Pharmaceutical combination of pkmyt1 inhibitor and use thereof

Abstract

The present disclosure provides a pharmaceutical combination comprising a PKMYT1 inhibitor having a novel structure or a pharmaceutically acceptable salt thereof and a second therapeutically active agent, and use thereof in treating cancers.

Description

Drug combinations of PKMYT1 inhibitors and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority and the benefits of Chinese patent application No. CN202410175801.5 filed with the State Intellectual Property Office of China on February 7, 2024, Chinese patent application No. CN202410635952.4 filed with the State Intellectual Property Office of China on May 21, 2024, and Chinese patent application No. CN202510121786.0 filed with the State Intellectual Property Office of China on January 24, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure belongs to the field of medicine, and particularly relates to a pharmaceutical combination containing a PKMYT1 inhibitor compound, a pharmaceutically acceptable salt thereof, and pharmaceutical uses thereof. Background Art

[0004] Cyclin E is a cyclin of the cyclin-dependent kinase 2 (CDK2) family. It binds to CDK2 in the G1 phase to form an activated CDK2-Cyclin E complex, promoting the transition from the G1 phase to the S phase of the cell cycle, where DNA replication initiates. The Cyclin E1 (CCNE1) gene encodes the primary protein of Cyclin E and thus plays a crucial role in regulating the G1-S phase transition of the cell cycle. Studies have suggested that overexpression of CCNE1 protein can lead to increased expression of the encoded Cyclin E protein, thereby enhancing the activity of the CDK2-Cyclin E complex, triggering premature cell cycle transitions, increasing DNA replication stress, and genomic instability. Amplification of CCNE1 is prevalent in various tumor types, particularly in highly malignant gynecological and gastrointestinal cancers, such as ovarian cancer (HGSOC), uterine cancer, and gastroesophageal cancer, and is associated with resistance to cytotoxic and targeted therapies.

[0005] CCNE1 itself is not considered a druggable target. Current research focuses on multi-targeted CDK inhibitors that act on its downstream cyclin, CDK2. Therefore, the lack of treatment options for CCNE1-amplified tumors makes the development of new therapies for this type of tumor a critical unmet need. To identify therapeutic targets for CCNE1-amplified tumors, a collaboration between Mount Sinai Hospital, the University of Toronto, and Repare Therapeutics in the United States discovered that CCNE1 amplification and PKMYT1 inhibition constitute a synthetic lethal pair. This finding was published in Nature on April 20, 2022. They conducted a genome-scale CRISPR-Cas9-based synthetic lethality screen in a cell model of CCNE1 amplification. Their results revealed that PKMYT1 is essential in CCNE1-amplified cells but not in otherwise healthy cells with normal CCNE1 levels. Therefore, PKMYT1 is a synthetic lethal gene for CCNE1, and PKMYT1 inhibitors could be used to treat CCNE1-amplified tumors. Furthermore, PP2A is a phosphatase associated with replication fork stress. Inactivating mutations in PPP2R1A increase replication fork stress in cells, thereby increasing reliance on PKMYT1 for G2 / M phase regulation. The protein encoded by the FBXW7 gene is the target protein recognition component of the cullin-RING ubiquitin ligase. The FBXW7 protein targets CCNE1 through the ubiquitin-dependent protein degradation pathway. Therefore, inactivating FBXW7 mutations lead to elevated CCNE1 levels. PKMYT1 inhibitors may also be used to treat neoplastic diseases in which FBXW7 mutations are inactivating.

[0006] PKMYT1 kinase, also known as MYT1, whose full name is "Membrane-associated tyrosine and threonine-specific cdc2 inhibitory kinase", is a member of the WEE family of kinases. During cell cycle transitions, it phosphorylates the threonine 14 site of CDK1 kinase, rendering the CDK1-Cyclin B complex inactive, negatively regulating the cell cycle checkpoints from G2 to M, and has an important impact on tumor cell proliferation, migration, and xenograft tumor formation.

[0007] Based on the discovery of a synthetic lethal relationship between CCNE1 and PKMYT1, a research team at Mount Sinai Hospital in Toronto developed the selective PKMYT1 inhibitor RP-6306. When used in combination with gemcitabine in a CCNE1 amplification model, RP-6306 demonstrated single-agent activity and durable tumor regression. RP-6306 treatment resulted in selective, unplanned activation of CDK1 in CCNE1-overexpressing cells, promoting premature mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis, at least in part, through premature activation of the MMB–FOXM1 mitotic transcriptional program. They concluded that PKMYT1 inhibition is a promising strategy for treating CCNE1-amplified cancers.

[0008] Patent application PCT / CN2023 / 134116 describes a variety of compounds with novel structures and good PKMYT1 inhibitory activity. To further explore their efficacy and expand their clinical uses, the present disclosure provides a combination containing such compounds, in order to enrich the types of clinical drugs and increase drug accessibility. Summary of the Invention

[0009] The present disclosure provides a drug combination comprising a novel PKMYT1 inhibitor or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof. The combination can be used to treat cancers mediated by one or more mutations, such as CCNE1 amplification, FBXW7 inactivating mutations, and PPP2R1A inactivating mutations. The drug combination exhibits excellent synergistic effects.

[0010] The PKMYT1 inhibitor is S-2-amino-1-(4-fluoro-3-hydroxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide, having the structure shown in the following formula (I):

[0011] All contents described in patent application PCT / CN2023 / 134116 are incorporated by reference into the scope of disclosure of this disclosure.

[0012] In a first aspect, the present disclosure provides the following scheme, which comprises the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in combination for preparing a medicament for treating cancers mediated by one or more mutations such as CCNE1 amplification, FBXW7 inactivation mutation, and PPP2R1A inactivation mutation.

[0013] In another preferred embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof can be administered simultaneously, sequentially or intermittently.

[0014] In another preferred embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are administered to the subject in the form of a compound preparation.

[0015] In another preferred embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof can be independently administered to the subject via any route of administration selected from oral, parenteral, rectal, dermal, nasal or inhalation routes, intratumoral administration, etc.

[0016] In another preferred embodiment, the cancer is selected from solid tumors.

[0017] In another preferred embodiment, the cancer is selected from breast cancer, ovarian cancer, gastric cancer, esophageal cancer, lung cancer, endometrial cancer, uterine carcinosarcoma, cervical cancer, colorectal cancer, and bladder cancer.

[0018] In a second aspect, the present disclosure provides a pharmaceutical combination comprising

[0019] The first component: a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, and

[0020] The second component: gemcitabine or a pharmaceutically acceptable salt thereof,

[0021] In some embodiments, the pharmaceutical combination is a fixed combination.

[0022] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the fixed combination are present in the same pharmaceutical composition.

[0023] In some embodiments, the pharmaceutical combination is a non-fixed combination.

[0024] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition.

[0025] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition, and the pharmaceutical composition of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of gemcitabine or a pharmaceutically acceptable salt thereof are present in the same medicine bag.

[0026] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition, and the pharmaceutical composition of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of gemcitabine or a pharmaceutically acceptable salt thereof are not present in the same medicine bag.

[0027] In another preferred embodiment, the first component and the second component may exist independently in the same dosage form or in different dosage forms, or the first component and the second component may exist in the same preparation.

[0028] In some embodiments, the weight ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to gemcitabine or a pharmaceutically acceptable salt thereof in the pharmaceutical combination (based on the weight of the corresponding free compound, compound of formula (I) : gemcitabine) is about 1:20 to about 20:1, for example, about 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1, or a range of any of the foregoing ratios.

[0029] In some embodiments, the weight ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to gemcitabine or a pharmaceutically acceptable salt thereof in the drug combination (based on the weight of the corresponding free compounds, compound of formula (I): gemcitabine) is about 1:10 to about 15:1; or about 0.4:1 to about 15:1; or about 1.2:1 to about 15:1; or about 1.2:1 to about 12:1; or about 1.2:1 to about 8:1.

[0030] In a third aspect, the present disclosure provides a drug kit comprising the drug combination of the second aspect.

[0031] In some embodiments of the kit of the present disclosure, the first component and the second component may exist independently in the same dosage form or in different dosage forms, or the first component and the second component may exist in the same formulation.

[0032] In some embodiments, the kit further includes instructions for the combined use of the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof.

[0033] In a fourth aspect, the drug combination of the second aspect or the drug kit of the third aspect is used in the preparation of a drug for treating cancers mediated by one or more mutations including CCNE1 amplification, FBXW7 inactivation mutation, and PPP2R1A inactivation mutation.

[0034] In another preferred embodiment, the cancer is selected from: solid tumors.

[0035] In another preferred embodiment, the cancer is selected from breast cancer, ovarian cancer, gastric cancer, esophageal cancer, lung cancer, endometrial cancer, uterine carcinosarcoma, cervical cancer, colorectal cancer, and bladder cancer.

[0036] In a fifth aspect, the present disclosure provides a method for treating cancer, comprising administering to a subject a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof.

[0037] In a sixth aspect, the present disclosure provides a method for treating cancer, the method comprising administering a therapeutically effective amount of the pharmaceutical combination of the present disclosure to a subject in need thereof, the pharmaceutical combination being as described above.

[0038] In a seventh aspect, the present disclosure provides the pharmaceutical combination of the present disclosure for use in treating cancer, the pharmaceutical combination being as described above.

[0039] In an eighth aspect, the present disclosure provides a kit of the present disclosure for treating cancer, the kit being as described above.

[0040] In a ninth aspect, the present disclosure provides use of the pharmaceutical combination or kit of the present disclosure in treating cancer.

[0041] In another preferred embodiment of any of the above aspects, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof can be administered simultaneously, sequentially or intermittently.

[0042] In another preferred embodiment of any of the above aspects, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are administered to the subject in the form of a combination preparation.

[0043] In some embodiments of any of the above aspects, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are separately prepared into pharmaceutical compositions.

[0044] In another preferred embodiment of any of the above aspects, the cancer is selected from cancers mediated by one or more mutations selected from CCNE1 amplification, FBXW7 inactivation mutation, PPP2R1A inactivation mutation, and the like.

[0045] In another preferred embodiment of any of the above aspects, the cancer is selected from solid tumors.

[0046] In another preferred embodiment of any of the above aspects, the cancer is selected from breast cancer, ovarian cancer, gastric cancer, esophageal cancer, lung cancer, endometrial cancer, uterine carcinosarcoma, cervical cancer, colorectal cancer, and bladder cancer.

[0047] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 0.01 mg / kg to about 50 mg / kg (calculated by weight of the compound of formula (I)), for example, about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg. or a range formed by any of the foregoing values.

[0048] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 0.1 mg / kg to about 25 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0049] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 1 mg / kg to about 20 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0050] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 1 mg / kg to about 5 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0051] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 1.5 mg / kg to about 5 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0052] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 1.5 mg / kg or about 5 mg / kg or about 10 mg / kg or about 15 mg / kg or about 20 mg / kg each time (calculated by the weight of the compound of formula (I)).

[0053] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 0.01 mg / kg to about 200 mg / kg (calculated by weight of the compound of formula (I)), for example, about 0.01 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg or 200 mg / kg, or a range thereof.

[0054] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 0.1 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0055] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 1 mg / kg to about 50 mg / kg (calculated by weight of the compound of formula (I)).

[0056] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2 mg / kg to about 20 mg / kg (calculated by weight of the compound of formula (I)).

[0057] In some embodiments of any of the foregoing aspects, the dose of gemcitabine or a pharmaceutically acceptable salt thereof is about 0.1 mg / kg to about 100 mg / kg (calculated by weight of gemcitabine) per dose, for example, about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, or a range of any of the foregoing values.

[0058] In some embodiments of any of the above aspects, the dose of gemcitabine or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 30 mg / kg (calculated as the weight of gemcitabine) per dose.

[0059] In some embodiments of any of the above aspects, the dose of gemcitabine or a pharmaceutically acceptable salt thereof is about 10 mg / kg to about 30 mg / kg (calculated as the weight of gemcitabine) per dose.

[0060] In some embodiments of any of the above aspects, the dose of gemcitabine or a pharmaceutically acceptable salt thereof is about 15 mg / kg to about 25 mg / kg (calculated as the weight of gemcitabine) per dose.

[0061] In some embodiments of any of the aforementioned aspects, the dose of gemcitabine or a pharmaceutically acceptable salt thereof is about 20 mg / kg (calculated as the weight of gemcitabine) per administration.

[0062] In some embodiments of any of the aforementioned aspects, the dose of gemcitabine or a pharmaceutically acceptable salt thereof is about 18 mg / kg (calculated as the weight of gemcitabine) per administration.

[0063] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof is from about 0.1 mg / kg to about 200 mg / kg (calculated as gemcitabine weight), for example, about 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, or 200 mg / kg, or a range thereof.

[0064] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 1 mg / kg to about 150 mg / kg.

[0065] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 5 mg / kg to about 100 mg / kg.

[0066] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 10 mg / kg to about 80 mg / kg.

[0067] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 10 mg / kg to about 70 mg / kg.

[0068] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 10 mg / kg to about 60 mg / kg.

[0069] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 15 mg / kg to about 35 mg / kg.

[0070] In some embodiments of any of the foregoing aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 10 mg / kg to about 30 mg / kg.

[0071] In some embodiments of any of the aforementioned aspects, the average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof (calculated as weight of gemcitabine) is about 18 mg / kg.

[0072] In another preferred embodiment of any of the above aspects, the second component is the hydrochloride of gemcitabine.

[0073] Technical effects of the present disclosure:

[0074] The compound of formula (I) disclosed herein has one or more advantages including good PKMYT1 enzyme inhibitory activity, good kinase selectivity, high anti-tumor cell activity, long half-life, slow clearance rate, good metabolic stability, good solubility, and good tolerability.

[0075] The drug combination of the compound of formula (I) disclosed herein and gemcitabine has a significant synergistic effect, and has an exposure in plasma and tumors that is superior to that of Yangshen. The drug combination disclosed herein has good clinical application potential.

[0076] Definition and Description

[0077] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0078] As used herein, "CCNE1 overexpression" refers to CCNE1 expression levels that are higher than those in normal cells. Compared to normal cells, cells overexpressing CCNE1 exhibit higher CCNE1 activity. For example, normal diploid cells exhibit two copies of CCNE1, while cells overexpressing CCNE1 exhibit at least three copies. CCNE1 overexpression can be measured by identifying the expression level of the gene product in the cell (e.g., CCNE1 mRNA transcript count or CCNE1 protein level).

[0079] The “FBXW7 inactivating mutation” described in the present disclosure includes various types of mutations that inactivate FBXW7 gene expression, including but not limited to base insertion, deletion, mutation, substitution, chemical modification, etc.

[0080] The "PPP2R1A inactivating mutation" described in the present disclosure includes various types of mutations that inactivate PPP2R1A gene expression, including but not limited to base insertion, deletion, mutation, substitution, chemical modification, etc.

[0081] As used in this disclosure, unless otherwise indicated, "by weight" refers to the weight of the compound of formula (I), gemcitabine in free form.

[0082] The term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be therapeutic in terms of partial or complete stabilization or cure of a disease and / or side effects caused by the disease. As used herein, "treat" encompasses any treatment of a patient's disease, including: (a) suppressing the symptoms of the disease, i.e., arresting its progression; or (b) relieving the symptoms of the disease, i.e., causing regression of the disease or its symptoms.

[0083] The compound of formula (I) or its pharmaceutically acceptable salt can be administered by a variety of routes, including but not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, through inhalation, vaginal, intraocular, through topical administration, subcutaneous, intrafatty, intraarticular, intraperitoneal and intrathecal. In some specific embodiments, by oral administration. The therapeutically effective amount of the compound of formula (I) or its pharmaceutically acceptable salt is including but not limited to from about 0.0001 to 200mg / kg weight / day (calculated as free compound weight), for example, from 1.5 to 50mg / kg weight / day or 1.5 to 20mg / kg weight / day. The dosage frequency of the compound of formula (I) or its pharmaceutically acceptable salt is determined by the needs of individual patients, including the severity, the response of the disease, the toxicity associated with any treatment, the age and health status of the patient, for example, once a day or twice, or more times a day. Dosing can be intermittent, for example, wherein over a period of several days, the subject receives a daily dose of the compound of Formula (I) or a pharmaceutically acceptable salt thereof, followed by a period of several or more days during which the patient does not receive a daily dose of the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0084] Gemcitabine or its pharmaceutically acceptable salt can be administered by multiple approaches, and this approach includes but is not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, through inhalation, vagina, intraocular, through topical administration, subcutaneous, intrafatty, intraarticular, intraperitoneal and intrathecal.In some specific embodiments, by intraperitoneal administration.The amount of giving gemcitabine can be determined according to the severity of the disease, the response of the disease, the toxicity related to any treatment, the age and health status of the patient.For example, gemcitabine or its pharmaceutically acceptable salt can be used once a week, and each treatment effective amount includes but is not limited to from about 0.1mg / kg to 200mg / kg (calculated as free compound), for example from 1mg / kg to 100mg / kg or 10mg / kg to 60mg / kg.In some embodiments, gemcitabine or its pharmaceutically acceptable salt is used once a week with injection preparation (for example solution type injection).

[0085] The term "pharmaceutical combination" refers to the simultaneous, concurrent or sequential use of two or more active ingredients.

[0086] The term "fixed combination" means that the active ingredients (e.g., a compound of Formula (I) or gemcitabine) are administered to a subject simultaneously in a fixed total dose or dose ratio, or in the form of a single entity, pharmaceutical composition or formulation. In some embodiments, for example, they are present in the same tablet, capsule, injection or bag.

[0087] The term "non-fixed combination" refers to two or more active ingredients administered to an individual as independent entities (e.g., pharmaceutical compositions, pharmaceutical preparations) simultaneously, concurrently or sequentially and without specific time limits, wherein the active ingredients administered to the individual reach a therapeutically effective level. An example of a non-fixed combination is cocktail therapy, e.g., administration of two, three or more active ingredients. In a non-fixed combination, the individual active ingredients may be packaged, sold or administered as completely independent pharmaceutical compositions. The "non-fixed combination" also includes the combined use of "fixed combinations" or a "fixed combination" with any one or more independent entities of the active ingredients.

[0088] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure, or pharmaceutical combinations thereof, or salts thereof, and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure, or pharmaceutical combinations thereof, to a subject.

[0089] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, dispersants, water, and saline.

[0090] The pharmaceutical compositions of the present disclosure can be prepared by combining the compounds of the present disclosure with suitable pharmaceutically acceptable excipients.

[0091] The pharmaceutical composition of the present disclosure can be manufactured by methods well known in the art, such as conventional dispersion methods, mixing methods, dissolution methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[0092] The term "single dose" refers to the smallest packaging unit containing a specific amount of a drug. For example, if a box of medicine contains seven capsules, each capsule is a single dose; or if each bottle of injection is a single dose. The term "multiple dose" refers to a combination of multiple single doses.

[0093] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present disclosure, which are prepared from the compounds of the present disclosure having specific substituents and relatively non-toxic acids or bases. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a neat solution or a suitable inert solvent. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a neat solution or a suitable inert solvent. The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing acid radicals or base groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0094] The term "therapeutically effective amount" refers to a sufficient amount of a compound or pharmaceutically acceptable salt of the present disclosure to treat a disorder at a reasonable benefit / risk ratio applicable to any medical treatment and / or prevention. However, it should be recognized that the total daily dosage of the pharmaceutically acceptable salts and compositions of the compounds of Formula I disclosed herein must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field. For example, it is practice in the art to start the dose of the compound at a level lower than that required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0095] The term "administering" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including but not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, by inhalation, vaginal, intraocular, topical, subcutaneous, intrafatty, intraarticular, intraperitoneal, and intrathecal. In some specific embodiments, administration is by oral administration.

[0096] Administration

[0097] The following does not limit the mode of administration of the disclosed pharmaceutical combination.

[0098] The active ingredients in the pharmaceutical combination of the present disclosure can be formulated separately, or some or all of them can be formulated together. In one embodiment, the pharmaceutical combination of the present disclosure can be formulated into a pharmaceutical composition suitable for single or multiple administrations.

[0099] The active ingredients in the pharmaceutical combination of the present disclosure may be administered separately, or some or all of them may be administered together. The ingredients in the pharmaceutical combination of the present disclosure may be administered substantially at different times, or some or all of them may be administered substantially simultaneously.

[0100] The active ingredients of the pharmaceutical combination of the present disclosure can be administered independently, or part or all of them together in various suitable routes, including but not limited to oral or intraperitoneal administration. In some embodiments, the active ingredients of the pharmaceutical combination of the present disclosure can be administered independently, or part or all of them together orally or by injection, such as intraperitoneal injection or intravenous injection.

[0101] The active ingredients in the pharmaceutical combination of the present disclosure may each independently, or some or all of them may contain a pharmaceutically acceptable carrier and / or excipient.

[0102] The pharmaceutical combination of the present disclosure may further comprise an additional therapeutic agent. In one embodiment, the additional therapeutic agent may be a therapeutic agent known in the art for treating cancer.

[0103] In some embodiments, an effective amount of a compound of formula (I) of the present disclosure or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof can be administered to an individual in need thereof simultaneously, sequentially, or at intervals. The term "subject" primarily refers to humans. Further, the present disclosure provides another approach in which the drug combination of the present disclosure is administered to non-human animals, including but not limited to vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs.

[0104] "Synergy" or "synergistic effect" means that when two drugs are used together, their total effect is greater than the sum of the effects of each drug when used alone, and their actions are in the same direction, achieving a mutually reinforcing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG1 shows that the compound of formula (I) combined with gemcitabine synergistically inhibits the proliferation of HCC1569 cells.

[0106] Figure 2 shows the tolerability of the compound of formula (I) in BALB / c Nude mice. DETAILED DESCRIPTION

[0107] The present disclosure is described in detail below by way of examples, but these examples are intended to illustrate and do not limit the scope of the present disclosure. Similarly, the present disclosure is not limited to any specific preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions made to the technical features of the present disclosure, or corresponding improvements, still fall within the scope of protection of the present disclosure.

[0108] Preparation Example 1 Preparation of the compound of formula (I)

[0109] 2-Amino-1-(4-fluoro-3-hydroxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide

[0110] In a one-necked flask, 6-chloro-5-(trifluoromethyl)pyridin-2-amine (39 g, 0.19 mol) was dissolved in dioxane / water (10:1). Methylboric acid (23.4 g, 0.39 mol), potassium carbonate (82 g, 0.59 mol), and PdCl2(dppf) (8 g, 0.011 mol) were then added. The reaction system was replaced with nitrogen and stirred at 110°C for 16 hours. LCMS analysis indicated the disappearance of the starting material and the formation of the product. The reaction mixture was quenched with purified water and extracted with ethyl acetate (700 mL x 3). The organic phases were combined, washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to yield 6-methyl-5-(trifluoromethyl)pyridin-2-amine (24 g).

[0111] In a single-necked flask, 6-methyl-5-(trifluoromethyl)pyridin-2-amine (24 g, 0.14 mol) was dissolved in acetonitrile (300 ml), and N-bromosuccinimide (29.13 g, 0.164 mol) was added in batches under ice bath. The reaction system was stirred at room temperature for 16 hours. LCMS showed that the starting material disappeared and the product was generated. The reaction solution was poured into 500 ml of water. It was extracted with ethyl acetate (400 ml × 3), and the organic phases were combined and washed with saturated brine (100 ml × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give 3-bromo-6-methyl-5-(trifluoromethyl)pyridin-2-amine (26.3 g). MS (ESI) M / Z: 257.0 [M+H + ]. 1 H NMR (400MHz, CDCl3) δ8.04(s,1H),5.30(s,1H,-NH2),2.73–2.59(m,3H).

[0112] In a three-necked flask, 3-bromo-6-methyl-5-(trifluoromethyl)pyridin-2-amine (26.3 g, 0.103 mol) was dissolved in bromoform, and bromine (19.9 g, 0.124 mol) was slowly added dropwise at 0 degrees Celsius. Tert-butyl nitrite (32.61 g, 0.31 mol) was then slowly added dropwise to the reaction solution at 0 degrees Celsius. The mixture was reacted at room temperature for 3 hours. LCMS detected that the raw material disappeared and the product was generated. The reaction solution was quenched by adding ice water, and the organic phase was washed three times with saturated sodium bicarbonate solution. The obtained organic phase was concentrated under reduced pressure using an oil pump to remove bromoform. The obtained mixture was purified by silica gel column chromatography to give 2,3-dibromo-6-methyl-5-(trifluoromethyl)pyridine (26.5 g). MS (ESI) M / Z: 320.0 [M+H + ].

[0113] In a single-necked flask, 2,3-dibromo-6-methyl-5-(trifluoromethyl)pyridine (15.1 g, 0.047 mol) was dissolved in ethylene glycol dimethyl ether (150 mL). 4-Fluoro-3-methoxy-2,6-dimethylaniline (8.8 g, 0.053 mol), cesium carbonate (38.75 g, 0.12 mol), Pd2dba3 (4.34 g, 4.74 mmol), and XantPhos (5.48 g, 9.48 mmol) were added at room temperature. The reaction system was replaced with nitrogen. The reaction system was reacted at 110°C for 12 hours. LCMS analysis indicated the disappearance of the starting material and the formation of the product. The reaction solution was quenched with purified water and the mixture was extracted with ethyl acetate (400 mL x 3). The organic phases were combined, washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give 3-bromo-N-(4-fluoro-3-methoxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)pyridin-2-amine (10.5 g). MS (ESI) M / Z: 407.1 [M+H] + .

[0114] To a 250 ml three-necked flask, sodium tert-butoxide (12.42 g, 129.3 mmol) was added. Ethylene glycol dimethyl ether (30 ml) was then added dropwise under nitrogen. A solution of malononitrile (8.53 g, 129.3 mmol) in ethylene glycol dimethyl ether (5 ml) was then added dropwise at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 30 minutes. Then, under nitrogen, a solution of 3-bromo-N-(4-fluoro-3-methoxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)pyridin-2-amine (10.5 g, 25.86 mmol) in ethylene glycol dimethyl ether (30 ml) and PdCl2(dppf) (1.89 g, 2.586 mmol) were added. The reaction system was allowed to react at 110°C for 16 hours. LCMS analysis indicated the formation of product, and the reaction mixture was poured into ice water. The reaction mixture was extracted with ethyl acetate (300 ml x 3), and the organic phases were combined, washed with saturated brine (50 ml x 3 times), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give 2-amino-1-(4-fluoro-3-methoxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (5.7 g, yield 56.3%). MS (ESI) M / Z: 393.2 [M+H] + .

[0115] 2-Amino-1-(4-fluoro-3-methoxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (5.7 g, 14.55 mmol) was dissolved in concentrated sulfuric acid (50 mL) under ice. The reaction mixture was stirred at room temperature for 2 hours. After LCMS monitoring showed the disappearance of the starting material, the reaction mixture was poured into ice water and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (3 times with 50 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give 2-amino-1-(4-fluoro-3-methoxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (4.4 g). 1 H NMR (400MHz, DMSO) δ8.28 (s, 1H), 7.26 (d, J = 12.2Hz, 1H), 7.19 (s, 2H), 6.94 (s, 2H), 3.87 ( d,J=1.0Hz,3H),2.45(d,J=1.6Hz,3H),1.82(s,3H),1.78(s,3H).MS(ESI)M / Z:411.3[M+H] + .

[0116] To a 250 mL three-necked flask, add 2-amino-1-(4-fluoro-3-methoxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (4.4 g, 10.7 mmol) and anhydrous dichloromethane (40 mL). A 1N dichloromethane solution of BBr3 (107 mL) was added dropwise under an ice bath, and the reaction mixture was stirred at room temperature for 2 hours. LCMS analysis indicated the disappearance of the starting material and the formation of the product. The reaction mixture was quenched with ice water and the pH was adjusted to 9 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The resulting residue was purified by acetonitrile slurrying to give 2-amino-1-(4-fluoro-3-hydroxy-2,6-dimethylphenyl)-6-methyl-5-(trifluoromethyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (3.1 g). 1 H NMR(400MHz,DMSO)δ9.63(s,1H),8.27(s,1H),7.19-7.07(m,3H),6.93(s,2 H),2.45(d,J=1.5Hz,3H),1.77(s,3H),1.72(s,3H).MS(ESI)M / Z:397.1[M+H + ].

[0117] SFC method: Instrument: SFC-150mg / m (waters), chiral column: Daicel OZ (25*250mm, 10μm), temperature: 30°C, mobile phase: CO2 / MeOH [0.2% NH3 (7M MeOH)] = 65 / 35, flow rate: 100 ml / min, back pressure: 100 bar, detection wavelength: 214 nm, cycle time: 10 min.

[0118] Compound of formula (I): 2.13 mg. SFC Rt = 1.770 min. [α] D 20 =+42.4°(0.10018,MeOH). 1 H NMR(400MHz,DMSO)δ9.63(s,1H),8.26(s,1H),7.19–7.05(m,3H),6.92(s,2 H),2.45(d,J=1.6Hz,3H),1.77(s,3H),1.72(s,3H).MS(ESI)M / Z:397.1[M+H + ].

[0119] Biological activity test methods

[0120] The positive control drug RP-6306 has the following structure. The specific preparation method can refer to the preparation steps of Example 182 in patent WO2021195781A1.

[0121] Biological Test Example 1: Enzyme Inhibitory Activity of Compounds

[0122] The enzymatic activity of the compound was verified using the ADP-GLO method

[0123] Experimental Materials:

[0124] PKMYT1 enzyme protein was purchased from Thermo Scientific (Cat. No. A33387), and ADP-GLO kit was purchased from Promega (Cat. No. V9103).

[0125] PKMYT1 enzyme activity reaction buffer: 70mM HEPES, 3mM MgCl2, 3mM MnCl2, 50μg ml-1PEG20000, 3μM sodium orthovanadate, 1.2mM DTT.

[0126] Experimental steps:

[0127] Using an ultra-micropipette, add the test compound (3.33 mM, dissolved in DMSO) starting at 10 μM and then dilute it in a 1:3 gradient (10,000 nM, 3,333 nM, 1,111 nM, 370 nM, 123 nM, 41 nM, 13.7 nM, 4.6 nM, 1.5 nM, 0.5 nM) to a 384-well reaction plate. Set up two replicates for each concentration. Set up 16 wells each for positive and negative controls: replicate wells containing 1 μM of the positive compound for the positive control and replicate wells containing DMSO for the negative control. Centrifuge the plate at 2,500 rpm for 1 minute.

[0128] Add 5 μL of the prepared enzyme solution to each well of the reaction plate for a final enzyme concentration of 10 nM. Centrifuge at 1000 rpm for 1 minute. Incubate at 37°C for 15 minutes.

[0129] Add 5 μL of the prepared ATP substrate solution to each well of the reaction plate for a final ATP concentration of 5 μM. Centrifuge at 1000 rpm for 1 minute. Seal the plate with aluminum foil and place in an incubator at 37°C for 90 minutes.

[0130] Add 10uL ADP-glo TMUse a reagent to stop the kinase reaction and deplete unused ATP, leaving only ADP and a very low background ATP. Centrifuge at 1000 rpm for 1 minute. Seal the plate with aluminum foil and incubate at room temperature for 60 minutes.

[0131] Add 20 μL of kinase assay reagent to convert ADP to ATP. Introduce luciferase and luciferin to detect ATP. Centrifuge at 1000 rpm for 1 minute. Seal the plate with aluminum foil and incubate at room temperature for 60 minutes.

[0132] ADP-GLO luminescence signal values ​​were read and analyzed using a PHERAstar microplate reader. The average inhibition rate of the positive control replicates was set as 100% relative inhibition rate; the average inhibition rate of the negative control replicates was set as 0% relative inhibition rate. The ADP-GLO readings were converted to relative inhibition rates, and the compound IC values ​​were fitted using a four-parameter model. 50 .

[0133] Experimental results:

[0134] This indicates that the compound of formula (I) has good enzyme inhibitory activity. For specific results, see Table 1.

[0135] Table 1 PKMYT1 enzyme activity test results

[0136] Biological Test Example 2. Cytostatic Activity of Compounds

[0137] The biological activity of the compounds was verified using the CTG assay (HCC1569).

[0138] Experimental Materials:

[0139] HCC1569 cells were purchased from ATCC (Cat. No. CRL-2330) and cultured in a 37°C, 5% CO2 cell culture incubator. HCC1569 complete culture medium: RPMI-1640 liquid medium (Cat. No. Gibico 11875-093), 20% FBS (Cat. No. Gibico 10099-141), and 1% Pen Strep (Cat. No. Gibico 15070-063).

[0140] Test compound: the compound of formula (I) disclosed herein.

[0141] Experimental steps:

[0142] 75cm 2HCC1569 cells in a culture flask were digested with 2 mL of trypsin for 2-3 minutes, then neutralized with 2 mL of 1640 complete medium. Centrifuge at 1200 rpm for 5 minutes. Resuspend the cells in 4 mL of 1640 complete medium. Count 500 μL of the cell suspension using a Vi-CELL-XR cell counter.

[0143] Using a Multidrop instrument, 1000 HCC1569 cells were seeded per well (50 μL 1640 Growth Media) in a 384-well plate, and drugs were added 24 hours later.

[0144] Using an ultra-micropipette, the test compound (concentration: 3.33 mM, dissolved in DMSO) was diluted with 10 gradients (10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM, 13.7 nM, 4.6 nM, 1.5 nM, 0.5 nM) in a 1:3 ratio with the highest starting concentration of 10 uM for drug treatment. Two replicate wells were set for each concentration; 14 wells each for positive and negative controls were set, the positive control was a replicate well of 10 uM positive compound, and the negative control was a replicate well of DMSO.

[0145] After drug addition, cells were placed in a 37°C incubator and cultured for 5 days. After 5 days, 25 μl of CTG buffer was added to each well for CTG assay, and the plate was read using a microplate reader. The CTG readings were analyzed. The average inhibition rate of the positive control replicates was set as 100% relative inhibition; the average inhibition rate of the negative control replicates was set as 0%. The CTG readings were converted to relative inhibition rates, and the inhibition rate (inhibition %) of the cells at each test compound concentration was calculated using the following formula:

[0146] Inhibition%=(bx) / (ba)*100%;

[0147] a=CTG value (highest concentration), b=CTG value (blank well), x=CTG value (test well value).

[0148] IC was analyzed using GraphPad PRISM 8. 50 Perform calculations.

[0149] (1) Statistically analyze the concentrations and inhibition rates corresponding to 10000nM, 3333nM, 1111nM, 370nM, 123nM, 41nM, 13.7nM, 4.6nM, 1.5nM, and 0.5nM. Calculate the statistics using Log10 (A compound concentration). (2) Input the data into GraphPad PRISM 8 and select Analysis. (3) Select Nonlinear regression (curve fit). (4) Select Log (inhibitor) vs. response-Variable slope. (5) Select the calculation formula and calculate it according to the following formula; Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope)), X: Dose or concentration logarithm, Y: Response value, Top and Bottom: Peak and bottom values. (6) Fit the data to obtain IC 50 (7) Adjust the fitting conditions according to the specific data. Adjust the constraints of Bottom, Top and HillSlope appropriately. The curve that best fits the actual situation has been achieved.

[0150] The experimental results show that the compound of formula (I) has good cell inhibitory activity. The specific results are shown in Table 2

[0151] Table 2 CTG test results

[0152] Biological Test Example 3: Study on the Metabolic Stability of Compounds in Mouse Liver Microsomes

[0153] The concentration of the parent drug in the incubation system was determined using LC / MS / MS, and the intrinsic clearance of the test compound in the microsomal system was calculated to evaluate its stability. The test concentration of the test compound and the positive control compound was 1 μM.

[0154] 1. Transfer 25 μL of NADPH (10 mM) or phosphate buffered saline (100 mM, pH 7.4) to the liver microsome incubation system and add 2 μL of the test substance or verapamil at a concentration of 200 μM. Prepare duplicates for samples with NADPH; prepare single replicates for samples without NADPH.

[0155] 2. Take 30 μl of the suspension at 0.5, 5, 15, 30, and 60 minutes, add 180 μl of acetonitrile containing internal standard to terminate the reaction, and vortex for 10 minutes.

[0156] 3. Centrifuge at 3220g for 20 minutes to precipitate the protein. Refrigerate the plate at 4°C for 30 minutes, then re-centrifuge at 3220g for 20 minutes. Transfer 100 μl of the supernatant to the sample plate and add 100 μl of pure water to mix thoroughly before analysis by UPLC-MS / MS.

[0157] 4. All data calculations were performed using Microsoft Excel. Peak areas were detected by extracting ion spectra. The in vitro half-life (t 1 / 2 ).

[0158] In vitro half-life (t 1 / 2 ) calculated by slope: in vitro t 1 / 2 =0.693 / k

[0159] In vitro clearance Cl int (Unit: μl / min / mg) Calculation: in vitro C Lint = kV / N; V = incubation volume per well (400 μl); V = incubation volume per well (400 μl). Specific results are shown in Table 3.

[0160] Table 3 Results of mouse liver microsome metabolic stability test

[0161] Conclusion: Compared with RP-6306, the compound of formula (I) has a significantly longer half-life, a slower clearance rate, and better metabolic stability.

[0162] Tissue distribution of the compound in Biological Test Example 4

[0163] OVCAR3 tumor cells (ATCC, catalog number HTB-161) were cultured in RPMI 1640 medium containing 20% ​​inactivated fetal bovine serum, supplemented with 0.01 mg / mL insulin and 1% penicillin-streptomycin. The cells were cultured in a 37°C, 5% CO2 incubator. The medium was changed every other day, and the cells were passaged every 3 to 4 days after confluence. Under sterile conditions, the in vitro cultured OVCAR3 cell suspension was centrifuged and then added to adjust the cell concentration to 1×10 8 ceils / mL, an equal volume of Matrigel was added and inoculated subcutaneously on the back of the right hind forelimb of mice (BALB / c nude mice, female, Beijing Weitong Lihua Experimental Animal Co., Ltd.) (0.1 mL / mouse). 31 days after inoculation, the average tumor volume was 100-150 mm 3 Mice were randomly divided into groups according to tumor size and body weight, and the administration of test compounds was started.

[0164] The experiment was divided into a solvent control group, a positive control group, and a test group, with 5-6 mice in each group. The specific experimental protocol and the frequency of dosing for each group are shown in the table below. For the test compound-administered group, the test compound was mixed in 0.5% methylcellulose and administered orally twice daily. The experiment was terminated after 28 days of dosing. PK plasma (EDTA-K2 anticoagulation) samples were collected (0.5h, 1h, 2h, and 6h after dosing). Mice were euthanized and tumor samples were collected 2h and 6h after the last dose.

[0165] Tumor concentration test method:

[0166] All tumor samples were added to pure water at a ratio of 1:3 (3 mL of solvent was added per g of tumor) and ground using a frozen homogenizer to obtain tumor sample solutions.

[0167] Standard curve preparation method:

[0168] The test compound powder was diluted in a gradient with 50% acetonitrile in water to prepare a series of working solutions. 3 μL of the working solution (10, 20, 40, 100, 200, 1000, 2000, 10,000, 20,000 ng / mL) was added to 57 μL of a blank Balb / c nude mouse tumor sample solution to obtain calibration standard solutions ranging from 0.5 to 1000 ng at concentrations of 0.5, 1, 2, 5, 10, 50, 100, 500, and 1000 ng / mL, respectively, in a total volume of 60 μL.

[0169] Quality control samples were prepared in the same manner as the calibration standards: 3 μL of working solution (30, 60, 120, 1000, 8000, 16000 ng / mL) was added to 57 μL of blank Balb / c nude mouse tumor sample solution to obtain 6 quality control samples with concentrations of 1.5 ng / mL, 3 ng / mL, 6 ng / mL, 50 ng / mL, 400 ng / mL and 800 ng / mL, respectively, in a total volume of 60 μL.

[0170] Take 30 μL of sample (including standard solution, quality control sample, and test sample) and add 200 μL of protein precipitant containing acetonitrile to precipitate protein. Vortex for 30 seconds, then centrifuge at 3900 rpm at 4°C for 15 minutes. Aspirate the supernatant and dilute it 3-fold with water. 5 μL of the diluted supernatant is loaded onto the LC / MS / MS system for quantitative analysis.

[0171] Dosage and frequency of each group

[0172] The experimental results are shown in Table 4:

[0173] Table 4 Tumor tissue concentration test results of the compounds

[0174] The above experimental results show that the tumor tissue concentration of the compound of formula (I) 2h and 6h after administration is significantly better than that of the positive drug, indicating that the compound of formula (I) has potential for better anti-tumor activity.

[0175] Metabolic stability of compounds in biological test example 5 in human liver cells

[0176] Prepare several 96-well sample precipitation plates, named T0, T15, T30, T60, T90, T120, T240, T0-MC, T240-MC, and blank matrix. Remove the recovery medium and incubation medium in advance and place them in a 37°C water bath to preheat. Remove the frozen hepatocytes from the liquid nitrogen tank and immediately immerse them in a 37°C water bath (about 90 seconds). After the frozen portion is thawed and loosened, pour them into centrifuge tubes containing 40mL of recovery medium and gently invert to resuspend the cells in the recovery medium. Centrifuge at 100×g for 5 minutes at room temperature, remove the supernatant, resuspend the hepatocytes in an appropriate volume of incubation medium, and calculate the cell viability using trypan blue staining. 198 μL of hepatocyte suspension (0.51×106 cells / mL) was added to the preheated incubation plate. For the culture medium control group, 198 μL of incubation medium without hepatocytes was added to the T0-MC and T120-MC incubation plates. All incubation plates were preincubated in a 37°C incubator for 10 minutes.

[0177] Add 2 μL of the test and control compound working solutions, mix thoroughly, and immediately place the plate on a plate shaker in the incubator. Start the timer to initiate the reaction. Prepare two replicates for each compound at each time point. Incubate at 37°C, saturated humidity, and 5% CO2.

[0178] In the assay system, the final concentration of the test article was 1 μM, the final concentration of the control article was 3 μM, the final concentration of hepatocytes was 0.5 × 10⁶ cells / mL, and the final concentration of total organic solvent was 0.96%, including 0.1% DMSO. At the end of the incubation period, the incubation plates were removed, and 25 μL of the mixture of compound and control compound with cells was added to a sample plate containing 125 μL of stop solution (200 ng / mL tolbutamide and labenolol in acetonitrile). For the blank sample plate, 25 μL of the incubation medium without hepatocytes was added directly. All sample plates were sealed and shaken on a plate shaker at 600 rpm for 10 minutes, followed by centrifugation at 3220 × g for 20 minutes. The supernatants of the test article and control articles were diluted 1:3 with ultrapure water. All samples were mixed and analyzed by LC / MS / MS.

[0179] The experimental results are shown in Table 5:

[0180] Table 5 Human HMS metabolic stability of compounds

[0181] The experimental results disclosed herein show that RP-6306 exhibits significant gender differences in in vitro human hepatocyte metabolism, with male hepatocytes exhibiting significantly lower metabolic stability than female hepatocytes. However, the compound of formula (I) exhibits no gender differences in in vitro human hepatocyte metabolic stability and exhibits good stability, reducing the risk of gender-related effects on drug efficacy and safety, and facilitating more convenient clinical applications.

[0182] Biological Test Example 6: Investigation of Compound Kinase Spectrum Selectivity

[0183] 1. Purpose of the experiment

[0184] Through 435 kinase activity inhibition experiments,

[0185] 2. Experimental Materials

[0186] Eurofins was commissioned to test compounds for inhibition against a panel of 435 kinases.

[0187] Kinases tested

[0188] 3. Experimental methods

[0189] Common enzyme activity test methods such as radioactive element methods and HTRF

[0190] 4. Experimental steps

[0191] First, the enzyme solution is diluted to a working concentration according to the dilution buffer recipe for each kinase. Next, the compounds are prepared, all prepared in 100% DMSO at 50x the final assay concentration. The compound is added to the assay wells as the first component in the reaction, followed by the remaining components, as detailed in the general assay protocol below. In the standard KinaseProfiler service, there is no pre-incubation step between the compound and the kinase before the reaction begins. Our positive control wells contain all reaction components except for the reference compound, which contains DMSO (final concentration of 2%), indicating 0% inhibition. Our blanks contain all reaction components with the reference inhibitor substituted for the compound of interest, indicating 100% inhibition. The experimental procedure is described using AAK1(h) as an example among 435 kinases. AAK1(h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM substrate, 10 mM magnesium acetate, and [γ-33P-ATP] (specific activity and concentration determined as needed). The reaction is initiated by adding the Mg / ATP mixture. After incubation at room temperature for 120 minutes, the reaction was stopped by adding phosphoric acid to a final concentration of 0.5%. Aliquots of the stopped reaction were spotted onto filters and washed four times with 0.425% phosphoric acid for 4 minutes each and once in methanol before drying and scintillation counting.

[0192] 5. Experimental Results

[0193] The experimental results are shown in Table 6. The results show that at high concentrations, the compound of formula (I) inhibits less than 20% of the kinases with an inhibition rate greater than 50%, indicating that the compound of formula (I) has good kinase selectivity and good safety.

[0194] Table 6 Kinase spectrum inhibition experiment

[0195] Biological Test Example 7 In vivo pharmacodynamic study of the compound of formula (I) combined with gemcitabine in an OVCAR3 transplanted tumor model

[0196] 1. Purpose of the experiment

[0197] The in vivo efficacy of the compound of formula (I) in combination with gemcitabine was evaluated in a BALB / c Nude mouse model of subcutaneous human ovarian cancer OVCAR3 cell xenograft tumors.

[0198] 2. Experimental Materials

[0199] 2.1 Experimental animals and breeding environment

[0200] Species: Mouse

[0201] Strain: BALB / c Nude mice

[0202] Arrival age: 6-8 weeks

[0203] Gender: Female

[0204] Weight: 18-22 grams

[0205] Supplier: Shanghai Lingchang Biotechnology Co., Ltd.

[0206] Animal Certificate: 20230003002222

[0207] 2.2 Compound Information

[0208] Table a. Compound information Note: 1. Gemcitabine hydrochloride is gemcitabine hydrochloride

[0209] 3. Experimental methods and steps

[0210] 3.1 Cell culture

[0211] Human ovarian cancer OVCAR3 cells (ATCC, HTB161) were cultured as monolayers in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C in a 5% CO2 incubator. Routine passage was performed twice weekly. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0212] 3.2 Tumor cell inoculation and grouping

[0213] will contain 10×10 6 100 μL of PBS and 100 μL of Matrigel (1:1) were mixed in equal volumes and then subcutaneously inoculated on the back of the right forelimb of each mouse. The average tumor volume reached 142 mm 3 The experimental groups and dosing schedule are shown in the table below.

[0214] Table b. Experimental animal groups and dosing regimens Note: 1. N: number of mice per group; 2. Dosing volume: 10 μL / g based on mouse body weight; 3. Vehicle: 0.5% methylcellulose (400 cp); 4. BID: twice-daily dosing; D: number of days; 5. po: oral administration; ip: intraperitoneal injection.

[0215] 3.3 Preparation of test substances

[0216] Table c. Preparation method of test substances Note: Gently mix thoroughly before administration. RP-6306 and the compound of formula (I) should be prepared twice weekly. Gemcitabine hydrochloride should be prepared immediately upon use.

[0217] 3.4 Daily observation of experimental animals

[0218] The use and welfare of experimental animals were carried out in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight change (measured twice weekly), and any physical signs or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0219] 3.5 Tumor Measurement and Experimental Indicators

[0220] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0221] The tumor inhibition efficacy of a compound was evaluated using the tumor inhibition rate (TGI) (%) or the tumor inhibition rate (T / C) (%). TGI (%) was calculated as follows: TGI (%) = [1 - (average tumor volume of a treatment group at the end of dosing - average tumor volume of the treatment group at the start of dosing) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%. Tumor proliferation rate (T / C) (%) was calculated as follows: T / C% = TVT (average tumor volume of the treatment group) / TVC (average tumor volume of the negative control group) × 100%.

[0222] 3.6 Statistical analysis

[0223] Statistical analysis was performed using GraphPad Prism 8.3.0 software, based on the mean and standard error (SEM) of tumor volume at the end of the study. Statistical analysis was performed using one-way ANOVA or T-test when variance was equal (p>0.05); Kruskal-Wallis or Mann-Whitney nonparametric tests were used when variance was unequal (p<0.05). A p<0.05 was considered significant.

[0224] 4. Experimental Results and Analysis

[0225] The tumor suppression effect is shown in Table 7 and Figure 1 below. The experimental results show that the combination of the compound of formula (I) and Gemcitabine has a synergistic inhibitory effect on the proliferation of OVCAR3 subcutaneous xenograft tumors, and is superior to the positive control group; and the combination of the compound of formula (I) and gemcitabine disclosed in the present invention has a stronger anti-tumor effect, reduces the dosage, and improves the effectiveness and safety of the drug.

[0226] Table 7 Tumor inhibition rate Note: 1. The p-value was analyzed by Kruskal-Wallis non-parametric test with the Vehicle group as the control based on the D28 tumor volume of different groups: *p < 0.05; **p < 0.01; ***p < 0.001; 2. The p-value was analyzed by Mann-Whitney non-parametric test with the Gemcitabine hydrochloride group as the control based on the D28 tumor volume of different groups: *p < 0.05; **p < 0.01; 3. Tumor proliferation rate T / C (%) = T (Average tumor volume of the treatment group) / TV C (Average tumor volume of the negative control group) × 100%. 4. The CI value was calculated based on the D28 tumor proliferation rate (T / C%) of the combination group and the corresponding single drug group. The calculation formula is as follows: Combination Index (CI) = AB(T / C%) / A(T / C%)*B(T / C%); when 0.3 < CI < 0.7, it is considered that there is an obvious synergistic effect between the two drugs; when 0.7 ≤ CI < 0.85, it is considered that there is a weak synergistic effect between the two drugs; when 0.85 ≤ CI < 1, it is considered that there is an additive effect between the two drugs;

[0227] 5. PK results

[0228] On the 28th day of administration, plasma samples were collected at 0.5 h, 1 h, 2 h, 4 h, and 8 h after administration. For each administration group, 3 mice were collected at each time point, and 40 - 50 μL of whole blood was collected for each sample, anticoagulated with EDTA-K2, and about 20 μL of plasma was obtained and frozen quickly. On the 29th day of administration, tumor samples were collected 2 h after administration. About 50 - 100 mg of tumor was collected from each mouse and frozen quickly. The contents of RP-6306 and the compound of formula (I) in plasma and tumor were detected respectively, and AUC was calculated.

[0229] Table d. Content of RP-6306 in plasma and tumor samples Note: T / P Ratio: Ratio of concentration in tumor to concentration in plasma

[0230] Table 8 Content of the compound of formula (I) in plasma and tumor samples

[0231] As can be seen from Tables 7 and 8, the exposure of the compound of formula (I) in plasma and tumor tissue in subcutaneous xenografts of OVCAR3 cells, whether used alone or in combination, was superior to that of the RP-6306 control group.

[0232] Biological Test Example 8: Cell Proliferation Inhibition Test Using a Compound in Combination with Gemcitabine

[0233] 1. Purpose of the experiment

[0234] The proliferation inhibitory activity of the compound of formula (I) in combination with gemcitabine on breast cancer cell lines was studied by CTG (Cell Titer-Glo Luminescent viability assay) to evaluate the combined effect of the two.

[0235] 2. Experimental Materials

[0236] 2.1 Cell Information

[0237] Cell Information

[0238] 2.2 Sample Information

[0239] Compound of formula (I) (self-made);

[0240] Gemcitabine hydrochloride (purchased from Selleck chem)

[0241] 2.3 Other reagents and consumables

[0242] Other reagents and consumables information

[0243] 3. Experimental Methods

[0244] 3.1 Experimental steps

[0245] (1) Cell inoculation

[0246] The day before compound treatment, cells were collected and centrifuged, counted using a cell counter, and diluted to the specified concentration using the corresponding culture medium according to the counting results. Cells were added to the corresponding 96-well cell culture plates as required using a multichannel liquid dispensing system and cultured in a 37°C incubator overnight.

[0247] (2) Preparation of compound working solution (10× final concentration) and dosing

[0248] The compound was diluted in a series of steps: the starting concentration of the compound of formula (I) was 1000 nM, and the dilution series was repeated 2-fold for a total of 5 concentrations; the starting concentration of gemcitabine was 100 nM, and the dilution series was repeated 3-fold for a total of 5 concentrations. For dosing, the compound working solution at each concentration was added to the designated wells according to the pre-set plate layout. Treatment was continued for 7 days.

[0249] (3) Detection

[0250] After the incubation, remove the culture plate, equilibrate to room temperature for 30 minutes, and take out the Cell The reagent was equilibrated to room temperature for 30 minutes and mixed. 80 μL Cell The reagent was shaken on a microplate constant temperature shaker at 200 rpm for 10 minutes. The above operations should be carried out under light-proof conditions. The luminescence signal was detected using an Envision microplate reader (PerkinElmer).

[0251] 3.2 Data Analysis

[0252] SynergyFinder online analysis software (https: / / synergyfinder.fimm.fi / ) was used to evaluate drug synergy, and the ZIP (Zero-inflated Poisson) regression mixed model was used for calculation.

[0253] 4. Experimental Results

[0254] It is generally accepted in the art that a ZIP score greater than 10 indicates synergistic effects. The ZIP score for the combination of the compound of formula (I) and gemcitabine is 19.51. See Figure 1 for a specific dose-dependency matrix. These experimental results demonstrate that the combination of the two drugs exhibits a synergistic anti-tumor effect.

[0255] Biological Test Example 9 In Vivo Tolerance Study in Mice

[0256] 1. Purpose of the experiment

[0257] The tolerability of the compound of formula (I) was evaluated in BALB / c Nude mice.

[0258] 2. Experimental Materials

[0259] 2.1 Experimental animals and breeding environment

[0260] Species: Mouse

[0261] Strain: BALB / c Nude mice

[0262] Arrival age: 6-8 weeks

[0263] Gender: Female

[0264] Weight: 18-22 grams

[0265] Supplier: Beijing Weitonglihua Laboratory Animal Co., Ltd.

[0266] Animal Certificate: 20230003028486

[0267] 2.2 Compound Information

[0268] Table a. Compound information Note: 1. Gemcitabine hydrochloride is gemcitabine hydrochloride

[0269] 3. Experimental methods and steps

[0270] 3.1 Experimental Grouping

[0271] The experimental groups and dosing schedules are shown in the table below.

[0272] Table b. Experimental animal groups and dosing regimens Note: 1. N: number of mice per group; 2. Dosing volume: 10 μL / g based on mouse body weight; 3. Vehicle 1: 0.5% methylcellulose (400 cp); Vehicle 2: normal saline; 4. BID: twice-daily dosing; D: days; QW: once-weekly dosing; 5. po: oral administration; ip: intraperitoneal injection.

[0273] 3.2 Preparation of test substances

[0274] Table c. Preparation method of test substances Note: Gently mix the drug thoroughly before administration. The compound of formula (I) and Gemcitabine hydrochloride should be prepared daily.

[0275] 3.3 Daily observation of experimental animals

[0276] The animals' health and mortality were monitored daily. Routine inspections included observing the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual inspection only), weight change (measured daily), physical signs, or other abnormalities. The number of deaths and adverse reactions within each group was recorded based on the number of animals in each group.

[0277] 3.4 Experimental indicators

[0278] The mice were weighed daily and the BWC (%) was calculated as follows: BWC (%) = (body weight of a treatment group on the day of dosing - body weight of the treatment group at the start of dosing) / body weight of the treatment group at the start of dosing × 100%. A BWC < -10% indicates that the mice are intolerant to the compound.

[0279] 4. Experimental Results and Analysis

[0280] The changes in mouse body weight are shown in Table 9 and Figure 2. The experimental results showed that the compound of formula (I) was well tolerated in Balb / c Nude mice at both 15 mg / kg and 20 mg / kg doses alone; and the compound of formula (I) at 10 mg / kg and in combination with Gemcitabine hydrochloride at 20 mg / kg was also well tolerated in Balb / c Nude mice.

[0281] Table 9 Mouse body weight Note: 1. BWC% was calculated based on the body weight of mice on day 6 after administration; 2. Vehicle 1: 0.5% methylcellulose (400 cp), Vehicle 2: normal saline;

[0282] Experimental Example 1 TS-FeSSIF & TS-FeSSGF Solubility Test

[0283] 1. Preparation of FeSSIF

[0284] Prepare buffer B: Dissolve 4.040 g of sodium hydroxide, 8.650 g of glacial acetic acid, and 11.874 g of sodium chloride in approximately 900 ml of ultrapure water. Adjust the pH of the solution to 5.0 with 1 mol / L sodium hydroxide or 1 mol / L hydrochloric acid. Then dilute the solution to 1000 mol with ultrapure water at room temperature.

[0285] Add powder: Add 11,200 g of FaSSIF, FeSSIF, and FaSSGF powder to approximately 500 mL of Buffer B. Stir until the powder is completely dissolved. Then dilute the solution to 1000 mL with Buffer B at room temperature.

[0286] Ready to use: Use within 48 hours at room temperature or within 24 hours at 37°C.

[0287] 2. Preparation of FeSSGF

[0288] Prepare buffer D: dissolve 1.220 g of sodium acetate, 0.514 g of glacial acetic acid, and 6.926 g of sodium chloride in approximately 500 mL of ultrapure water.

[0289] Mix with milk: Mix with milk in an equal volume (1:1) and adjust the pH of the solution to 5.0 with 1 mol / L hydrochloric acid.

[0290] Ready to use: Use within 48 hours at room temperature or within 24 hours at 37°C.

[0291] 3. Solubility determination:

[0292] Stock solutions of test compounds and control compounds were prepared in DMSO (Solarbio S&T Co., LTD) at a concentration of 10 mmol / L.

[0293] First, 50 microliters of stock solution (10 mmol / L) of each sample was added to a vial of an uncovered solubility sample plate. The assay was performed in duplicate. DMSO was then evaporated using a centrifugal vacuum evaporator. 500 microliters of buffer were added to dissolve the samples separately. A stirring bar was placed on each vial and sealed with a PTFE / silicone stopper. The sample plate was then transferred to an Eppendorf Thermomixer Comfort plate shaker and shaken at 1100 rpm for 24 hours at 25°C. Afterwards, the stirring bar was removed using a large magnet and the sample was transferred from the solubility sample plate to a filter plate. The supernatant was centrifuged at 4,000 rpm and 25°C for 30 minutes. 350 microliters of aliquots were transferred from the supernatant. The tip was placed in acetonitrile for 5 seconds and then in water for 5 seconds. The first 25 microliters were discarded and 300 microliters were distributed to another 96-bottle glass insert plate and centrifuged again (4,000 rpm, 25°C, 30 minutes). Remove a 5 μL aliquot of the supernatant and 5 μL of DMSO, then add 490 μL of a 1:1 mixture of water and acetonitrile containing an internal standard. Based on peak shape, dilute the solution with a certain ratio of ultrapure water. The dilution factor varies depending on the solubility value and LC-MS signal response.

[0294] Add 50 μL of stock solution (10 mmol / L) of each sample to a vial of an uncovered solubility plate. The assay was performed in duplicate. The DMSO was then evaporated using a centrifugal vacuum evaporator. 500 μL of DMSO was added to dissolve the sample. A stirring bar was placed on each vial and sealed with a molded PTFE / silicone stopper. The solubility plate was then transferred to an Eppendorf Thermomixer Comfort plate shaker and shaken at 1100 rpm for 2 hours at 25°C. After 2 hours, each compound should be completely dissolved. Take a 10 μL aliquot and add 990 μL of a 1:1 mixture of water and acetonitrile containing an internal standard to 10 μL. The concentration of the standard sample may vary depending on the LC / MS signal response.

[0295] All calculations were performed using Microsoft Excel. Samples were analyzed and quantified using LC / MS / MS based on standards of known concentrations. The solubility of the test compound was calculated as follows: [Sample] = (Area ratio Sample ×DF Sample ×[STD]) / Area ratio STD , DF represents the dilution factor.

[0296] The experimental results are shown in Table 10:

[0297] Table 10 Solubility test results

[0298] The compound of formula (I) has good solubility, which is beneficial to in vivo absorption and the development of subsequent preparations.

Claims

1. Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in combination for preparing a medicament for treating cancer mediated by one or more mutations selected from CCNE1 amplification, FBXW7 inactivating mutation, and PPP2R1A inactivating mutation.

2. The use according to claim 1, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are administered simultaneously, sequentially or intermittently.

3. The use according to claim 1 or 2, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are administered to a subject in the form of a compound preparation.

4. The use according to any one of claims 1 to 3, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof are independently administered to the subject via any one of oral, parenteral, rectal, dermal, nasal or inhalation routes, and intratumoral administration.

5. The use according to any one of claims 1 to 4, wherein the cancer is selected from solid tumors; Preferably, the cancer is selected from breast cancer, ovarian cancer, gastric cancer, esophageal cancer, lung cancer, endometrial cancer, uterine carcinosarcoma, cervical cancer, colorectal cancer, and bladder cancer.

6. A pharmaceutical combination comprising the following active ingredients, The first component: a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and The second component: gemcitabine or a pharmaceutically acceptable salt thereof, 7. The pharmaceutical combination according to claim 6, wherein the first component and the second component exist independently in the same dosage form or in different dosage forms, or the first component and the second component exist in the same preparation.

8. The pharmaceutical combination of claim 6, wherein: The drug combination is a fixed combination; or The drug combination is a non-fixed combination; Optionally, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the fixed combination are present in the same pharmaceutical composition; Optionally, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition; Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition, and the pharmaceutical composition of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of gemcitabine or a pharmaceutically acceptable salt thereof are present in the same medicine bag; Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition, and the pharmaceutical composition of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of gemcitabine or a pharmaceutically acceptable salt thereof are not present in the same medicine bag; Preferably, the second component is the hydrochloride salt of gemcitabine.

9. The pharmaceutical combination according to any one of claims 6 to 8, wherein: The weight ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to gemcitabine or a pharmaceutically acceptable salt thereof (calculated based on the weight of the corresponding free compound, compound represented by formula (I): gemcitabine) is 1:20 to 20:1, 1:10 to 15:1, 0.4:1 to 15:1, 1.2:1 to 12:1 or 1.2:1 to 8:

1.

10. A pharmaceutical kit comprising the pharmaceutical combination according to any one of claims 6 to 9.

11. The kit according to claim 10, wherein the first component and the second component are present independently in the same dosage form or in different dosage forms, or the first component and the second component are present in the same preparation; Optionally, the kit further comprises instructions for the combined use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and gemcitabine or a pharmaceutically acceptable salt thereof.

12. Use of the pharmaceutical combination according to any one of claims 6 to 9 or the pharmaceutical kit according to claim 10 or 11 in the preparation of a medicament for treating cancer mediated by one or more mutations selected from CCNE1 amplification, FBXW7 inactivating mutation, and PPP2R1A inactivating mutation.

13. The use according to claim 12, wherein the cancer is selected from: solid tumors; Preferably, the cancer is selected from breast cancer, ovarian cancer, gastric cancer, esophageal cancer, lung cancer, endometrial cancer, uterine carcinosarcoma, cervical cancer, colorectal cancer, and bladder cancer.

14. The use according to any one of claims 1 to 5 and 12 to 13, wherein: The average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 0.01 mg / kg to 200 mg / kg, 0.1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, or 2 mg / kg to 20 mg / kg, calculated as the weight of the compound of formula (I); and / or The average weekly dose of gemcitabine or a pharmaceutically acceptable salt thereof is 0.1 mg / kg to 200 mg / kg, 5 mg / kg to 100 mg / kg, 10 mg / kg to 80 mg / kg, 10 mg / kg to 60 mg / kg, 15 mg / kg to 35 mg / kg, or 10 mg / kg to 30 mg / kg, calculated as the weight of gemcitabine.

Images