Pharmaceutical compositions and uses thereof for the prevention or treatment of tumors

Abstract

According to an embodiment of the technology disclosed in this application, the present invention relates to a pharmaceutical composition for preventing or treating a tumor, comprising a small molecule kinase inhibitor that blocks the transforming growth factor-β (TGF-β) signaling pathway, wherein administration of the small molecule kinase that blocks the TGF-β signaling pathway in combination with at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor provides superior tumor therapeutic or tumor growth inhibitory effects in patients in need of tumor treatment or tumor growth inhibition compared to administration of the small molecule kinase inhibitor, the immune checkpoint regulator, or the receptor tyrosine kinase inhibitor alone.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating tumors, a method for preventing or treating tumors and the use of the pharmaceutical composition, in which a small molecule kinase inhibitor that blocks the transforming growth factor-β (TGF-β) signaling pathway is administered in combination with at least one of an immune checkpoint control agent and a receptor tyrosine kinase inhibitor. [Background technology]

[0002] Tumor cells are characterized by the fact that they secrete substances that inactivate T cells, thereby evading immune cell attack, modifying the tumor microenvironment, dividing rapidly, and allowing tumor cells to leave the tissue in which they were produced and spread to other tissues. On the one hand, tumor cells create their own blood vessels to obtain the nutrients they need for rapid growth and metastasis (tumor angiogenesis).

[0003] Regulatory T cells are known to be key cells involved in immune evasion of tumor cells, and several treatment strategies have been developed to reduce the number of regulatory T cells in tumor tissues. For example, methods have been developed to reduce T cells, including chemotherapy drugs for controlling immunosuppressive responses by administering cyclophosphamide (CP) at low concentrations, and immune checkpoint control agents such as antibodies targeting molecules such as CD25, CTLA-4, or PD-1, or chemokine receptors specifically expressed by regulatory T cells, including ipilimumab (Yervoy®), which targets CTLA-4, and pembrolizumab (Keytruda®) and nivolumab (Opdivo), which target programmed cell death protein-1 (PD-1). TM ) has been approved for sale by the U.S. Food and Drug Administration (FDA).

[0004] On the one hand, vascular endothelial growth factor receptors (VEGFR) 1-3, platelet-derived growth factor receptor (PDGFR-α), reconstituted during transfection (RET) receptor and fibroblast growth factor receptors (FGFR) 1-4 are receptor tyrosine kinases known to be involved in tumor cell division and angiogenesis. Therefore, drugs that selectively inhibit receptor tyrosine kinases have been developed, and methods for enhancing anti-cancer treatment effects by using a combination of receptor tyrosine kinase selective inhibitors and immune checkpoint control agents are also being actively researched. Furthermore, in 2021, the FDA approved the combination of the receptor tyrosine kinase inhibitor lenvatinib (Lenvima®) and the anti-PD-1 antibody pembrolizumab for the treatment of advanced kidney cancer and advanced endometrial cancer.

[0005] However, there are tumor patients who do not respond to treatment with immune checkpoint regulators and / or receptor tyrosine kinase inhibitors. Due to the low response rate of immune checkpoint regulators, which are only effective in about 20%-30% of cancer patients, the development of combination therapeutic agents to increase the response rate of immune checkpoint regulators and maximize their effectiveness is being actively carried out. Furthermore, even if previously developed tumor therapeutic agents are used, adverse effects such as weight loss in subjects threaten the quality of life of tumor patients. Therefore, there is a need to develop novel treatments that have fewer adverse effects and have excellent effects in suppressing tumor growth. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent 2220275 Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors have discovered that administering to a subject a small molecule kinase inhibitor that blocks the TGF-β signaling pathway in combination with at least one selected from an immune checkpoint control agent and a receptor tyrosine kinase inhibitor has a better effect of suppressing tumor growth and is safer than administering a small molecule kinase inhibitor that blocks the TGF-β signaling pathway, a receptor tyrosine kinase inhibitor or an immune checkpoint control agent alone, thereby completing the present invention. [Means for solving the problem]

[0008] In one embodiment of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of tumors, comprising a small molecule kinase inhibitor that blocks the TGF-β signaling pathway, wherein the small molecule kinase inhibitor that blocks the TGF-β signaling pathway is administered in combination with at least one of an immune checkpoint control agent and a receptor tyrosine kinase inhibitor.

[0009] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating tumors is provided, comprising a small molecule kinase inhibitor that blocks the signaling pathway of TGF-β and at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor.

[0010] In another embodiment of the present invention, the present invention provides a combination for preventing or treating tumors comprising a small molecule kinase inhibitor that blocks the TGF-β signaling pathway and at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor.

[0011] In another embodiment of the present invention, the present invention provides a method of preventing or treating a tumor in a subject, comprising administering to the subject a small molecule kinase inhibitor that blocks the TGF-β signaling pathway and at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor.

[0012] In one embodiment of the invention, the invention provides the use of a small molecule kinase inhibitor that blocks the TGF-β signaling pathway, administered in combination with at least one of an immune checkpoint control agent and a receptor tyrosine kinase inhibitor, in the manufacture of a medicament for the prevention or treatment of a tumor.

[0013] In one embodiment of the present invention, the present invention provides a kit for preventing or treating tumors, comprising a small molecule kinase inhibitor that blocks the TGF-β signaling pathway and an instruction manual directing the administration of a combination of a small molecule kinase inhibitor that blocks the TGF-β signaling pathway with at least one immune checkpoint regulator and a receptor tyrosine kinase inhibitor. Effect of the Invention

[0014] Administration of a small molecule kinase inhibitor that blocks the TGF-β signaling pathway in combination with at least one selected from an immune checkpoint regulator and a receptor tyrosine kinase inhibitor to a subject has superior effects in inhibiting tumor growth compared to the use of the small molecule kinase inhibitor, immune checkpoint regulator or tyrosine kinase inhibitor alone. [Brief description of the drawings]

[0015] [Figure 1] The figure shows the effect of co-administration of compound 1 and anti-PD-1 antibody, control group (vehicle), administration of compound 1 alone, and administration of anti-PD-1 antibody alone on suppressing tumor volume increase in a melanoma mouse model.

[0016] [Diagram 2] 1 shows the changes in body weight during the test period in the control group (vehicle) and the test drug administration groups (Compound 1 + anti-PD-1 antibody, Compound 1 and anti-PD-1 antibody) in a melanoma mouse model.

[0017] [Diagram 3]The tumor volumes of the control group (vehicle + anti-IgG2b antibody) and the test drug groups (vehicle + anti-PD-L1 antibody, Compound 1 (15 mg / kg / day) + anti-PD-L1 antibody, Compound 1 (30 mg / kg / day) + anti-PD-L1 antibody, LY2157299 (75 mg / kg / day) + anti-PD-L1 antibody, and LY2157299 (150 mg / kg / day) + anti-PD-L1 antibody) in the melanoma mouse model are shown as mean ± SEM.

[0018] [Figure 4] Figure 2 shows the tumor volumes in the control group (vehicle + anti-IgG2b antibody) and test drug groups (vehicle + anti-PD-L1 antibody, compound 1 (15 mg / kg / day) + anti-PD-L1 antibody, compound 1 (30 mg / kg / day) + anti-PD-L1 antibody, LY2157299 (75 mg / kg / day) + anti-PD-L1 antibody, and LY2157299 (150 mg / kg / day) + anti-PD-L1 antibody) on day 22 after cell line inoculation in a melanoma mouse model.

[0019] [Diagram 5] Figure 2 shows the changes in body weight during the study period in the control group (vehicle + anti-IgG2b antibody) and the test drug administration groups (vehicle + anti-PD-L1 antibody, Compound 1 (15 mg / kg / day) + anti-PD-L1 antibody, Compound 1 (30 mg / kg / day) + anti-PD-L1 antibody, LY2157299 (75 mg / kg / day) + anti-PD-L1 antibody, and LY2157299 (150 mg / kg / day) + anti-PD-L1 antibody) in a melanoma mouse model.

[0020] [Figure 6] This shows the effect of co-administration of compound 1 and anti-CTLA-4 antibody, the control group (vehicle), administration of compound 1 alone, and administration of anti-CTLA-4 antibody alone in a melanoma mouse model on inhibiting tumor volume increase, and the difference in tumor volume measured 15 days after drug administration.

[0021] [Figure 7]1 shows changes in body weight during the test period in a melanoma mouse model for the control group (vehicle), the group administered with Compound 1 alone, the group administered with an anti-CTLA-4 antibody alone, and the group administered with Compound 1 and an anti-CTLA-4 antibody together.

[0022] [Figure 8] This shows the effect of co-administration of Compound 1 and anti-PD-1 antibody, the control group (vehicle), administration of Compound 1 alone, and administration of anti-PD-1 antibody alone in a mouse model of colorectal cancer, in suppressing tumor volume increase, and the difference in tumor weight measured 26 days after drug administration.

[0023] [Figure 9] This shows the effect of co-administration of Compound 1 and anti-CTLA-4 antibody, the control group (vehicle), administration of Compound 1 alone, and administration of anti-CTLA-4 antibody alone in a mouse model of colorectal cancer in suppressing tumor volume increase, and the difference in tumor volume measured 26 days after drug administration.

[0024] [Figure 10] 1 shows the effect of suppressing tumor volume increase in a mouse model of colorectal cancer by co-administration of compound 1, anti-PD-1 antibody, and anti-CTLA-4 antibody, co-administration of compound 1 and anti-CTLA-4 antibody, a control group (vehicle), administration of compound 1 alone, administration of anti-PD-1 antibody alone, administration of anti-CTLA-4 antibody alone, and co-administration of anti-PD-1 antibody and anti-CTLA-4 antibody.

[0025] [Figure 11] 1 shows the effect of suppressing tumor volume increase in a mouse model of colorectal cancer by co-administration of compound 1 and lenvatinib, co-administration of compound 1, lenvatinib and an anti-PD-1 antibody, a control group (vehicle), administration of lenvatinib alone, administration of an anti-PD-1 antibody alone, co-administration of lenvatinib and an anti-PD-1 antibody, and administration of compound 1 alone.

[0026] [Figure 12]1 shows the changes in tumor volume during the test period in a colorectal cancer mouse model for the control group (vehicle) and the test drug administration groups (anti-CTLA-4 antibody, lenvatinib, lenvatinib + anti-PD1 antibody, lenvatinib + anti-CTLA-4 antibody, compound 1, compound 1 + anti-CTLA-4 antibody, and compound 1 + lenvatinib + anti-CTLA-4 antibody).

[0027] [Figure 13] 1 shows the changes in body weight during the test period in the control (vehicle) and test drug administration groups (compound 1, lenvatinib, anti-CTLA-4 antibody, anti-PD1 antibody, compound 1 + anti-CTLA-4 antibody, compound 1 + anti-PD-1 antibody, compound 1 + lenvatinib, lenvatinib + anti-PD1 antibody, lenvatinib + anti-CTLA-4 antibody, anti-PD-1 antibody + anti-CTLA-4 antibody, compound 1 + lenvatinib + anti-PD-1 antibody, compound 1 + lenvatinib + anti-CTLA-4 antibody, compound 1 + anti-PD-1 antibody + anti-CTLA-4 antibody) in a mouse model of colorectal cancer.

[0028] [Figure 14] The figure shows the effect of co-administration of compound 1 and anti-PD-1 antibody, control group (vehicle), administration of compound 1 alone, and administration of anti-PD-1 antibody alone on suppressing tumor volume increase in a mouse sarcoma model.

[0029] [Figure 15] The tumor remission rates in a mouse sarcoma model are shown for co-administration of Compound 1 and anti-CTLA-4 antibody, a control group (vehicle), administration of Compound 1, and administration of anti-CTLA-4 antibody.

[0030] [Figure 16] This shows the changes in body weight during the test period in the control group (vehicle) and the test drug administration groups (anti-PD-1 antibody, compound 1, anti-CTLA-4 antibody, compound 1 + anti-PD-1 antibody, and compound 1 + anti-CTLA-4 antibody) in a sarcoma mouse model.

[0031] [Figure 17]This shows the effect of co-administration of Compound 1 and anti-PD-1 antibody, the control group (vehicle), administration of Compound 1 alone, and administration of anti-PD-1 antibody alone in a breast cancer mouse model, in suppressing tumor volume increase, and the difference in tumor volume measured 23 days after cell line inoculation.

[0032] [Figure 18] This shows the effect of co-administration of compound 1 and anti-CTLA-4 antibody, the control group (vehicle), administration of compound 1 alone, and administration of anti-CTLA-4 antibody alone in a breast cancer mouse model on suppressing tumor volume increase, and the difference in tumor volume measured 23 days after cell line inoculation.

[0033] [Figure 19] The graph shows the changes in body weight during the test period in the control group (vehicle) and the test drug administration groups (anti-PD-1 antibody, compound 1, anti-CTLA-4 antibody, compound 1 + anti-PD-1 antibody, and compound 1 + anti-CTLA-4 antibody) in a breast cancer mouse model.

[0034] [Figure 20] The figures show the survival rates of mice in a bladder cancer mouse model in which compound 1 was co-administered with an anti-PD-1 antibody, a control group (vehicle), and mice administered compound 1 and an anti-PD-1 antibody.

[0035] [Figure 21] The graph shows changes in body weight during the test period in the control group and the test drug administration groups (Compound 1, anti-PD-1 antibody, and Compound 1 + anti-PD-1 antibody) of a bladder cancer mouse model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] One embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of tumors comprising a small molecule kinase inhibitor that blocks the transforming growth factor-β (TGF-β) signaling pathway, wherein the small molecule kinase inhibitor that blocks the TGF-β signaling pathway is administered in combination with at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor.

[0037] The term "small molecule kinase inhibitors that block the signal transduction pathway of TGF-β" refers to small molecule kinase inhibitors that can inhibit the signal transduction pathway of TGF-β, such as TGF type I receptor kinase inhibitors or kinase inhibitors that dually inhibit TGF-β type I receptor and vascular endothelial growth factor receptor (VEGFR)-2. Preferably, the small molecule kinase inhibitors that block the signal transduction pathway of TGF-β can be the novel 2-pyridyl-substituted imidazole derivatives disclosed in Korean Patent 1938368 or pharma- ceutically acceptable salts or solvates thereof. For example, small molecule kinase inhibitors that block the TGF-β signaling pathway include 1-[6-(6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-2,3-dihydro-imidazo[1,2-a]imidazol-1-yl]-ethanone, 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxaline, 6 -[2-(6-methyl-pyridin-2-yl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrimidin-3-yl]-quinoxaline, 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoline, 6-[2-(6-methyl-pyridin-2-yl)-5,6,7,8-tetrahydro-imidazo[1,2- a]pyrimidin-3-yl]-quinoline, 2-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-thieno[3,2-c]pyridine, 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-benzothiazole, 5-benzo[b]thiophene-5- yl-6-(6-methyl-pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine, 5-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-benzoxazole, 4-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoline, 5-benzo[1,3]dioxol-5-yl-6-(6-methyl-pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 5-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-6-(6-methyl-pyridin-2-yl)-2,3-dihydro-1H-imidazo[1, 2-a]imidazole, 7-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-2-pyrazol-1-yl-quinoxaline, dimethyl-(2-{7-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxaline-2-yloxy}-ethyl)-amine, 2-methoxy-7-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a ]imidazol-3-yl]-quinoxaline, 5-(3,5-dimethoxyphenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, N,N-dimethyl-4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)aniline, 4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)benzonitrile, 2-methyl-6-(6- (6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)quinoline, 4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)aniline, N-(4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)phenyl)acetamide, N-(4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)phenyl)methanesulfonamide, tert-butyl (4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)phenyl)carbamate, 5-(4-(4-methylpiperazin-1-yl)phenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 4-(4-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl) phenyl)morpholine, 6-(6-methylpyridin-2-yl)-5-(m-tolyl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 5-(4-methoxyphenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 6-(6-methylpyridin-2-yl)-5-(4-(trifluoromethyl)phenyl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 6-(6-methylpyridin-2-yl)-5-(4-(methylthio)phenyl)-2,3-di Hydro-1H-imidazo[1,2-a]imidazole, 5-(3-fluoro-4-methoxyphenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 5-(4-fluorophenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole, 1-acetyl-6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carboxylic acid ester ethyl ester, 6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carboxylic acid ethyl ester, [6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazol-2-yl]-methanol, 1-acetyl-6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carbonitrile, 6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carbonitrile, 6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carboxylic acid amide, (6-(6-methylpyridin-2 More preferably, the small molecule kinase inhibitor that blocks the TGF-β signaling pathway may be a compound selected from the group consisting of the following compounds: N-((6-(6-methylpyridin-2-yl)-5-(thieno[3,2-c]pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-2-yl)methyl)acetamide, or a pharma- ceutically acceptable salt thereof. More preferably, the small molecule kinase inhibitor that blocks the TGF-β signaling pathway may be a compound selected from the group consisting of the following compounds: 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxaline;6-[2-(6-methyl-pyridin-2-yl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrimidin-3-yl]-quinoxaline;6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoline;2-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-thienoline 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-benzothiazole; 6-(6-(6-methyl-pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine; and dimethyl-(2-{7-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxalin-2-yloxy}-ethyl)-amine.

[0038] As used herein, the term "pharmaceutically acceptable salts" refers to salts suitable for use in contact with human and animal tissues. Examples of suitable salts with such inorganic and organic acids include, but are not limited to, acetic acid, citric acid, formic acid, fumaric acid, hydrochloric acid, lactic acid, maleic acid, malic acid, methane-sulfonic acid, nitric acid, phosphoric acid, p-toluenesulfonic acid, succinic acid, sulfuric acid, tartaric acid, trifluoroacetic acid, and the like. Additionally, pharmaceutically acceptable salts can be acid or base addition salts formed by the reaction of inorganic bases, such as sodium hydroxide, ammonium hydroxide, or potassium hydroxide, with organic bases, such as mono-, di-, tri-alkyl and aryl amines or substituted ethanolamines.

[0039] In one embodiment of the present invention, the small molecule kinase inhibitor that blocks the TGF-β signaling pathway is 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxaline, 6-[2-(6-methyl-pyridin-2-yl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrimidin-3-yl]-quinoxaline, 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoline, 2-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[ 1,2-a]imidazol-3-yl]-thieno[3,2-c]pyridine, 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-benzothiazole, 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or dimethyl-(2-{7-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxalin-2-yloxy}-ethyl)-amine.

[0040] On the one hand, TGF-β inhibits cell proliferation and induces apoptosis in normal environments, but is present in high concentrations in the tumor microenvironment, inhibits immunity, promotes cancer cell metastasis, and increases tumor density and hardness through fibrosis of cancer stroma, promotes angiogenesis, blocks the delivery of anticancer drugs and the infiltration of immune cells into tumor tissues, thereby causing drug resistance. Therefore, small molecule kinase inhibitors that block the signaling pathway of TGF-β not only inhibit the immune evasion and metastasis of cancer cells, but also inhibit the fibrosis of tumor stroma caused by TGF-β, normalize tumor vascular function, thereby increasing the intratumoral delivery of drugs used in combination and the infiltration rate of immune cells into tumors, and also increase the oxygen concentration in the microenvironment around the tumor. Furthermore, the small molecule kinase inhibitors blocking the TGF-β signaling pathway of the present invention may be used in combination with various anti-cancer treatments / anti-cancer drugs, such as immune checkpoint control agents (e.g., drugs that inhibit immune checkpoint proteins to suppress regulatory T cells, drugs that bind to immune checkpoint proteins to activate effector T cells, etc.), chemotherapy, targeted therapy, nanoparticles, and radiation therapy. When the small molecule kinase inhibitors blocking the TGF-β signaling pathway of the present invention are used in combination with immune checkpoint control agents, the anti-cancer efficacy can be improved by increasing the infiltration rate of immune cells into tumors, and immune cells are the target of immune checkpoint control agents. Furthermore, when the small molecule kinase inhibitors blocking the TGF-β signaling pathway of the present invention are used in combination with chemotherapy, targeted therapy, or nanoparticles, the small molecule kinase inhibitors blocking the TGF-β signaling pathway of the present invention can increase the efficiency of drug delivery and show improved anti-cancer efficacy, and when the small molecule kinase inhibitors blocking the TGF-β signaling pathway of the present invention are used in combination with radiation therapy, the small molecule kinase inhibitors can increase the oxygen concentration in the tumor microenvironment and show improved efficacy of tumor treatment / suppression.

[0041] Immune checkpoint refers to a group of signal molecules processed by immune cells, which can control and manage the persistence of immune responses while maintaining self-tolerance, and includes signal molecules involved in inhibitory pathways that maintain self-tolerance and aid immune responses and signal molecules involved in stimulatory pathways. Furthermore, the effectiveness of immune responses is determined by a delicate balance between co-stimulatory and co-inhibitory signals.

[0042] Immune checkpoint protein refers to both the protein involved in the pathway that inhibits immune response and the protein involved in the pathway that activates immune response.Immune checkpoint protein can be the protein involved in the signal transduction pathway that inhibits the activity of regulatory T cells, or the protein involved in the signal transduction pathway that directly stimulates effector T cells or memory T cells.In addition, immune checkpoint control can be for immune tolerance inhibition or immune system activation, and can include inhibition of the entire signal transduction pathway that activates regulatory T cells that are involved in immune tolerance to increase anti-tumor immune response, or stimulation of the signal transduction pathway that activates effector T cells or memory T cells. Examples of immune checkpoint proteins include T cell receptors, including, but not limited to, programmed cell death protein ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T cell membrane protein 3 (corresponding to T cell immunoglobulin mucin receptor 3: TIM3), OX40, V-domain immunoglobulin suppressor of T cell activation (VISTA), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), 4-1BB (CD137), glucocorticoid-induced tumor necrosis factor-related protein (GITR), adenosine A2a receptor (A2aR), and the like.

[0043] Thus, an immune checkpoint regulator may be an anti-immune checkpoint protein antibody or antigen-binding fragment thereof, an aptamer, a fusion protein bound to an immune checkpoint protein, or a small molecule compound that binds to an immune checkpoint protein or is involved in the mechanisms associated with its inhibition or activation.

[0044] Furthermore, the immune checkpoint control agent may be a substance that controls a signal transduction pathway, in which an immune checkpoint protein selected from the group consisting of PD-L1, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, OX40, VISTA, TIGIT, 4-1BB, GITR, and A2aR is involved. Furthermore, the immune checkpoint control agent may be a substance that inhibits signal transduction or reduces expression of a T cell receptor selected from the group consisting of PD-L1, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, OX40, VISTA, TIGIT, 4-1BB, GITR, and A2aR. Thus, immune checkpoint control agents can inhibit regulatory T cells or activate effector or memory T cells by inhibiting signaling or reducing expression via PD-L1, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, OX40, VISTA, TIGIT, 4-1BB, GITR, A2aR, or combinations thereof. Additionally, immune checkpoint control agents can be PD-L1 inhibitors, PD-1 inhibitors, CTLA-4 inhibitors, OX40 agonists, or combinations thereof.

[0045] Specifically, the immune checkpoint control agent may be at least one selected from the group consisting of an aptamer, peptide, antibody, and antigen-binding fragment of an antibody that specifically binds to PD-L1 protein; at least one selected from the group consisting of an aptamer, peptide, antibody, and antigen-binding fragment of an antibody that specifically binds to PD-1 protein; at least one selected from the group consisting of an aptamer, peptide, antibody, and antigen-binding fragment of an antibody that specifically binds to OX40 protein; and at least one selected from the group consisting of an aptamer, peptide, antibody, and antigen-binding fragment of an antibody that specifically binds to CTLA-4 protein. More specifically, the immune checkpoint control agent may be an anti-PD-L1 antibody or an antigen-binding fragment thereof; an anti-PD-1 antibody or an antigen-binding fragment thereof; an anti-OX40 antibody or an antigen-binding fragment thereof; or an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

[0046] The PD-L1 inhibitor may be, but is not limited to, a neutralizing antibody or antigen-binding fragment thereof that binds to PD-L1, an aptamer, or a fusion protein containing an antibody capable of binding to PD-L1, or a small molecule compound that inhibits the function of PD-L1. Examples of PD-L1 inhibitors include, but are not limited to, BMS-936559 (MDX1105, Bristol Myers Squibb), MEDI4736 (MedImmune, AstraZeneca), MPDL3280A (Roche), CK-301 (Checkpoint Therapeutica), MSB0010718C (Merck), and the like.

[0047] The PD-1 inhibitor may be, but is not limited to, a neutralizing antibody or antigen-binding fragment thereof that binds to PD-1, an aptamer or a fusion protein comprising an antibody capable of binding to PD-1, or a small molecule compound that inhibits the function of PD-1. Examples of PD-1 inhibitors include, but are not limited to, AMP-224 (Amplimmune, GlaxoSmith Klein), AMP-514 (MEDI0680, Amplimmune, GlaxoSmith Klein), nivolumab (Opdivo, Bristol Myers Squibb), pembrolizumab (Keytruda, Merck), pidilizumab (Cure Tech), BGB-A317 (BeiGene, Celgene), PF-06801591 (Pfizer), PDR001 (Novartis), TSR-042 (Tesaro, GlaxoSmithKline), MGA012 (Incyte / MacroGenics), IBI308 (Innovent, Eli Lilly), BI754091 (Boehringer Ingelheim), and the like.

[0048] The CTLA-4 inhibitor may be, but is not limited to, a neutralizing antibody or its antigen-binding fragment that binds to CTLA-4, an aptamer, or a fusion protein that contains an antibody that can bind to CTLA-4, or a small molecule compound that inhibits the function of CTLA-4. Examples of CTLA-4 inhibitors include, but are not limited to, ipilimumab (Yervoy, Bristol Myers Squibb), tremelimumab (Pfizer), etc.

[0049] OX40 agonist can be, but is not limited to, a neutralizing antibody or its antigen-binding fragment that binds to OX40, an aptamer or a fusion protein that contains an antibody that can bind to OX40, or a small molecule compound that activates the function of OX40. Examples of OX40 agonist include, but are not limited to, ABBV-368 (Abbvie), GSK3174998 (GlaxoSmithKline), MOXR0916 (Genentech), INCAGN01949 (Incyte Biosciences International), IBI101 (Innovent), BMS-986178 (Bristol Myers Squibb), PF-04518600 (Pfizer) and the like.

[0050] The term "receptor tyrosine kinase" as used herein refers to a transmembrane protein that penetrates the cell membrane and is involved in the cell signal transduction system that controls cell proliferation, cell migration, cell cycle and cell differentiation. Furthermore, the term "receptor tyrosine kinase inhibitor" as used herein can be an anti-receptor tyrosine kinase antibody or its antigen-binding fragment, an aptamer, a fusion protein, or a small molecule compound that binds to a receptor tyrosine kinase protein or is involved in a mechanism related to inhibition. Furthermore, the receptor tyrosine kinase inhibitor can be an inhibitor of at least one tyrosine kinase selected from VEGF receptor, FGF receptor, PDGFRα, KIT, RET and c-Met. Specifically, the receptor tyrosine kinase inhibitor can be an antibody or its antigen-binding fragment, an aptamer, or a fusion protein containing an antibody that can bind to at least one tyrosine kinase selected from VEGF receptor, FGF receptor, PDGFRα, KIT, RET and c-Met, or a small molecule compound that selectively inhibits tyrosine kinase. Furthermore, the receptor tyrosine kinase inhibitor may be a compound that inhibits the function of at least one tyrosine kinase selected from VEGF receptor, FGF receptor, PDGFRα, KIT, RET and c-Met, or may be at least one selected from an aptamer, a peptide, an antibody and an antigen-binding fragment of an antibody that specifically selects for said tyrosine kinase protein, including, but not limited to, cabozantinib, lenvatinib, vandetanib, selpercatinib, pralsetinib, etc.

[0051] On the one hand, the term "antibody" as used herein may be a neutralizing antibody having the ability to neutralize a target protein.

[0052] The term "pharmaceutical composition" as used herein includes any product resulting directly or indirectly from the combination of multiple components in specified amounts and products containing specific components in predetermined amounts or ratios. In particular, it includes any carrier containing one or more active ingredients and inactive ingredients, as well as any product resulting directly or indirectly from the combination, complex formation or aggregation or degradation of one or more ingredients or the interaction or other type of reaction of one or more ingredients of any two or more ingredients.

[0053] As used herein, the term "tumor" refers to a cellular disorder characterized by uncontrolled or disregulated cell proliferation, reduced cell differentiation, inappropriate ability to invade surrounding tissues and / or establish new growths at ectopic locations. Additionally, tumors include, but are not limited to, solid tumors and blood-borne tumors, and further include diseases of the skin, tissues, organs, bone, cartilage, blood and blood vessels, or primary and metastatic tumors.

[0054] Further, tumors include, but are not limited to, (1) leukemias, including acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, myelodysplastic syndromes, preleukemic conditions, and chronic myelomonocytic leukemia (CMML); (2) chronic leukemias, including but not limited to chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; (3) lymphomas, including but not limited to Hodgkin's disease and non-Hodgkin's disease; (4) smoldering polyomas, including but not limited to ... (5) bone and connective tissue sarcomas, including but not limited to osteosarcoma, osteogenic sarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic carcinoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; (6) glioma, glioblastoma, astrocytoma, brain stem glioma, ependymoma, oligodendroma, and sarcoma of the thymus. (7) brain tumors, including but not limited to glioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pinealocytoma, pineoblastoma, oligodendroglioma, and primary cerebral lymphoma; (8) breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, ductal carcinoma, medullary breast carcinoma, mucinous breast carcinoma, tubular breast carcinoma, papillary breast carcinoma, Paget's disease, and inflammatory breast carcinoma; (9) adrenal gland carcinoma, including but not limited to pheochromocytoma and adrenocortical carcinoma; (10) thyroid cancer, including but not limited to papillary or follicular thyroid carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma. (10) pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatinoma, and carcinoid or islet cell tumors; (11) pituitary cancer, including but not limited to Cushing's disease, prolactinoma, acromegaly, and diabetes insipidus; (12) eye cancer, including but not limited to ocular melanoma, e.g., iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; (13) vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma;(14) vulvar carcinoma, including but not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; (15) cervical carcinoma, including but not limited to squamous cell carcinoma and adenocarcinoma; (16) endometrial carcinoma, (17) uterine carcinoma, including but not limited to uterine sarcoma; (18) ovarian carcinoma, including but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; (19) ovarian carcinoma, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma. (20) gastric cancer, including but not limited to adenocarcinoma, fungus-like (polypoid), ulcerating, superficial spreading, generalized spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; (21) colon cancer; (22) rectal cancer; (23) liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; (24) gallbladder carcinoma, including but not limited to adenocarcinoma; (25) cholangiocarcinoma, including but not limited to papillary, nodular, and generalized; (26) non-small cell lung carcinoma, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung carcinoma. (27) testicular cancer, including but not limited to germinal tumor, seminoma, anaplastic, classical (classical), spermatocytic, non-seminomatous, embryonal carcinoma, teratocarcinoma, and trophoblastic (yolk sac tumor); (28) prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; (29) penile cancer; (30) oral cancer, including laryngeal carcinoma, pharyngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma, and squamous cell carcinoma; (31) basal carcinoma; (32) salivary gland carcinoma, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; (33) including but not limited to squamous cell carcinoma and verrucous carcinoma. (34) skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, and acral lentigo melanoma; (35) kidney cancer, including but not limited to renal cell carcinoma, adenocarcinoma, adenocarcinoma, fibrosarcoma, and transitional cell carcinoma (renal pelvis and / or ureter); (36) Wilms' tumor; (37) bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma; (38) head and neck cancer (mouth, nose, throat, larynx, paranasal sinuses, or salivary gland cancer, head and neck squamous cell carcinoma); (39) hepatocellular carcinoma;and (40) appendix cancer, bronchial cancer, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), malignant peripheral nerve sheath tumor (MPNST) and urethral cancer, as well as other cancers not included therein (see Fishman et al., 1985, Medicine, 2nd Ed., JB Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America). On the other hand, the tumor may be a metastatic tumor, an unresectable tumor or a locally advanced tumor. Furthermore, the tumor may be selected from melanoma, sarcoma, brain tumor, breast cancer, adrenal gland cancer, thyroid cancer, pancreatic cancer, pituitary cancer, glioblastoma, eye cancer, vaginal cancer, vulvar cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, basal carcinoma, salivary gland cancer, pharyngeal cancer, skin cancer, kidney cancer, Wilms' tumor, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, appendix cancer, bronchial carcinoma, choriocarcinoma, chordoma, ependymoma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer and urethral cancer. In some embodiments of the present invention, the tumor may be a solid tumor.;

[0055] As used herein, the term "prevention" refers to any action that prevents or delays the onset of cancer, and the term "treatment" refers to any action that improves or beneficially alters the symptoms of cancer.

[0056] The term "subject", as used herein, refers to an animal, preferably a mammal, and most preferably a human.

[0057] In one embodiment of the present invention, the subject who needs tumor treatment or tumor growth inhibition can be, but is not limited to, the subject who is at risk of developing or experiencing tumor recurrence.In another embodiment of the present invention, the subject who needs tumor treatment or tumor growth inhibition can be, but is not limited to, the subject who is being treated with at least one anticancer treatment selected from, but is not limited to, surgery, radiation therapy and chemotherapy.Furthermore, the subject who needs tumor treatment or tumor growth inhibition can be, but is not limited to, the subject who is being administered at least one chemotherapy selected from the group consisting of receptor tyrosine kinase inhibitors and immune checkpoint control agents.

[0058] The terms "in combination," "combination," or "combined administration" as used herein should be understood to include administering a small molecule kinase inhibitor that blocks the TGF-β signaling pathway in combination with an immune checkpoint regulator and / or a receptor tyrosine kinase inhibitor at the same time or separately at different times. When a small molecule kinase inhibitor that blocks the TGF-β signaling pathway, an immune checkpoint regulator, and / or a receptor tyrosine kinase inhibitor are administered individually, their administration cycles may be different from each other, and their administration routes may be different from each other.

[0059] As used herein, the term "pharmaceutical acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., a substance that may be administered in a composition without causing any undesirable biological effects in a subject or interacting in a deleterious manner with any other components of the pharmaceutical composition in which it is contained.

[0060] The term "pharmaceutical acceptable carrier" as used herein refers to carriers and auxiliary substances that are compatible with other components of the formulation, such as diluents or additives. Examples of pharmaceutical acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. Carriers are selected to minimize any degradation of the active ingredient and to minimize any adverse effects on the subject, as is well known to those of skill in the art. The pH of the solution is preferably about 5 to 8, more preferably about 7 to about 7.5. Carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing an antibody, where the matrix is ​​in the form of a shaped article, e.g., a film, liposome, or microparticle. It will be apparent to those of skill in the art that some carriers are more preferred depending, for example, on the route of administration and concentration of the composition to be administered. Additionally, pharmaceutical acceptable carriers are known to those of skill in the art, and most are standard carriers for drugs administered to humans, typically including solutions such as sterile water, saline, and buffers at physiological pH.

[0061] In other embodiments of the present invention, the pharmaceutical compositions of the present invention may also include thickeners, diluents, buffers, preservatives, surface active agents, and the like.

[0062] Pharmaceutical compositions for parenteral injection include pharma- ceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, glycerol, polyols (e.g., propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also include preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity can be ensured by the inclusion of various antimicrobial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, and the like. The inclusion of isotonic agents, such as sugars, sodium chloride, and the like, can also be desirable. Prolonged absorption of injectable pharmaceutical forms can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are prepared by forming microencapsulated matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending on the drug-to-polymer ratio and the nature of the particular polymer used, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by the inclusion of a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use.

[0063] The pharmaceutical composition for oral administration may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. The pharmaceutical composition for oral administration may contain one or more optional agents, such as sweeteners, such as fructose, aspartame or saccharin; flavorings, such as peppermint, wintergreen oil or cherry coloring, and preservatives, to provide a pharmacologic formulation that is easy to swallow. Furthermore, in tablet or pill form, the composition may be coated to delay disintegration and absorption in the digestive tract, thereby providing a sustained action over an extended period of time. A selectively permeable membrane surrounding an osmotically active driving compound is also suitable for orally administered compounds of the present invention. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which expands and forces the drug or drug composition through an opening. These delivery platforms can provide an essentially zero order delivery profile, as opposed to the sharp profiles of immediate release formulations. A time-delay material, such as glycerol monostearate or glycerol stearate, may also be used. Oral compositions can include standard vehicles, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.

[0064] For oral liquid pharmaceutical compositions, formulations such as suspensions, elixirs and solutions, suitable carriers, additives or diluents include water, saline, alkylene glycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol) oils, alcohol, slightly acidic buffers of pH 4 to pH 6 (e.g., about 5.0 mM to about 50.0 mM acetic acid, citric acid, ascorbic acid), and the like. Additionally, flavoring agents, preservatives, coloring agents, bile salts, acylcarnitines, and the like may be added. Furthermore, in some embodiments of the present invention, pharmaceutical compositions for oral administration may be in the form of tablets, capsules, and the like formulated in a conventional manner.

[0065] In the present invention, a therapeutically effective amount of a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β refers to at least the minimum dose of the small molecule kinase inhibitor disclosed herein required to achieve the desired therapeutic effect, including a dose sufficient to reduce tumor symptoms, tumor growth, etc. Furthermore, a therapeutically effective amount of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor refers to at least the minimum dose of the immune checkpoint regulator and a receptor tyrosine kinase inhibitor disclosed herein required to achieve the desired therapeutic effect, including a dose sufficient to reduce tumor symptoms, tumor growth, etc. The efficacy of the combination of a small molecule kinase inhibitor and at least one selected from an immune checkpoint regulator and a receptor tyrosine kinase inhibitor disclosed herein in treating a tumor is determined by observing the improvement of the subject based on one or more clinical symptoms and / or physiological indicators associated with the condition. Improvement of the tumor may also be indicated by a reduced need for concomitant treatment.

[0066] In another embodiment of the present invention, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β can be administered once a day, twice a day or three times a day, but is not limited thereto.Preferably, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β can be administered twice a day at the same dose or different doses.More preferably, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β can be administered twice a day at the same dose.

[0067] In certain embodiments of the invention, the small molecule kinase inhibitor, immune checkpoint regulator, and receptor tyrosine kinase inhibitor may each independently be administered to a subject in need of treatment, including, but not limited to, topically, parenterally, orally, intravenously, intramuscularly, subcutaneously, or via aerosol.

[0068] In certain embodiments of the invention, small molecule kinase inhibitors that block the TGF-β signaling pathway may be administered in combination with immune checkpoint regulators.

[0069] In another embodiment of the invention, small molecule kinase inhibitors that block the TGF-β signaling pathway may be administered in combination with receptor tyrosine kinase inhibitors.

[0070] In other embodiments of the invention, small molecule kinase inhibitors that block the TGF-β signaling pathway may be administered in combination with receptor tyrosine kinase inhibitors and immune checkpoint regulators.

[0071] In one embodiment of the present invention, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor are each contained as active ingredients in separate preparations, and can be administered simultaneously or at different times.Specifically, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor can be administered separately at different times.

[0072] Additionally, in other aspects of the invention, the small molecule kinase inhibitors may be administered orally.

[0073] Furthermore, the immune checkpoint regulators and receptor tyrosine kinase inhibitors may be administered by known administration methods, dosages and administration cycles for the corresponding immune checkpoint regulators and receptor tyrosine kinase inhibitors in the field to which the present invention belongs.

[0074] In another embodiment of the present invention, the present invention relates to a pharmaceutical composition for treating tumors, comprising a small molecule kinase inhibitor that blocks the TGF-β signaling pathway for treating tumor patients receiving immune checkpoint control agents and / or receptor tyrosine kinase inhibitors.

[0075] In another embodiment of the present invention, the present invention may be a pharmaceutical composition for treating a tumor comprising a small molecule kinase inhibitor that blocks the signaling pathway of TGF-β, wherein the small molecule kinase inhibitor that blocks the signaling pathway of TGF-β is administered in combination with at least one selected from an immune checkpoint control agent and a receptor tyrosine kinase inhibitor, thereby enhancing the effect of the immune checkpoint control agent and / or the receptor tyrosine kinase inhibitor or enhancing the effect of the small molecule kinase inhibitor that blocks the signaling pathway of TGF-β.

[0076] In one embodiment of the present invention, the present invention can be a combination or pharmaceutical composition for preventing or treating tumor, comprising at least one of small molecule kinase inhibitors that block TGF-β signaling pathway and immune checkpoint regulators and receptor tyrosine kinase inhibitors.In this case, small molecule kinase inhibitors that block TGF-β signaling pathway, immune checkpoint regulators and receptor tyrosine kinase inhibitors can be administered simultaneously, sequentially or separately, and the type, tumor type, administration method and dose of small molecule kinase inhibitors that block TGF-β signaling pathway, immune checkpoint regulators and receptor tyrosine kinase inhibitors are as defined above.

[0077] The term "combination" as used herein refers to a product produced by mixing or blending two or more active ingredients, including both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, for example, at least one selected from a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, an immune checkpoint regulator, and a receptor tyrosine kinase inhibitor, are all administered simultaneously to a patient in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, for example, at least one selected from a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, an immune checkpoint regulator, and a receptor tyrosine kinase inhibitor, are administered to a subject as separate entities, together, simultaneously, or sequentially without any specific time limit, where such administration provides a therapeutically effective level of the active ingredients in the patient's body. Non-fixed combination also applies to cocktail therapy, for example, administration of three or more active ingredients.

[0078] In one embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of melanoma, colorectal cancer, bladder cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with a PD-1 inhibitor (preferably an anti-PD-1 antibody or a fragment thereof).

[0079] In an embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of melanoma, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with a PD-L1 inhibitor (preferably an anti-PD-L1 antibody or a fragment thereof).

[0080] In another embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of melanoma, colorectal cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof).

[0081] In another embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with lenvatinib.

[0082] In one embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with lenvatinib and a PD-1 inhibitor (preferably an anti-PD-1 antibody or a fragment thereof).

[0083] In another embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with lenvatinib and a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof).

[0084] In one embodiment of the present invention, the pharmaceutical composition or combination of the present invention may be a pharmaceutical composition or combination for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, which may be administered in combination with a PD-1 inhibitor (preferably, an anti-PD-1 antibody or a fragment thereof) and a CTLA-4 inhibitor (preferably, an anti-CTLA-4 antibody or a fragment thereof).

[0085] In another embodiment of the present invention, the present invention can be a method for preventing or treating tumor in a subject, comprising administering to the subject at least one of a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β and an immune checkpoint regulator and a receptor tyrosine kinase inhibitor.In this case, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor can be administered simultaneously, sequentially or separately, and the type, tumor type, administration method and dose of the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor that blocks the signal transduction pathway of TGF-β are as defined above.

[0086] In one embodiment of the present invention, the present invention may be a method for preventing or treating melanoma, colorectal cancer, bladder cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof and a PD-1 inhibitor (preferably an anti-PD-1 antibody or a fragment thereof).

[0087] In one embodiment of the present invention, the present invention may be a method for preventing or treating melanoma in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof and a PD-L1 inhibitor (preferably an anti-PD-L1 antibody or a fragment thereof).

[0088] In another embodiment of the present invention, the present invention may be a method for preventing or treating melanoma, colorectal cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof and a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof).

[0089] In another embodiment of the present invention, the present invention may be a method for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof and lenvatinib.

[0090] In one embodiment of the present invention, the present invention may be a method for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, lenvatinib, and a PD-1 inhibitor (preferably an anti-PD-1 antibody or a fragment thereof).

[0091] In another embodiment of the present invention, the present invention may be a method for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, lenvatinib and a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof).

[0092] In one embodiment of the present invention, the present invention may be a method for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer in a subject, comprising administering to the subject 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, a PD-1 inhibitor (preferably, an anti-PD-1 antibody or a fragment thereof), and a CTLA-4 inhibitor (preferably, an anti-CTLA-4 antibody or a fragment thereof).

[0093] Furthermore, in another embodiment of the present invention, the present invention relates to the use of a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β administered in combination with at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor in the manufacture of a medicament for preventing or treating a tumor, in which the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor can be administered simultaneously, sequentially or separately, and the type, tumor type, administration method and dosage of the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor are as defined above.

[0094] In one embodiment of the present invention, the present invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, administered in combination with a PD-1 inhibitor (preferably an anti-PD-1 antibody or a fragment thereof), for the manufacture of a medicament for the prevention or treatment of melanoma, colorectal cancer, bladder cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer.

[0095] In one embodiment of the invention, the invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, administered in combination with a PD-L1 inhibitor (preferably an anti-PD-L1 antibody or a fragment thereof) for the manufacture of a medicament for the prevention or treatment of melanoma.

[0096] In another embodiment of the present invention, the present invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutically acceptable salt thereof, administered in combination with a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof), for the manufacture of a medicament for the prevention or treatment of melanoma, colorectal cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer.

[0097] In another embodiment of the present invention, the present invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, administered in combination with lenvatinib, for the manufacture of a medicament for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer.

[0098] In one embodiment of the present invention, the invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, administered in combination with lenvatinib and a PD-1 inhibitor (preferably, an anti-PD-1 antibody or a fragment thereof), for the manufacture of a medicament for the prevention or treatment of melanoma, colorectal cancer, head and neck squamous cell carcinoma or liver cancer.

[0099] In another embodiment of the present invention, the present invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof administered in combination with lenvatinib and a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof) for the manufacture of a medicament for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer.

[0100] In one embodiment of the present invention, the present invention may be the use of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutically acceptable salt thereof, administered in combination with a PD-1 inhibitor (preferably an anti-PD-1 antibody or a fragment thereof) and a CTLA-4 inhibitor (preferably an anti-CTLA-4 antibody or a fragment thereof), for the manufacture of a medicament for the prevention or treatment of colorectal cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer.

[0101] Furthermore, the present invention may be a kit for preventing or treating a tumor, comprising a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β and an instruction manual instructing administration of at least one combination selected from a small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β and an immune checkpoint regulator and a receptor tyrosine kinase inhibitor. In this case, the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor may be administered simultaneously, sequentially or separately, and the type, tumor type, administration method and dose of the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β, the immune checkpoint regulator and the receptor tyrosine kinase inhibitor are as defined above.

[0102] In one embodiment of the present invention, the present invention may be a kit for preventing or treating melanoma, colorectal cancer, bladder cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof; and an instruction manual instructing the administration of a combination of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof and a PD-1 inhibitor (preferably, an anti-PD-1 antibody or a fragment thereof).

[0103] In one embodiment of the present invention, the present invention may be a kit for preventing or treating melanoma, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof; and an instruction manual instructing the administration of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof and a PD-L1 inhibitor (preferably, an anti-PD-L1 antibody or a fragment thereof) in combination.

[0104] In another embodiment of the present invention, the present invention may be a kit for preventing or treating melanoma, colorectal cancer, sarcoma, breast cancer, cervical cancer, head and neck squamous cell carcinoma or liver cancer, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof; and an instruction manual directing the administration of a combination of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof and a CTLA-4 inhibitor (preferably, an anti-CTLA-4 antibody or a fragment thereof).

[0105] In another embodiment of the present invention, the present invention may be a kit for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof; and an instruction manual instructing the administration of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof in combination with lenvatinib.

[0106] In one embodiment of the present invention, the present invention may be a kit for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof; and an instruction manual instructing the administration of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutical acceptable salt thereof, in combination with lenvatinib and a PD-1 inhibitor (preferably, an anti-PD-1 antibody or a fragment thereof).

[0107] In another embodiment of the present invention, the present invention may be a kit for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof; and an instruction manual instructing the administration of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharmaceutically acceptable salt thereof, in combination with lenvatinib and a CTLA-4 inhibitor (preferably, an anti-CTLA-4 antibody or a fragment thereof).

[0108] In one embodiment of the present invention, the present invention may be a kit for preventing or treating colorectal cancer, cervical cancer, head and neck squamous cell carcinoma, or liver cancer, comprising 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutically acceptable salt thereof; and an instruction manual instructing the administration of 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine or a pharma- ceutically acceptable salt thereof with a PD-1 inhibitor (preferably, an anti-PD-1 antibody or a fragment thereof) and a CTLA-4 inhibitor (preferably, an anti-CTLA-4 antibody or a fragment thereof) in combination.

[0109] Method of the invention Working Example Hereinafter, the present invention will be described in more detail with reference to the following examples.However, in the following examples, compound 1 (6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazole[1,5-a]pyridine phosphate) is used as a small molecule kinase inhibitor that blocks TGF-β signaling pathway or its pharmacologic acceptable salt to illustrate the present invention, but the scope of the present invention is not limited to the corresponding compound.

[0110] Example 1. Antitumor effect in melanoma mouse model Example 1.1. Combination of anti-PD-1 antibody and Compound 1 Example 1.1.1 Preparation of Melanoma Mouse Model and Test Drugs Forty female C57BL / 6 mice, aged 6–8 weeks, were purchased from OrientBio and incubated at 1 × 10 6 B16F10 cell line (KCLB, #80008) was injected subcutaneously into the right flank of each mouse, thereby implanting the cancer cell line. The day of B16F10 cell line implantation was defined as day 0, and tumor volumes were measured on day 9. Mice were classified according to the mean and deviation of tumor volumes on day 9. A 0.9% physiological saline solution was prepared as a vehicle, and a mixed solution of mouse anti-PD-1 antibody (BioXCell, clone #RMP1-14) and PBS, and a mixed solution of compound 1 and the vehicle were prepared.

[0111] Example 1.1.2. Test drug administration method, measured variables and statistical analysis The mice classified in Example 1.1.1 were assigned to a control group (oral administration of vehicle), oral administration of Compound 1, intraperitoneal administration of anti-PD-1 antibody, or a combination of anti-PD-1 antibody and Compound 1. Drug administration was started on the 9th day after B16F10 cell line implantation, and the test was completed on the 24th day after drug administration. The dose and administration method of the drug administered to each group are shown in Table 1 below. When administered orally (po) and intraperitoneally (ip), the dose per individual was 5mL / kg in both cases. [Table 1] Mouse tumor volumes and body weights were measured twice weekly during the study period after drug administration. Mouse tumor volumes were calculated as (tumor length (L) x tumor width (W) x tumor width (W)) ÷ 2, where tumor length (L) is the longest tumor dimension and tumor width (W) is the longest tumor dimension perpendicular to L. On the other hand, if the tumor volume of an individual mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued. The tumor volume changes in each group were statistically analyzed using two-way analysis of variance.

[0112] Example 1.1.3. Tumor growth inhibitory effect and body weight change When comparing the tumor volumes on the 15th day after drug administration, it was found that the tumor volume in the anti-PD-1 antibody alone group was 45% lower than that measured in the control group, the tumor volume in the compound 1 alone group was 44% lower than that measured in the control group, while the tumor volume in the anti-PD-1 antibody and compound 1 combination group was 74% lower than that measured in the control group. Furthermore, when the tumor volumes on the 15th day after drug administration were examined, it was found that the tumor volumes in the compound 1 and anti-PD-1 antibody combination group were statistically significantly different (i.e., p<0.05 and p<0.001) compared with the anti-PD-1 antibody alone group or the compound 1 alone group (see FIG. 1). There was no statistically significant difference in weight change between the control and drug-treated groups throughout the study period (see Figure 2).

[0113] Example 1.2. Combination of anti-PD-L1 antibodies with Compound 1 Example 1.2.1 Preparation of Melanoma Mouse Model and Test Drugs Ninety 8-week-old female C57BL / 6 mice were purchased from OrientBio, and melanoma mouse models were prepared by the method used for preparing melanoma mouse models in Example 1.1.1. The day of B16F10 cell line implantation was set as day 0, and tumor volumes were measured on day 4. Mice were classified according to the mean and deviation of tumor volumes on day 4. The medium and PBS used in Example 1.1.1 were used as carriers to prepare a mixed solution of mouse anti-IgG2b antibody (BioXCell) and PBS, a mixed solution of mouse anti-PDL1 antibody (BioXCell) and PBS, a mixed solution of compound 1 and medium, and a mixed solution of LY2157299 (Chembo Pharma) and medium.

[0114] Example 1.2.2. Test drug administration method, measured variables and statistical analysis The mice classified in Example 1.2.1 were assigned to a control group (administered in combination with a vehicle, anti-IgG2b antibody, and a mixture of PBS), a vehicle and anti-PDL1 antibody group, an anti-PDL1 antibody and Compound 1 group (low dose: 15 mg / kg / day, high dose: 30 mg / kg / day), or an anti-PDL1 antibody and LY2157299 group (low dose: 75 mg / kg / day, high dose: 150 mg / kg / day), and the drugs were administered from the 4th to the 21st day after the day of B16F10 cell line transplantation, and the test was terminated on the 22nd day. The dose and administration method of the drug administered to each group are shown in Table 2 below, and the dose per individual was 10 mL / kg for both oral (po) and intraperitoneal (ip) administration. [Table 2] The tumor volumes of the mice were measured at 2-day intervals from the day of drug administration, and the tumor volumes were measured by autopsy at the end of the study. In this case, the mouse tumor volumes were calculated by the same method as the tumor volume calculation method in Example 1.1.2. In addition, the mouse body weights were measured on the 4th, 7th, 11th, 14th, 18th and 21st days from the day of B16F10 cell line implantation. The tumor volume changes and body weight changes of each group were statistically analyzed using Tukey's multiple comparison after ANOVA test. When comparing the tumor volumes on the 22nd day from the day of B16F10 cell line implantation, the statistically significant differences were compared with the control group, where the vehicle and anti-IgG2b antibody were statistically analyzed using Dunnett's multiple comparison after ANOVA test. The statistically significant differences between the mouse anti-PD-L1 antibody, test drug, single treatment group and the combination treatment group of compound 1 and anti-PD-L1 antibody on the 22nd day from the day of B16F10 cell line implantation were analyzed by Student's t-test.

[0115] Example 1.2.3. Tumor growth inhibitory effect and body weight change Except for the low-dose LY2157299 and anti-PD-L1 antibody combination group, the anti-PD-L1 antibody alone group, the low-dose Compound 1 and anti-PD-L1 antibody combination group, the high-dose Compound 1 and anti-PD-L1 antibody combination group, and the high-dose LY2157299 and anti-PD-L1 antibody combination group were found to have significant tumor growth inhibitory effects compared to the control group from day 7 of drug administration (i.e., day 10 from the day of B16F10 cell line implantation) to day 19 (day 22 from the day of cell line implantation) (see Figure 3). Specifically, when the difference in tumor volume in the control group was statistically analyzed using ANOVA test and Tukey's multiple comparison, the difference was found to be statistically significant ( * :p<0.05; ** :p<0.01; *** :p<0.001). In particular, at the end of the study, 22 days after the B16F10 cell line implantation, only the group administered the combination of high-dose Compound 1 and anti-PD-L1 antibody showed a statistically significant reduction in tumor volume compared to the control group ( * , p<0.05) (see Figure 4), which was a statistically significant reduction ( # , p<0.05, one-tailed Student's t-test) are also shown (see Figures 3 and 4). There was no statistically significant difference in body weight change between the control group and the drug-treated group over the entire study period (see Figure 5).

[0116] Example 1.3. Combined Administration of Anti-CTLA-4 Antibody and Compound 1 Example 1.3.1 Preparation of Melanoma Mouse Model and Test Drugs Forty female C57BL / 6 mice, aged 6–8 weeks, were purchased from OrientBio and incubated at 1 × 10 6 B16F10 cell line (KCLB, #80008) was injected subcutaneously into the right flank of each mouse, thereby implanting the cancer cell line. The day of B16F10 cell line implantation was defined as day 0, and tumor volumes were measured on day 9. Mice were classified according to the mean and deviation of tumor volumes on day 9. A 0.9% physiological saline solution was prepared as a vehicle, and a mixed solution of mouse anti-CTLA-4 antibody (BioXCell, clone #9D9) and PBS and a mixed solution of Compound 1 and vehicle were prepared as test drugs.

[0117] Example 1.3.2. Test drug administration method, measured variables and statistical analysis The mice classified in Example 1.3.1 were assigned to a control group (oral administration of vehicle), oral administration of Compound 1, intraperitoneal administration of anti-CTLA-4 antibody, or a combination administration of anti-CTLA-4 antibody and Compound 1, and drug administration was started from the 9th to 21st day after the B16F10 cell line implantation date, and the test was completed on the 22nd day. The dose and administration method of the drug administered to each group are shown in Table 3 below, and the dose per individual was 5mL / kg in both oral (po) and intraperitoneal (ip) administrations. [Table 3] The tumor volume and body weight of the mice were measured twice a week during the test period after drug administration. In this case, the tumor volume of the mice was calculated by the same method as that of Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued. The tumor volume and body weight changes in each group were statistically analyzed using two-way ANOVA and one-tailed t-test.

[0118] Example 1.3.3. Tumor growth inhibitory effects and body weight changes During the drug administration period, the group administered the combination of compound 1 and anti-CTLA-4 antibody was found to have superior tumor growth inhibitory effects compared to the vehicle administration group, the compound 1 alone administration group, or the anti-CTLA-4 antibody alone administration group (see Figure 6(a)). Furthermore, when the tumor volume was examined 15 days after drug administration, the tumor volume in the group administered the combination of compound 1 and anti-CTLA-4 antibody was found to be statistically significantly different (i.e., p<0.01) compared to the group administered anti-CTLA-4 antibody alone or the group administered compound 1 alone (see Figure 6(b)). There was no statistically significant difference in body weight change between the control and drug-treated groups over the entire study period (see Figure 7).

[0119] Example 2. Antitumor effects in a mouse model of colorectal cancer Example 2.1 Preparation of colorectal cancer mouse model and test drugs One hundred and seventy female BALB / C mice aged 6–8 weeks were purchased from OrientBio, and the cancer cell lines were inoculated into the right flank of each mouse at 7.5 × 10 5 CT26 cell line (mouse colon cancer cell) was implanted by subcutaneous injection. The day of CT26 cell line implantation was set as day 0, and the tumor volume was measured on day 7. The average tumor volume on day 7 was 50 mm 3 It was found that 110 mice were selected after excluding those with tumor volumes significantly larger or smaller than the average tumor volume. A 0.9% physiological saline solution was prepared as a vehicle, and a mixture of mouse anti-PD-1 antibody (BioXCell, clone #RMP1-14) and PBS, a mixture of mouse anti-CTLA-4 antibody (BioXCell, clone #9D9) and PBS, a mixture of lenvatinib and vehicle, and a mixture of compound 1 and vehicle were prepared.

[0120] Example 2.2. Test drug administration method, measured variables and statistical analysis The mice selected in Example 2.1 were assigned to the groups listed in Table 4 below and administered vehicle or drug, with drug administration starting on day 7 from the date of cell line inoculation and the study ending on day 27 from the date of drug administration. [Table 4] When administered orally (po) and intraperitoneally (ip), the dose was 5 mL / kg in both cases. The tumor volume and body weight of the mice were measured twice a week during the test period after drug administration. In this case, the tumor volume of the mice was calculated by the same method as that of Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued. The tumor volume changes and body weight changes in each group were statistically analyzed using two-way analysis of variance, Bonferroni test and one-tailed t-test.

[0121] Example 2.3. Tumor growth inhibitory effect Example 2.3.1. Combination Administration of Compound 1 with Anti-PD-1 Antibody and Compound 1 with Anti-CTLA-4 Antibody The tumor volume of the anti-PD-1 antibody and compound 1 combination group was measured to be lower than that of the anti-PD-1 antibody alone, compound 1 alone, or vehicle-treated control group (see FIG. 8(a)). Furthermore, when comparing the tumor volumes measured on the 26th day from the date of drug administration, the tumor volume of the anti-PD-1 antibody alone group was 4.8% lower than that of the control group, the tumor volume of the compound 1 alone group was 59% lower than that of the control group, while the tumor volume of the anti-PD-1 antibody and compound 1 combination group was 77% lower than that of the control group. Furthermore, when comparing the tumor weights (g) measured on the 26th day from the date of drug administration, the tumor body of the anti-PD-1 antibody and compound 1 combination group was found to be the smallest, and the difference in tumor weight between the compound 1 alone group and the anti-PD-1 antibody and compound 1 combination group was found to be statistically significant (see FIG. 8(b)). Furthermore, the tumor volume of the group administered the combination of high-dose anti-CTLA antibody and compound 1 was measured to be lower than that of the group administered high-dose anti-CTLA antibody alone, the group administered compound 1 alone, or the control group administered only the vehicle throughout the entire test period (see FIG. 9(a)). Furthermore, when comparing the tumor volumes measured on the 26th day after the drug administration date, it was found that the tumor volume of the group administered high-dose anti-CTLA-4 antibody alone was 27% lower than that of the control group, the tumor volume of the group administered compound 1 alone was 59% lower than that of the control group, and the tumor volume of the group administered the combination of high-dose anti-CTLA-4 antibody and compound 1 was 77% lower than that of the control group (see FIG. 9(b)). Furthermore, the tumor volumes observed in the groups administered the combination of anti-PD-1 antibody, low-dose anti-CTLA-4 antibody and compound 1 were found to be lower than the tumor volumes observed in the groups administered the vehicle alone, the group administered the anti-PD-1 antibody alone, the group administered the low-dose anti-CTLA-4 antibody alone, the group administered compound 1 alone, the group administered the combination of anti-PD-1 antibody and high-dose anti-CTLA-4 antibody, and the group administered the combination of compound 1 and low-dose anti-CTLA-4 antibody (see Figure 10). Furthermore, the tumor volumes in the group treated with the combination of anti-PD-1 antibody, low-dose anti-CTLA-4 antibody and Compound 1 were 88% lower than the tumor volume in the control group treated with vehicle alone, and the tumor volumes observed in the group treated with anti-PD-1 antibody alone, the group treated with low-dose anti-CTLA-4 antibody alone, the group treated with Compound 1 alone, the group treated with the combination of Compound 1 and low-dose anti-CTLA-4 antibody, and the group treated with the combination of anti-PD-1 antibody and high-dose anti-CTLA-4 antibody were 4.8%, 23%, 59%, 60% and 63%, respectively, lower than the tumor volume in the control group treated with vehicle alone (see Figure 10).

[0122] Example 2.3.2. Combination of Compound 1 and Lenvatinib or Combination of Compound 1, Lenvatinib and Anti-PD-1 Antibody and / or Anti-CTLA-4 Antibody The tumor volume in the group administered the combination of lenvatinib and compound 1 was measured to be lower than the tumor volume in the group administered the combination of anti-PD-1 antibody alone, the group administered the combination of lenvatinib alone, the group administered the combination of compound 1 alone, the group administered the combination of lenvatinib and anti-PD-1 antibody, or the control group administered only with the vehicle throughout the entire test period, and it was found that the tumor volume in the group administered the combination of lenvatinib, compound 1, and anti-PD-1 antibody was lower than the tumor volume in the group administered the combination of lenvatinib and compound 1 (see FIG. 11). Furthermore, the tumor volume in the group administered the combination of lenvatinib, anti-CTLA-4 antibody, and compound 1 was measured to be lower than the tumor volume in the group administered the combination of anti-CTLA-4 antibody alone, the group administered the combination of lenvatinib and anti-PD-1 antibody, the group administered the combination of lenvatinib and anti-CTLA-4 antibody, and the control group throughout the entire test period (see FIG. 12). Furthermore, when comparing the tumor volumes measured 26 days after the day of drug administration, the tumor volume reductions in the compound 1 alone administration group, lenvatinib alone administration group, anti-PD-1 antibody alone administration group, anti-CTLA-4 antibody alone administration group, lenvatinib and anti-PD1 antibody combination administration group, and lenvatinib and anti-CTLA-4 antibody combination administration group were 59%, 30%, 4.8%, 23%, 41%, and 56%, respectively, compared to the tumor volume in the control group administered with the vehicle only. On the other hand, when comparing the tumor volumes measured 26 days after drug administration, the tumor volume reductions in the group administered with the combination of lenvatinib and compound 1 and the group administered with the combination of lenvatinib, compound 1, and anti-PD-1 antibody were found to be 68% and 90%, respectively, compared to the tumor volume in the control group administered with the vehicle alone. Furthermore, the tumor volume reduction in the group administered with the combination of lenvatinib, compound 1, and anti-CTLA-4 antibody was 82% compared to the tumor volume in the control group administered with the vehicle alone. In summary, it is confirmed that the use of compound 1 in combination with at least one selected from lenvatinib, an anti-PD-1 antibody, and an anti-CTLA-4 antibody exhibits superior anti-cancer effects compared to the use of lenvatinib, an anti-PD-1 antibody, or an anti-CTLA-4 antibody alone, or administration of a vehicle alone.

[0123] Example 2.4. Weight change There was no statistically significant difference in body weight change between the drug-treated groups and the control group that received only the vehicle during the entire study period (see FIG. 13).

[0124] Example 2.5. In vitro studies to determine whether the mechanisms of action of Compound 1 and lenvatinib overlap To determine whether the mechanisms of action of compound 1 and lenvatinib overlap, the restoration of the inhibited IFN-γ secretion function of T cells was measured under the condition of T cell receptor stimulation alone. Changes in IFN-γ secretion in hPBMCs were measured by treating human peripheral blood mononuclear cells (hPBMCs) with vehicle, with anti-CD3 and anti-CD28 antibodies (T cell stimulants) alone, with TGF-β (a T cell function inhibitor) alone, or with lenvatinib and / or compound 1 plus TGF-β, anti-CD3 and anti-CD28 antibodies. First, human peripheral blood mononuclear cells (hPBMCs) were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES and 1×NEAA under conditions of 37° C. in a 5% carbon dioxide humidified incubator. A 6-well plate was coated with anti-CD3 monoclonal antibody (OKT3) overnight at 4°C. The antibody solution in each well of the 6-well plate was aspirated and washed with PBS. hPBMCs were seeded on the coated 6-well plate. To increase the reactivity of hPBMCs, the culture was carried out for 5 days at 37°C in a 5% carbon dioxide humidified incubator. The 5-day cultured hPBMCs were seeded at 1 × 10 5 hPBMCs were seeded at a cell / well concentration. hPBMCs seeded in a 96-well plate were treated with lenvatinib or compound 1, the hPBMCs were cultured for 1 hour, and then TGF-β was added to it and the hPBMCs were cultured for 1 hour. hPBMCs were treated with anti-CD3 antibody / anti-CD28 antibody-coated microbeads at a cell:bead ratio of 2:1, and the hPBMCs were stimulated for 72 hours. After 72 hours, the supernatants were collected and conventional IFN-γ ELISA was performed. The IFN-γ content produced in each group was measured. Data analysis was performed using Prism 5 (GraphPad Software, Inc.), and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison. ELISA analysis showed that stimulation with anti-CD3 and anti-CD28 antibodies increased IFNγ secretion in human PBMCs, and TGF-β treatment inhibited IFNγ secretion in human PBMCs. Furthermore, treatment with lenvatinib, anti-CD3, anti-CD28, and TGF-β increased IFNγ secretion in human PBMCs, and treatment with compound 1, anti-CD3, anti-CD28, and TGF-β increased IFNγ secretion in human PBMCs. Furthermore, treatment with compound 1, lenvatinib, anti-CD3, anti-CD28, and TGF-β showed an increase in IFNγ secretion, similar to stimulation with anti-CD3 and anti-CD28. Therefore, it was confirmed that the mechanisms of action of the two drugs act independently. On the other hand, lenvatinib is known to have poor anticancer effects in an anergic environment of endothelial cells accompanied by a decrease in binding proteins such as VCAM-1 produced on the surface of vascular endothelial cells. Therefore, the combination of compound 1 with lenvatinib, which has a different mechanism of action, complements and improves the anticancer effects of lenvatinib.

[0125] Example 3. Antitumor effect in a mouse sarcoma model Example 3.1 Preparation of Sarcoma Mouse Model and Test Drugs Sixty female BALB / C mice, aged 6–8 weeks, were purchased from Vital River Laboratories Research Model and Services Company. 6 The WEHI-164 cell line (mouse sarcoma cell line) was subcutaneously injected into the right flank of each mouse to implant the cancer cell line. The day of implantation of the WEHI-164 cell line was designated as day 0, and the tumor volume was measured on day 7. The average tumor volume on day 7 was 53 mm 3 It was found that mice were selected to maintain a constant mean and deviation for each group by excluding individuals with tumor volumes significantly larger or smaller than the mean tumor volume. A 0.9% physiological saline solution was prepared as the vehicle, and a mixed solution of anti-PD-1 antibody (BioXCell) and PBS, a mixed solution of anti-CTLA-4 antibody (BioXCell) and PBS, and a mixed solution of compound 1 and the vehicle were prepared as test drugs.

[0126] Example 3.2. Test drug administration method, measured variables and statistical analysis The mice selected in Example 3.1 were assigned to a control group, an anti-PD-1 antibody administration group, a compound 1 administration group, an anti-CTLA-4 antibody administration group, a combination of compound 1 and anti-PD-1 antibody administration group, or a combination of compound 1 and anti-CTLA-4 antibody administration group, respectively. The drugs were administered from day 7 to day 23 after the day of WEHI-164 cell line implantation. The doses and administration methods of the drugs administered to each group are shown in Table 5 below, and the doses were 5mL / kg in both cases when administered orally (po) and intraperitoneally (ip). [Table 5] The changes in tumor volume and body weight of the mice were measured twice a week during the test period after drug administration, and the tumor volume was measured using the same method as in Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued. Differences in tumor volumes during the study were statistically analyzed using one-tailed t-tests, ANOVA and Tukey's multiple comparison test.

[0127] Example 3.3. Tumor growth inhibitory effect and remission rate The tumor volume of the group administered the combination of anti-PD-1 antibody and compound 1 was smaller than that of the group administered the combination of anti-PD-1 antibody alone, the group administered the combination of compound 1 alone, or the vehicle-administered control group (see FIG. 14). Furthermore, the tumor volume of the group administered the combination of anti-PD-1 antibody alone measured on the 16th day from the start of drug administration was 64% smaller than that of the control group, and the tumor volume of the group administered the combination of compound 1 alone was 71% smaller than that of the control group. However, the tumor volume of the group administered the combination of anti-PD-1 antibody and compound 1 was astonishingly 98% smaller than that of the control group, and the difference in tumor volume between the group administered the combination of anti-PD-1 antibody and compound 1 and the group administered the combination of compound 1 alone or the group administered the anti-PD-1 antibody alone was found to be statistically significant (p<0.05) (see FIG. 14). Furthermore, the complete tumor remission rate was 20% when anti-CTLA-4 antibody was administered alone compared to the vehicle control group, 30% when compound 1 was administered alone compared to the vehicle control group, and 80% when anti-CTLA-4 antibody and compound 1 were administered in combination compared to the vehicle control group. The difference in complete tumor remission rate between the anti-CTLA-4 antibody and compound 1 combination group and the anti-CTLA-4 antibody alone or compound 1 alone group was statistically significant (p<0.05), and therefore the combination of anti-CTLA-4 antibody and compound 1 has an excellent effect on tumor growth inhibition (see FIG. 15). There was no statistically significant difference in body weight change between the drug-treated groups and the control group that received only the vehicle during the entire study period (see FIG. 16).

[0128] Example 4. Antitumor effect in a mouse model of breast cancer Example 4.1 Preparation of breast cancer mouse model and test drugs Sixty female BALB / C mice, aged 6–8 weeks, were purchased from Vital River Laboratories Research Model and Services Company. 5 The 4T1 cell line (mouse breast cancer cells) was subcutaneously injected into the right flank of each mouse to implant the cancer cell line. The day of implantation of the 4T1 cell line was designated as day 0, and the tumor volume was measured on day 6. The average tumor volume on day 6 was 52 mm 3 It was found that mice were selected to maintain a constant mean and deviation for each group by excluding individuals with tumor volumes significantly larger or smaller than the mean tumor size. A 0.9% physiological saline solution was prepared as the vehicle, and a mixed solution of anti-PD-1 antibody (BioXCell) and PBS, a mixed solution of anti-CTLA-4 antibody (BioXCell) and PBS, and a mixed solution of compound 1 and the vehicle were prepared as test drugs.

[0129] Example 4.2. Test drug administration method, measured variables and statistical analysis The mice selected in Example 4.1 were assigned to a control group, an anti-PD-1 antibody administration group, a compound 1 administration group, an anti-CTLA-4 antibody administration group, a combination of compound 1 and anti-PD-1 antibody administration group, or a combination of compound 1 and anti-CTLA-4 antibody administration group. The drugs were administered from day 6 to day 30 after the day of 4T1 cell line implantation. The doses and administration methods of the drugs administered to each group are shown in Table 6 below, and the doses were 5mL / kg in both cases when administered orally (po) and intraperitoneally (ip). [Table 6] The changes in tumor volume and body weight of the mice were measured twice a week during the test period after drug administration, and the tumor volume was measured using the same method as in Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued. Differences in tumor volumes were statistically analyzed using ANOVA and Tukey's multiple comparison test.

[0130] Example 4.3. Tumor growth inhibitory effect and remission rate The tumor volume of the anti-PD-1 antibody and compound 1 combination group was shown to be smaller than that of the anti-PD-1 antibody alone, compound 1 alone, or vehicle-treated control group (see FIG. 17(a)). Furthermore, the tumor volume of the anti-PD-1 antibody alone group measured on day 23 from the date of cell line inoculation was increased by 2.6% compared to that of the control group, and the tumor volume of the compound 1 alone group was reduced by 16% compared to that of the control group (see FIG. 17(b)). On the other hand, the tumor volume of the anti-PD-1 antibody and compound 1 combination group was found to be reduced by an astonishing 35% compared to that of the control group, and the difference in tumor volume between the control group and the anti-PD-1 antibody and compound 1 combination group was found to be less than p-value 0.001 when analyzed using two-way ANOVA and less than p-value 0.01 when analyzed using one-way ANOVA and Tukey's multiple comparison test, resulting in a statistically significant reduction in tumor volume compared to the control group (see FIG. 17). Furthermore, compared to the tumor volume of the vehicle-treated control group measured on day 23 from the date of cell line inoculation, the tumor volume of the anti-CTLA-4 antibody alone was reduced by 17%, the tumor volume of the compound 1 alone was reduced by 16%, while the tumor volume of the anti-CTLA-4 antibody and compound 1 combination group was reduced by an astonishing 46% compared to the tumor volume of the vehicle-treated control group. In terms of tumor volume measured on day 23 from the date of cell line inoculation, the tumor volume difference between the control group and the anti-CTLA-4 antibody and compound 1 combination group was found to be less than p-value 0.001 when analyzed using two-way ANOVA and one-way ANOVA and Tukey's multiple comparison test, resulting in a statistically significant reduction in tumor volume compared to the control group (see FIG. 18). There was no statistically significant difference in body weight change between the drug-treated groups and the vehicle-only control group during the entire study period (see FIG. 19).

[0131] Example 5. Antitumor effect in a mouse model of bladder cancer Example 5.1 Preparation of Bladder Cancer Mouse Model and Test Drugs Seven-week-old C3H / HeN female mice were purchased from OrientBio. 5 The MBT2 cell line (mouse bladder cancer cells) was subcutaneously injected into the right flank of each mouse to implant the cancer cell line. The day of MBT2 cell line implantation was designated as day 0, and the tumor volume was measured on day 6. The average tumor volume on day 6 was 50 mm 3 It was found that mice were selected to maintain a constant mean and deviation for each group by excluding individuals with tumor volumes significantly larger or smaller than the mean tumor size. A 0.9% physiological saline solution was prepared as the vehicle, and a mixed solution of anti-PD-1 antibody (BioXCell) and PBS and a mixed solution of Compound 1 and the vehicle were prepared as test drugs.

[0132] Example 5.2. Test drug administration method, measured variables and statistical analysis The mice selected in Example 5.1 were assigned to a control group, an anti-PD-1 antibody-administered group, a compound 1-administered group, or a combination of compound 1 and anti-PD-1 antibody-administered group, respectively. The drugs were administered from day 6 to day 28 after the day of MBT2 cell line implantation. The doses and administration methods of the drugs administered to each group are shown in Table 7 below, and the doses were 10 mL / kg in both cases when administered orally (po) and intraperitoneally (ip). [Table 7] The tumor volume and body weight of the mice were measured three times a week during the study period after drug administration. The tumor volume was measured in the same manner as in Example 1.1.2. On the other hand, when the tumor volume of each mouse was 1,500 mm 3 Mice were sacrificed, the study terminated, and classified as responders to the drug when tumor volume was less than one-quarter of the tumor volume at the sacrifice criteria. Survival rates were determined by the date of death of individual mice or when tumor volume for individual mice reached 1,500 mm 3 Survival rates were calculated using the date of sacrifice over 10 days, and significant differences in survival rates were analyzed using Kaplan-Meier survival curve (survival curve) analysis (Prism 5.0).

[0133] Example 5.3. Survival rate and body weight change The results of determining the proportion of drug responders based on tumor volume measured 30 days after the date of cell line inoculation are shown in Table 8 below. [Table 8] The group treated with the combination of anti-PD-1 antibody and Compound 1 showed a higher responder rate than the group treated with anti-PD-1 antibody alone, the group treated with Compound 1 alone, or the vehicle control group (Table 8, Figure 20), and the survival rate was significantly increased compared to the vehicle control group in terms of Kaplan-Meier survival rate (log-rank test, p-value = 0.0042) (see Figure 20). There was no statistically significant difference in body weight change between the drug-treated groups and the vehicle-only control group during the entire study period (see FIG. 21).

[0134] Example 6. Antitumor effect in animal models of cervical cancer A 0.9% physiological saline solution was prepared as a vehicle, and a mixed solution of mouse anti-CTLA-4 antibody and PBS, a mixed solution of mouse anti-PD-1 antibody and PBS, a mixed solution of vehicle and lenvatinib, and a mixed solution of compound 1 and vehicle were prepared. Purchase 6-8 week old female C57BL / 6 mice and incubate at 3 × 10 6 The U14 cell line was subcutaneously injected into the flank of each mouse to implant the cancer cell line. The day of implantation of the U14 cell line was designated as day 0, and the tumor size was measured on day 7. The mice were then divided into groups according to the deviation of the average tumor volume on day 7 from 50 mm 3 The mice were classified so as not to cause any difference in the number of mice. The classified mice were assigned to the groups listed in Table 9 below, and were administered with a vehicle or a drug. Drug administration was started on the 7th day from the day of cell line inoculation, and the test was terminated on the 27th day from the day of starting drug administration. The mice were administered orally (po) and intraperitoneally (ip), and the administration volume was 5 mL / kg in both cases. [Table 9] The tumor volume and body weight of the mice were measured twice a week during the test period after drug administration. In this case, the tumor volume of the mice was calculated by the same method as that of Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued.

[0135] Example 7. Antitumor effect in an animal model of head and neck squamous cell carcinoma A 0.9% physiological saline solution was prepared as a vehicle, and a mixed solution of mouse anti-CTLA-4 antibody and PBS, a mixed solution of mouse anti-PD-1 antibody and PBS, a mixed solution of vehicle and lenvatinib, and a mixed solution of compound 1 and vehicle were prepared. Purchase 6-8 week old female C57BL / 6 mice and incubate at 3 × 10 6 The MOC1 cell line was subcutaneously injected into the flank of each mouse to implant the cancer cell line. The day of implantation of the MOC1 cell line was designated as day 0, and the tumor size was measured on day 7. The mice were then divided into groups according to the deviation of the average tumor volume on day 7 from that of each group to 50 mm3 The mice were classified so as not to cause any difference in the number of mice. The classified mice were assigned to the groups listed in Table 10 below, and were administered with a vehicle or a drug. Drug administration was started on the 7th day from the day of cell line inoculation, and the test was terminated on the 27th day from the day of starting drug administration. The mice were administered orally (po) and intraperitoneally (ip), and the administration volume was 5 mL / kg in both cases. [Table 10] The tumor volume and body weight of the mice were measured twice a week during the test period after drug administration. In this case, the tumor volume of the mice was calculated by the same method as that of Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued.

[0136] Example 8. Antitumor effect in an animal model of liver cancer A 0.9% physiological saline solution was prepared as a vehicle, and a mixed solution of mouse anti-CTLA-4 antibody and PBS, a mixed solution of mouse anti-PD-1 antibody and PBS, a mixed solution of vehicle and lenvatinib, and a mixed solution of compound 1 and vehicle were prepared. Purchase 6-8 week old female BALB / 6 mice and incubate at 1 × 10 6 H22 cell line (mouse liver cancer cell line) was subcutaneously injected into the flank of each mouse to implant the cancer cell line. The day of implantation of H22 cell line was designated as day 0, and tumor size was measured on day 7. Mice were divided into groups with a deviation of 50 mm for the average tumor volume on day 7. 3 The mice were classified so as not to cause any difference in the dose. The classified mice were assigned to the groups listed in Table 11 below, and were administered with a vehicle or a drug. Drug administration was started on the 7th day from the day of cell line inoculation, and the test was terminated on the 27th day from the day of initiation of drug administration. The administration was orally (po) and intraperitoneally (ip), and the administration volume was 5 mL / kg in both cases. [Table 11] The tumor volume and body weight of the mice were measured twice a week during the test period after drug administration. In this case, the tumor volume of the mice was calculated by the same method as that of Example 1.1.2. On the other hand, when the tumor volume of each mouse was 3,000 mm 3 When the seroconversion factor was exceeded, the corresponding mice were sacrificed and the study was discontinued.

Claims

**Claim 1** A pharmaceutical composition for the prevention or treatment of tumors, comprising a small molecule kinase inhibitor that blocks the signal transduction pathway of transforming growth factor-β (TGF-β), wherein the small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β is administered in combination with at least one of an immune checkpoint inhibitor and a receptor tyrosine kinase inhibitor. **Claim 2** The small molecule kinase inhibitor that blocks the signal transduction pathway of TGF-β is: 1) 1-[6-(6-methyl-pyridin-2-yl)-5-quinoxalin-6-yl-2,3-dihydro-imidazo[1,2-a]imidazol-1-yl]-ethanone; 2) 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoxaline; 3) 6-[2-(6-methyl-pyridin-2-yl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrimidin-3-yl]-quinoxaline; 4) 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoline; 5) 6-[2-(6-methyl-pyridin-2-yl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrimidin-3-yl]-quinoline; 6) 2-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-thieno[3,2-c]pyridine; 7) 6-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-benzothiazole; 8) 5-benzo[b]thiophen-5-yl-6-(6-methyl-pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 9) 6-(6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridine; 10) 5-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-benzoxazole; 11) 4-[2-(6-methyl-pyridin-2-yl)-6,7-dihydro-5H-imidazo[1,2-a]imidazol-3-yl]-quinoline; 12) 5 - Benzo[1,3]dioxol - 5 - yl - 6 - (6 - methyl - pyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazole; 13) 5 - (2,3 - Dihydro - benzo[1,4]dioxin - 6 - yl) - 6 - (6 - methyl - pyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazole; 14) 7 - [2 - (6 - methyl - pyridin - 2 - yl) - 6,7 - dihydro - 5H - imidazo[1,2 - a]imidazol - 3 - yl] - 2 - pyrazol - 1 - yl - quinoxaline; 15) Dimethyl - (2 - {7 - [2 - (6 - methyl - pyridin - 2 - yl) - 6,7 - dihydro - 5H - imidazo[1,2 - a]imidazol - 3 - yl] - quinoxalin - 2 - yloxy} - ethyl) - amine; 16) 2 - Methoxy - 7 - [2 - (6 - methyl - pyridin - 2 - yl) - 6,7 - dihydro - 5H - imidazo[1,2 - a]imidazol - 3 - yl] - quinoxaline; 17) 5 - (3,5 - Dimethoxyphenyl) - 6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazole; 18) N,N - Dimethyl - 4 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)aniline; 19) 4 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)benzonitrile; 20) 2 - Methyl - 6 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)quinoline; 21) 4 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)aniline; 22) N - (4 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)phenyl)acetamide; 23) N - (4 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)phenyl)methanesulfonamide; 24) tert - Butyl(4 - (6 - (6 - methylpyridin - 2 - yl) - 2,3 - dihydro - 1H - imidazo[1,2 - a]imidazol - 5 - yl)phenyl)carbamate; 25) 5-(4-(4-Methylpiperazin-1-yl)phenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 26) 4-(4-(6-(6-Methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-5-yl)phenyl)morpholine; 27) 6-(6-Methylpyridin-2-yl)-5-(m-tolyl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 28) 5-(4-Methoxyphenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 29) 6-(6-Methylpyridin-2-yl)-5-(4-(trifluoromethyl)phenyl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 30) 6-(6-Methylpyridin-2-yl)-5-(4-(methylthio)phenyl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 31) 5-(3-Fluoro-4-methoxyphenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 32) 5-(4-Fluorophenyl)-6-(6-methylpyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazole; 33) 1-Acetyl-6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carboxylic acid ethyl ester; 34) 6-(6-Methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carboxylic acid ethyl ester; 35) [6-(6-Methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazol-2-yl]-methanol; 36) 1-Acetyl-6-(6-methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carbonitrile; 37) 6-(6-Methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carbonitrile; 38) 6-(6-Methyl-pyridin-2-yl)-5-thieno[3,2-c]pyridin-2-yl-2,3-dihydro-1H-imidazo[1,2-a]imidazole-2-carboxamide; 39) (6-(6-Methylpyridin-2-yl)-5-(thieno[3,2-c]pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-2-yl)methanamine; and 40) N-((6-(6-Methylpyridin-2-yl)-5-(thieno[3,2-c]pyridin-2-yl)-2,3-dihydro-1H-imidazo[1,2-a]imidazol-2-yl)methyl)acetamide The pharmaceutical composition according to claim 1, which is a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1, wherein a small molecule kinase inhibitor that blocks the TGF-β signaling pathway is administered in combination with an immune checkpoint regulator.

4. The pharmaceutical composition according to claim 1 or 3, wherein the immune checkpoint regulator is at least one selected from the group consisting of a programmed death-ligand 1 (PD-L1) inhibitor, a programmed cell death protein 1 (PD-1) inhibitor, and a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor.

5. The immune checkpoint regulator is 1) at least one selected from the group consisting of an aptamer, a peptide, an antibody, and an antigen-binding fragment of an antibody that specifically binds to the PD-L1 protein; 2) at least one selected from the group consisting of an aptamer, a peptide, an antibody, and an antigen-binding fragment of an antibody that specifically binds to the PD-1 protein; and 3) at least one selected from the group consisting of an aptamer, a peptide, an antibody, and an antigen-binding fragment of an antibody that specifically binds to the CTLA-4 protein The pharmaceutical composition according to claim 1 or 3, which is at least one selected from the group consisting of.

6. The pharmaceutical composition according to claim 1 or 3, wherein the immune checkpoint regulator is an anti-PD-L1 antibody or an antigen-binding fragment thereof; an anti-PD-1 antibody or an antigen-binding fragment thereof; or an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

7. The pharmaceutical composition according to claim 1, wherein a small molecule kinase inhibitor that blocks the TGF-β signaling pathway is administered in combination with a receptor tyrosine kinase inhibitor.

8. The pharmaceutical composition according to claim 1 or 7, wherein the receptor tyrosine kinase inhibitor is an inhibitor of at least one tyrosine kinase selected from VEGFR, FGFR, PDGFRα, KIT, RET, and c-Met.

9. The pharmaceutical composition according to claim 1 or 7, wherein the receptor tyrosine kinase inhibitor is at least one selected from a compound that inhibits the function of at least one tyrosine kinase selected from VEGFR, FGFR, PDGFRα, KIT, RET, and c-Met, or an aptamer, peptide, antibody, and antigen-binding fragment of an antibody that specifically selects for a tyrosine kinase protein.

10. The pharmaceutical composition according to claim 7, wherein a small molecule kinase inhibitor that blocks the TGF-β signaling pathway is further administered in combination with an immune checkpoint regulator.

11. The pharmaceutical composition according to claim 1, wherein the tumor is selected from the group consisting of melanoma, sarcoma, brain tumor, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancer, glioblastoma, eye cancer, vaginal cancer, vulvar cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, basal cell carcinoma, salivary gland cancer, pharyngeal cancer, skin cancer, kidney cancer, Wilms tumor, bladder cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, appendiceal cancer, bronchial cancer, choriocarcinoma, chordoma, epithelioma, gastrointestinal stromal tumor (GIST), neuroendocrine cancer, and urethral cancer.

12. The pharmaceutical composition according to claim 1, wherein the tumor is a solid tumor.

13. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is for oral administration.

14. The pharmaceutical composition according to claim 1, wherein a small molecule kinase inhibitor that blocks the TGF-β signaling pathway is administered separately or simultaneously with at least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor.

15. A small molecule kinase inhibitor that blocks the TGF-β signaling pathway; and At least one of an immune checkpoint regulator and a receptor tyrosine kinase inhibitor A combination for the prevention or treatment of tumors.

16. A small molecule kinase inhibitor that blocks the TGF-β signaling pathway; and At least one of an immune checkpoint control agent and a receptor tyrosine kinase inhibitor A pharmaceutical composition for preventing or treating tumors, comprising the same.

17. Use of a small molecule kinase inhibitor that blocks the TGF-β signaling pathway, which is administered in combination with at least one of an immune checkpoint control agent and a receptor tyrosine kinase inhibitor, in the manufacture of a medicament for preventing or treating tumors.

18. A small molecule kinase inhibitor that blocks the TGF-β signaling pathway; and An instruction manual for instructing administration in combination of a small molecule kinase inhibitor that blocks the TGF-β signaling pathway with at least one of an immune checkpoint control agent and a receptor tyrosine kinase inhibitor A kit for preventing or treating tumors, comprising the same.

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