Pharmaceutical combination comprising KRAS inhibitor, composition comprising same, and use thereof

Abstract

A pharmaceutical combination comprising a KRAS inhibitor, a composition comprising same, and a use thereof. The pharmaceutical combination A comprises a substance X and a substance Y, wherein the substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is one or more of fedratinib, ruxolitinib, and baricitinib; the substance Y is a compound comprising a fragment as shown in formula I or a pharmaceutically acceptable salt thereof. The combined use of the KRAS inhibitor and the JAK inhibitor has a synergistic effect, and the application prospect is good.

Description

A drug combination containing a KRAS inhibitor, a composition containing the same, and its application

[0001] This application claims the benefit of Chinese patent application No. 2023112633729, filed on September 27, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to a drug combination containing a KRAS inhibitor, a composition containing the same and applications thereof. Background Art

[0003] Pancreatic cancer is the fourth leading cause of cancer death worldwide, with a five-year survival rate of less than 9%. Its incidence ranks among the top 10 of all malignant tumors, and its incidence is steadily increasing. Generally speaking, the incidence of pancreatic cancer is higher in developed countries than in developing countries. Pancreatic cancer is a highly malignant digestive system tumor, with an estimated 120,000 new cases and deaths in China in 2022. By 2040, the rate of new cases and deaths from pancreatic cancer is projected to increase by 88.8%. Its mortality rate ranks sixth among all cancers. Surgery currently remains the only potentially curative treatment for pancreatic cancer. Although there are currently no targeted or immunotherapeutic drugs for pancreatic cancer, clinical research on related monoclonal antibodies is ongoing. KRAS point mutations are closely associated with pancreatic cancer. Studies have reported that the KRAS mutation rate in pancreatic ductal adenocarcinoma is approximately 90%, with codon 12 being a hotspot for mutation. Therefore, detecting this point mutation has important clinical value in the diagnosis, prognosis, and monitoring of pancreatic cancer.

[0004] Colorectal cancer is one of the most common malignant tumors of the digestive tract, with its incidence and mortality ranking third and fourth among urban malignant tumors, respectively. Compared with other advanced malignant tumors of the digestive tract, the 5-year survival rate of colorectal cancer patients has been significantly improved due to the widespread use of early diagnosis and treatment, the discovery of new molecular targets, and the use of corresponding targeted drugs. The launch of the targeted drug cetuximab has made great progress in the treatment of colorectal cancer, but approximately 40% of colorectal cancer patients have mutations in the KRAS gene, and these patients cannot benefit from cetuximab treatment, so the treatment of colorectal cancer still faces huge challenges. Exploring new mechanisms of colorectal cancer occurrence and treatment combinations remains an important issue that needs to be addressed in colorectal cancer clinical practice.

[0005] Non-small cell lung cancer (NSCLC) has the highest mortality rate among all lung cancers in my country. NSCLC accounts for over 80% of lung cancers, and most patients are diagnosed at an advanced stage. Therefore, chemotherapy remains the mainstay of treatment. However, chemotherapy-based treatments over the past decade have not yielded significant improvements in the efficacy of NSCLC. KRAS gene mutations are considered a poor prognostic factor in NSCLC. In NSCLC, most studies suggest that patients with KRAS G12C mutations in lung adenocarcinomas have significantly worse overall survival compared to patients with non-mutated KRAS. The KRAS gene mutation rate in NSCLC patients is 9.8%, of which 29.5% harbor G12C mutations. KRAS-mutated NSCLC has undergone treatment through chemotherapy, chemotherapy combined with anti-angiogenic drugs, chemotherapy combined with immunotherapy, and targeted therapies. Amgen's KRAS small molecule inhibitor is approved for NSCLC, but it is ineffective in treating patients with drug resistance or insensitivity.

[0006] New treatment options are needed for KRAS-mutant solid tumors, such as combination therapies targeting different pathways. One promising approach is to use drug combinations as multi-targeted agents. In optimal scenarios, this could yield additive efficacy, such as synergy, potentially mitigating the potential for undertreatment by individual drugs when used alone.

[0007] Compound 1 It is a highly selective small molecule covalent inhibitor of the KRAS G12C mutation, forming an irreversible covalent bond with the unique cysteine ​​residue of KRAS G12C, locking the protein in an inactive state to prevent downstream signaling without affecting wild-type KRAS. Currently, two similar products have been approved by the FDA: AMG510 and MRTX849. Compound 1 has better KRAS inhibition ability.

[0008] With the discovery of abnormal biological functions of the Janus kinase / signal transducer and activator of transcription (JAK / STAT) pathway, a series of targeted drugs targeting this pathway have been clinically developed. Fedratinib is the second targeted drug approved by the US Food and Drug Administration for the treatment of myelofibrosis after ruxolitinib. Fedratinib is an oral JAK2 selective inhibitor with a strong inhibitory effect on JAK2. Its inhibitory effects on JAK1, TYK2, and JAK3 are approximately 30, 100, and 300 times weaker than JAK2, respectively. In addition, it still has the ability to inhibit FLT3 and RET activity. (Wernig G, Kharas MG, Okabe R, et al. Efficacy of TG101348, a selective JAK2 inhibitor in treatment of a murine model of JAK2 V617F-induced polycythemia vera [J]. Cancer Cell, 2008, 13(4): 311-320).

[0009] Fidatinib is a new JAK inhibitor that, by inhibiting the (JAK / STAT) pathway, has demonstrated some efficacy against myelofibrosis symptoms in clinical studies. Due to its ability to inhibit the activity of FLT3, RET, and JAK2, it has a superior effect in inhibiting tumor cell proliferation and has inhibitory effects on some tumor cells.

[0010] However, the combination of fidatinib and KRAS inhibitors has not yet been used to treat solid tumors, especially KRAS mutant solid tumors.

[0011] Summary of the Invention

[0012] The technical problem addressed by the present invention is to provide a drug combination containing a KRAS inhibitor, a composition containing the same, and its use. The KRAS inhibitor of the present invention can be used in combination with a JAK inhibitor. Furthermore, compared with the use of either fidartinib or a KRAS inhibitor alone, the drug combination of the present invention exhibits a synergistic effect and can be used to treat KRAS mutant tumors, showing promising application prospects.

[0013] The present invention provides a pharmaceutical composition A comprising: substance X and substance Y;

[0014] Wherein, the substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is Fidatinib Ruxolitinib and baricitinib One or more of;

[0015] The substance Y is a compound containing a fragment as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0016] in,

[0017] n1 is 0, 1, 2, 3, or 4;

[0018] R 1 is H or halogen;

[0019] Each R 2 are independently C1-C6 alkyl or C1-C6 alkyl substituted by one or more CN;

[0020] Ring A is a benzene ring or a 6-8 membered heterocyclic ring; the heterocyclic ring in the 6-8 membered heterocyclic ring is a saturated or unsaturated monocyclic ring, the heteroatom is N and / or O, and the number of the heteroatoms is 1, 2 or 3;

[0021] Ring B is an unsaturated 6-8 membered heterocyclic ring; in the unsaturated 6-8 membered heterocyclic ring, the heteroatoms are N and / or O, and the number of the heteroatoms is 1, 2 or 3.

[0022] In some embodiments, in Formula I, for Wherein, n3 is 0, 1 or 2;

[0023] n4 is 0 or 1;

[0024] n5 is 0, 1, 2, 3, or 4;

[0025] Each R 3 are independently C1-C6 alkyl or -NR a R b ; preferably methyl or isopropyl;

[0026] Each R 4 independently halogen, C1-C6 alkyl or C1-C6 alkyl substituted by one or more halogens; preferably fluorine, chlorine or -CF3;

[0027] Each R 5 are independently -OH, halogen, C1-C6 alkyl, -NR a R b or C1-C6 alkyl substituted by one or more halogens; preferably -OH, -NH2, -CF3, fluorine, chlorine or methyl;

[0028] R a is H or C1-C6 alkyl;

[0029] R b is H or C1-C6 alkyl.

[0030] In some embodiments, in the pharmaceutical combination A, the compound containing the fragment shown in Formula I is Preferably More preferably

[0031] In some embodiments, in the drug combination A, the JAK inhibitor is fidatinib.

[0032] In some embodiments, the drug combination A has a synergistic effect.

[0033] In some embodiments, in the pharmaceutical combination A, the molar ratio of the substance X to the substance Y may be (0.1-500):1, preferably (5-500):1, and more preferably 5:1, 23:1, 25:1, 125:1, 200:1 or 250:1.

[0034] In some embodiments, the active ingredients of the pharmaceutical combination A consist of substance X as described above and substance Y as described above.

[0035] In some embodiments, the active ingredients of the drug combination A are fidatinib and composition.

[0036] In some embodiments, the active ingredients of the drug combination A are fidatinib and composition.

[0037] In some embodiments, the active ingredients of the drug combination A are fidatinib and composition.

[0038] The present invention provides a pharmaceutical composition B comprising: substance X, substance Y and pharmaceutical excipients;

[0039] Wherein, the substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is Fidatinib Ruxolitinib and baricitinib One or more of;

[0040] The substance Y is a compound containing a fragment as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0041] Among them, n1, R 1 、R2 , Ring A and Ring B are as described above.

[0042] In some embodiments, in the pharmaceutical composition B, the JAK inhibitor is fidatinib.

[0043] In some embodiments, the pharmaceutical composition B has a synergistic effect.

[0044] In some embodiments, the content of the substance X in the pharmaceutical composition B is preferably 50-600 mg; more preferably 100-400 mg; for example, 200 mg.

[0045] In some embodiments, in the pharmaceutical composition B, the molar ratio of the substance X to the substance Y may be (0.1-500):1, preferably (5-500):1, and more preferably 5:1, 23:1, 25:1, 125:1, 200:1 or 250:1.

[0046] In some embodiments, the active ingredient of the pharmaceutical composition B consists of substance X as described above and substance Y as described above.

[0047] In some embodiments, the active ingredients of the pharmaceutical composition B are composed of Fidatinib and composition.

[0048] In some embodiments, the active ingredients of the pharmaceutical composition B are composed of Fidatinib and composition.

[0049] In some embodiments, the active ingredients of the pharmaceutical composition B are composed of Fidatinib and composition.

[0050] In some embodiments, the pharmaceutical composition B may be a separate pharmaceutical composition.

[0051] In some embodiments, the pharmaceutical composition B can be prepared into various suitable dosage forms according to different administration methods, including gastrointestinal dosage forms (such as oral dosage forms) and parenteral dosage forms (such as injection dosage forms, respiratory tract dosage forms, mucosal dosage forms or cavity dosage forms), preferably capsules, tablets, pills, granules, powders or oral liquids.

[0052] In some embodiments, the pharmaceutical composition B is preferably presented in the form of an oral dosage form.

[0053] The present invention provides a pharmaceutical composition C comprising:

[0054] The first pharmaceutical composition C-1 comprises a substance X and a pharmaceutical excipient, wherein the substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is Fidatinib Ruxolitinib and baricitinib One or more of; and,

[0055] The second pharmaceutical composition C-2 comprises substance Y and pharmaceutical excipients, wherein substance Y is a compound containing a fragment as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0056] Among them, n1, R 1 、R 2 , Ring A and Ring B are as described above.

[0057] In some embodiments, the JAK inhibitor is fidatinib.

[0058] In some embodiments, the pharmaceutical composition C has a synergistic effect.

[0059] In some embodiments, the first pharmaceutical composition C-1 can be a separate pharmaceutical composition; and the second pharmaceutical composition C-2 can be a separate pharmaceutical composition.

[0060] In some embodiments, the content of the substance X in the first pharmaceutical composition C-1 is preferably 50-600 mg; more preferably 100-400 mg; for example, 200 mg.

[0061] In some embodiments, in the pharmaceutical composition C, the molar ratio of the substance X to the substance Y may be (0.1-500):1, preferably (5-500):1, and more preferably 5:1, 23:1, 25:1, 125:1, 200:1 or 250:1.

[0062] In some embodiments, the pharmaceutical composition C preferably consists of a first pharmaceutical composition C-1 and a second pharmaceutical composition C-2.

[0063] In some embodiments, the first pharmaceutical composition C-1 is preferably composed of the substance X as described above and pharmaceutical excipients, more preferably composed of Fidatinib and pharmaceutical excipients.

[0064] In some embodiments, the second pharmaceutical composition C-2 is preferably composed of the substance Y as described above and a pharmaceutical excipient, more preferably composed of any of the following compounds and a pharmaceutical excipient: For example, by and pharmaceutical excipients.

[0065] In some embodiments, the first pharmaceutical composition C-1 can be prepared into various suitable dosage forms according to different administration methods, including gastrointestinal dosage forms (e.g., oral dosage forms) and parenteral dosage forms (e.g., injection dosage forms, respiratory tract dosage forms, mucosal dosage forms, or cavity dosage forms), preferably capsules, tablets, pills, granules, powders, or oral liquids; for example, presented in the form of oral dosage forms.

[0066] In some embodiments, the second pharmaceutical composition C-2 can be prepared into various suitable dosage forms according to different administration methods, including gastrointestinal dosage forms (such as oral dosage forms) and parenteral dosage forms (such as injection dosage forms, respiratory tract dosage forms, mucosal dosage forms or cavity dosage forms), preferably capsules, tablets, pills, granules, powders or oral liquids; for example, presented in the form of oral dosage forms.

[0067] In some embodiments, the first pharmaceutical composition C-1 is in the form of an oral dosage form, and the second pharmaceutical composition C-2 is in the form of an oral dosage form.

[0068] The present invention provides a combination medicine kit D, which comprises:

[0069] A first container comprising the first pharmaceutical composition C-1 as described above; and

[0070] A second container comprises the second pharmaceutical composition C-2 as described above.

[0071] The present invention also provides the use of the above-mentioned drug combination A, drug composition B or drug composition C in the preparation of a medicament for a proliferative disease; the proliferative disease is preferably cancer or a tumor; the cancer or tumor is more preferably one or more of intestinal cancer, pancreatic cancer, non-small cell lung cancer, solid tumor, melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial carcinoma, bladder cancer, small cell lung cancer, sarcoma, intestinal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, renal cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell tumor, Wilms' tumor and hepatocellular carcinoma; for example, intestinal cancer, pancreatic cancer, non-small cell lung cancer or a solid tumor.

[0072] The present invention also provides the use of the above-mentioned pharmaceutical composition A, pharmaceutical composition B or pharmaceutical composition C in the preparation of a drug for a proliferative disease carrying a KRAS mutation;

[0073] The proliferative disease is preferably a proliferative disease carrying a KRAS mutant; the KRAS mutant in the proliferative disease with a KRAS mutant is more preferably one or more of A146T, Q61L, Q61K, G12D, G12C, G13D, G12V, G12R and G13C; for example, G12C.

[0074] In some embodiments, the proliferative disease carrying a KRAS mutant can be KRAS G12C mutant colorectal cancer, KRAS G12C mutant pancreatic cancer, KRAS G12C mutant ovarian cancer, KRAS G12C mutant non-small cell lung cancer, or KRAS G12C mutant solid tumor.

[0075] The present invention also provides a method for preventing and / or treating a proliferative disease, comprising administering a therapeutically effective amount of the above-described pharmaceutical combination A, pharmaceutical composition B or pharmaceutical composition C to a subject (e.g., a human) or patient in need thereof; the proliferative disease is as described above.

[0076] The present invention also provides a method for preventing and / or treating a proliferative disease carrying a KRAS mutant, comprising administering a therapeutically effective amount of the above-described drug combination A, drug composition B, or drug composition C to a subject (e.g., a human) or patient in need thereof; the proliferative disease carrying a KRAS mutant is as described above.

[0077] In the above-mentioned pharmaceutical combination A, pharmaceutical composition B, pharmaceutical composition C, their uses and their methods of treatment:

[0078] In some embodiments, the substance X and substance Y can be administered simultaneously or separately.

[0079] The “simultaneous administration” means, for example, that substance X and substance Y are contained in separate pharmaceutical compositions and administered simultaneously; or, alternatively, a “separate pharmaceutical composition containing substance X” and a “separate pharmaceutical composition containing substance Y” are administered simultaneously.

[0080] "Separate administration" means, for example, that a "separate pharmaceutical composition containing substance X" and a "separate pharmaceutical composition containing substance Y" are administered separately at different times, for example, one of the "separate pharmaceutical composition containing substance X" and the "separate pharmaceutical composition containing substance Y" is administered first, and the other is administered subsequently. The separate administration may be close in time or remote in time.

[0081] Whether administered simultaneously or separately, the administration regimens (including administration routes, administration doses, administration intervals, etc.) of substance X and substance Y may be the same or different, and may be adjusted as needed by those skilled in the art to provide the optimal therapeutic effect.

[0082] In some embodiments, the substance X can be administered gastrointestinal tract (e.g., orally, sublingually, or rectally), injected (e.g., subcutaneously, intradermally, intravenously, intraperitoneally, or intramuscularly), respiratory tract, or topically (e.g., mucosa or skin); preferably, it can be administered gastrointestinal tract, such as orally.

[0083] In some embodiments, the substance Y can be administered via the gastrointestinal tract (e.g., orally, sublingually, or rectally), injected (e.g., subcutaneously, intradermally, intravenously, intraperitoneally, or intramuscularly), respiratory tract, or topically (e.g., mucosa or skin); preferably, it can be administered via the gastrointestinal tract, such as orally.

[0084] In some embodiments, the substance X is preferably administered orally; and, the substance Y is preferably administered orally.

[0085] In some embodiments, the total daily dosage of the substance X can be administered according to clinical needs; the total daily dosage is preferably in the range of 50-1000 mg; for example, 200 mg, 400 mg or 600 mg.

[0086] In some embodiments, the administration frequency of the substance X can be adjusted according to clinical needs; preferably once a day or twice a day; for example, once a day.

[0087] In some embodiments, the total daily dosage of the substance X is preferably in the range of 50-1000 mg, such as 200 mg, 400 mg or 600 mg; and the administration frequency is preferably once a day or twice a day, such as once a day.

[0088] In some embodiments, the substance X is administered orally at the total dosage and frequency described above.

[0089] In some embodiments, the substance X is administered orally at a dose of 200 mg to 1000 mg, once daily or twice daily.

[0090] The term "pharmaceutically acceptable salt" refers to salts prepared from compounds of the present invention with relatively nontoxic, pharmaceutically acceptable acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in neat solution or in a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent.

[0091] The term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and in the preparation of prescriptions. They are all substances, other than the active ingredient, contained in a pharmaceutical preparation. For a complete list, see Part IV of the Pharmacopoeia of the People's Republic of China (2015 edition) or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0092] The term "content" refers to the mass of a component contained in a specific substance. For example, "the content of substance X in pharmaceutical composition B" refers to the mass of component X contained in pharmaceutical composition B; "the content of substance X in the first pharmaceutical composition C-1" refers to the mass of component X contained in the first pharmaceutical composition C-1.

[0093] The term "treat" refers to any of the following: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that leads to a disease; or (3) slowing the progression of one or more biological manifestations of a disease.

[0094] The term "therapeutically effective amount" refers to the amount of a compound administered to a subject that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the compound, the type of disease, the severity of the disease, the age of the subject, etc., but can be adjusted by those skilled in the art as appropriate.

[0095] The term "subject" refers to any animal that has been or is about to be treated, preferably a mammal, most preferably a human. Mammals include but are not limited to cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0096] In the structure fragment This refers to the bond through which the structural fragment is connected to the rest of the molecule. For example, It refers to pyridyl.

[0097] The term "one or more" means 1, 2, 3, 4 or 5, for example 1, 2 or 3.

[0098] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0099] The term "alkyl" refers to a linear or branched, saturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

[0100] The term "heterocycle" refers to a cyclic, saturated or unsaturated group having a specified number of ring atoms (e.g., 5-12 members, 6-8 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S, such as N and / or O). When the "heterocycle" is an unsaturated heterocycle (group), it has one or more (e.g., 1, 2, or 3) carbon-carbon sp 2 Double bond. Heterocycles include but are not limited to:

[0101] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0102] The reagents and raw materials used in the present invention are commercially available.

[0103] The positive progress of the present invention is that: (1) the present invention has one or more of the following advantages: KRAS inhibitors can be used in combination with JAK inhibitors, have a good inhibitory effect on cancer cell proliferation (particularly on the proliferation of KRAS mutant cancer cells), can be used to treat KRAS mutant tumors including pancreatic cancer, non-small cell lung cancer, intestinal cancer and solid tumors, and have good application prospects. Furthermore, the combination of KRAS inhibitors and JAK inhibitors is significantly more effective than the use of JAK inhibitors and KRAS inhibitors alone, exerting a synergistic effect.

[0104] The term "synergistic" as used herein refers to the effect of two therapeutic agents (e.g., Fidatinib and Compound 1) that produces an effect (e.g., slowing the progression of symptoms of a proliferative disease, particularly cancer, or its syndrome) that is greater than the effect of simply adding each agent on its own. Furthermore, in some embodiments, the inhibitory effect of the combination of the KRAS inhibitor and the JAK inhibitor of the present invention in an anti-proliferation experiment of KRAS G12C mutant tumor cells satisfies one or more of the following: that is, within the same observation window (72 hours), relative to 100% growth capacity of the cell sample treated with the vehicle DMSO, the combined treatment can significantly reduce the cell proliferation capacity as follows:

[0105] (a) The cell proliferation rate of MIA-PACA2 was 13.2%;

[0106] (b) The cell proliferation rate to MIA-PACA2_R / Compound 1-0.5 μM was 28.6%;

[0107] (c) The cell proliferation rate of NCI-H358 was 29.2%;

[0108] (d) The cell proliferation rate of SW1463 was 38.0%.

[0109] (2) On the other hand, the combination of the KRAS inhibitor and the JAK inhibitor of the present invention overcomes the problem of drug resistance to KRAS drugs in people receiving KRAS G12C drug treatment, and further solves the problem of drug resistance of KRAS mutant tumors including pancreatic cancer, non-small cell lung cancer, intestinal cancer and solid tumors (Zhao, Y., et al., Diverse alterations associated with resistance to KRAS (G12C) inhibition. Nature, 2021. 599 (7886): p. 679-683.). BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 shows the results of the synergistic inhibition of the proliferation of different cancer cells with KRAS G12C mutations by compound 1 and fidatinib in vitro, wherein A shows the combined inhibitory effect of compound 1 (250 nM) and different concentrations of fidatinib (0.039 μM-1.25 μM) on the proliferation of MIA-PACA2 cells; B shows the combined inhibitory effect of compound 1 (10 nM) and different concentrations of fidatinib (0.63 μM-2.5 μM) on the proliferation of MIA-PACA2 cells resistant to compound 1; C shows the combined inhibitory effect of compound 1 (10 nM) and different concentrations of fidatinib (0.63 μM-2.5 μM) on the proliferation of NCI-H358 cells; D shows the combined inhibitory effect of different concentrations of compound 1 (12.5 nM-50 nM) and different concentrations of fidatinib (2.5 μM-10 μM) on the proliferation of MIA-PACA2 cells resistant to compound 1.

[0111] Figure 2 shows the experimental results of the effect of the combination of Compound 1 and Fidatinib on the early apoptosis of MIA-PACA2 cells, Compound 1-resistant MIA-PACA2 cells, NCI-H358 cells and SW1463 cells.

[0112] FIG3 shows the results of clone formation experiments of MIA-PACA2 cells, compound 1-resistant MIA-PACA2 cells, NCI-H358 cells, and SW1463 cells.

[0113] Figures 4 and 5 show the results of protein immunoblotting of MIA-PACA2 cells.

[0114] FIG6 shows the experimental results of the combination of Compound 1 and Fidatinib on the changes in tumor volume in a mouse model of human non-small cell lung cancer.

[0115] FIG7 shows the experimental results of the combined use of compound 1 and fidartinib on the changes in mouse body weight in a human non-small cell lung cancer model.

[0116] FIG8 shows the experimental results of the combination of compound 1 and fidartinib on the changes in tumor volume in a mouse model of human pancreatic cancer.

[0117] FIG9 shows the experimental results of the combined use of compound 1 and Fidatinib on the changes in mouse body weight in a human pancreatic cancer model.

[0118] FIG10 shows the combined inhibitory effect of compound 1 (0.25 μM) and different concentrations of tofacitinib (0.04 μM-1.25 μM) on the proliferation of SW837 cells.

[0119] FIG11 shows the combined inhibitory effect of compound 1 (0.25 μM) and different concentrations of tofacitinib (0.04 μM-1.25 μM) on the proliferation of Calu-1 cells.

[0120] FIG12 shows the combined inhibitory effect of compound 1 (0.25 μM) and different concentrations of tofacitinib (0.04 μM-1.25 μM) on the proliferation of MIA-PACA2 cells.

[0121] FIG13 shows the combined inhibitory effect of compound 1 (0.25 μM) and different concentrations of tofacitinib (0.04 μM-1.25 μM) on the proliferation of NCI-H358 cells. DETAILED DESCRIPTION

[0122] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0123] The experimental groups involved in the following examples are shown in Table 1:

[0124] Table 1

[0125] Example 1 Anti-proliferation experiment of KRAS G12C mutant tumor cells

[0126] Experimental Materials:

[0127] Cells: MIA-PACA2 cells, NCI-H358 cells, SW1463 cells, and DMEM / F12 culture medium were purchased from Wuhan Punosai Biotechnology Co., Ltd.

[0128] MIA-PACA2_R / Compound 1-0.5μM refers to a MIA-PACA2 cell line resistant to Compound 1. This cell line was obtained by long-term (3 weeks) culture with a low concentration (0.5μM) of Compound 1. Culture conditions consisted of DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% carbon dioxide. During drug treatment, the medium was changed twice weekly.

[0129] Reagents: CCK8 reagent, DMEM, RPMI 1640, fetal bovine serum (FBS), 0.25% EDTA trypsin, PBS, and penicillin-streptomycin (P / S) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai Solaibao Biotechnology Co., Ltd.; Fedratinib was purchased from Haoyuan Bio (HY-10409); Compound-1 was synthesized by Wuhan WuXi AppTec Co., Ltd.

[0130] Key instrument: TECAN infiniti200 FPLEX microplate reader

[0131] Solution preparation

[0132] Drug dilution:

[0133] Weigh compound 1 and Fedratinib, dissolve the drugs to 50 mM with DMSO reagent, dilute them with DMSO reagent from the 50 mM stock solution to 200 times the final concentration, and then dilute them with culture medium to 100 times the final concentration;

[0134] Complete medium:

[0135] Prepare DMEM:FBS:P / S at a ratio of 89:10:1.

[0136] Experimental methods:

[0137] 1. Seed 2000 cells in a 96-well plate, add 100 μL of complete culture medium per well, and culture in a cell culture incubator at 37°C for 24 hours;

[0138] 2. Add 1 μL of the drug to be tested to the cells and culture them in a cell culture incubator for 72 hours;

[0139] 3. Add CCK solution (10% of the total volume of culture medium) to each well;

[0140] 4. Continue culturing in the cell culture incubator for 1 hour;

[0141] 5. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0142] Data Analysis: Raw data were converted to cell proliferation rates using the equation (Sample-Min) / (Max-Min) x 100%. Figure 1 shows the difference in cell proliferation between the single-drug and combination-treated groups. Table 2 shows the combined inhibitory effects of different drug combinations and dose combinations in various cell models, including MIA-PACA2 cells, Compound 1-resistant MIA-PACA2 cells, NCI-H358 cells, and SW1463 cells.

[0143] Table 2 Results of antiproliferative experiments on tumor cells with KRAS G12C mutation

[0144] Experimental conclusion: The combination of compound 1 of the present invention and Fedratinib has stronger tumor proliferation inhibitory activity than single drugs, and the combined effect is less than the theoretical synergistic effect threshold.

[0145] Example 2 Apoptosis Experiment of Tumor Cells with KRAS G12C Mutation

[0146] Experimental Materials:

[0147] Cells: MIA-PACA2 cells, NCI-H358 cells, SW1463 cells, and DMEM / F12 culture medium were purchased from Wuhan Punosai Biotechnology Co., Ltd.

[0148] Reagents: DMEM, fetal bovine serum (FBS), 0.25% EDTA trypsin, PBS, and penicillin-streptomycin (P / S) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai Solaibao Biotechnology Co., Ltd.; V / PI apoptosis kit was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd.; ACEA NovoCyte series flow cytometer was purchased from Agilent Technologies Co., Ltd.;

[0149] Solution preparation

[0150] Drug dilution:

[0151] Take 10 mM compound 1 and Fedratinib stock solutions and dilute them with DMSO to the desired concentration;

[0152] Complete medium:

[0153] Prepare DMEM:FBS:P / S at a ratio of 89:10:1.

[0154] Experimental methods:

[0155] 1. Inoculate 1×10 5 Each well contained 1 mL of complete culture medium and cultured in a 37°C cell culture incubator for 24 hours.

[0156] 2. Add 10 μL of the drug to be tested to the cells and culture for 4 hours and 24 hours depending on the cell status;

[0157] 3. Wash the cells once with PBS and add 0.2 mL of trypsin to digest for 2 minutes;

[0158] 4. Resuspend the cells with 1 mL of complete culture medium, transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend with 1 mL of complete culture medium, and culture in a 37°C cell culture incubator for 0.5 hours;

[0159] 5. Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, wash once with 1 mL of pre-chilled PBS, centrifuge and discard the supernatant, then add 100 μL of 1× Annexin V Binding Buffer and gently resuspend the cells.

[0160] 6. Add 5 μL V, mix gently;

[0161] 7. Add 5 μL of PI staining solution and mix gently;

[0162] 8. Incubate at 4°C in the dark for 30 minutes;

[0163] 9. After incubation, add 400 μL of 1× Annexin V binding buffer to resuspend the cells and immediately detect apoptosis signals on the instrument;

[0164] Data analysis: The early apoptosis ratio of the samples was analyzed using ACEA NovoExpress flow cytometry software, and graphed using GraphPad Prism software. Figure 2 shows the difference in early apoptosis between the single-drug treatment group and the combination treatment group.

[0165] Table 3

[0166] Experimental conclusion: The combination of compound 1 of the present invention and Fedratinib has a significant effect in promoting tumor cell apoptosis in tumor cells carrying KRAS G12C.

[0167] Example 3 KRAS G12C mutant tumor cell clone formation experiment

[0168] Experimental Materials:

[0169] Cells: MIA-PACA2 cells, NCI-H358 cells, SW1463 cells, and DMEM / F12 culture medium were purchased from Wuhan Punosai Biotechnology Co., Ltd.

[0170] Reagents: DMEM, fetal bovine serum (FBS), 0.25% EDTA trypsin, PBS, and penicillin-streptomycin (P / S) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai Solaibao Biotechnology Co., Ltd.; crystal violet staining solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0171] Solution preparation

[0172] Drug dilution:

[0173] Take 10 mM compound 1 and Fedratinib stock solutions and dilute them with DMSO to the desired concentration;

[0174] Complete medium:

[0175] Prepare DMEM:FBS:P / S at a ratio of 89:10:1.

[0176] Experimental methods:

[0177] 1. Inoculate 1×10 5 Each well contained 2 mL of complete culture medium and cultured in a 37°C cell culture incubator for 24 hours.

[0178] 2. Add 20 μL of the drug to be tested to the cells and culture them in a 37°C cell culture incubator for another 48 hours;

[0179] 3. Remove the cells and wash twice with pre-cooled PBS;

[0180] 4. Fix with pre-cooled 4% paraformaldehyde (Biosharp, 4%, Cat. No. BL539A) for 10 minutes;

[0181] 5. Discard the paraformaldehyde, equilibrate to room temperature, add crystal violet staining solution, stain for 30 minutes, and observe whether the color is formed;

[0182] 6. Discard the staining solution and rinse with purified water three times;

[0183] 7. After drying at room temperature, take photos and analyze;

[0184] Data Analysis: Under brightfield conditions, a piece of white paper was placed under the six-well plate, and photos were taken with a camera and plotted. OD590 spectrophotometric analysis of selected images (3E, 3G) was used to quantify the relative number of colonies (Figures 3F, 3H). The results are shown in Figure 3.

[0185] Experimental conclusion: In tumor cells carrying KRAS G12C mutations, the combination of compound 1 and Fedratinib significantly inhibited the formation of tumor cell clones compared to the monotherapy group (Figure 3A-D). The combination of two other KRAS G12C inhibitors, MRTX849 and AMG510, with Fedratinib also significantly enhanced the tumor clone inhibition effect (Figure 3E-H). This experiment shows that not only compound 1 can be used in combination with Fedratinib, but other representative KRAS G12C inhibitors also have similar effects.

[0186] Example 4 Western Blot Experiment of Compound 1 and Fedratinib

[0187] Experimental Materials:

[0188] Cells: MIA-PACA2 cells were purchased from Wuhan Punosai Biotechnology Co., Ltd.

[0189] Reagents: DMEM, fetal bovine serum (FBS), 0.25% EDTA trypsin, PBS, penicillin-streptomycin (P / S), 6× loading buffer, and protein marker (10-180 kDa) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai Solaibao Biotechnology Co., Ltd.; pErk1 / 2 (T202 / Y204) antibody, pSTAT3 (Y705) antibody, and pJAK2 (Y1007 / 1008) antibody were purchased from Cell Signaling Technology (CST); GAPDH antibody, ammonium persulfate (APS), glycine, Tris, and SDS were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; goat anti-rabbit IgG (HRP) antibody and RIPA protein lysis buffer were purchased from Lanjieke Technology Co., Ltd.; 20× TBS, 10× electroporation buffer, 30% acrylamide, 1.5 M Tris-HCl pH 8.8, 1.0 M Tris-HCl pH 6.8 and Tween-20 were purchased from Shanghai Solaibao Biotechnology Co., Ltd.; BCA protein quantification kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; skim milk powder was purchased from Inner Mongolia Yili Industrial Group Co., Ltd.; PVDF membrane and ECL luminescence colorimetric solution were purchased from Merck Millipore; methanol was purchased from Hangzhou Mico Chemical; Bio-Rad electrophoresis and blotting system was purchased from Bio-Rad, USA; and chemiluminescence / fluorescence image analysis system was purchased from Shanghai Tianneng Technology Co., Ltd.

[0190] Solution preparation

[0191] 10× SDS-PAGE electrophoresis buffer: Dissolve 30.2 g of Tris powder, 144 g of glycine, and 10 g of SDS in 1 mL of deionized water. Dilute to 1× electrophoresis buffer with deionized water before use.

[0192] 1× TBST buffer: Take 50 mL of 20× TBS, dilute it in 1 L of deionized water, and add 1 mL of Tween-20;

[0193] 5% blocking solution: 2.5 g skim milk powder dissolved in 50 mL of 1× TBST;

[0194] Drug dilution: Take 10 mM compound 1 and Fedratinib stock solutions and dilute them with DMSO to the desired concentration;

[0195] Complete medium:

[0196] Prepare DMEM:FBS:P / S at a ratio of 89:10:1.

[0197] Experimental methods:

[0198] 1. Seed 2 × 10 cells in a 12-well plate 5 MIA-PACA2 cells were cultured in 1 mL of complete medium per well in a 37°C cell culture incubator for 24 hours.

[0199] 2. Prepare 1.5mm thick protein glue;

[0200] 3. Add 10 μL of the drug to be tested to the cells and culture them in a 37°C cell culture incubator for another 2 hours;

[0201] 4. Remove the cells, wash once with PBS, and discard the supernatant;

[0202] 5. Add 200 μL RIPA protein lysis buffer, lyse on ice, and transfer to a 1.5 mL centrifuge tube;

[0203] 6. Centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0204] 7. Take the supernatant, quantify the protein by BCA method, and add 6× loading buffer to prepare the sample;

[0205] 8. Protein electrophoresis;

[0206] 9. Dilute 10× electrotransfer buffer to 1× electrotransfer buffer, remove the protein gel, and transfer the membrane using the wet transfer method;

[0207] 10. After transfer, block with 5% blocking solution at room temperature for 1 hour;

[0208] 11. Wash three times with 1× TBST buffer and incubate with antibodies;

[0209] 12. Incubate with ECL luminescent solution, detect chemiluminescence, and take photos;

[0210] Data Analysis: The Tianneng Chemiluminescence / Fluorescence Image Analysis System was used to develop and photograph the samples. The results are shown in Figures 4 and 5.

[0211] Experimental conclusion: In tumor cells carrying the KRAS p.G12C mutation, the combination of compound 1 and Fedratinib can inhibit the phosphorylation level of STAT3 compared with the single-drug group, and has a stronger inhibitory effect on the phosphorylation of ERK, an important downstream regulatory protein of KRAS, and has a significant blocking effect on downstream signal activation, inducing cell apoptosis.

[0212] Example 5 Pharmacological and efficacy study of compound 1 combined with Fedratinib in a mouse model of human non-small cell lung cancer

[0213] Experimental animals:

[0214] Balb / c nude female mice, 5 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0215] Modeling method and grouping: NCI-H358 non-small cell lung cancer cells (Human Non-small Cell Lung Cancer Cells) were isolated and established from the tumor tissue of a patient before chemotherapy in 1981. This cell line carries the KRAS p.G12C heterozygous mutation.

[0216] NCI-H358 cells were cultured in RPMI-1640 (15% FBS + 1% Penicillin-Streptomycin Solution) at 37°C with 5% CO2. After cell digestion and counting, the cell suspension was mixed with Matrigel (Corning HC 354248) in a 1:1 ratio and kept on ice. 1*10E7 cells were inoculated subcutaneously in the right armpit of mice. Eleven days after inoculation, the tumor volume reached an average of 200 mm. 3 Start group dosing.

[0217] Table 4

[0218] Dosing treatment and data analysis: Dosing was stopped on the 21st day of inoculation, and endpoint statistical analysis was performed on the 24th day to compare the changes in mouse tumor volume. The results are shown in Figures 6 and 7. The tumor in the control group showed a growth trend, with an average growth of 276.5%. The tumor in the GEC5 group remained stable with no obvious tumor shrinkage effect. The FED7 group had no obvious tumor shrinkage effect and could not maintain tumor stability. Comb7 significantly shrank the tumor, with an average tumor shrinkage rate of 33.9%. From the overall efficacy analysis, it was found that the GEC5 group and the Comb7 group had significant tumor inhibitory effects compared with the control group (GEC5: pvalue = **, Comb7: p value = ***), and the FED7 group had no significant inhibitory effect on tumor growth. The Comb7 group had a significant inhibitory effect on tumor growth compared with the GEC5 monotherapy group and the FED7 group (p value = **).

[0219] * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001

[0220] Experimental conclusion:

[0221] In the NCI-H358 non-small cell lung cancer (CDX) mouse model, GEC5 monotherapy significantly inhibited tumor growth, with a 50.58% inhibition rate, compared to 100% tumor growth in the control group. Tumor volume remained stable and showed no reduction. Fedratinib monotherapy had no significant inhibitory effect, with a 28.96% inhibition rate. Comb7, in combination with GEC5, significantly inhibited tumor proliferation, with a 73.57% inhibition rate and significantly superior tumor reduction to GEC5 and Fedratinib monotherapy. Compared to the control group, which had a 100% inhibition rate in TGI tumor growth, safety data showed no significant changes in animal body weight at the end of dosing on day 24 post-inoculation in the Fedratinib, GEC5, and Comb7 monotherapy groups, indicating a lack of toxicity.

[0222] Example 6 Pharmacological and efficacy experiments of compound 1 combined with Fedratinib in a mouse pancreatic cancer tumor model

[0223] Experimental animals:

[0224] Balb / c nude female mice, 5 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0225] Modeling and grouping: MIA-PACA2 cells were purchased from Wuhan Pronocell Biotechnology Co., Ltd. and cultured in DMEM (10% FBS + 1% Penicillin-Streptomycin Solution) at 37°C with 5% CO2. After cell digestion and counting, the cells were kept on ice throughout the process. 5*10E6 cells / cell were inoculated subcutaneously in the right axilla of mice. 30 days after inoculation, the tumor volume reached an average of 200 mm 3 Start group medication around 12 o'clock.

[0226] Table 5

[0227] Data Analysis:

[0228] After 3 days of drug treatment, Comb7 demonstrated a significant tumor growth inhibitory effect, with superior tumor suppression ability compared to the GEC5 and FED7 groups. Drug administration was discontinued on the 10th day after treatment, endpoint statistics were obtained on the 11th day, and the changes in tumor volume in mice before and after treatment were analyzed and compared on the 12th day. The results are shown in Figures 8 and 9. Tumors in the control group showed a significant growth trend, with an average growth rate of 162.7%. Tumor volume in the GEC5 group remained stable with no growth. The FED7 group had poor tumor suppression ability, with a tumor growth trend. The Comb7 combination treatment group showed significant tumor shrinkage, with an average tumor shrinkage rate of 78.0%. Overall efficacy analysis showed no significant difference in tumor suppression between the GEC5 and FED7 groups and the control group (p value > 0.05). The Comb7 group demonstrated a significant tumor growth inhibitory effect compared to the control group (p value < 0.0001****).

[0229] Experimental conclusion: In the MIA-PACA2 human pancreatic cancer cell subcutaneous tumor model, the combination of compound 1 of the present invention and Fedratinib has a tumor growth inhibitory effect.

[0230] Comparative Example: The combination of Compound 1 and Tofacitinib showed no obvious synergistic effect in inhibiting the proliferation of different cell lines with KRAS G12C mutation.

[0231] Experimental Materials:

[0232] Cells: MIA-PACA2 cells, NCI-H358, Calu-1, SW837 cells, and DMEM / F12 culture medium were purchased from Wuhan Punosai Biotechnology Co., Ltd.

[0233] Reagents: DMEM, RPMI 1640, fetal bovine serum (FBS), 0.25% EDTA trypsin, PBS, and penicillin-streptomycin (P / S) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai Solaibao Biotechnology Co., Ltd.; Tofacitinib (MB4155) was purchased from Dalian Meilun Biotechnology Co., Ltd.; and CTG kit (HY-K0302) was purchased from MCE Company.

[0234] Solution preparation

[0235] Drug dilution:

[0236] Weigh compound 1 and Tofacitinib, dissolve the drugs to 50 mM with DMSO reagent, dilute them with DMSO reagent from the 50 mM stock solution to 200 times the final concentration, and then dilute them with culture medium to 100 times the final concentration;

[0237] Complete medium:

[0238] Prepare DMEM:FBS:P / S at a ratio of 89:10:1.

[0239] Experimental methods:

[0240] 1. Seed 5000 cells in a 96-well plate, add 100 μL of complete culture medium per well, and culture in a cell culture incubator at 37°C for 24 hours;

[0241] 2. Add 1 μL of the drug to be tested to the cells and culture them in a cell culture incubator for 72 hours;

[0242] 3. Add 10% of the total volume of culture medium to each well of CTG solution;

[0243] 4. Continue culturing in the cell culture incubator for 1 hour;

[0244] 5. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0245] Data analysis: The raw data were converted into cell proliferation rate using the equation (Sample-Min) / (Max-Min)×100%.

[0246] Experimental Conclusions: As shown in Figures 10-13, using a cell proliferation assessment method similar to that in Example 1, Compound 1 produced approximately 50% growth inhibition at a concentration of 250 nM in various human tumor cells carrying the KRAS p.G12C mutation, such as SW837 and Calu-1 cells. Compound 1 produced approximately 70% growth inhibition at a concentration of 250 nM in MIA-PACA2 cells, and approximately 90% growth inhibition at a concentration of 250 nM in NCI-H358 cells. Tofacitinib alone had no growth inhibitory effect in these cells at any of the three different concentrations. Accordingly, the combination of Compound 1 with different concentrations of the JAK inhibitor Tofacitinib did not produce any synergistic or additive effects in these cells.

[0247] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A pharmaceutical combination A, comprising: substance X and substance Y; in, The substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is one or more of fidatinib, ruxolitinib and baricitinib; The substance Y is a compound containing a fragment as shown in formula I or a pharmaceutically acceptable salt thereof; in, n1 is 0, 1, 2, 3 or 4; R 1 is H or halogen; Each R 2 is independently C1-C6 alkyl or C1-C6 alkyl substituted by one or more CN; Ring A is a benzene ring or a 6-8 membered heterocyclic ring; the heterocyclic ring in the 6-8 membered heterocyclic ring is a saturated or unsaturated monocyclic ring, the heteroatom is N and / or O, and the number of the heteroatom is 1, 2 or 3; Ring B is an unsaturated 6-8 membered heterocyclic ring; in the unsaturated 6-8 membered heterocyclic ring, the heteroatom is N and / or O, and the number of the heteroatom is 1, 2 or 3.

2. The pharmaceutical combination A according to claim 1, characterized in that: In Formula I, for Wherein, n3 is 0, 1 or 2; n4 is 0 or 1; n5 is 0, 1, 2, 3 or 4; Each R 3 are independently C1-C6 alkyl or -NR a R b ; preferably Methyl or isopropyl; Each R 4 independently halogen, C1-C6 alkyl or C1-C6 alkyl substituted by one or more halogens; preferably fluorine, chlorine or -CF3; Each R 5 are independently -OH, halogen, C1-C6 alkyl, -NR a R b or C1-C6 alkyl substituted by one or more halogens; preferably -OH, -NH2, -CF3, fluorine, chlorine or methyl; R a is H or C1-C6 alkyl; R b It is H or C1-C6 alkyl.

3. The pharmaceutical combination A according to claim 2, characterized in that: The compound containing the fragment shown in formula I is Preferably More preferably 4. The pharmaceutical combination A according to claim 3, characterized in that: It meets one or more of the following conditions: (1) In the drug combination A, the JAK inhibitor is Fidatinib; (2) In the drug combination A, the molar ratio of the substance X to the substance Y is (0.1-500):1, preferably (5-500):1, more preferably 5:1, 23:1, 25:1, 125:1, 200:1 or 250:1; and (3) The drug combination A has a synergistic effect.

5. The pharmaceutical combination A according to claim 4, characterized in that: The active ingredients of the drug combination A are composed of the substance X and the substance Y; Preferably, the drug combination A is any of the following combinations: (1) Fidatinib and composition; (2) Fidatinib and composition; (3) Fidatinib and composition.

6. A pharmaceutical composition B comprising: substance X, substance Y and pharmaceutical excipients; in, The substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is one or more of fidatinib, ruxolitinib and baricitinib; The substance Y is a compound containing a fragment as shown in formula I or a pharmaceutically acceptable salt thereof; Among them, n1, R 1 , R 2 , Ring A and Ring B are as described in any one of claims 1 to 3.

7. The pharmaceutical composition B according to claim 6, characterized in that It meets one or more of the following conditions: (1) In the pharmaceutical composition B, the JAK inhibitor is Fidatinib; (2) In the drug combination B, the content of the substance X is preferably 50-600 mg; more preferably 100-400 mg; for example 200 mg; (3) In the pharmaceutical composition B, the molar ratio of the substance X to the substance Y may be (0.1-500):1, preferably (5-500):1, more preferably 5:1, 23:1, 25:1, 125:1, 200:1 or 250:1; (4) The pharmaceutical composition B is a single pharmaceutical composition; (5) The dosage form of the pharmaceutical composition B is a gastrointestinal dosage form and a parenteral dosage form; the gastrointestinal dosage form is preferably an oral dosage form; the parenteral dosage form is preferably an injection dosage form, a respiratory dosage form, a mucosal dosage form or a cavity dosage form; more preferably an oral dosage form; for example, a capsule, a tablet, a pill, a granule, a powder or an oral solution; and (6) The pharmaceutical composition B has a synergistic effect.

8. The pharmaceutical composition B according to claim 7, characterized in that The active ingredients of the pharmaceutical composition B are composed of the substance X and the substance Y; Preferably, the active ingredient of the pharmaceutical composition B is any combination of the following: (1) Fidatinib and composition; (2) Fidatinib and composition; (3) Fidatinib and composition.

9. A pharmaceutical composition C, comprising: A first pharmaceutical composition C-1, comprising a substance X and a pharmaceutical excipient, wherein the substance X is a JAK inhibitor or a pharmaceutically acceptable salt thereof; the JAK inhibitor is one or more of fidatinib, ruxolitinib and baricitinib; and The second pharmaceutical composition C-2 comprises a substance Y and a pharmaceutical excipient, wherein the substance Y is a compound containing a fragment as shown in Formula I or a pharmaceutically acceptable salt thereof; in, n1、R 1 , R 2 , Ring A and Ring B are as described in any one of claims 1 to 3.

10. The pharmaceutical composition C according to claim 9, characterized in that It meets one or more of the following conditions: (1) The JAK inhibitor is Fidatinib; (2) The first pharmaceutical composition C-1 is a single pharmaceutical composition; the second pharmaceutical composition C-2 is a single pharmaceutical composition; (3) In the first pharmaceutical composition C-1, the content of the substance X is preferably 50-600 mg; more preferably 100-400 mg; for example 200 mg; (4) In the pharmaceutical composition C, the molar ratio of the substance X to the substance Y is (0.1-500):1, preferably (5-500):1, more preferably 5:1, 23:1, 25:1, 125:1, 200:1 or 250:1; (5) The pharmaceutical composition C consists of a first pharmaceutical composition C-1 and a second pharmaceutical composition C-2; (6) The first pharmaceutical composition C-1 is composed of the substance X and pharmaceutical excipients, preferably consisting of Fidatinib and pharmaceutical excipients. become; (7) The second pharmaceutical composition C-2 is composed of the substance Y and a pharmaceutical excipient, preferably any of the following compounds and a pharmaceutical excipient: More preferably by and pharmaceutical excipients; (8) The dosage form of the first pharmaceutical composition C-1 is a gastrointestinal dosage form or a parenteral dosage form; the gastrointestinal dosage form is preferably an oral dosage form; the parenteral dosage form is preferably an injection dosage form, a respiratory dosage form, a mucosal dosage form or a cavity dosage form; more preferably an oral dosage form; for example, a capsule, a tablet, a pill, a granule, a powder or an oral liquid; (9) The dosage form of the first pharmaceutical composition C-2 is a gastrointestinal dosage form or a parenteral dosage form; the gastrointestinal dosage form is preferably an oral dosage form; the parenteral dosage form is preferably an injection dosage form, a respiratory dosage form, a mucosal dosage form or a cavity dosage form; more preferably an oral dosage form; for example, a capsule, a tablet, a pill, a granule, a powder or an oral liquid; and (10) The pharmaceutical composition C has a synergistic effect.

11. The pharmaceutical composition C according to claim 10, characterized in that: In the pharmaceutical combination C, the first pharmaceutical composition C-1 is presented in the form of an oral dosage form, and the second pharmaceutical composition C-2 is presented in the form of an oral dosage form.

12. A combination kit D, comprising: A first container comprising a first pharmaceutical composition C-1 according to any one of claims 9 to 11; and, A second container comprising the second pharmaceutical composition C-2 according to any one of claims 9 to 11.

13. Use of the pharmaceutical combination A according to any one of claims 1 to 5, the pharmaceutical composition B according to any one of claims 6 to 8, or the pharmaceutical composition C according to any one of claims 9 to 11 in the preparation of a medicament for a proliferative disease; The proliferative disease is preferably one or more of intestinal cancer, pancreatic cancer, non-small cell lung cancer, solid tumors, melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial carcinoma, bladder cancer, small cell lung cancer, sarcoma, intestinal adenocarcinoma, gastrointestinal stromal tumors, gastroesophageal cancer, renal cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell tumor, Wilms tumor and hepatocellular carcinoma; for example, intestinal cancer, pancreatic cancer, non-small cell lung cancer or solid tumors.

14. Use of the pharmaceutical combination A according to any one of claims 1 to 5, the pharmaceutical composition B according to any one of claims 6 to 8, or the pharmaceutical composition C according to any one of claims 9 to 11 in the preparation of a drug for a proliferative disease carrying a KRAS mutant; The KRAS mutant in the proliferative disease with KRAS mutant is preferably one or more of A146T, Q61L, Q61K, G12D, G12C, G13D, G12V, G12R and G13C; for example, G12C; The KRAS mutant proliferative disease is more preferably KRAS G12C mutant colorectal cancer, KRAS G12C mutant pancreatic cancer, KRAS G12C mutant ovarian cancer, KRAS G12C mutant non-small cell lung cancer or KRAS G12C mutant solid tumor.

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