol, powder, liquid, emulsion, suspension, syrup, injection, etc.) suitable for rectal, nasal, pulmonary, vaginal, topical (local), oral, or parenteral (including subcutaneous, implant, intravenous, and intramuscular) administration.

[0035] The pharmaceutical compositions of the present invention are stable, low-toxicity, and safe to use. The dosage of compound A used in the combination of pharmaceutical compositions of the present invention varies depending on the patient's condition, weight, age, type of compound, administration method, administration route, etc., but is adjusted to achieve the desired effect. For example, the daily dosage of compound A used in the combination of pharmaceutical compositions of the present invention, when administered orally, is typically about 0.01 to about 20 mg per kg of body weight, preferably about 0.01 to about 10 mg, and more preferably about 0.01 to about 5 mg. This dosage can be administered once or in multiple doses, such as two or three times. The dosage of the antitumor agent used in the combination of pharmaceutical compositions of the present invention also varies depending on the patient's condition, weight, age, type of compound, administration method, administration route, etc., but is adjusted to achieve the desired effect. In the combination of pharmaceutical compositions of the present invention, the compounding ratio between compound A and the antitumor agent can be appropriately selected depending on the patient, administration route, target disease, symptoms, specific combination of drugs, etc. For example, when the patient is a human, 0.01 to 100 parts by weight of the antitumor agent can be used per 1 part by weight of compound A. In the present disclosure, the term "effective amount" refers to an amount of a compound or a combination of one or more compounds that induces a desired biological response or pharmacological effect when administered simultaneously or sequentially.

[0036] The pharmaceutical composition of the present invention may also comprise a commercial package containing a description explaining the dosage and administration method for administering the CLK inhibitor comprising Compound A or a derivative thereof in combination or in combination with the antitumor agent.

[0037] In this specification, each specific feature described in one embodiment relating to each aspect of the present invention may be combined in any manner to form a new embodiment, and it should be understood that such a new embodiment may also be included in each aspect of the present invention.

[0038] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0039] Example 1: Effect of Combination of Compound A and Antitumor Agent In order to determine whether or not there is a synergistic or additive effect between Compound A and an antitumor agent in the combination of the pharmaceutical composition of the present invention, first, the dose-response curves of each compound alone were determined, and then the concentrations of Compound A and the antitumor agent were compared to the IC values ​​of the compounds for the target cells. ５０ The cell growth inhibitory effects were measured when the compounds were mixed at three fixed ratios: 50%:50% (1:1), 80%:20% (4:1), and 20%:80% (1:4). The presence or absence of synergistic or additive effects on cell growth inhibition was determined by curve shift analysis, and the degree of synergistic or additive effects was subsequently quantified using the Combination Index (CI). All cell line stocks used in this assay were subcultured in ATCC-recommended medium and prepared by freezing according to the ATCC-recommended protocol.

[0040] (i) Preparation of Compound Single Agent and Mixture Compound A and the antitumor agent were each stored in powder form under the conditions recommended by the supplier. The powder was then weighed using a calibrated balance, dissolved in 100% DMSO, and stored at room temperature. However, cisplatin, melphalan, and oxaliplatin were dissolved in water before use. The compound stocks prepared in this way were used at their respective IC concentrations on the day of the experiment. ５０ The compounds were further diluted with DMSO to a concentration 3160 times higher than that of the original solution, and then mixed at three fixed ratios (1:1, 1:4, 4:1). ５０ Since the final expected growth inhibitory effect is matched based on the IC value, all compound stock solutions are assumed to have theoretically equal potency. ５０ Antitumor drug group in which IC was not calculated ５０ For compounds (not available), the compounds were not mixed at the fixed ratio, but were mixed with compound A at a 1:1 ratio so that the final concentrations would be the fixed concentrations shown in Tables 2 and 4. The concentrations of compound A before this mixing were ５０The results were 10.00 times, 3.16 times, 1.00 times, 0.32 times, 0.10 times, 0.03 times, and 0.01 times of the original amount.

[0041] Next, in experiments using human promyelocytic leukemia cell line HL-60 and human lung cancer cell line NCI-H661, IC ５０ Compound A and an antitumor agent were mixed at a fixed ratio of 3160x the original concentration, and the compound stock was diluted with DMSO at a 3:1 ratio to prepare seven compound dilutions. After further dilution to 31.6x with sterilized 20 mM Hepes buffer (pH 7.4), 5 μl of the compound or mixture was added to 45 μl of cell culture medium in duplicate in a 384-well plate assay. The final DMSO concentration after addition of the compound or mixture was 0.4%. The final assay concentration ranges for the compound alone were 10 μM, 3.16 μM, 1.00 μM, 0.32 μM, 0.10 μM, 0.03 μM, and 0.01 μM. In the case of the human promyelocytic leukemia-derived cell line MV-4-11, the compound was diluted using MultiDrop (Thermo) to prepare a compound solution with a concentration 10 times the final concentration. Specifically, compound A and the antitumor agent were mixed at the above-mentioned fixed ratio, and the IC ５０ Nine compound dilutions were prepared by diluting the compound at a 2-fold common ratio (3-fold common ratio for Venetoclax) with a maximum volume of 160-fold (810-fold for Venetoclax only). 50 μl of cell culture medium was placed in a 96-well V-bottom plate for compound preparation, and Compound A and the antitumor agent were added to the prepared concentrations using a MultiDrop. The DMSO concentration in each well was normalized with DMSO to 1% in all wells. 10 μl of the compound single agent or mixture was added in triplicate to 90 μl of cell culture medium in a 96-well plate assay. The final DMSO concentration after addition of the compound single agent or mixture was 0.1% (0.2% for Venetoclax only). The compound single agent was prepared at the same concentration as each compound contained in the mixture.

[0042] (ii) Cell Proliferation Assay In experiments with the human promyelocytic leukemia cell-derived cell line HL-60 and the human lung cancer cell line NCI-H661, stocks of the human promyelocytic leukemia cell-derived cell line HL-60 and the human lung cancer cell line NCI-H661 were thawed and diluted with appropriate medium. Cell numbers of 200 to 3,200 cells, depending on the cell line used, were dispensed into 384-well plates in 45 μl of medium, and the outer edge of each 384-well plate was filled with phosphate-buffered saline. The seeded cells were incubated at 37°C in 5% CO ２ After 24 hours of incubation under humidified conditions, 5 μl of the compound solution or mixture prepared above was added and the cells were further incubated for 72 hours. However, for EPZ-6438, JQ1, MLN-4924, MLN-7243, MPI-0479605, and NMS-P715, the incubation was continued for 120 hours. After incubation, the plate was left at room temperature for 30 minutes, after which 25 μl of ATPlite1Step™ solution (Perkin-Elmer) was added to each well and shaken for 2 minutes. After shaking for 10 minutes at room temperature in the dark, luminescence was measured using a multimode Envision reader (Perkin-Elmer). For experiments using the human promyelocytic leukemia-derived cell line MV-4-11, a stock of the human promyelocytic leukemia-derived cell line MV-4-11 was thawed, diluted with the appropriate medium, and dispensed into a 96-well plate at a cell count of 20,000 in 90 μl of medium. Each well was filled to the rim with phosphate-buffered saline. 10 μl of the compound solution or mixture prepared above was added and cultured for an additional 72 hours. After incubation, 100 μl of CellTiter-Glo™ 2.0 Reagent (Promega) was added to each well, and the plate was shaken in the dark at room temperature for 30 minutes. Luminescence was measured using an EnSight multimode plate reader (Perkin-Elmer).

[0043] (iii) Measurement at time 0 of incubation: For the HL-60 and NCI-H661 cell lines, 45 μl of cells were dispensed in quadruplicate, and 5 μl of DMSO-containing Hepes buffer and 25 μl of ATPlite 1Step™ solution (Perkin-Elmer) were added and mixed for 2 minutes. After 10 minutes of incubation at room temperature in the dark, luminescence was measured. For the MV-4-11 cell line, 90 μl of cells were dispensed in X quadruplicate, and 100 μl of CellTiter-Glo™ was added to each well. The cells were shaken in the dark at room temperature for 30 minutes, and luminescence was measured using an EnSight multimode plate reader (Perkin-Elmer).

[0044] (iv) Cell proliferation adjustment. Cell division times for all cell lines were calculated from the proliferation signals at t = 0 h and t = 72 h for untreated cells. A deviation of 0.5-2.0 fold from the historical mean cell division time was considered an invalid assay.

[0045] (v) Maximum luminescence For the HL-60 and NCI-H661 cell lines, the maximum luminescence was measured in each 384-well plate in the presence of 0.4% DMSO, without any compound, after 72 hours of culture. The values ​​for n = 14 were averaged to calculate the maximum luminescence. This average value is defined as the luminescence after t = 72 hours in the untreated group. For the MV-4-11 cell line, the maximum luminescence was measured in each 96-well plate in the presence of 0.1% DMSO, without any compound, after 72 hours of culture. The values ​​for n = 3 were averaged to calculate the maximum luminescence. This average value is defined as the luminescence after t = 72 hours in the untreated group.

[0046] (vi) Dose-response curves In this assay, accurate IC ５０Therefore, the dose-response signal for each single compound was represented by the following four-parameter logistic curve using XL-fit5 (IDBS): luminescence = bottom + (top - bottom) / (1 + 10(logIC50 - log[cpd])·hill) (where cpd is the concentration of the test compound, hill is the Hill coefficient, and bottom and top are the minimum and maximum values ​​of the curve, respectively.) Figures 1, 2, and 3 show the IC values ​​of each single compound for each cell line (HL-60, NCI-H661, and MV-4-11, respectively). ５０ Indicates the value.

[0047] (vii) Curve Shift Analysis This assay allows visual confirmation of synergistic effects. The concentrations of the mixture of compound A (cpd1) and the antitumor agent (cpd2) were determined based on the concentrations of each compound alone and the IC ５０ The "units" were calculated using the following formula: [mix] = [cpd1] / IC ５０，ｃｐｄ１ +[cpd2] / IC ５０，ｃｐｄ２ (wherein mix represents the concentration of the mixture, cpd1 represents the concentration of compound A, and cpd2 represents the concentration of the antitumor agent.) The dose-response signal of the mixture was expressed by the following four-parameter logistic curve using XL-fit5 (IDBS): luminescence = bottom + (top - bottom) / (1 + 10 (logX - log[mix]) * hill) (wherein hill represents the Hill coefficient, X represents the inflection point of the curve, and bottom and top represent the minimum and maximum values ​​of the curve, respectively.) Here, in the formula, the mixture [mix] is ５０ Since it is expressed as a value of IC ５０ When the IC values ​​are comparable, the curves of the individual compounds overlap, and their inflection points have a value of 1. ５０ The values ​​are the IC ５０ The value determined for the value.

[0048] In a mixture without synergy, the curve overlaps with the curves of each compound alone. In a mixture with synergy, the curve shifts to the left and the IC ５０In other words, the mixture exhibits a stronger cell growth inhibitory effect than would be expected based on each compound alone, which is a good index for evaluating synergistic effects. The mixture of compound A and the antitumor agent exhibited a lower IC value at any ratio between 1:4 and 4:1. ５０ The curve shifted to the left, indicating a synergistic effect compared to when each compound was used alone.

[0049] (viii) Determination of CI CI is one of the most widely used quantitative indicators of synergy. CI is used to evaluate the concentration required to achieve a certain "specific effect." For example, a CI of 0.1 indicates that the compound concentration required to achieve a specific effect is one-tenth of the concentration expected to achieve the same level of effect based on data from the single compound. In another example, a combination (a potent compound and a weak compound) with a CI of 0.1 means that the weak compound improves the effective concentration of the potent compound by 10 times. As mentioned above, CI is defined for a certain effect (effect F) (e.g., F = 0.75 for a 75% effect).

[0050] Therefore, the concentration required to achieve effect F by combining compound A with an antitumor agent was compared with the concentrations required for each compound alone (referred to as cpd1 and cpd2, respectively). Ｆ was calculated by the following formula: CI Ｆ = [cpd1] Ｆ / IC Ｆ，ｃｐｄ１ + [cpd2] Ｆ / IC Ｆ，ｃｐｄ２ In the formula, [cpd1] ０．７５ where F is the concentration of compound A alone in the mixture that produces a 75% inhibitory effect (F = 0.75), and IC ０．７５、ｃｐｄ１ is the IC of Compound A alone ７５The range of F values ​​(0 to 1.0) was calculated using the Calcusyn™ program for the results of HL-60 and NCI-H661 cell lines, and using the GraphPad Prism 9 program for the results of MV-4-11 cell line. A CI of less than 1 indicates synergy, and in particular, a CI of less than 0.3 indicates strong synergy.

[0051] For each combination of compound A and an antitumor agent, CI was plotted as a function of F. CI was evaluated as shown in Table 1. An additive effect was observed when 0.9≦CI≦1.1, and a synergistic effect was observed when CI<0.9.

[0052]

[0053] The CI values ​​calculated for each combination of Compound A and an antitumor agent in HL-60 cells are shown in Tables 2 and 3 below.

[0054] The results for NCI-H661 cells are shown in Tables 4 and 5 below.

[0055] The results for MV-4-11 cells are shown in Table 6 below.

[0056] As can be seen from the above results, in both hematopoietic tumors and solid tumors, the cell proliferation inhibitory effect of the combination of Compound A and an antitumor agent was found to be more pronounced than when Compound A or the antitumor agent was used alone.

[0057] Example 2: Combined Effect of Compound A and Anti-PD-1 Antibody To determine whether or not a combination of pharmaceutical compositions of the present invention has an additive or synergistic antitumor effect, Compound A was combined with an anti-mouse PD-1 antibody as an antitumor agent and co-administered to subcutaneous tumor-bearing mice. The changes in tumor volume during the administration period of subcutaneously transplanted tumors were compared between the control group and the drug-administered group and evaluated using the efficacy index T / C. To assess the combined effect, the changes in tumor volume for each individual were statistically analyzed by two-way analysis of variance to determine whether or not an additive or synergistic effect was present. MC38 cells (a mouse colon cancer cell line) and 7- to 9-week-old female C57BL / 6 mice were prepared by Crown Bioscience, Inc., a testing contractor, and were used.

[0058] (i) Preparation of drug solution for administration Compound A was prepared as a suspension at a concentration of 1.25 mg / mL using 0.5% methylcellulose solution. Anti-mouse PD-1 antibody (CD279; BioXCell) was diluted with PBS to a concentration of 1 mg / mL.

[0059] (ii) Subcutaneous Implantation Model Test of MC38 Cells A cell suspension of cultured MC38 cells, a mouse colon cancer cell line, was subcutaneously injected into the abdominal cavity of 7- to 9-week-old female C57BL / 6 mice at a dose of 1 × 10 per mouse. ６ The tumors were subcutaneously transplanted at a concentration of 0.1 mL of cells in PBS. The tumor volume was measured using a vernier caliper and calculated using the formula: tumor volume = (tumor major axis x tumor minor axis x tumor minor axis) / 2. When the tumor volume was approximately 100 mm ３When the mice reached 100 mg / kg, they were divided into four groups, G1 to G4, with 10 mice per group (G1: control group, G2: compound A administration group, G3: anti-PD-1 antibody administration group, G4: compound A + anti-PD-1 antibody combination administration group). The compound A administration group (G2) was orally administered (PO) at a dose of 12.5 mg / kg twice daily (BID), twice weekly (BIW) for 12 days, while the anti-PD-1 antibody administration group (G3) was intraperitoneally administered at a dose of 10 mg / kg once daily, twice weekly for 12 days. The combination administration group (G4) received compound A (12.5 mg / kg) and anti-PD-1 antibody (10 mg / kg), while the control group (G1) was orally administered a 0.5% methylcellulose solution. The drug administration volume was 10 mL / kg. After grouping, tumor volume and body weight were measured three times weekly. The results of measuring the tumor volume and body weight are shown in Figures 4 and 5, respectively. The administration period is indicated by a bold black line.

[0060] (iii) Evaluation of antitumor effect Statistical analysis was performed using two-way analysis of variance using EZR (ver. 1.54) software on the amount of change in tumor volume during the administration period for each group and each individual. The T / C value (%) calculated by the following formula was used as an index of antitumor effect: T / C value (%) = (change in tumor volume in drug-administered group) / (change in tumor volume in control group) × 100. The results are shown in Table 7 below.

[0061]

[0062] The T / C value (%) on day 12 in the combined administration group (G4) was 48.6%, demonstrating a stronger antitumor effect of the combined use of compound A and anti-PD-1 antibody compared to the groups (G2, G3) in which compound A and anti-PD-1 antibody were administered alone.

[0063] Example 3: Combined Effect of Compound A with an Antitumor Agent Selected from the Group Consisting of Quizartinib, Midostaurin, Cytarabine, and Azacitidine (Two-Drug Combination) To determine whether or not a combination of pharmaceutical compositions of the present invention has an additive or synergistic antitumor effect, Compound A was combined with an antitumor agent selected from the group consisting of quizartinib, midostaurin, cytarabine, and azacitidine and co-administered to subcutaneous tumor-bearing mice. The change in tumor volume during the administration period of the subcutaneously transplanted tumor was compared between the control group and the drug-administered group and evaluated using the efficacy index T / C. To assess the combined effect, the change in tumor volume for each individual was statistically analyzed for the presence or absence of an additive or synergistic effect by two-way analysis of variance. MV-4-11 cells (human acute myeloid leukemia cell line) were purchased from ATCC, and 6-week-old female BALB / cA Jcl-nu / nu mice purchased from CLEA Japan, Inc. were used.

[0064] (i) Preparation of drug solutions for administration Compound A was prepared as a suspension at a concentration of 0.625 mg / mL using 0.5% methylcellulose solution. Quizartinib was prepared as a suspension at a concentration of 0.03 mg / mL using 2-hydroxypropyl-beta-cyclodextrin. Midostaurin was prepared as a suspension at a concentration of 1 mg / mL using 0.5% methylcellulose solution. Cytarabine was diluted with physiological saline to prepare a solution at a concentration of 2.5 mg / mL. Azacitidine was diluted with physiological saline to prepare a solution at a concentration of 0.5 mg / mL.

[0065] (ii) Subcutaneous transplantation model test of MV-4-11 cells A cell suspension of cultured human acute myeloid leukemia cell line MV-4-11 cells was subcutaneously injected into the abdominal cavity of 6-week-old female BALB / cA Jcl-nu / nu mice at a dose of 3 × 10 per mouse. ６ The tumors were subcutaneously transplanted at a ratio of 0.1 mL / 0.1 cells (a 1:1 mixture of HBSS and Matrigel). The tumor volume was measured using a caliper and calculated using the formula: tumor volume = (tumor major axis x tumor minor axis x tumor minor axis) / 2. When the tumor volume was approximately 130 mm ３When the mice reached this age, they were divided into 10 groups, G1 to G10, with 5 animals per group (G1: control group, G2: compound A administration group, G3: quizartinib administration group, G4: midostaurin administration group, G5: cytarabine administration group, G6: azacitidine administration group, G7: compound A + quizartinib combination administration group, G8: compound A + midostaurin combination administration group, G9: compound A + cytarabine combination administration group, G10: compound A + azacitidine combination administration group). The compound A group (G2) received oral administration (PO) at a dose of 6.25 mg / kg twice daily (BID), twice weekly (BIW) for 14 days; the quizartinib group (G3) received oral administration (PO) at a dose of 0.3 mg / kg once daily (QD), 5 days on, 2 days off per week for 14 days; and the midostaurin group (G4) received oral administration (PO) at a dose of 10 mg / kg once daily. The cytarabine group (G5) received 25 mg / kg of ... The combination administration group (G7) received Compound A (6.25 mg / kg) and quizartinib (0.3 mg / kg), the combination administration group (G8) received Compound A (6.25 mg / kg) and midostaurin (10 mg / kg), the combination administration group (G9) received Compound A (6.25 mg / kg) and cytarabine (25 mg / kg), the combination administration group (G10) received Compound A (6.25 mg / kg) and azacitidine (5 mg / kg), and the control group (G1) was orally administered a 0.5% methylcellulose solution. The drug administration volume was 10 mL / kg. After grouping, tumor volume and body weight were measured twice a week. The tumor volume and body weight measurements are shown in Figures 6 and 7, respectively.

[0066] (iii) Evaluation of antitumor effect As an index of antitumor effect, the T / C value (%) calculated by the following formula was used: T / C value (%) = (change in tumor volume in drug-administered group) / (change in tumor volume in control group) × 100 The results are shown in Table 8 below.

[0067] The T / C values ​​(%) on day 15 in the combined administration groups (G7, G8, G9, and G10) were -7.0%, -19.6%, -9.2%, and 4.2%, respectively, demonstrating a stronger antitumor effect of the combined use of compound A and the antitumor agent compared to the groups (G2, G3, G4, G5, and G6) in which compound A and the antitumor agent used were administered alone.

[0068] Example 4: Combination Effect of Compound A with Venetoclax and Azacitidine (Triple Combination) To determine whether or not a combination of pharmaceutical compositions of the present invention provides an additive or synergistic antitumor effect, Compound A was combined with venetoclax and azacitidine as antitumor agents and co-administered to subcutaneous tumor-bearing mice. The change in tumor volume during the administration period of subcutaneously transplanted tumors was compared between the control group and the drug-administered group and evaluated using the efficacy index T / C. To assess the effect of the combination, the change in tumor volume for each individual was statistically analyzed for the presence or absence of an additive or synergistic effect by two-way analysis of variance. MV-4-11 cells (human acute myeloid leukemia cell line) were purchased from ATCC, and 6-week-old female BALB / cA Jcl-nu / nu mice purchased from CLEA Japan, Inc. were used.

[0069] (i) Preparation of drug solutions for administration Compound A was prepared as a suspension at a concentration of 0.625 mg / mL using 0.5% methylcellulose solution. Venetoclax was prepared as solutions at concentrations of 2.5 and 5 mg / mL using Phosal 50PG / PEG 400 / Ethanol. Azacitidine was diluted with saline to prepare a solution at a concentration of 0.5 mg / mL.

[0070] (ii) Subcutaneous transplantation model test of MV-4-11 cells A cell suspension of cultured human acute myeloid leukemia cell line MV-4-11 cells was subcutaneously injected into the abdominal cavity of 6-week-old female BALB / cA Jcl-nu / nu mice at a dose of 3 × 10 per mouse. ６ The tumors were subcutaneously transplanted at a ratio of 0.1 mL / cell (a 1:1 mixture of HBSS and Matrigel). Tumor volume was measured using a caliper and calculated using the formula: tumor volume = (tumor major axis x tumor minor axis x tumor minor axis) / 2. When the tumor volume was approximately 110 mm ３When the animals reached this age, they were divided into 9 groups, G1 to G9, with 5 animals per group (G1: control group, G2: compound A administration group, G3 and G4: venetoclax administration group, G5: azacitidine administration group, G6 and G7: venetoclax + azacitidine combination administration group, G8 and G9: compound A + venetoclax + azacitidine combination administration group). The compound A group (G2) was orally administered (PO) at a dose of 6.25 mg / kg twice daily (BID), twice weekly (BIW) for 14 days; the venetoclax groups (G3 and G4) were orally administered (PO) at doses of 25 and 50 mg / kg, respectively, once daily (QD), on a 5-day-on-week, 2-day-off schedule for 14 days; and the azacitidine group (G5) was subcutaneously administered (SC) at a dose of 5 mg / kg once daily (QD), twice weekly for 14 days. The combination group (G6) received Compound A (6.25 mg / kg) and venetoclax (25 mg / kg), the combination group (G7) received Compound A (6.25 mg / kg) and venetoclax (50 mg / kg), the combination group (G8) received Compound A (6.25 mg / kg), venetoclax (25 mg / kg), and azacitidine (5 mg / kg), the combination group (G9) received Compound A (6.25 mg / kg), venetoclax (50 mg / kg), and azacitidine (5 mg / kg), and the control group (G1) was orally administered a 0.5% methylcellulose solution. The drug administration volume was 10 mL / kg. After grouping, tumor volume and body weight were measured twice weekly. The tumor volume and body weight measurements are shown in Figures 8 and 9, respectively.

[0071] (iii) Evaluation of antitumor effect As an index of antitumor effect, the T / C value (%) calculated by the following formula was used: T / C value (%) = (change in tumor volume in drug-administered group) / (change in tumor volume in control group) × 100 The results are shown in Table 9 below.

[0072] The T / C value (%) on day 15 in the compound A monotherapy group (G2) was 1.8%. The T / C values ​​(%) on day 15 in the venetoclax and azacitidine dual-drug combination groups (G6 and G7) were 41.3% and 38.5%, respectively, demonstrating a stronger antitumor effect than the venetoclax or azacitidine monotherapy groups (G3, G4, and G5). However, in the compound A, venetoclax, and azacitidine triple-drug combination groups (G8 and G9), the T / C values ​​(%) on day 15 were -25.7% and -26.1%, respectively, demonstrating a significantly stronger antitumor effect than the venetoclax and azacitidine dual-drug combination group. Furthermore, complete tumor disappearance was observed in 3 of 5 and 5 of 5 patients, respectively, in the triple-drug combination groups (G8 and G9).

[0073] Example 5: Combined effect of compound A with an antitumor agent selected from the group consisting of niraparib, docetaxel, carboplatin, and paclitaxel (two-drug combination) In order to determine whether or not a combination of pharmaceutical compositions of the present invention has an additive or synergistic effect in the antitumor effect, compound A was combined with an antitumor agent selected from the group consisting of niraparib, docetaxel, carboplatin, and paclitaxel, and these were co-administered to subcutaneous tumor-bearing mice. The change in tumor volume during the administration period of the subcutaneously transplanted tumor was compared between the control group and the drug-administered group, and evaluated using the efficacy index T / C. OVCAR-8 cells (human ovarian cancer cell line) and 8-9 week-old female BALB / c nude mice were prepared by Crown Bioscience, Inc., a testing contractor, and were used.

[0074] (i) Preparation of Drug Solutions for Administration Compound A was prepared as a 1.25 mg / mL suspension using 0.5% methylcellulose solution. Niraparib was prepared as a 3.75 mg / mL solution in 10% DMSO / 40% PEG 300 / 5% Tween 80 / 45% saline (volume ratio). Docetaxel was prepared by diluting 0.3 mL of 40 mg / mL docetaxel (Taxotere®) with 0.9 mL of 13% ethanol to prepare 1.2 mL of a 10 mg / mL solution, and then diluting 1.0 mL with 4 mL of saline to prepare a 2 mg / mL solution. Carboplatin was diluted with saline from a 10 mg / mL administration solution to prepare a 5 mg / mL solution. Paclitaxel was diluted with saline from a 6 mg / mL administration solution to prepare a 3 mg / mL solution.

[0075] (ii) Subcutaneous Implantation Model Test of OVCAR-8 Cells A cell suspension of cultured OVCAR-8 cells, a human ovarian cancer cell line, was subcutaneously implanted into the abdominal cavity of 8-week-old female BALB / c nude mice at a rate of 1 × 10 per mouse. ７ The tumors were subcutaneously transplanted at a ratio of 0.1 mL / cell (a 1:1 mixture of PBS and Matrigel). Tumor volume was measured using a caliper and calculated using the formula: tumor volume = (tumor major axis x tumor minor axis x tumor minor axis) / 2. When the tumor volume was approximately 160 mm ３When the time reached 100 mg / kg, the mice were divided into 10 groups, G1 to G10, with 6 individuals per group (G1: control group, G2: compound A administration group, G3: niraparib administration group, G4: docetaxel administration group, G5: carboplatin administration group, G6: paclitaxel administration group, G7: compound A + niraparib combination administration group, G8: compound A + docetaxel combination administration group, G9: compound A + carboplatin combination administration group, G10: compound A + paclitaxel combination administration group). The Compound A group (G2) received oral (PO) administration at a dose of 12.5 mg / kg twice daily (BID), twice weekly (BIW) for 21 days; the niraparib group (G3) received oral (PO) administration at a dose of 37.5 mg / kg once daily (QD), daily for 21 days; and the docetaxel group (G4) received oral (PO) administration at a dose of 20 mg / kg once daily (QD), once weekly (QW). The carboplatin group (G5) received 50 mg / kg of ... The combination administration group (G7) received Compound A (12.5 mg / kg) and Niraparib (37.5 mg / kg), the combination administration group (G8) received Compound A (12.5 mg / kg) and Docetaxel (20 mg / kg), the combination administration group (G9) received Compound A (12.5 mg / kg) and Carboplatin (50 mg / kg), the combination administration group (G10) received Compound A (12.5 mg / kg) and Paclitaxel (15 mg / kg), and the control group (G1) received a 0.5% methylcellulose solution orally. The drug administration volume was 5 mL / kg for Paclitaxel and 10 mL / kg for the others. After grouping, tumor volume and body weight were measured twice a week. The tumor volume and body weight measurements are shown in Figures 10 and 11, respectively.

[0076] (iii) Evaluation of antitumor effect As an index of antitumor effect, the T / C value (%) calculated by the following formula was used: T / C value (%) = (change in tumor volume in drug-administered group) / (change in tumor volume in control group) × 100 The results are shown in Table 10 below. The T / C values ​​(%) on day 22 in the compound A combination administration groups (G7, G8, G9 and G10) were 44.2%, -22.3%, 35.2% and 28.4%, respectively, demonstrating a stronger antitumor effect due to the combination of compound A and the antitumor agent compared to the groups (G2, G3, G4, G5 and G6) in which the antitumor agent and compound A were administered alone.

[0077] The combination of the pharmaceutical composition of the present invention has a significantly superior antitumor activity and is therefore useful for the prevention and / or treatment of cancer.

Claims

1. The following formula (I): A combined agent for preventing or treating cancer or precancerous lesions, comprising a first component which is a CLK inhibitor consisting of 1 - ((5 - (1R) - 1 - fluoroethyl) - 1,3,4 - oxadiazol - yl)methyl) - 6 - (4 - methoxypyrrolo[2,1 - f][1,2,4]triazin - 5 - yl) - 2 - methyl - 1H - imidazo[4,5 - b]pyridine, a salt thereof or a hydrate thereof, and a second component consisting of at least one antitumor agent.

2. The combined agent according to claim 1, wherein the first component and the second component are administered simultaneously or sequentially.

3. The combined agent according to claim 1, wherein the anti-tumor agent of the second component is selected from hormonal therapy agents, chemotherapy agents, immunotherapy agents, and molecular target drugs.

4. The combined agent according to claim 1, wherein the anti-tumor agent of the second component is selected from the group consisting of antimetabolites, CDK inhibitors, anticancer antibiotics, proteasome inhibitors, topoisomerase I inhibitors, ALK inhibitors, alkylating agents, PI3K inhibitors, adrenocortical hormone drugs, plant-derived anticancer agents, mTOR inhibitors, BCL-2 inhibitors, Akt inhibitors, HER2 inhibitors, EGF receptor inhibitors, PARP inhibitors, CSF inhibitors, FLT3 inhibitors, Aurora inhibitors, MEK (MEK1 / 2) inhibitors, Raf inhibitors, PLK inhibitors, NEDD8 inhibitors, UAE inhibitors, differentiation inducers, cytotoxic drugs, IDH1 / 2 inhibitors, VEGFR inhibitors, and anti-PD-1 antibodies.

5. The anti-tumor agent of the second component is 5-fluorouracil, abemaciclib, actinomycin D, all-trans retinoic acid, AZD6738, BAY1895344, bortezomib, camptothecin, ceritinib, crizotinib, cyclophosphamide, cytarabine, dactolisib, dasatinib, decitabine, dexamethasone, dinaciclib, docetaxel, doxorubicin, duvelisib, entinostat, epirubicin, etoposide, everolimus, gemcitabine, ibrutinib, imatinib, ipatasertib, tucatinib, KU-60019, lapatinib, masitinib, mercaptopurine, methotrexate, mitomycin C, mitoxantrone, MK-1775, momelotinib, navitoclax, niraparib, olaparib, palbociclib, pexidartinib, PHA-793887, quizartinib, ruxolitinib, SCH900776, SN-38, topotecan, tozasertib, trametinib, UMI-77, VE-822,The combination agent according to claim 1, which is at least one anti-tumor agent selected from the group consisting of vemurafenib, venetoclax, vinblastine, vincristine, volasertib, cisplatin, melphalan, oxaliplatin, EPZ-6438, JQ1, MLN-4924, MLN-7243, MPI-0479605, NMS-P715, carboplatin, paclitaxel, azacitidine, hydroxyurea,ivosidenib, enasidenib, gilteritinib, sorafenib, lenvatinib, regorafenib, midostaurin, and rucaparib.

6. The combined agent according to claim 1, wherein the anti-tumor agent of the second component is an anti-PD-1 antibody.

7. The combined agent according to claim 1, which is used for the prevention or treatment of cancer or pre-cancerous lesions selected from the group consisting of colorectal cancer, lung cancer, mesothelioma, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, breast cancer, ovarian cancer, testicular tumor, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, gestational choriocarcinoma, brain tumor, retinoblastoma, skin cancer, sarcoma, malignant bone tumor, bladder cancer, acute myeloid leukemia, myelodysplastic syndrome, and other blood cancers.

8. The combined agent according to claim 1, which is used for the prevention or treatment of myelodysplastic syndrome, acute myeloid leukemia, or ovarian cancer.

9. The following formula (I): A pharmaceutical composition for preventing or treating cancer or precancerous lesions, comprising a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof, wherein the pharmaceutical composition is administered to a patient in need thereof, simultaneously or sequentially, in combination with an antitumor agent.

10. A pharmaceutical composition for preventing or treating cancer or a pre-cancerous lesion, comprising an anti-tumor agent, wherein the pharmaceutical composition is administered to a patient in need thereof simultaneously or sequentially in combination with a CLK inhibitor consisting of 1-(((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof, represented by the following formula (I): A pharmaceutical composition.

11. The following formula (I): A method for treating cancer or a precancerous lesion, comprising simultaneously or sequentially administering to a patient in need of treatment for cancer an effective amount of a CLK inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof, or a hydrate thereof, and an effective amount of an antitumor agent.

12. The following formula (I): An effective amount of a CLK inhibitor consisting of 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof is administered to a patient in need of treatment for cancer, which comprises a method for treating cancer or a precancerous lesion, further comprising administering an effective amount of an antitumor agent, a method for treating cancer or a precancerous lesion.

13. Use of a CLK inhibitor consisting of 1-(((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof, for the manufacture of a therapeutic agent for cancer or a pre-cancerous lesion, administered in combination with an anti-tumor agent. ​ 14. The following formula (I): Use of an antitumor agent for producing a therapeutic agent for cancer or a pre-cancerous lesion, which is administered in combination with a CLK inhibitor comprising 1-(((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof.

15. The following formula (I): A first component which is a CLK inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof, and a second component comprising at least one antitumor agent selected from the group consisting of quizartinib, midostaurin, cytarabine, venetoclax and azacitidine. The combined agent according to claim 1 for preventing or treating myelodysplastic syndrome, acute myeloid leukemia or their premalignant lesions, which is obtained by combining the two components.

16. The following formula (I): A first component which is a CLK inhibitor comprising 1-((5-(1R)-1-fluoroethyl)-1,3,4-oxadiazol-yl)methyl)-6-(4-methoxypyrrolo[2,1-f][1,2,4]triazin-5-yl)-2-methyl-1H-imidazo[4,5-b]pyridine, a salt thereof or a hydrate thereof, and a second component comprising at least one antitumor agent selected from niraparib, docetaxel, carboplatin and paclitaxel, combined together, for preventing or treating ovarian cancer or a pre-cancerous lesion thereof, the combined agent according to claim 1.

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