CSF1R kinase inhibitors and uses thereof

Abstract

The present invention provides CSF1R kinase inhibitor compounds or pharmaceutically acceptable salts thereof, and their use in medicine for treating CSF1R kinase signaling pathway-related diseases or in immunomodulation.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese Patent Application No. 202011007689.2, filed on September 23, 2020, the entire contents of which are incorporated herein by reference in their entirety and for all purposes.

[0002] The present invention relates generally to the pharmaceutical field, and specifically to CSF1R kinase inhibitors and their use (as CSF1R kinase inhibitor pharmaceuticals) in treating CSF1R kinase signaling pathway-associated diseases or improving tumor immunosuppressive conditions. [Background technology]

[0003] The tumor microenvironment, which cannot be separated as a functional whole, plays an important role in tumor progression. Many stromal cells in the microenvironment, such as tumor-associated macrophages (TAMs), dendritic cells (DCs), regulatory T cells (Tregs), fibroblasts, and killer T cells, promote tumor progression through interactions with tumor cells.

[0004] Among them, tumor-associated macrophages (TAMs) are important microenvironmental stromal cells. The proportion of macrophages in some tumor tissues can reach 50%, and macrophages in gliomas account for more than 30% of the tumor weight. Macrophages are the most important antigen-presenting cells in the body and exist at every stage of tumor development. They are called tumor-associated macrophages (TAMs). Classically activated macrophages (M1 type) are widely involved in the innate and adaptive immunity of the body, exert antigen-presenting effects, and have obvious killing and inhibiting effects on tumors, while alternatively activated macrophages (M2 type) exert immunosuppressive effects and play an important role in promoting tumor growth, promoting angiogenesis, and invading and metastasis. Macrophage colony-stimulating factor (CSF1) is a classical tumor-promoting factor that can recruit macrophages to tumor regions, promote the interaction between tumor cells and macrophages, and release various tumor-promoting growth factors (e.g., VEGF, matrix metalloproteinases, etc.) into the microenvironment, thereby promoting tumor growth and metastasis. CSF1 exerts its biological effects by binding to its sole cell surface receptor CSF1R. After binding to its receptor CSF1R, CSF1 exerts its function by inducing a signal cascade reaction and activating multiple downstream signal pathways. Research has revealed that CSF1 and CSF1R are abnormally elevated in many types of malignant tumors and are closely related to tumor development, progression and poor prognosis. Therefore, CSF1R has become an important target for controlling tumor-associated macrophages in the tumor microenvironment, and the research and development of CSF1R small molecule inhibitors has attracted increasing attention.

[0005] Currently, many CSF1R small molecule inhibitors are in the stage of clinical research and development, and the fast-developing selective CSF1R inhibitors include pexidartinib (also known as PLX3397) and BLZ945. PLX3397 was approved in August 2019 for the treatment of tendon sheath giant cell tumor (TGCT). TGCT is a benign soft tissue tumor caused by overexpression of CSF1. As a CSF1R inhibitor, PLX3397 reduces the number of TAMs in tumors by blocking the CSF1 / CSF1R signal pathway, and induces repolarization of TAMs and reduces the number of M2-type TAMs, demonstrating that CSF1R is a very promising tumor treatment target. However, the toxicity and side effects of this drug are relatively large, and a black box warning is attached to the drug label, suggesting that this drug has a risk of severe and potentially fatal liver damage. In addition, commercially available drugs such as Pazopanib, Imatinib, and Sunitinib have also been reported to have CSF1R inhibitory activity, but they are all broad-spectrum inhibitors and have no confirmed clinical indications by targeting CSF1R. In addition, no highly selective CSF1R inhibitors have been approved for the treatment of tumors. It can be seen that the research and development of safe and effective drugs targeting CSF1R for use in high-risk solid tumors has not yet been successful. Therefore, further research is needed to meet clinical needs.

[0006] Immune checkpoint drugs, including CTLA-4 antibodies and anti-PD-1 / anti-PD-L1 antibodies, are the most important developments in cancer treatment in the past decade. These immunotherapies restore antitumor immunity, reverse immune escape, and promote tumor cell death. Their applications have expanded, and they have reshaped many of the standard treatments used up to now. However, it is important to note that the immune system may be overactivated, and immune-related adverse events caused by this activation are on the rise. It has been reported that up to 60% of patients treated with the anti-CTLA-4 antibody ipilimumab (Yervoy) develop immune-related adverse events, of which 10-30% are severe (level 3-4) immune-related adverse events, which are also dose-dependent. Approximately 10% of patients treated with anti-PD-1 antibodies develop immune-related adverse events of level 3 or higher, including fatigue, headache, arthralgia, skin rash, pruritus, pneumonia, diarrhea and / or colitis, hepatitis, and endocrine disorders. The combined administration of anti-CTLA-4 and anti-PD-1 antibodies increases the incidence and severity of immune-related adverse events. Some patients treated with the anti-PD-L1 antibody avelumab (Bavencio) have experienced infusion-related reactions, the severity of which is mainly level 1-3. These adverse reactions are usually dose-related, and lowering the dose can reduce or alleviate the adverse reactions, but often leads to a decrease in the antitumor efficacy. How to enhance the antitumor efficacy of immune checkpoint drugs or exert antitumor effects under low-dose conditions is a technical issue that needs to be resolved as soon as possible. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Chinese Patent Application No. 202011007689.2 Summary of the Invention [Means for solving the problem]

[0008] Chinese Patent No. 201410062209.0 (CN104860885B, the entire contents of which are incorporated herein by reference in their entirety and for all purposes) discloses compounds of general formula (A), in particular compounds having a structure as shown in formula (I): [ka]

[0009] In CN104860885B, these compounds have been reported as VEGFR inhibitors with excellent activity that can inhibit angiogenesis of tumor cells. The present inventors have been conducting research and development, focusing on the compound represented by formula (I), and have found that such compounds are also potent inhibitors of CSF1R kinase, inhibit tumor-associated macrophages, and upregulate CD8 + We found that it activates T cells, antagonizes the tumor immunosuppressive microenvironment, enhances the antitumor efficacy of immune checkpoint drugs, and shows significant efficacy in multiple human- or mouse-derived subcutaneous tumor models and brain orthotopic tumor models.

[0010] Current clinical studies have shown that CSF1R selective inhibitors can act on TAMs to exert antitumor effects, but it is difficult to inhibit tumor growth with strong effects when used alone, and combined administration is an effective strategy to enhance the antitumor efficacy of such inhibitors. In addition, VEGF / VEGFR targeted drugs have a defect that the efficacy is difficult to sustain, and one of the main reasons for this is that TAMs in the tumor microenvironment produce angiogenesis-promoting factors and enzymes such as VEGF, which promote the production of neovascularization in tumors, thereby significantly reducing the therapeutic effect of VEGF / VEGFR targeted drugs. Based on this, many studies have found that the combination of VEGF / VEGFR targeted drugs and CSF1R targeted drugs can effectively and synergistically exert antitumor effects. Therefore, the development of selective inhibitors that simultaneously target VEGFR and CSF1R is undoubtedly of great value, which not only has the dual effect of inhibiting tumor angiogenesis and inhibiting the function of TAMs in the tumor microenvironment, but also effectively avoids the attenuation of the therapeutic effect of VEGFR targeted drugs caused by TAMs in the microenvironment. Therefore, developing a VEGFR / CSF1R dual-targeting inhibitor is expected to provide a new therapeutic strategy for tumor treatment.

[0011] In general, the present invention provides various uses of compounds within the scope of general formula (A) or pharma- ceutically acceptable salts thereof as CSF1R kinase inhibitors, including pharmaceutical uses, disease treatment methods, pharmaceutical compositions and uses thereof, etc. The specific contents are described in the following aspects.

[0012] The compounds of the present invention represented by general formula (A) are as follows: [ka] [In the formula, R 1 is located at any one of the 5-8 positions on the naphthalene ring, and has the following structure: [ka] Here, R 4is selected from the group consisting of hydrogen, halogen, C1-C3 alkyl, and C1-C3 alkoxy; [ka] is located at any of the 1-4 positions on the naphthalene ring, R 3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted 5-10 membered heteroaryl containing 1-5 heteroatoms selected from N, O and S, in which case the substituents are 1-3 substituents, and the substituents are each independently selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halo C1-C3 alkyl, halo C1-C3 alkoxy, hydroxy, amino, nitro, and halogen; R 2 R at positions 1 to 8 on the naphthalene ring 1 and [ka] and is hydrogen or a halogen.

[0013] In some embodiments of general formula (A), R 3 is selected from the group consisting of hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted 5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O and S, in which case the substituents are 1-3 substituents, each of which is independently selected from the group consisting of C1-C3 alkyl, methoxy, trifluoromethyl, trifluoromethoxy, hydroxy, amino, nitro, F, Cl and Br; R 2 is hydrogen, F, Cl or Br. In some embodiments of general formula (A), R 4 is selected from the group consisting of hydrogen, F, Cl, Br, methyl, and methoxy.

[0014] In some embodiments, the compound of general formula (A) is specifically a compound represented by the following general formula (B): [ka] [In the formula, [ka] is located at any of the 1-4 positions on the naphthalene ring, R 3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted 5-10 membered heteroaryl containing 1-5 heteroatoms selected from N, O and S, in which case the substituents are 1-3 substituents, and the substituents are each independently selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halo C1-C3 alkyl, halo C1-C3 alkoxy, hydroxy, amino, nitro, and halogen; R 2 R at positions 1 to 8 on the naphthalene ring 1 and [ka] is hydrogen or halogen; Z is C(R 5 )=CH, S or O; Y is NH, NMe, O, CH=C(R 6 ) or CH=N, R 5 is selected from the group consisting of hydrogen, halogen, C1-C3 alkyl, and C1-C3 alkoxy; R 6 is selected from the group consisting of hydrogen, pyrazolyl, pyrazolyl substituted with C1-C3 alkyl, and pyrazolyl substituted with hydroxy C1-C3 alkyl.

[0015] In some embodiments of general formula (B), R 5 is selected from the group consisting of hydrogen, F, Cl, Br, methyl, and methoxy; R6 is selected from the group consisting of hydrogen, pyrazolyl, methyl-substituted pyrazolyl, and hydroxyethyl-substituted pyrazolyl.

[0016] In some embodiments, the compound of general formula (A) is specifically a compound represented by the following general formula (C), (D), (E) or (F). [ka] [In the formula, [ka] is located at the 1st or 2nd position on the naphthalene ring, R 3 is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted 5-10 membered heteroaryl containing 1-5 heteroatoms selected from N, O and S, in which case the substituents are 1-3 substituents, and the substituents are each independently selected from C1-C3 alkyl, C1-C3 alkoxy, halo C1-C3 alkyl, halo C1-C3 alkoxy, hydroxy, amino, nitro and halogen; R 2 R at positions 1 to 8 on the naphthalene ring 1 and [ka] is hydrogen or halogen; R 4 is selected from the group consisting of hydrogen, halogen, C1-C3 alkyl, and C1-C3 alkoxy; V is S or O; W is N or C(R 7 ) and R 7 is selected from the group consisting of hydrogen, pyrazolyl, pyrazolyl substituted with C1-C3 alkyl, and pyrazolyl substituted with hydroxy C1-C3 alkyl.

[0017] In some embodiments of general formula (C), (D), (E) or (F), R 4 is selected from hydrogen, F, Cl, Br, methyl, and methoxy; R 7 is selected from the group consisting of hydrogen, pyrazolyl, methyl-substituted pyrazolyl, and hydroxyethyl-substituted pyrazolyl.

[0018] In one specific embodiment, the compound of general formula (A) is a compound represented by the following formula (I) (herein also referred to as "Compound I", which is a leading compound of the present invention): [ka]

[0019] According to one aspect, the present invention provides the use of a compound of general formula (A) to (F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating a disease associated with the CSF1R kinase signaling pathway (as a CSF1R kinase inhibitor medicament).

[0020] The CSF1R kinase signaling pathway-related diseases according to the present invention include cancer or tumor, hyperplasia, immune disorder, inflammation, and the like. In some embodiments, the disease is cancer or tumor. In some specific embodiments, the cancer or tumor is a CSF1 / CSF1R-dependent cancer or tumor or a tumor rich in TAMs. In some more specific embodiments, the CSF1 / CSF1R-dependent cancer or tumor includes CSF1 / CSF1R-dependent leukemia, giant cell tumor of tendon sheath, and the like, and the tumor rich in TAMs includes, but is not limited to, glioma, metastatic brain tumor, or colorectal cancer.

[0021] According to another aspect, the present invention provides the use of a compound of the general formulae (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, in the manufacture of an immunomodulatory drug. In some embodiments, the immunomodulation is immunoenhancement. In some specific embodiments, the immunoenhancement is amelioration of tumor immunosuppressive state. In some more specific embodiments, the amelioration of tumor immunosuppressive state is characterized by inhibition of survival of M2-biased macrophages, reversal of the macrophage M2-biased polarized phenotype, and CD8 upregulation due to the macrophage M2-biased polarized phenotype. + the inhibitory effect of T cells and / or reshaping the tumor immune microenvironment.

[0022] According to another aspect, the present invention also provides use of a compound of any of the general formulae (A) to (F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting M2-biased macrophage proliferation.

[0023] According to another aspect, the present invention also provides the use of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating or inhibiting a tumor insensitive to an immune checkpoint drug. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor includes, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0024] According to another aspect, the present invention also provides a use of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for enhancing the antitumor efficacy of an immune checkpoint drug. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor includes, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0025] According to another aspect, the present invention also provides the use of the compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, in the manufacture of an antitumor medicament for combination with an immune checkpoint drug. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor includes, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0026] Furthermore, in the above-mentioned use, the medicament contains a therapeutically effective amount of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, and, if necessary, a pharma- ceutically acceptable excipient or carrier.

[0027] The administration method of the medicament of the present invention is not particularly limited, and representative administration methods include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and local administration.In such cases, the medicament of the present invention can be prepared into various clinically acceptable dosage forms, such as oral dosage forms, injection dosage forms, local administration dosage forms or external dosage forms.

[0028] The medicament of the present invention may be used alone or in combination with other therapeutic ingredients in clinical practice. In some embodiments, the other therapeutic ingredients are selected from an antitumor drug or an immunomodulatory agent. In some embodiments, the medicament of the present invention may be used in combination with an immune checkpoint drug. In some specific embodiments, the immune checkpoint drug includes an anti-PD-1 antibody or an anti-PD-L1 antibody, etc. For ease of clinical use, the compound of general formula (A)-(F) or a pharmaceutical acceptable salt thereof according to the present invention, particularly compound I or a pharmaceutical acceptable salt thereof, may be combined with other therapeutic ingredients to prepare a compounded drug or a pharmaceutical combination product. In some embodiments, the other therapeutic ingredients are an antitumor drug or an immunomodulatory agent. In some specific embodiments, the other therapeutic ingredients are immune checkpoint drugs. In some more specific embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody, etc.

[0029] According to another aspect, the present invention also provides a method of using said medicament, which comprises administering a therapeutically effective amount of a compound of the present invention of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, to an individual in need of treatment. The individual may be a mammal, for example a human.

[0030] According to another aspect, the present invention provides a method for treating a disease associated with the CSF1R kinase signaling pathway, comprising administering a therapeutically effective amount of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, to an individual in need of such treatment. The individual may be a mammal, such as a human. The CSF1R kinase signaling pathway-associated disease according to the present invention includes cancer or tumor, hyperplasia, immune disorder, inflammation, and the like. In some embodiments, the disease is a cancer or tumor. In some specific embodiments, the cancer or tumor is a CSF1 / CSF1R-dependent cancer or tumor, or a tumor rich in TAMs. In some more specific embodiments, the CSF1 / CSF1R-dependent cancer or tumor includes CSF1 / CSF1R-dependent leukemia, giant cell tumor of tendon sheath, and the like, and the tumor rich in TAMs includes, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0031] According to another aspect, the present invention also provides a method for immunomodulation, comprising administering a therapeutically effective amount of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, to an individual in need thereof. The individual may be a mammal, e.g., a human. In some embodiments, the immunomodulation is immunoenhancement. In some specific embodiments, the immunoenhancement is amelioration of tumor immunosuppressive state. In some more specific embodiments, the amelioration of tumor immunosuppressive state is characterized by inhibition of survival of M2-biased macrophages, reversal of the macrophage M2-biased polarized phenotype, and suppression of CD8 expression by the macrophage M2-biased polarized phenotype. + the inhibitory effect of T cells and / or reshaping the tumor immune microenvironment.

[0032] According to another aspect, the present invention also provides a method for treating or inhibiting a tumor insensitive to an immune checkpoint drug, comprising administering a therapeutically effective amount of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, to an individual in need of such treatment or inhibition. The individual may be a mammal, such as a human. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor may be, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0033] According to another aspect, the present invention also provides a method for enhancing the antitumor efficacy of an immune checkpoint drug, comprising administering a therapeutically effective amount of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, to an individual undergoing or about to undergo tumor treatment with an immune checkpoint drug. The individual may be a mammal, for example a human. In some embodiments, the immune checkpoint drug is a PD-1 antibody or an anti-PD-L1 antibody, etc.

[0034] According to another aspect, the present invention also provides a method for treating or inhibiting a tumor, comprising administering to an individual in need thereof a therapeutically effective amount of a compound of general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, in combination with an immune checkpoint drug. The individual may be a mammal, e.g., a human. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody, etc. In some embodiments, the tumor includes, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0035] It should be noted that the combined administration according to the present invention includes any suitable mode of pharmaceutical combination, including preparing two or more pharmacologic active ingredients in a single pharmaceutical composition and administering the same, or preparing two or more pharmacologic active ingredients in separate pharmaceutical compositions and administering them simultaneously or sequentially.

[0036] According to another aspect, the present invention also provides a compound of the above general formulas (A) to (F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, as a CSF1R kinase inhibitor.

[0037] According to another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, for use in treating a CSF1R kinase signaling pathway-associated disease in a subject. The CSF1R kinase signaling pathway-associated disease according to the present invention includes cancer or tumor, hyperplasia, immune disorder, inflammation, and the like. In some embodiments, the disease is cancer or tumor. In some specific embodiments, the cancer or tumor is a CSF1 / CSF1R-dependent cancer or tumor or a tumor rich in TAMs. In some more specific embodiments, the CSF1 / CSF1R-dependent cancer or tumor includes CSF1 / CSF1R-dependent leukemia, tendon sheath giant cell tumor, and the like, and the tumor rich in TAMs includes, but is not limited to, glioma, metastatic brain tumor, or colorectal cancer.

[0038] According to another aspect, the present invention provides a pharmaceutical composition for immunomodulation comprising a therapeutically effective amount of a compound of the general formulae (A)-(F) or a pharma- ceutical acceptable salt thereof, in particular compound I or a pharma- ceutical acceptable salt thereof. In some embodiments, the immunomodulation is immunoenhancement. In some specific embodiments, the immunoenhancement is amelioration of tumor immunosuppressive state. In some more specific embodiments, the amelioration of tumor immunosuppressive state is characterized by inhibition of survival of M2-biased macrophages, reversal of the macrophage M2-biased polarized phenotype, and CD8 upregulation due to the macrophage M2-biased polarized phenotype. + the inhibitory effect of T cells and / or reshaping the tumor immune microenvironment.

[0039] According to another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by any one of the general formulas (A) to (F) or a pharma- ceutical acceptable salt thereof, in particular compound I or a pharma- ceutical acceptable salt thereof, for use in inhibiting the proliferation of macrophages of an M2-polarized phenotype.

[0040] According to another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, for treating or inhibiting tumors insensitive to immune checkpoint drugs. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the tumor includes, but is not limited to, glioma, metastatic brain tumor, or colorectal cancer.

[0041] According to another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, for enhancing the antitumor efficacy of an immune checkpoint drug. In some embodiments, the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody, etc. In some embodiments, the tumor includes, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0042] According to another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the general formula (A)-(F) or a pharma- ceutically acceptable salt thereof, particularly compound I or a pharma- ceutically acceptable salt thereof, in combination with an immune checkpoint drug for treating or inhibiting a tumor in an individual. The individual may be a mammal, for example a human. In some embodiments, the immune checkpoint drug is preferably an anti-PD-1 antibody, an anti-PD-L1 antibody. In some embodiments, the tumor may be, but is not limited to, a glioma, a metastatic brain tumor, or a colorectal cancer.

[0043] According to another aspect, the present invention also provides a combination pharmaceutical or pharmaceutical combination product comprising a therapeutically effective amount of a compound of the above general formulas (A) to (F) or a pharma- ceutically acceptable salt thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, and another therapeutic ingredient, for treating or inhibiting a disease associated with the CSF1R kinase signaling pathway in a subject.

[0044] The CSF1R kinase signaling pathway-related diseases of the present invention include cancer or tumor, hyperplasia, immune disorder, inflammation, and the like. In some embodiments, the disease is cancer or tumor. In some specific embodiments, the cancer or tumor is a CSF1 / CSF1R-dependent cancer or tumor, or a tumor rich in TAMs. In some more specific embodiments, the CSF1 / CSF1R-dependent cancer or tumor includes CSF1 / CSF1R-dependent leukemia, giant cell tumor of tendon sheath, and the like, and the tumor rich in TAMs includes, but is not limited to, glioma, metastatic brain tumor, or colorectal cancer.

[0045] In some embodiments, the other therapeutic component is an anti-tumor agent or an immunomodulatory agent. In some embodiments, the other therapeutic component is an immune checkpoint agent. In some specific embodiments, the immune checkpoint agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0046] The pharmaceutical combination product of the present invention includes not only a product form in which two or more pharmacoactive ingredients are blended as a single pharmaceutical composition, but also a set product form in which, for example, two or more pharmacoactive ingredients are blended separately as separate pharmaceutical compositions and exist in a physically separated form in the product.

[0047] The therapeutically effective amount according to the present invention refers to a dosage considered to be pharma- ceutical effective, i.e., an amount of an active compound sufficient to significantly improve the condition without causing serious side effects. The daily dosage for a human weighing 60 kg is usually 0.01 to 2000 mg, preferably 1 to 500 mg, 5 to 500 mg, or 5 to 200 mg. Exemplary effective doses are, for example, 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, and 500 mg. Preferably, the above daily doses may be converted to the compounds represented by general formulas (A)-(F), particularly compound I. The compound may be administered in a single dose once a day, or in multiple doses a day, or at intervals. The dosage of the anti-PD-1 antibody or anti-PD-L1 antibody will vary depending on the specific type of antibody, the type and stage of cancer, etc., and a single dosage may be 0.5 mg / kg to 30 mg / kg, preferably 1 to 20 mg / kg; for example, in the case of a human weighing 60 kg, a single dosage will usually be 1 mg to 1800 mg, for example, 50 mg to 1200 mg, or 100 mg to 900 mg, 150 mg to 600 mg, or 200 mg to 500 mg. Exemplary single doses are, for example, 60 mg, 100 mg, 120 mg, 150 mg, 180 mg, 210 mg, 240 mg, 270 mg, 300 mg, 330 mg, 360 mg, 400 mg, 500 mg, 600 mg, 900 mg, 1200 mg, etc., and interval administration is, for example, once every 3 days, once every 5 days, once a week, once every 10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, etc., for example, once every 3 to 7 days, or once every 1 to 6 weeks. The specific dose and frequency of administration must take into account factors such as the route of administration and the health condition of the patient, which can be determined by a skilled physician based on ordinary skill.The administration route is not particularly limited, and representative administration routes include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous administration) and topical administration.

[0048] In the context of this specification, the CSF1 / CSF1R-dependent cancer or tumor refers to a cancer or tumor in which CSF1 / CSF1R is highly expressed or highly activated. The expression or activation level of CSF1 / CSF1R in cancer or tumor tissues and / or cells is detected by a person skilled in the art using a conventional detection method in the art (including but not limited to enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry, Western blotting, tissue chips, genetic testing, etc.). The expression or activation level is 130% or more, preferably 150% or more, preferably 175% or more, preferably 200% or more, preferably 250% or more, or preferably 300% or more of the normal level. The normal level may be the expression or activation level of CSF1 / CSF1R in the corresponding tissues and / or cells of the general population, or the expression or activation level of CSF1 / CSF1R in the cancer surrounding tissues and / or cells of the same patient.

[0049] In the context of the present specification, the tumor rich in TAMs refers to a tumor in which TAMs are abundantly infiltrated in the tumor tissue, and a person skilled in the art can detect the surface markers of TAMs or count TAMs using conventional detection methods in the technical field (including but not limited to enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry, Western blotting, tissue chips, genetic testing, etc.), and if the expression level of the surface markers of TAMs in the tumor tissue is different from the expression level of the corresponding surface markers in the pericancerous tissue, or the TAM count is 130% or more, preferably 150% or more, preferably 175% or more, preferably 200% or more, preferably 250% or more, or preferably 300% or more than that of the pericancerous tissue, it is recognized as having abundant TAMs infiltration, and the tumor is recognized as a tumor rich in TAMs. In addition to the representative markers of macrophages (e.g., F4 / 80), the surface markers of TAMs include, but are not limited to, general TAMs surface markers, tumor-induced macrophage surface markers, and tumor-inhibitory macrophage surface markers. The general TAMs surface markers include, but are not limited to, CD14, CD11c, CD68, and / or CD11b, and are preferably CD68 and / or CD11b. The tumor-induced macrophage surface markers include, but are not limited to, CD206, CSF1R, CSF1, CD115, PPARG, ARG1, CD163, CD301, Dectin-1, PDL2, Fizz1, CD204, PD-L1, Arginase-I, YM1, MGL2, Osteopontin, MMPs, or CCR2, and are preferably CD206. The surface markers of the tumor-inhibitory macrophages include, but are not limited to, IL1a, IL1b, IL6, NOS2, TLR2, TLR4, CD80, CD86, MHC-II, CD38, CD40, CD64, HLA-DR (CD74) or CD169, and preferably CD86 and / or MHC-II.The difference in the expression level of the surface marker means that, when the surface marker is a surface marker for general TAMs, the expression level of the surface marker in the tumor tissue is 130% or more, preferably 150% or more, preferably 200% or more of the expression level of the corresponding surface marker in the pericance tissue; when the surface marker is a surface marker for tumor-induced macrophages (e.g., CD206, etc.), the expression level of the surface marker in the tumor tissue is 130% or more, preferably 150% or more, preferably 200% or more of the expression level of the corresponding surface marker in the pericance tissue; and, preferably, when the surface marker further includes a surface marker for tumor-inhibitory macrophages (e.g., CD86 and / or MHC-II, etc.), the expression level of the surface marker for tumor-inhibitory macrophages in the tumor tissue is 80% or less, preferably 50% or less of the expression level of the corresponding surface marker in the pericance tissue.

[0050] In the present specification, when a tumor is described as being insensitive to an immune checkpoint drug, it means that the tumor inhibition rate is less than 50% when the tumor is treated with a normal dose of the immune checkpoint drug. When treated with an immune checkpoint drug at the lower end of the normal dose range, the tumor inhibition rate is preferably less than 30%, preferably less than 20%, and more preferably less than 10%. In one embodiment of the present invention, the tumor inhibition rate is expressed as a tumor volume inhibition rate TGI (%), calculated by TGI (%) = 100 x {1 - [(V last treatment day - V day 0) / (V last control day - V control day 0)], where V is the tumor volume, and calculated by V = 1 / 2 x a x b 2 where a and b are the length and width of the tumor, respectively.

[0051] In the context of this specification, anti-PD-1 antibodies include, but are not limited to, CD279, nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, etc. Anti-PD-L1 antibodies include, but are not limited to, CD274, durvalumab, atezolizumab, etc.

[0052] The numerical values ​​or numerical ranges according to the present invention can vary within a range acceptable to a person skilled in the art, for example, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% of the numerical value or numerical range.

[0053] A "subject," "patient," or "individual" according to the present invention includes all members of the animal kingdom, including, but not limited to, mammals (e.g., mice, rats, cats, monkeys, dogs, horses, pigs, etc.) and humans. Preferably, a subject according to the present invention is a human. Unless otherwise indicated, the terms "patient," "subject," or "individual" can be used interchangeably.

[0054] In the context of the present specification, the compounds of the general formulas (A) to (F) or pharma- ceutically acceptable salts thereof, in particular compound I or a pharma- ceutically acceptable salt thereof, are not particularly limited, and preferably include hydrochloride, sulfate, phosphate, nitrate, hydrofluoride, hydrobromide, formate, acetate, picrate, citrate, maleate, methylsulfonate, ethylsulfonate, trifluoromethylsulfonate, p-toluenesulfonate, and the like.

[0055] In the present invention, the alkyl is preferably an aliphatic alkyl, and may be a linear alkyl, a branched alkyl, a spirocycloalkyl, a bridged cycloalkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, a cycloalkynyl, an alkoxyalkyl, an alkoxyacylalkyl, or a cycloalkylalkyl, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohex ... These include xanyl, allyl, propargyl, cyclobutenyl, cyclohexenyl, and expressions such as "C1-C8" contemplate the corresponding group having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, e.g., "C1-C8 alkyl" means alkyl having 1, 2, 3, 4, 5, 5, 6, 7, or 8 carbon atoms, and "C2-C10 alkenyl" means alkenyl having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0056] In the present invention, said cycloalkyl may be a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably the cycloalkyl contains 3 to 10 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentenyl, cyclohexyl, cyclooctyl, etc. Polycyclic cycloalkyl includes spiro, fused, and bridged ring cycloalkyl.

[0057] The heteroaromatic group refers to a heteroaromatic system containing 1-4 heteroatoms and 5-14 ring atoms, the heteroatoms including oxygen, sulfur, and nitrogen. The heteroaryl is preferably 5- or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl can be fused to an aryl, heterocyclic, or cycloalkyl ring, where the ring attached to the parent structure is a heteroaryl ring.

[0058] Unless otherwise indicated, the structural formulae depicted herein are intended to include all tautomeric, optical, and stereoisomeric forms (e.g., astereomers, diastereomers, geometric, or conformational isomers), such as the R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, and (Z), (E) conformers. Thus, all single stereochemical isomers, tautomers, or mixtures of enantiomers, diastereomers, geometric isomers, conformations, or tautomers of the compounds of the present invention are within the scope of the present invention.

[0059] The term "tautomer" means that structural isomers with different energies can be interconverted across a low energy barrier. For example, proton tautomers (proton shift) include tautomers due to proton migration, such as 1H-indazole and 2H-indazole, and 1H-benzo[d]imidazole and 3H-benzo[d]imidazole, and valence tautomers include tautomers due to recombination of some of the bonded electrons.

[0060] In vivo and in vitro studies according to the present invention demonstrate (see detailed results in the Examples below): (1) Compound I according to the present invention can significantly inhibit CSF1R kinase activity in vitro. (2) Compound I according to the present invention significantly inhibited the in vitro proliferation of CSF1-stimulated mouse-derived macrophages (BMDM) and dose-dependently inhibited the CSF1-stimulated phosphorylation of CSF1R and the activation of the downstream signal molecule AKT, demonstrating that inhibition of CSF1R effectively inhibits the proliferation of primary macrophages. (3) Compound I of the present invention can significantly inhibit the expression of macrophage marker F4 / 80 and M2 macrophage marker CD206 in tumor tissues of a nude mouse transplanted tumor model of a glioma cell line U87MG and a nude mouse subcutaneously transplanted tumor model of a colorectal cancer cell line HT-29. Compound I was shown to exert antitumor activity by inhibiting the survival of CSF-1-induced macrophages as a CSF1R kinase inhibitor and reversing the polarized phenotype of macrophages with M2 bias.

[0061] (4) As a result of in vivo efficacy experiments, the test substance Compound I at 10 mg / kg was orally administered twice daily for three consecutive weeks, and significantly inhibited the growth of the subcutaneously transplanted human glioma tumor in nude mice U87MG. The T / C percentage obtained on the 21st day was 11.91%, while the positive control drug Axitinib at 40 mg / kg administered in a similar manner gave a T / C percentage of 30.27% on the 21st day. In this model, the tumor growth inhibitory effect of Compound I was significantly superior to that of Axitinib. In the test substance Compound I, 20mg / kg, 10mg / kg and 5mg / kg groups, oral administration twice daily for three consecutive weeks significantly delayed the growth of human colorectal cancer HT-29 nude mouse subcutaneously transplanted tumors, and the inhibitory effect increased with increasing dose, with the T / C percentages obtained on the 21st day being 17.38%, 31.27% and 42.70%, respectively. Among them, in the 20mg / kg group of Compound I, the growth of the tumors carried by the mice was almost completely stagnated during the three weeks of experimental treatment, which was significantly superior to the positive control drug Axitinib 40mg / kg group (the T / C percentage obtained on the 21st day was 48.88%).

[0062] (5) In a mouse astrocytoma DBT immunologically normal mouse tumor model, compound I at 5 mg / kg alone had no significant inhibitory activity against the tumor. The anti-PD-1 antibody (10 mg / kg) monotherapy group had a certain inhibitory effect on tumor growth, but the tumor continued to grow slowly. When compound I was administered twice daily at 5 mg / kg in combination with anti-PD-1 antibody, tumor growth was significantly inhibited, tumor volume hardly increased, and a significant difference was observed between the combination group and the anti-PD-1 antibody monotherapy group at the end of the experiment. The results showed that the combination with compound I could enhance the tumor inhibitory effect of anti-PD-1 antibody in the DBT tumor model. In addition, flow cytometry results showed that the combination group had a significantly higher CD8 in tumor tissue than the CD8 in tumor tissue. + The number of T cells was significantly increased compared with the blank control formulation group and the anti-PD-1 antibody monotherapy group, suggesting that Compound I could reverse the tumor-inhibitory immune microenvironment.

[0063] (6) In the U87MG brain orthotopic tumor transplant model, the survival times of mice in each experimental treatment group with Compound I were all extended to different degrees, with the median survival times of the 40 mg / kg and 20 mg / kg groups being 58.5 days and 54.0 days, respectively, both of which were significantly extended (41.0 days) compared with the 40 mg / kg group of the positive control drug Axitinib. (7) As a result of the initial therapeutic effect of clinical trials, Compound I showed a good therapeutic effect against solid tumors such as gliomas, especially high-grade gliomas (specific data are not shown in the present specification).

[0064] From the above research results, compound I or a pharma- ceutical acceptable salt thereof according to the present invention can improve tumor immunosuppressive state by reforming tumor microenvironment, exert antitumor therapeutic effects, and enhance the antitumor efficacy of immune checkpoint drugs, and is expected to have a promising clinical application. [Brief description of the drawings]

[0065] [Figure 1]1. Effects of Compound I and control compound PLX3397 on CSF1R phosphorylation and downstream signaling pathways in BMDMs. [Diagram 2] FIG. 1 shows the inhibitory effect of compound I on the number and polarity of macrophages in U87MG nude mouse transplanted tumors. A shows the quantitative statistical analysis of F4 / 80 immunohistochemistry, and B shows the quantitative statistical analysis of CD206 immunohistochemistry. [Diagram 3] The inhibitory effect of compound I on the number and polarity of macrophages in HT-29 nude mouse transplanted tumors. Figure A shows the quantitative statistical analysis of F4 / 80 immunohistochemistry, and Figure B shows the quantitative statistical analysis of CD206 immunohistochemistry. [Figure 4] The sensitivity of compound I to immune checkpoint drugs is improved. Figure A shows the growth inhibition effect of compound I combined with anti-PD-1 antibody on mouse DBT subcutaneously transplanted glioma tumors. Figure B shows the effect of compound I combined with anti-PD-1 antibody on the content of CD8+ T cells in tumor tissue. [Diagram 5] Effect of Compound I on survival time of U87MG brain orthotopic tumor-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The present invention will be further described below with reference to specific examples. Note that these examples are only used to illustrate the present invention, and are not used to limit the scope of the present invention. Experimental methods without specific conditions described in the following examples are usually performed according to general conditions or according to conditions suggested by manufacturers.

[0067] Source or production of experimental materials Compound I: self-produced by Shanghai Runshi Pharmaceutical Technology Co., Ltd. All positive control compounds, reagents and raw materials used in the experiments were either commercially purchased or self-prepared. Preparation of the test substances (Compound I and positive control compound) for in vitro experiments: After weighing, add DMSO and mix for 10 min. -2mol / L and diluted to the required concentration when used.

[0068] The test substance Compound I formulation for in vivo experiments is formulated and prepared as follows: [Table 1]

[0069] Example 1: Effect of Compound I on CSF1R kinase activity 1.ELISA method Enzyme reaction substrate Poly(Glu,Tyr) 4:1 The antibody was diluted to 20 μg / mL in potassium ion-free PBS (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.2-7.4), coated onto a microtiter plate, and reacted at 37°C for 12-16 hours. The plate was washed with T-PBS (potassium ion-free PBS containing 0.1% Tween-20) and dried for use. 2 , 0.5 mM MnCl 2 , 0.2 mM Na 3 VO 4 ATP solution (final concentration 5 μM) diluted with 1 mM DTT, test compound or solvent control, and recombinant CSF1R kinase protein were added in order per well to start the reaction. After reaction at 37°C for 1 hour and washing the plate with T-PBS, antibody PY99 dilution was added and reaction was continued for 0.5 hours on a shaker at 37°C, and the plate was washed with T-PBS. Horseradish peroxidase-labeled goat anti-mouse secondary antibody dilution was added and reaction was continued for 0.5 hours at 37°C. After washing the plate, 2 mg / mL OPD color development solution (0.03% H 2 O 2 The reaction was carried out at 25°C for 1-10 minutes in the dark, and then diluted with 0.1M citric acid-sodium citrate buffer (pH 5.4) containing 2M H 2 SO 4The reaction was stopped by adding 0.01% CI 0.01 to 0.01% and the plate was read using a tunable microplate reader at 490 nm. The inhibition rate was calculated as follows:

number

[0070] 2. Experimental Results Compound I significantly inhibited CSF1R kinase activity, an important target in tumor-associated macrophages, and IC 50 The IL-10 activity was 19.3±3.4nM, which is comparable to that of the commercially available drug pexidartinib (PLX3397), suggesting that it may be promising for reshaping the tumor microenvironment and antagonizing tumors.

[0071] [Table 2]

[0072] Example 2: Effects of Compound I on survival of primary macrophages and the intracellular CSF1R signaling pathway 1. Experimental Method 1.1 Isolation and culture of bone marrow-derived macrophages (BMDM) After euthanasia, mice (BALB / c) were immersed in 75% alcohol for 3-5 min. The tibia and femur were removed using ophthalmic forceps and ophthalmic scissors on a clean bench. The joints on both sides of the tibia and femur were cut off, and bone marrow cells were extracted using a sterile buffer. The bone marrow cells were filtered through a 70 μm nylon membrane and centrifuged to remove the supernatant. Red blood cell lysis solution was added, and after leaving it for 3-4 min, it was added to a sterile buffer to stop lysis. After centrifugation, the supernatant was discarded to obtain bone marrow cell precipitate. It was cultured in 1640 medium (with FBS and 10 ng / mL CSF1) for 5-7 days to obtain macrophages, which were identified using flow cytometry.

[0073] 1.2 Cell proliferation inhibition experiment The detection was performed using a CCK8 cell counting kit. Bone marrow-derived macrophages induced with CSF1 for 5-7 days were inoculated into a 96-well culture plate and cultured overnight, and compounds with different concentrations were added for 72 h, and a blank control group was set up. Then, 10 μL of CCK8 reagent was added to each well, and the wells were left in an incubator for 4-12 h, and the cells were read with a microplate reader, and the measurement wavelength was 450 nm. The inhibition rate of cell growth by the compounds was calculated using the following formula: Inhibition rate % = (OD value of blank control group - OD value of treatment group) / OD value of blank control group x 100% I C 50 Values ​​were calculated using the four-parameter method, and each experiment was repeated three times independently.

[0074] 1.3 Western blot experiment Bone marrow-derived macrophages induced with CSF1 for 5-7 days were centrifuged, the original culture medium was discarded, 2 mL of serum-free culture medium was added, and after 6 h of starvation, different concentrations of compound I and positive drug PLX3397 were added for 2 h, and CSF1 factors were added at a final concentration of 50 ng / mL for 15 min stimulation, the culture medium was discarded, and the cells were washed three times with pre-cooled PBS, and 1×SDS gel and loading buffer were added to dissolve the cells. The cell lysate was heated in a boiling water bath for 15 min and then stored at -20°C.

[0075] The above protein samples were subjected to SDS-PAGE electrophoresis, and then the proteins were transferred to a cellulose nitrate membrane using a semi-dry electrotransfer system. The cellulose nitrate membrane was placed in a blocking solution (5% skim milk powder diluted with TBS-T) and blocked at room temperature for 2 h. The membrane was then placed in a primary antibody and incubated overnight at 4 °C. It was washed three times with TBS-T for 15 min each. The membrane was placed in a secondary antibody dilution solution and incubated at room temperature for 1 to 2 h. The membrane was washed three times as above, and then colored with a coloring reagent and developed.

[0076] 2. Experimental Results 2.1 Growth inhibitory effect of compound I on BMDM CCK8 kit was used to detect the inhibitory activity of compound I on the in vitro proliferation of BMDM cells and IC 50 The results are shown in Table 2. Compound I significantly inhibited the proliferation of CSF1-stimulated BMDM cells, with a mean IC 50 was 0.093±0.024 μM, which was slightly weaker than the positive drug PLX3397 (0.030±0.009 μM).

[0077] [Table 3]

[0078] 2.2 Detection of the target inhibitory activity of compound I on BMDMs Western blotting experimental method was used to detect the inhibitory effect of compound I on CSF1R and signal pathway in mouse primary macrophages. The experiment was independently repeated three times, and all experimental results were quantified. According to the results shown in Figure 1 (experimental data were statistically analyzed using t-test, ** indicates P<0.01 compared with blank control group, *** indicates P<0.001 compared with blank control group), CSF1 stimulation can activate CSF1R, which is expressed as an increase in phosphorylated CSF1R (p-CSF1R) expression and activation of downstream signal molecule AKT (increased phosphorylated AKT (p-AKT) level). When compound I was administered, the phosphorylation levels of CSF1-stimulated CSF1R and AKT were dose-dependently inhibited, and the CSF1R phosphorylation inhibition rate reached 57.3% and the AKT phosphorylation inhibition rate reached 64.4% at a concentration of 100 nM. The inhibitory activity of compound I was comparable to that of the positive drug PLX3397.

[0079] Example 3: Growth inhibitory effect of Compound I on human glioma U87MG tumors transplanted into nude mice 1. Experimental animals: BALB / c nude mice, female, age: 3-4 weeks, Shanghai Institute of Materia Medica, Chinese Academy of Sciences 2. Experimental Method Human glioma U87MG cell line was injected subcutaneously into the right axilla of nude mice at 5 × 10 6 The cells were inoculated at a cell inoculation dose of 1 / mouse, transplanted to form tumors, and then passaged for one generation in nude mice.

[0080] The tumor tissue at its peak growth stage was approximately 1.5 mm 3 The tumor was cut into pieces and inoculated subcutaneously into the right axilla of nude mice under aseptic conditions. The diameter of the tumor was measured using a caliper and found to be approximately 90 mm. 3 When the mice had grown to an average volume of 1000 mg / kg, the animals were randomly divided into groups. Compound I was orally administered to the 40 mg / kg, 20 mg / kg, and 10 mg / kg groups twice daily for 21 consecutive days, and the blank control formulation group was orally administered the same amount of the blank control formulation twice daily for 21 consecutive days. The positive control drug Axitinib was orally administered to the 40 mg / kg group twice daily for 21 consecutive days. The vehicle group was given the same amount of water for injection. The diameter of the implanted tumors was measured twice a week throughout the experiment, and the mice were weighed at the same time. The formula for calculating tumor volume (TV) was TV=1 / 2×a×b. 2 where a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) was calculated from the measurement results, and the formula is RTV = V t / V 0 Here, V 0 When administered in separate cages (i.e., d 0 ) is the tumor volume measured at t is the tumor volume at each measurement. The evaluation index of antitumor activity is the relative tumor growth rate T / C (%), calculated by the formula T / C (%) = (T RTV / C RTV ) × 100%, and T RTV :Treatment group RTV, C RTV : Solvent group RTV.

[0081] 3. Experimental Results The results are shown in Table 3. In the blank control formulation group, a formulation with the exact same formulation as Compound I (except that it does not contain Compound I) was intragastrically administered twice a day for 21 consecutive days. As a result, there was no obvious effect on the growth of the human glioma U87MG nude mouse subcutaneously transplanted tumor, and the T / C percentage obtained on the 21st day was 79.86%.

[0082] The test substance Compound I was orally administered twice daily at 40 mg / kg, 20 mg / kg and 10 mg / kg for 3 weeks, and the administration significantly inhibited the growth of human glioma U87MG subcutaneously implanted tumors in nude mice, with the T / C percentages on the 21st day being 9.77%, 8.14% and 11.91%, respectively.

[0083] The positive control drug Axitinib 40mg / kg group was orally administered twice a day for 21 consecutive days, and had a significant inhibitory effect on the growth of human glioma U87MG subcutaneously transplanted tumors in nude mice, with a T / C percentage of 30.27% on day 21. In this model, Compound I at 10mg / kg had a better tumor growth inhibitory effect than Axitinib at 40mg / kg. All mice in each group were in good condition throughout the experiment.

[0084] [Table 4]

[0085] Example 4: Growth inhibitory effect of Compound I on human colorectal cancer HT-29 tumors transplanted into nude mice 1. Experimental animals: BALB / c nude mice, female, age: 6 weeks, purchased from Shanghai Lingchang Biotechnology Co., Ltd. 2. Experimental Method Human colorectal cancer HT-29 cell line was injected subcutaneously into the right axilla of nude mice at a dose of 5 × 10 6 The cells were inoculated at a cell inoculation dose of 1 / mouse, transplanted to form tumors, and then passaged for one generation in nude mice.

[0086] The tumor tissue at its peak growth stage was approximately 1.5 mm 3 The tumor was cut into pieces and inoculated subcutaneously into the right axilla of nude mice under aseptic conditions. The diameter of the tumor was measured using a caliper and found to be approximately 120 mm. 3When the mice had grown to an average volume of 1000 mg / kg, the animals were randomly divided into groups. Compound I was orally administered to the 20 mg / kg, 10 mg / kg, and 5 mg / kg groups twice daily for 21 consecutive days, and the blank control formulation group was orally administered the same amount of the blank control formulation twice daily for 21 consecutive days. The positive control drug Axitinib was orally administered to the 40 mg / kg group twice daily for 21 consecutive days. The vehicle group was given the same amount of water for injection. The diameter of the implanted tumors was measured twice a week throughout the experiment, and the mice were weighed at the same time. The formula for calculating tumor volume (TV) was TV=1 / 2×a×b. 2 where a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) was calculated from the measurement results, and the formula is RTV = V t / V 0 Here, V 0 When administered in separate cages (i.e., d 0 ) is the tumor volume measured at t is the tumor volume at each measurement. The evaluation index of antitumor activity is the relative tumor growth rate T / C (%), calculated by the formula T / C (%) = (T RTV / C RTV ) × 100%, and T RTV :Treatment group RTV, C RTV : Solvent group RTV.

[0087] 3. Experimental results The experimental results are shown in Table 4. In the blank control formulation group, the blank control formulation was intragastrically administered twice a day for 21 consecutive days. As a result, there was no effect on the growth of human colorectal cancer HT-29 nude mouse subcutaneously transplanted tumors, and the T / C percentage obtained on the 21st day was 87.68%.

[0088] The 20mg / kg, 10mg / kg and 5mg / kg groups of test substance Compound I significantly delayed tumor growth under the same experimental treatment regime, and the inhibitory effect increased with increasing dose, with the T / C percentages obtained on the 21st day being 17.38%, 31.27% and 42.70%, respectively. Among them, in the 20mg / kg group of Compound I, the growth of the tumors carried by the mice was almost completely halted during the 3 weeks of experimental treatment.

[0089] The positive control drug Axitinib, 40 mg / kg group, was orally administered twice daily for 21 consecutive days, partially inhibiting the growth of human colorectal cancer HT-29 subcutaneously implanted tumors in nude mice, with a T / C percentage of 48.88% on the 21st day, equivalent to that of the 5 mg / kg group of Compound I.

[0090] [Table 5]

[0091] Example 5: Effects of Compound I on macrophage-related indicators (F4 / 80, CD206) in human glioblastoma cell line U87MG nude mouse transplanted tumor model and human colorectal cancer HT-29 nude mouse subcutaneously transplanted tumor model 1. Experimental Method After the in vivo experiment, the tumor tissues subcutaneously implanted in U87MG and HT-29 nude mice were harvested, fixed in 4% paraformaldehyde, and paraffin sections were prepared.

[0092] The expression of the different proteins was evaluated using the multiplicative quick score method (MQS). This evaluation method considers both the staining intensity and the cell staining range to estimate the percentage of positive cells and gave a range score of 1 to 6 (1 = 1%-4%, 2 = 5%-19%, 3 = 20%-39%, 4 = 40%-59%, 5 = 60%-79%, 6 = 80%-100%). The average intensity of positive stained cells was assigned an intensity score of 0 to 3 points (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining). The range score was then multiplied by the intensity score to calculate a total score A (minimum 0, maximum 18).

[0093] When analyzing the changes of macrophage marker F4 / 80 and M2-type macrophage marker CD206 in U87MG tumor tissues, quantitative statistics (vehicle: n=6, blank control formulation: n=6, compound I 10mg / kg: n=6, compound I 20mg / kg: n=6, compound I 40mg / kg: n=6, Axitinib: n=4) were performed for all sections, and the experimental data were analyzed for significant differences using t-test, where * indicates P<0.05 compared with blank control formulation group, ** indicates P<0.01 compared with blank control formulation group, and *** indicates P<0.001 compared with blank control formulation group.

[0094] Quantitative statistics (vehicle: n=12, blank control formulation: n=6, Compound I at 5 mg / kg: n=6, Compound I at 10 mg / kg: n=6, Compound I at 20 mg / kg: n=6, Axitinib: n=6) were performed on all sections when analyzing the changes of macrophage marker F4 / 80 and M2-type macrophage marker CD206 in HT-29 tumor tissues. The experimental data were analyzed for significant differences using t-test, with * indicating P<0.05 compared to the blank control formulation group, ** indicating P<0.01 compared to the blank control formulation group, and *** indicating P<0.001 compared to the blank control formulation group.

[0095] II. Experimental results 2.1 Changes in macrophage marker F4 / 80 and M2-type macrophage marker CD206 in U87MG tumor tissue Compared with the blank control formulation group, the 40 mg / kg dose group of compound I significantly reduced the expression of F4 / 80 in macrophages and reduced the number of macrophages in tumor tissues (Figure A in Figure 2). Compound I also significantly inhibited the expression of M2 macrophage marker CD206 in a dose-dependent manner (Figure B in Figure 2), with the 10 mg / kg dose group being significantly inhibited, with an inhibition rate of 34.48%, and the 40 mg / kg dose group being inhibited by 70.69%. Meanwhile, Axitinib (40 mg / kg) did not significantly affect the expression of each macrophage-related marker, did not inhibit CSF1R kinase activity, and was consistent with not affecting macrophage function. These results suggest that compound I, as a CSF1R kinase inhibitor, exerts antitumor activity by inhibiting the survival of CSF-1-induced macrophages and reversing the polarized phenotype of macrophage M2 type bias.

[0096] 2.2 Changes in macrophage marker F4 / 80 and M2-type macrophage marker CD206 in HT-29 tumor tissue In the HT-29 nude mouse transplanted tumor model, compound I inhibited the expression of F4 / 80 and CD206 in a dose-dependent manner, and the inhibitory activity was significantly different from that of the blank control formulation group at a dose of 20 mg / kg. The positive drug Axitinib did not inhibit the expression of F4 / 80 and CD206, and the results were as shown in Figures A and B in Figure 3.

[0097] Example 6: Sensitization of Compound I to the Antitumor Efficacy of Immune Checkpoint Drugs 1. Experimental Method Under aseptic conditions, a mouse astrocytoma DBT cell suspension was injected subcutaneously into the right axilla of BALB / c mice. The diameter of the subcutaneously implanted tumor in BALB / c mice was measured using a caliper, and the tumor was approximately 100 to 200 mm. 3When the tumors grew to an average volume of 1000 mg / kg, the animals were randomly assigned to groups. Compound I (5 mg / kg, alone or in combination with anti-PD-1 antibody) was orally administered twice daily for 28 consecutive days. Anti-PD-1 antibody (10 mg / kg, alone or in combination with Compound I) (Bio X Cell, InVivoMAb anti-mouse PD-1 (CD279) (product code: BE0146)) was intraperitoneally injected once every 3 days for 28 consecutive days. The diameter of the implanted tumors was measured twice a week throughout the experiment, and the mice were weighed at the same time. The formula for calculating tumor volume (TV) was TV=1 / 2×a×b. 2 where a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) was calculated from the measurement results, and the formula is RTV = V t / V 0 where V 0 When administered in separate cages (i.e., d 0 ) is the tumor volume measured at t is the tumor volume at each measurement.

[0098] At the end of the experiment, the freshly harvested tumor was first chopped into pieces, and the tumor tissue mass was suspended in 2.5 mL of the enzyme solution prepared, and the tumor tissue was digested on a shaker at 37 ° C. After reacting for 30 to 60 minutes, it was filtered through a 70 μM filter to obtain a cell suspension. The cells were treated with red blood cell lysis solution for 10 minutes, centrifuged at 300 g for 5 minutes, resuspended in PBS, and counted. Next, flow antibody staining was performed, and the necessary cells were washed twice with PBS, suspended in a heavy suspension using 100 μL of PBS, and 0.5 μL of fluorescent antibody FVS510 was added and stained for 30 minutes at 4 ° C away from light, and then washed twice with 1 mL of flow loading buffer (#130-091-221-1, Miltenyi product) and centrifuged at 4 ° C and 300 g for 5 minutes. A blocking antibody dilution solution (1 μL of mouse anti-CD16 / 32 was added to every 200 μL of the above buffer) was prepared, and 200 μL of the antibody dilution solution was added to each sample for blocking for 30 minutes, and 1 μL of CD45 and CD8 antibodies were added to each sample and incubated at 4 ° C for 30 minutes. A single staining tube was placed in this step, and then the sample was washed twice with 1 mL of flow loading buffer in the same manner, and centrifuged at 4 ° C and 300 g for 5 minutes each time. Finally, 300 μL of flow loading buffer was added to make a heavy suspension, and flow analysis was performed with a Fortessa flow cytometer (BD).

[0099] All dose groups had an n=9, and the experimental data were analyzed for significant differences using t-test, where ns indicated no significant difference compared to the blank control formulation group, * indicated P<0.05 compared to the blank control formulation group, and ** indicated P<0.01 compared to the blank control formulation group.

[0100] 2. Experimental Results Compound I did not show a significant inhibitory effect on the in vivo growth of DBT tumors at a single dose of 5 mg / kg. The anti-PD-1 antibody (10 mg / kg) monotherapy group had a certain inhibitory effect on tumor growth, but the tumors still continued to grow slowly. When anti-PD-1 antibody was combined with compound I at a dose of 5 mg / kg twice a day, tumor growth was significantly inhibited and tumor volume was hardly increased. At the end of the experiment, the transplanted tumor volume in the combination group was 19.89% of that in the anti-PD-1 antibody monotherapy group. The results showed that the combination with compound I could enhance the tumor inhibitory effect of anti-PD-1 antibody in the DBT tumor model. In addition, the flow analysis showed that CD8 in tumor tissues was significantly inhibited and tumor volume was not significantly increased. + The number of T cells was significantly increased in both the blank control group and the anti-PD-1 antibody single agent group, suggesting that compound I can reverse the tumor-inhibitory immune microenvironment. Specific results are shown in Figure 4, Panels A and B.

[0101] Example 7: Effect of Compound I on survival time of mice orthotopically inoculated with human glioma U87MG 1. Experimental Animals BALB / cA nude mice, male, 7-8 weeks old, were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd.

[0102] 2. Experimental Method Nude mice were anesthetized by intraperitoneal injection of 50 mg / kg Zoletil 50, and the heads of the nude mice were fixed in a prone position in a stereotaxic apparatus. A vertical incision was made at the intersection of the medial canthal line and the sagittal midline of the head, and the skull was separated and exposed. A hole was drilled in the skull 2 ​​mm to the right and 0.5 mm anterior to the bregma in the horizontal direction. 5 × 10 U87MG cells were then implanted. 5Nude mice were inoculated at the right caudate nucleus of the brain at 100 mg / mouse / mouse. The incision was closed with sterile medical sutures, and the animals were kept warm until they woke up. On the 7th day after inoculation, the mice were randomly divided into groups (the day of inoculation was day 0), and treatment with different compounds was started on the 8th day. Compound I was orally administered twice a day at 40 mg / kg or 20 mg / kg until the mice died naturally. The positive control drug Axitinib was orally administered twice a day at 40 mg / kg until the mice died naturally. The vehicle group was given the same amount of water for injection. The blank control formulation group was given the same amount of blank control formulation. The mice were weighed twice a week throughout the experiment. The deaths of the mice were recorded during the experiment, and the survival rate was calculated. The experimental data was analyzed using Log-rank, and p≦0.05 was a significant difference.

[0103] 1. Experimental Results The experimental results are shown in Table 5 and Figure 5. Each experimental group began experimental treatment on the 8th day after inoculation. Mice in the solvent group began to die on the 27th day, and by the 48th day, all mice in this group had died, with a median survival time of 46 days. Mice in the blank control formulation group (Blank solvent) began to die on the 31st day, and by the 52nd day, the last mouse in this group had died, with a median survival time of 41 days, which was not significantly different from the solvent group.

[0104] In the positive control drug Axitinib 40 mg / kg group, tumor-bearing mice died one after another during the experimental treatment, with deaths in this group beginning on the 28th day, and all mice in this group having died by the 48th day. The median survival time of this group was 41 days, which was not significantly different from the vehicle group.

[0105] Compound I showed similar antitumor efficacy in U87MG nude mouse subcutaneous tumor model, and in U87MG brain orthotopic tumor model, the survival time of mice in each experimental treatment group of compound I was extended to different degrees. Mice in the experimental treatment group of 40 mg / kg compound I began to die one after another from the 43rd day, and the last mouse in this group died by the 75th day, with a median survival time of 58.5 days, significantly longer than the vehicle group, blank control formulation group and Axitinib group. Compound I 20 mg / kg group could similarly extend the survival time of the model mice, with a median survival time of 54 days.

[0106] [Table 6]

[0107] Full names and Japanese names of English abbreviations used herein: CSF1R: Colony-stimulating factor 1 receptor CSF-1: Colony-stimulating factor 1, Colony-stimulating factor 1 TAMs: Tumor-associated macrophages Treg: Regulatory T cells DC: Dendritic cells TGCT: tenosynovial giant cell tumor, tendon sheath giant cell tumor VEGF: Vascular endothelial growth factor VEGFR: Vascular endothelial growth factor receptor DBT: Mouse brain astroglioma AKT: protein kinase B, PKB, protein kinase B DMSO: Dimethylsulfoxide ELISA: enzyme linked immunosorbent assay Poly(Glu,Tyr)4:1: Polyglutamic acid tyrosine peptide (4:1) HEPES: 4-hydroxyethylpiperazineethanesulfonic acid DTT: DL-Dithiothreitol ATP: Adenosine triphosphate PY99: Anti-phosphotyrosine monoclonal antibody OPD: o-Phenylenediamine OD:optical density BALB / c: immunodeficient mouse substrain FBS: fetal bovine serum SDS: Sodium dodecyl sulfate SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis TBS-T: Triethanolamine buffered saline (containing 0.05% Tween-20) PBMC: Peripheral blood mononuclear cell BMDM: Bone marrow-derived macrophages PD-1: Programmed cell death protein 1 PD-L1: Programmed cell death 1 ligand 1 FMO: Fluorescence Minus One Log-rank: Log rank PBS: Phosphate buffer saline FVS510: Fixable viability stain 510, a dye for identifying cell death

Claims

1. A pharmaceutical composition for use in treating a disease associated with the CSF1R kinase signaling pathway, comprising a compound represented by the following formula (I) or a pharma- ceutical acceptable salt thereof, wherein the disease associated with the CSF1R kinase signaling pathway is CSF1 / CSF1R-dependent leukemia or tendon sheath giant cell tumor, or glioma, metastatic brain tumor, or colorectal cancer. 【Chemistry 1】

2. 1. A pharmaceutical composition for use in treating or inhibiting a tumor in an individual in combination with an immune checkpoint drug, comprising a compound of formula (I) below or a pharma- ceutical acceptable salt thereof, wherein the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody, and the tumor is a glioma, a metastatic brain tumor, or a colorectal cancer. 【Chemistry 2】

3. A pharmaceutical composition for use in treating or inhibiting a tumor insensitive to an immune checkpoint drug, comprising a compound of formula (I) below or a pharma- ceutical acceptable salt thereof, wherein the immune checkpoint drug is an anti-PD-1 antibody or an anti-PD-L1 antibody, and the tumor is a glioma, a metastatic brain tumor, or a colorectal cancer. 【Chemistry 3】

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