Pharmaceutical combination and use for treating tumors

Abstract

The present invention relates to a pharmaceutical combination product and a therapeutic use thereof. Specifically, the present invention provides a pharmaceutical combination product comprising a first pharmaceutically active ingredient and a second pharmaceutically active ingredient, wherein the first pharmaceutically active ingredient is a KRAS inhibitor. The present invention also provides a use for treating tumors, comprising administering the pharmaceutical combination product of the present invention to a subject in need.

Description

Pharmaceutical combinations and uses for treating tumors

[0001] CROSS-REFERENCE

[0002] This application claims priority to Chinese Patent Application No. 202410780431.8 entitled “Pharmaceutical combinations and uses for treating tumors” filed on June 17, 2024, the entire disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of medicinal chemistry. Specifically, the present application relates to the use of KRAS(G12C) inhibitors in combination with other drugs for treating tumors. BACKGROUND

[0004] RAS is the most frequently mutated gene family in human cancers, and cancers with RAS mutations account for about 25% of all cancer types. KRAS is the most frequently mutated subtype, accounting for 85% of RAS mutations. Mutations usually affect the interaction of KRAS protein with GTPase-activating proteins or the intrinsic GTPase activity of KRAS, increasing the proportion of KRAS bound to GTP in cells, leading to the continuous activation of RAS-RAF-MEK and PI3K-AKT signaling pathways, and thus promoting cell proliferation, survival, and migration, and ultimately leading to the occurrence of cancer. KRAS(G12C) mutation refers to the mutation of guanine to thymine in the 12th codon, resulting in the substitution of glycine at the 12th amino acid with cysteine. According to statistics, about 13% of lung adenocarcinomas, 3% of colorectal cancers, and 1-2% of other solid tumors (including pancreatic cancer, endometrial cancer, bladder cancer, ovarian cancer, and small cell lung cancer) carry KRAS(G12C) mutations.

[0005] KRAS(G12C) as a key driver of tumorigenesis has relatively poor clinical efficacy of its inhibitors compared to approved therapies targeting other classic oncogenic drivers. In terms of objective response rate, median progression-free survival, and patient benefit time, KRAS(G12C) small molecule inhibitors still have obvious room for improvement. In addition, primary and secondary drug resistance are also important factors affecting the antitumor activity of KRAS(G12C) inhibitors. Therefore, how to further improve the clinical efficacy of KRAS(G12C) inhibitors and reduce or delay the development of drug resistance is an important direction for future research.

[0006] Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase encoded by PTK2 gene, which usually regulates various biological functions of cells, including cell survival, migration and invasion, by transducing signals of cell adhesion. TCGA data shows that more than 20% of ovarian cancer, lung squamous cell carcinoma, esophageal cancer and uveal melanoma have increased PTK2 copy number, and the increased PTK2 copy number is associated with poor prognosis of patients. Protein kinase WEE1 is a member of the serine / threonine protein kinase family, which plays a role in cell cycle checkpoint by inhibiting CDC2 / cyclinB kinase to prevent cells from entering M phase from G2. Inhibition of WEE1 in cells can prevent DNA repair, increase DNA damage, cause mitotic catastrophe and cell death, and tumor cells usually characterized by cell cycle disorder and high level of DNA damage are more sensitive to WEE1 inhibition. Therefore, FAK and WEE1 have become popular targets for the development of tumor treatment drugs, but no FAK and WEE1 inhibitors have been approved for marketing due to unexpected clinical efficacy or safety problems.

[0007] In summary, how to enhance the antitumor efficacy of targeted drugs, reduce the dose and toxicity, and slow down or delay the emergence of drug resistance remains a problem to be solved in the field of tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0008] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings described in the following description only relate to some embodiments of the present application, but not limit the present application.

[0009] Fig. 1 and Fig. 2 are the changes of FAK phosphorylation level of cells in Example 1.

[0010] Fig. 3 is the synergistic coefficient of drug inhibition of cell proliferation in Example 2.

[0011] Fig. 4 is the effect of drug on cell colony formation in Example 2.

[0012] Fig. 5 is the blocking effect of drug on cell cycle progression in Example 3.

[0013] Fig. 6 is the induction effect of drug on cell apoptosis in Example 4.

[0014] Fig. 7 is the synergistic coefficient of drug inhibition of cell proliferation in Example 5.

[0015] Fig. 8 is the tumor growth curve and body weight change curve of tumor-bearing mice in Example 6.

[0016] Fig. 9 is the tumor growth curve and body weight change curve of tumor-bearing mice in Example 7.

[0017] Figure 10 is a plot of tumor growth curve and body weight change curve of tumor-bearing mice in Example 8.

[0018] Figure 11 is a plot of tumor growth curve and body weight change curve of tumor-bearing mice in Example 9.

[0019] Figure 12 is a plot of tumor growth curve and body weight change curve of tumor-bearing mice in Example 10.

[0020] Figure 13 is a plot of tumor growth curve and body weight change curve of tumor-bearing mice in Example 11. SUMMARY

[0021] In one aspect, the present application provides a pharmaceutical combination product comprising a first pharmaceutically active ingredient which is a KRAS inhibitor and a second pharmaceutically active ingredient.

[0022] In some embodiments, the second pharmaceutically active ingredient is a FAK inhibitor and / or a WEE1 inhibitor.

[0023] In some embodiments, the KRAS inhibitor is a KRAS(G12C) inhibitor.

[0024] In some embodiments, the product is used for treating abnormal cell growth, in particular cancer.

[0025] In some embodiments, the product further comprises one or more pharmaceutically acceptable excipients.

[0026] In some embodiments, the first and second pharmaceutically active ingredients are administered sequentially or simultaneously.

[0027] In another aspect, the present application also provides the use of a KRAS(G12C) inhibitor and a FAK inhibitor or a WEE1 inhibitor in the manufacture of a medicament for treating a tumor.

[0028] In yet another aspect, the present application provides a pharmaceutical combination product of a KRAS(G12C) inhibitor and a FAK inhibitor or a WEE1 inhibitor for use in treating a tumor in a subject.

[0029] In yet another aspect, the present application provides a method of treating a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of a KRAS(G12C) inhibitor and a FAK inhibitor or a WEE1 inhibitor.

[0030] In yet another aspect, the present application provides a kit or a pharmaceutically acceptable pharmaceutical combination product comprising a KRAS(G12C) inhibitor and a FAK inhibitor or a WEE1 inhibitor.

[0031] In yet another aspect, the present application provides use of a KRAS(G12C) inhibitor and a FAK inhibitor or a WEE1 inhibitor in the manufacture of a medicament for the combined treatment of a tumor.

[0032] Optionally, the KRAS(G12C) inhibitor is SY-5933 (Formula I) or a pharmaceutically acceptable salt thereof.

[0033] Optionally, the FAK inhibitor is Conteltinib (Formula II) or a pharmaceutically acceptable salt thereof.

[0034] Optionally, the WEE1 inhibitor is SY-4835 (Formula III), ZN-c3, Debio-0123, SC-0191, IMP-7068, APR-1051 or a pharmaceutically acceptable salt thereof, further preferably SY-4835 or a pharmaceutically acceptable salt thereof.

[0035] Optionally, the FAK inhibitor is SY-3505 (Formula IV) or a pharmaceutically acceptable salt thereof.

[0036] Optionally, the tumor is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastatic tumor caused by spindle cell carcinoma, anaplastic large cell lymphoma, anaplastic thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma, or a hematologic malignancy, such as acute myelocytic leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML).

[0037] Optionally, the tumor is preferably lung cancer, colorectal cancer, and pancreatic cancer. DETAILED DESCRIPTION

[0038] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the described embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0039] The present application can be implemented in other specific forms without departing from the essential attributes of the application. It is to be understood that any and all embodiments of the present application can be combined with one or more other embodiments or technical features from other embodiments to produce additional embodiments without departing from the scope of the present application. The present application includes such additional embodiments.

[0040] All publications and patents mentioned in the present application are hereby incorporated by reference in their entirety. In the event of a conflict in terminology, construction, or meaning between the present application and any publication or patent incorporated by reference, the terms, constructions, or meanings in the present application shall prevail.

[0041] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs. In the event that there is a plurality of definitions for a term herein, those in this document prevail. Where reference is made to a URL or other source of

[0043] Except in operating examples and where otherwise explicitly indicated, all numerical quantities in this description specifying amounts of materials, amounts of doses, and other quantitative values are to be understood as modified by the word "about" in all instances. It is also to be understood that the presentation of a range of values shall be considered to be a disclosure of all values and sub-ranges falling within the range. All ratios are weight / weight, unless otherwise stated.

[0044] The use of the terms "including", "containing", or "comprising" and the like, are used herein to mean that the specified element is included, but not to the exclusion of any other elements which can be added. The use of the terms "containing" or "comprising" or "including" is not intended to foreclose addition of more elements, but rather the acknowledgment of the presence of at least the referenced element. It is further to be understood that all terminology used herein is for the purpose of describing only the present application, and is not intended to limit the scope of the application in any manner.

[0045] Definitions

[0046] As used in the present application, the following terms and symbols have the meanings indicated below, unless otherwise stated in the context in which they are used. As used herein, the term "inhibitor" means an effective inhibitor, which can be suitable for mammals, particularly humans.

[0047] In some embodiments, the KRAS (G12C) inhibitor is SY-5933, or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, the FAK inhibitor is Conteltinib, or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, the WEE1 inhibitor is SY-4835, ZN-c3, Debio-0123, SC-0191, IMP-7068, APR-1051, or a pharmaceutically acceptable salt thereof, in some preferred embodiments, the WEE1 inhibitor is SY-4835, or a pharmaceutically acceptable salt thereof.

[0050] As used herein, "pharmaceutical combination" or "pharmaceutical combination product" can refer to either a fixed combination in one dosage unit form (e.g. all pharmaceutical active ingredients are present in one dosage form) or a kit-of-parts for combined administration where one pharmaceutical is intended to be administered with the other pharmaceutical(s).

[0051] As used herein, "co-therapy" or "co-drug" refers to the use of one pharmaceutical in conjunction with another pharmaceutical(s) for the treatment of a disease, including both the combination of one pharmaceutical with another pharmaceutical(s) and the combination of one pharmaceutical with instructions indicating that the pharmaceutical can be used in conjunction with another pharmaceutical(s).

[0052] As used herein, the term "treatment" refers to application of one or more pharmaceutical substances to a subject having a disease or symptoms of the disease, with the purpose to cure, relieve, alleviate, alter, change, improve, improve the condition of, or affect the disease or symptoms of the disease. In some embodiments, the disease is a tumor or cancer.

[0053] As used herein, the term "tumor" refers to an abnormal growth of tissue resulting from the deregulated control of locally organized cells that have lost their normal growth controls at the genetic level under the influence of various tumorigenic factors. The tumor includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, renal cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastatic tumor caused by spindle cell carcinoma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma, or a hematologic malignancy, such as acute myelocytic leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML). In some embodiments, the tumor is preferably selected from colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), and pancreatic cancer.

[0054] As used herein, the term "subject" or "subject" refers to mammals and non-mammals. Mammals refer to any member of the mammalian class, which includes, but is not limited to, humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cows, horses, sheep, goats and pigs; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds and the like. The term "subject" does not limit the particular age or gender. In some embodiments, the subject is a human.

[0055] As used herein, the term "pharmaceutically acceptable" means nontoxic, biologically tolerable, and suitable for administration to a subject.

[0056] As used herein, the term "pharmaceutically acceptable salt" refers to nontoxic, biologically tolerable, and suitable for administration to a subject, acid addition salts, including but not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, carbonic acid, boric acid, phosphoric acid, sulfuric acid, sulfurous acid, nitric acid and the like; and acid addition salts formed with organic acids such as formic acid, acetic acid, fumaric acid, maleic acid, furoic acid, tartaric acid, succinic acid, citric acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonic acid, benzoic acid, salicylic acid, stearic acid, and the like.

[0057] The amount of the KRAS(G12C) inhibitor and the FAK inhibitor or the WEE1 inhibitor required for treatment can vary not only with the particular agent selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician. In general, however, the dosage can range from about 0.1 to about 50 mg / kg of body weight per day.

[0058] The KRAS(G12C) inhibitor is administered at a dosage range of 50 mg / day - 2000 mg / day in adults. In a particular embodiment, SY-5933 or a pharmaceutically acceptable salt thereof is administered at a dosage of 200 mg / day - 1000 mg / day in adults.

[0059] The FAK inhibitor is administered at a dosage range of 50 mg / day - 2000 mg / day in adults. In a particular embodiment, Conteltinib, SY3505 or a pharmaceutically acceptable salt thereof is administered at a dosage of 300 mg / day - 800 mg / day in adults.

[0060] The WEE1 inhibitor is administered at a dosage range of 5 mg / day - 1000 mg / day in adults. In a particular embodiment, SY-4835 or a pharmaceutically acceptable salt thereof is administered at a dosage of 20 mg / day - 100 mg / day in adults.

[0061] Examples

[0062] The following examples are provided to further illustrate the present application. It will be understood that these examples are intended to be illustrative only and are not intended to limit the scope of the present application.

[0063] The experimental methods in the following examples, for which no specific conditions are indicated, were carried out in accordance with the usual conditions for such reactions or in accordance with the conditions recommended by the manufacturer.

[0064] The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

[0065] Example 1

[0066] Study of Conteltinib inhibition of FAK-induced activation by SY-5933

[0067] Experimental protocol:

[0068] 1) Study of SY-5933-induced activation of FAK

[0069] The effect of SY-5933 on KRAS(G12C) mutant non-small cell lung cancer,

[0070] Effect of Conteltinib on FAK protein phosphorylation levels in pancreatic cancer and colorectal cancer cells and non-G12C mutant non-small cell lung cancer cells

[0071] 2) Study of Conteltinib inhibiting FAK-induced activation caused by SY-5933

[0072] Effect of Conteltinib, SY-5933 and their combination on FAK phosphorylation in KRAS (G12C) mutant non-small cell lung cancer, pancreatic cancer and colorectal cancer cells was detected by Western blotting

[0073] Experimental materials:

[0074] SY-5933 and Conteltinib were synthesized by the Drug Chemistry Department of Shouyao Holdings (Beijing) Co., Ltd. The compounds were prepared into 10 mM stock solutions with DMSO and stored at -20℃ after aliquoting.

[0075] NCI-H358 cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; MIAPaCa-2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences; SW837 cells were purchased from Nanjing Keygen Biotech Co., Ltd. and were correctly identified by STR; A549 cells were purchased from ATCC.

[0076] DMEM liquid medium was purchased from Biological Industries, item number 06-1055-57-1ACS. RPMI-1640 was purchased from Biological Industries, item number 01-100-1ACS. F-12K medium was purchased from GIBCO, item number 21127-022. Fetal bovine serum FBS was purchased from Biological Industries, item number 04-001-1ACS. Penicillin-Streptomycin was purchased from GIBCO, item number 15070-063. 10× PBS phosphate buffer (pH = 7.2-7.4) was purchased from Solarbio, item number: P1022.

[0077] Pierce TM BCA Protein Assay Kit was purchased from Thermo, item number: 23225. 10× Cell Lysis Buffer was purchased from CST, item number: 9803S. Protease Inhibitor was purchased from Bimake, item number: B14001. Phosphatase inhibitor was purchased from Bimake, item number: B15001.

[0078] p-FAKY397 was purchased from Thermo, Cat# 44-624G. p-ERK(T202 / Y204) Rabbit mAb was purchased from CST, Cat# 4370. GAPDH(14C10) Rabbit mAb was purchased from CST, Cat# 3683. RAS Rabbit mAb was purchased from Abeam, Cat# ab108602. Anti-rabbit IgG, HRP-linked Antibody was purchased from CST, Cat# 7074P2.

[0079] Experimental methods:

[0080] NCI-H358 cells were cultured using RPMI-1640 medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin; MIAPaCa-2 cells and SW837 cells were cultured using DMEM medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin. A549 cells were cultured using F-12K medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin. Cells were counted and plated in 6-well cell culture plates, NCI-H358, MIAPaCa-2, SW837 and A549 cells were plated at 1.5x10 6 Cells were inoculated with 2 mL of medium per well and incubated at 37°C overnight. The next day, different concentrations of test compounds were added for 4 hours (NCI-H358, MIAPaCa-2, SW837 cells) or 24, 48 hours (A549 cells). The medium was aspirated, washed once with PBS, and the cells were trypsinized, the medium was terminated, and the cells were collected by centrifugation. 30 μL of cell lysis solution was added to each sample, and lysis was performed on ice for 20 minutes. Centrifugation was performed at 4°C at 12000 rpm for 10 minutes, and the supernatant was taken. Protein quantification was performed by the BCA method, and an equal amount of total protein was added to 5x electrophoresis loading buffer, and the sample was boiled at 105°C for 5 minutes.

[0081] Electrophoresis: 100-120V, 100 minutes. Membrane transfer: (PVDF) wet transfer 300mA, room temperature for 2 hours. Blocking: 5% skim milk incubated at room temperature for 1 hour. Primary antibody: the primary antibody was diluted with TBST according to the proportion, and incubated at 4°C overnight. Membrane washing: 5 minutes x 3 times. Secondary antibody, incubated at room temperature for 1 hour. Membrane washing: 5 minutes x 3 times, ECL method for protein detection.

[0082] Experimental results:

[0083] After treatment of KRAS(G12C) mutant pancreatic cancer cell MIAPaCa-2, non-small cell lung cancer cell NCI-H358 and colorectal cancer cell SW837 with SY-5933, the level of FAK Y397 phosphorylation in the cells increased, indicating that FAK was induced to be activated, and the induction was time-dependent; but similar phenomenon did not exist in KRAS(G12S) mutant non-small cell lung cancer cell A549 (as shown in Figure 1). The addition of FAK inhibitor Conteltinib significantly inhibited the increase in the level of FAK protein phosphorylation caused by SY-5933 (as shown in Figure 2).

[0084] Example 2

[0085] Study on synergistic inhibition of non-small cell lung cancer, pancreatic cancer and colorectal cancer cells with KRAS(G12C) mutation by combination of SY-5933 and Conteltinib

[0086] Experimental scheme:

[0087] 1) The inhibitory effect of combination of SY-5933 and Conteltinib on the proliferation of non-small cell lung cancer, pancreatic cancer and colorectal cancer cells with KRAS(G12C) mutation was detected by Cell Titer-Glo reagent, and the synergistic coefficient of the combination was calculated by Combenefit 2 software.

[0088] 2) The inhibitory effect of SY-5933, Conteltinib and their combination on the colony formation of KRAS(G12C) mutant pancreatic cancer and colorectal cancer cells was detected by crystal violet staining.

[0089] Experimental materials:

[0090] SY-5933 and Conteltinib were synthesized by the Pharmaceutical Chemistry Department of Shouyao Holdings (Beijing) Co., Ltd. The compounds were prepared into 10 mM stock solutions with DMSO, and stored at -20℃ after aliquoting.

[0091] NCI-H358 cells were purchased from the Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, MIAPaCa-2 cells were purchased from the China National Culture Collection Cell Bank, SW1463 cells were purchased from ATCC, and SW837 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and were correctly identified by STR.

[0092] DMEM medium was purchased from Biological Industries, item number 06-1055-57-1ACS. RPMI-1640 medium was purchased from Biological Industries, item number 01-100-1ACS. L-15 medium was purchased from VivaCell, item number C3070-0500. Fetal bovine serum FBS was purchased from Biological Industries, item number 04-001-1ACS. Penicillin-Streptomycin was purchased from GIBCO, item number 15070-063. 10x PBS buffer was purchased from Solarbio, pH 7.2-7.4, item number P1022.

[0093] Cell Titer-Glo was purchased from Promega, item number G7570. Crystal violet was purchased from ALADDIN, item number: 548-62-9. Paraformaldehyde was purchased from National Pharmaceutical Group, item number: 80096618.

[0094] Experimental method:

[0095] NCI-H358 cells were cultured using RPMI-1640 medium, MIAPaCa-2 cells and SW837 cells were cultured using DMEM medium, and SW1463 was cultured using L-15 medium. The above-mentioned culture medium was added with 10% fetal bovine serum and 1% Penicillin-Streptomycin.

[0096] The procedure of cell proliferation inhibition experiment is as follows: MIA PaCa-2, NCI-H358 and SW1463 cells are counted and plated in 96-well cell culture plates, 500 cells (MIA PaCa-2) and 2000 cells (NCI-H358 and SW1463) per well, respectively, 190 μL of medium is added to each well, and incubated at 37°C overnight. The next day, the combination of compounds is added, and the operation is as follows: the compounds Conteltinib and SY-5933 are gradient-diluted with DMSO, 4 μL of each concentration is added to 96 μL of serum-free medium, and shaken to mix. Then 5 μL of the suspended compound is taken by the gun and added to the test cells. The final concentration of compound Conteltinib for MIA PaCa-2 cells is 2000, 600, 200, 60, 20 and 0 nM, and the final concentration of compound SY-5933 is 6, 2, 0.6, 0.2, 0.06 and 0 nM. The final concentration of compound Conteltinib for NCI-H358 cells is 250, 125, 62.5, 31.25 and 0 nM, and the final concentration of compound SY-5933 is 5, 2.5, 1.25, 0.625 and 0 nM. The final concentration of compound Conteltinib for SW1463 cells is 10000, 3000, 1000, 300, 100 and 0 nM, and the final concentration of compound SY-5933 is 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 and 0 nM. After 5 days of compound action, the supernatant is removed, 25 μL of CellTiter-Glo reagent is added, shaken to mix for 3 minutes in the dark, and the luminescence signal is read. Finally, the synergistic effect of the combination of compound SY-5933 and Conteltinib on cell proliferation inhibition is calculated using Combenefit 2.

[0097] The procedure of colony formation experiment is as follows: MIAPaCa-2 and SW837 cells are counted and plated in 6-well plates, with the number of 500 cells / well and 4000 cells / well, respectively. After the 6-well plates are incubated in a 37°C incubator for 24 hours, drug treatment is performed, with the final concentrations of SY-5933: 0.3, 3 nM, Conteltinib: 30, 300 nM (MIAPaCa-2 cells) and SY-5933: 0.01, 0.1, 1 nM, Conteltinib: 10, 30 nM (SW837 cells). The 6-well plates are continuously cultured in a 37°C incubator, and after 3 days, fresh medium containing corresponding concentrations of drugs is added for continuous culture for 5 days. After the control well cells form appropriate size clones (at least 50 cells per clone), the medium is removed and washed with PBS twice, 4% paraformaldehyde (2 mL / well) is added for room temperature fixation for 10 minutes. After removing the paraformaldehyde and washing with PBS twice, 0.2% crystal violet staining solution (PBS preparation, 0.22 μm filter) is added for room temperature staining for 10 minutes. Carefully rinse with distilled water until the background is clean and free of color, and after drying, scanning is performed.

[0098] Experimental results:

[0099] After 5 days of combined use of SY-5933 and Conteltinib, the proliferation of KRAS (G12C) mutant pancreatic cancer cells MIA PaCa-2, non-small cell lung cancer cells NCI-H358 and colorectal cancer cells SW1463 is significantly inhibited, and the analysis by Combenefit 2 software shows that the combined use of SY-5933 and Conteltinib has a significant synergistic effect (as shown in Figure 3). The colony formation experiment shows that, compared with SY-5933 and Conteltinib alone, the combined use of the two has a stronger inhibitory effect on the colony formation of cells in pancreatic cancer cells MIA PaCa-2 and colorectal cancer cells SW837 (as shown in Figure 4).

[0100] Example 3

[0101] Blocking effect of combined use of SY-5933 and Conteltinib on cell cycle of pancreatic cancer cells MIA PaCa-2

[0102] Experimental materials:

[0103] SY-5933 and Conteltinib are synthesized by the Drug Development Department of Shouyao Holdings (Beijing) Co., Ltd., and the compounds are prepared into 10 mM stock solutions with DMSO, and stored at -20°C after aliquoting.

[0104] MIAPaCa-2 cells were purchased from China Academy of Sciences Typical Culture Collection Cell Library. DMEM liquid medium was purchased from Biological Industries, item number 06-1055-57-1ACS. Fetal bovine serum FBS was purchased from Biological Industries, item number 04-001-1ACS. Penicillin-Streptomycin was purchased from GIBCO, item number 15070-063. BrdU was purchased from MCE, item number HY-15910. Anti-BrdU-FITC antibody was purchased from BD, item number 347583. PI (Propidium iodide) was purchased from Sigma, item number 25535-16-4. RNase A was purchased from Solarbio, item number R1030.

[0105] Experimental method:

[0106] MIAPaCa-2 cells were cultured with DMEM medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin. Cells were plated in 6-well plates at a concentration of 5x10 5 / 2mL of the 6-well plate, and the following day, the combination compound was added for 48 hours. The final concentration of the compound was 3, 1, 0.3, 0nM SY-5933 or 300, 100, 0nM Conteltinib. 20μM BrdU was used to label the cells for 1h, and the cells were trypsinized and fixed with 70% ethanol overnight. 2x105 cells were taken and added with 2M HCl / 0.5% TrionX-100 for 30 minutes at room temperature. Neutralization was performed in 0.1M sodium tetraborate pH8.5. Washing was performed twice with 0.5% Tween20 / 1% BSA / PBS, and the remaining 50μL. 5μL of anti-BrdU-FITC antibody was added, and the cells were stained for 30 minutes at room temperature in the dark. Washing was performed twice with 0.5% Tween20 / 1% BSA / PBS, and 200μL of PBS containing 1μg / mL PI and 0.5mg / mL RNase A was added for staining at 37 degrees for 15 minutes before detection.

[0107] Experimental results:

[0108] As shown in Figure 5, in KRAS (G12C) mutant pancreatic cancer cells MIAPaCa-2, compared with single drug, SY-5933 combined with Conteltinib can make more tumor cells arrest in the G1 phase.

[0109] Example 4

[0110] Induction of apoptosis of pancreatic cancer cells MIAPaCa-2 by SY-5933 combined with Conteltinib

[0111] Experimental materials:

[0112] SY-5933 and Conteltinib were synthesized by the Drug Chemistry Department of Shouyao Holdings (Beijing) Co., Ltd. The compounds were prepared into 10 mM stock solutions with DMSO, and stored at -20 °C after aliquoting.

[0113] MIAPaCa-2 cells were purchased from the China Academy of Sciences Typical Culture Preservation Committee Cell Library. DMEM liquid medium was purchased from Biological Industries, item number 06-1055-57-1ACS. Fetal bovine serum FBS was purchased from Biological Industries, item number 04-001-1ACS. Penicillin-Streptomycin was purchased from GIBCO, item number 15070-063. PE Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences, item number 559763.

[0114] Experimental method:

[0115] MIAPaCa-2 cells were cultured using DMEM medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin, and the cells were plated in a 6-well plate at a concentration of 3 x 10 5 On the second day, different concentrations of SY-5933 (10, 3, 1, and 0 nM) and different concentrations of Conteltinib (300, 100, and 0 nM) were added for incubation for 24 hours. The above cells were collected in corresponding 1.5 mL centrifuge tubes, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended with cold PBS, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. The operation was performed according to the instructions of the PE Annexin V Apoptosis Detection Kit I, and the cells were resuspended with binding buffer. 1 x 10 5 / 100 μL of cells were added with 5 μL of PE Annexin V and 5 μL of 7-AAD, and incubated at room temperature for 15 minutes in the dark. 400 μL of binding buffer was added, and CytoFLEX flow cytometry was used for detection.

[0116] Experimental results:

[0117] As shown in Figure 6, after SY-5933 combined with Conteltinib was used to treat MIAPaca-2 cells for 24 hours, the cells were induced to undergo apoptosis. Compared with single drug, the combination of SY-5933 and Conteltinib had stronger activity in inducing cell apoptosis.

[0118] Example 5

[0119] Study of SY-5933 in combination with SY-4835 synergistically inhibiting the proliferation of KRAS(G12C) mutant pancreatic cancer and colorectal cancer cells

[0120] Experimental materials:

[0121] SY-5933 and SY-4835 were synthesized by the Pharmaceutical Chemistry Department of Shouyao Holdings (Beijing) Co., Ltd. The compounds were prepared into 10 mM stock solutions with DMSO, and stored at -20°C after aliquoting.

[0122] NCI-H358 cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, SW837 cells were purchased from Nanjing Keye Biotechnology Co., Ltd. and were correctly identified by STR, and SW1463 cells were purchased from ATCC.

[0123] DMEM liquid medium was purchased from Biological Industries, item number 06-1055-57-1ACS. RPMI-1640 medium was purchased from Biological Industries, item number 01-100-1ACS. L-15 medium was purchased from VivaCell, item number C3070-0500. Fetal bovine serum FBS was purchased from Biological Industries, item number 04-001-1ACS. Penicillin-Streptomycin was purchased from GIBCO, item number 15070-063. 10×PBS buffer was purchased from Solarbio, pH 7.2-7.4, item number P1022. Cell Titer-Glo was purchased from Promega, item number G7570.

[0124] Experimental method:

[0125] NCI-H358 cells were cultured using RPMI-1640 medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin. SW837 cells were cultured using DMEM medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin. SW1463 cells were cultured using L15 medium added with 10% fetal bovine serum and 1% Penicillin-Streptomycin. NCI-H358, SW837 and SW1463 cells were counted and plated in 96-well cell culture plates at 2000 cells per well, 190 μL medium was added to each well, and incubated at 37°C overnight. The next day, the combination compounds were added, as follows: compounds SY-4835 and SY-5933 were gradiently diluted with DMSO, 4 μL of each concentration was added to 96 μL of serum-free medium, and shaken to mix. Then, 5 μL of the suspended compounds was added to the test cells using an 8-channel pipette. The final concentration of compound SY-4835 for NCI-H358 cells was 3000, 1000, 300, 100, 30, 10, 3 and 0 nM, and the final concentration of compound SY-5933 was 300, 100, 300, 10, 3, 1, 0.3, 0.1, 0.03 and 0 nM. The final concentration of compound SY-4835 for SW837 cells was 10000, 3000, 1000, 300, 100 and 0 nM, and the final concentration of compound SY-5933 was 3000, 1000, 300, 100, 30, 10, 3, 1, 0.3 and 0 nM. The final concentration of compound SY-4835 for SW1463 cells was 10000, 3000, 1000, 300, 100 and 0 nM, and the final concentration of compound SY-5933 was 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 and 0 nM. After 5 days of compound treatment, the supernatant was removed, 25 μL of CellTiter-Glo reagent was added, and shaken to mix for 3 minutes in the dark. The luminescent signal was read, and finally, the synergistic effect of the combination of compound SY-5933 and SY-4835 on cell proliferation inhibition was calculated using Combenefit 2.

[0126] Experimental results:

[0127] After 5 days of combined action of SY-5933 and SY-4835, the proliferation of KRAS(G12C) mutant non-small cell lung cancer cells NCI-H358 and colorectal cancer cells SW837 and SW1463 was significantly inhibited, and analysis using Combenefit 2 software showed that the combination of SY-5933 and SY-4835 had a significant synergistic effect (as shown in Figure 7).

[0128] Example 6

[0129] In vivo anti-tumor efficacy study of SY-5933 combined with Conteltinib in a mouse subcutaneous xenograft tumor model of pancreatic cancer MIA PaCa-2 cells

[0130] Experimental materials:

[0131] Zinc alloy digital caliper (MNT-200, resolution: 0.01 mm, Shanghai Meinaite Industry Co., Ltd.), JEB2002 electronic balance (resolution: 0.01 g, Shanghai Puchun Metrological Instrument Co., Ltd.), XS105 electronic balance (resolution: 0.01 mg, Mettler-Toledo Instrument Co., Ltd.), AL204 electronic balance (resolution: 0.1 mg, Mettler-Toledo Instrument Co., Ltd.), Haier biological safety cabinet (model: HR40-IIA2, Qingdao Haier Special Electrical Appliance Co., Ltd.).

[0132] Hydroxypropyl methyl cellulose (batch number: PDR527640, Shanghai Kalon Coating Technology Co., Ltd.); Polysorbate 80 (batch number: 20210902, Nanjing Well Pharmaceutical Group Co., Ltd.); Sodium carboxymethyl cellulose (batch number: 20210302, Liaocheng Ahua Pharmaceutical Co., Ltd.)

[0133] SY-5933 (batch number: PCS0684-22001, content: 99.1%, Analysis and Process Department of First Pharmaceutical Holding (Beijing) Co., Ltd.); Conteltinib (batch number: PCS0273-22003M1, content: 100.3%, Analysis and Process Department of First Pharmaceutical Holding (Beijing) Co., Ltd.)

[0134] Female BALB / c-nude mice, 5 weeks old, 45, certificate number: SCXK(Jing)2021-0006, purchased from Beijing Vantianlihua Experimental Animal Technology Co., Ltd. The mice were fed in IVC independent air supply isolation cages, and the cage size was 325*210*180mm. The barrier environment conditions were as follows: temperature: 20-26℃; humidity: 40-70%; light cycle: 12 hours of light and 12 hours of darkness per day. Feed (Co 60 Sterilized feed, Beijing Kaoshehui Feed Co., Ltd.). Drinking water: purified water, sterilized by high-pressure steam. During the experiment, 5 mice were fed in 1 cage, and the animals were free to drink and eat. The cage, bedding, drinking water and feed were replaced twice a week, and the replaced cage and drinking bottle were sterilized by high-pressure steam. The cage, room walls and floor were cleaned with 84 disinfectant or new jieer disinfectant twice a week. The experimental animal use license number is SYXK(Jing)2022-0050.

[0135] Experimental method:

[0136] Human pancreatic cancer cell line MIAPaCa-2 was purchased from China Typical Culture Collection Center of Wuhan University School of Life Sciences. The cells were cultured in monolayer in DMEM medium supplemented with 10% fetal bovine serum in a 37°C 5% CO2 incubator. The cells were routinely digested and passaged with trypsin-EDTA 2-3 times a week. When the cells were in the exponential growth phase and the abundance was 80%-90%, the cells were collected and inoculated. When the tumors grew to 400-700 mm 3 , the tumors were passaged twice and stored for later use. The MIAPaCa-2 tumor mass was recovered and passaged twice and inoculated subcutaneously on the left and right sides of the back of each mouse.

[0137] When the average tumor volume was about 150 mm 3 , tumor-bearing mice with regular shape and uniform size were selected for the experiment. According to the random stratification method based on tumor volume and mouse weight, the mice were divided into 4 groups, namely, the model group, the SY-5933 (0.5 mpk) group, the Conteltinib (100 mpk) group, and the combination SY-5933 / Conteltinib group, with 10 tumor-bearing mice in each group. The grouping information is shown in Table 1. Drug administration was started on the same day of grouping, once a day, and the tumor diameter and body weight were measured 3 times a week during the period. The administration was continued for 14 days. At the end of the experiment, sampling (plasma and tumor tissue) was performed at 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after administration, and digital cameras were used to take pictures of the tumor-bearing mice and tumor mass. After heparin anticoagulation, the blood samples were centrifuged at 5200 rpm for 5 minutes to separate the plasma, which was stored at -80°C for SY-5933 and Conteltinib content determination. The tumor tissue was divided into three parts, quickly frozen in liquid nitrogen, and stored at -80°C for SY-5933 and Conteltinib content determination and Western Blotting detection of protein expression.

[0138] Table 1. Drug administration scheme of SY-5933 combined with Conteltinib in MIAPaca-2 subcutaneous xenograft tumor in nude mice

[0139] The relative tumor growth rate (T / C) and tumor growth inhibition rate (TGI) were mainly used as detection indicators. The evaluation criteria were as follows: T / C (%) > 40% was ineffective; T / C (%) ≤ 40% and P < 0.05 after statistical processing were effective. The tumor volume calculation formula was V = 0.5 × a × b 2 , where V was the tumor volume, a and b were the length and width of the tumor, respectively. The relative tumor volume (RTV) was calculated according to the tumor volume, and the calculation formula was: RTV = V t / V0, where V0 was the tumor volume measured at the time of grouping, and V tTumor volume was measured every time. The relative tumor growth rate was calculated according to the formula: T / C (%) = T RTV / C RTV *100%. (T RTV : treatment group RTV; C RTV : negative control group RTV). The tumor growth inhibition rate was calculated according to the formula: TGI (%) = (1- (tumor volume of treatment group - tumor volume when the treatment group was divided / tumor volume of control group - tumor volume when the control group was divided)) * 100%. The data was expressed as Mean ± SEM, and the significance between groups was compared by t test.

[0140] Experimental results:

[0141] The results are shown in Figure 8 (A is the tumor growth curve, and B is the animal weight curve). The human pancreatic cancer MIAPaCa-2 xenograft tumor model grew well, and at the end of the experiment (Day 14), the tumor in the model group grew to 999.0 ± 76.1 mm 3 . The tumor volumes in the SY-5933 (0.5 mg / kg) single drug, Conteltinib (100 mg / kg) single drug, and SY-5933 (0.5 mg / kg) + Conteltinib (100 mg / kg) groups were 755.4 ± 96.0 mm 3 , 627.8 ± 58.0 mm 3 , and 148.7 ± 19.6 mm 3 , respectively, as shown in Table 2. SY-5933 0.5 mg / kg showed a weak effect on inhibiting tumor growth, with TGI and T / C of 27.3% and 72.8% at the end of the experiment (Day 14), respectively. Conteltinib 100 mg / kg showed a weak effect on inhibiting tumor growth, with TGI and T / C of 41.7% and 62.1% at the end of the experiment (Day 14), respectively. At the same time, the SY-5933 combined with Conteltinib group showed a significantly better tumor inhibition effect than the single drug groups (p < 0.001), with TGI and T / C of 95.6% and 15.0% at the end of the experiment (Day 14), respectively, and showed a significant synergistic effect on inhibiting tumor growth. No mouse death caused by drugs was observed in the experiment, and the body weight of the mice in each drug administration group did not change significantly compared with the mice in the vehicle group, and the animal tolerance was good.

[0142] Table 2. Evaluation of the tumor inhibition effect of SY-5933 combined with Conteltinib in the MIAPaca-2 nude mouse subcutaneous xenograft tumor model (based on data on Day 14 after grouping and administration)

[0143] Note: 1. Data are mean ± standard error of mean, calculated according to the days after grouping administration

[0144] 2. ***: p<0.001, vs Vehicle group, T-test

[0145] 3. ***: p<0.001, vs SY-5933 + Conteltinib group, T-test

[0146] Example 7

[0147] In vivo anti-tumor efficacy study of SY-5933 combined with Conteltinib in non-small cell lung cancer NCI-H358 mouse subcutaneous xenograft tumor model

[0148] Experimental materials:

[0149] Zinc alloy digital caliper (MNT-200, resolution: 0.01 mm, Shanghai Meinaite Industry Co., Ltd.), JEB2002 electronic balance (resolution: 0.01 g, Shanghai Puchun Metrological Instrument Co., Ltd.), XS105 electronic balance (resolution: 0.01 mg, Mettler-Toledo Instrument Co., Ltd.), AL204 electronic balance (resolution: 0.1 mg, Mettler-Toledo Instrument Co., Ltd.), Haier biological safety cabinet (model: HR40-IIA2, Qingdao Haier Special Electrical Appliance Co., Ltd.).

[0150] Hydroxypropyl methyl cellulose (batch number: PDR527640, Shanghai Kalon Coating Technology Co., Ltd.); Polysorbate 80 (batch number: 20210902, Nanjing Well Pharmaceutical Group Co., Ltd.); Sodium carboxymethyl cellulose (batch number: 20210302, Liaocheng Ahua Pharmaceutical Co., Ltd. in Shandong)

[0151] SY-5933 (batch number: PCS0684-22001, content: 99.1%, Analysis and Preparation Technology Department of First Pharmaceutical Holding (Beijing) Co., Ltd.); Conteltinib (batch number: PCS0273-22003M1, content: 100.3%, Analysis and Preparation Technology Department of First Pharmaceutical Holding (Beijing) Co., Ltd.)

[0152] Female NOD SCID mice, 5 weeks old, 48, certificate number: SCXK(Jing)2021-0006, purchased from Beijing Vantianlihua Experimental Animal Technology Co., Ltd. The mice were fed in IVC independent air supply isolation cages, and the mouse cage specifications were 325*210*180 mm. The barrier environment conditions were: temperature: 20-26℃; humidity: 40-70%; light cycle: light on for 12 hours and light off for 12 hours per day. Feed (Co 60Sterilized feed, Beijing Kaocuo Feed Co., Ltd.). Drinking water: purified water, and sterilized by high-pressure steam. During the experiment, 5 mice were housed in one cage, and the animals were free to drink and eat. The cage, bedding, drinking water, and feed were replaced twice a week. The replaced cage and drinking bottle were sterilized by high-pressure steam after being washed. The cage, room walls, and floor were cleaned twice a week with 84 disinfectant or new jieerling. Experimental animal use license number: SYXK(Jing)2022-0050.

[0153] Experimental method:

[0154] Human non-small cell lung cancer cells NCI-H358 were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were cultured in vitro in a monolayer, and the culture conditions were RPMI-1640 medium with 10% fetal bovine serum, 37°C, 5% CO2 incubator. The cells were passaged by routine digestion with trypsin-EDTA 2-3 times a week. When the cells were in the exponential growth phase, the abundance was 80%-90%, the cells were collected and inoculated after counting. The NCI-H358 cell suspension mixed with PBS was 0.1 mL (containing 5.0 x 10 6 , 30% Matrigel) was subcutaneously injected into the subcutaneous part of the left and right sides of the back of each mouse.

[0155] When the average tumor volume was about 150 mm 3 , tumor-bearing mice with regular shape and uniform size were selected for the experiment. According to the random stratification method of tumor volume and mouse weight, they were divided into 4 groups, namely the model group, the SY-5933 (1 mpk) group, the Conteltinib (100 mpk) group, and the combined SY-5933 / Conteltinib group, with 10 tumor-bearing mice in each group. The grouping information is shown in Table 3. Drug administration began on the same day of grouping, once a day, and the tumor diameter and body weight were measured 3 times a week during the period. Continuous administration was performed until the 14th day. At the end of the experiment, sampling (plasma and tumor tissue) was performed at 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after drug administration, and digital cameras were used to take pictures of the tumor-bearing mice and tumor masses. After heparin anticoagulation, the blood samples were centrifuged at 5200 rpm for 5 minutes to separate the plasma, which was stored at -80°C for SY-5933 and Conteltinib content determination. The tumor tissue was divided into three parts, frozen in liquid nitrogen, and stored at -80°C for SY-5933 and Conteltinib content determination and Western Blotting detection of protein expression.

[0156] Table 3 SY-5933 combined with Conteltinib in NCI-H358 mouse subcutaneous xenograft tumor administration scheme

[0157] The relative tumor growth rate (T / C) and tumor growth inhibition rate (TGI) were used as the detection indexes. The evaluation criteria were as follows: T / C (%) > 40% was invalid; T / C (%) ≤ 40%, and P < 0.05 by statistical treatment was effective. The tumor volume calculation formula was V = 0.5 × a × b 2 , where V was the tumor volume, a and b were the length and width of the tumor, respectively. The relative tumor volume (RTV) was calculated according to the tumor volume, and the calculation formula was: RTV = V t / V0, where V0 was the tumor volume measured when the grouping was administered, and V t was the tumor volume at each time of measurement. The relative tumor growth rate calculation formula was: T / C (%) = T RTV / C RTV * 100%. (T RTV : RTV of the treatment group; C RTV : RTV of the negative control group). The tumor growth inhibition rate calculation formula was TGI (%) = (1- (tumor volume of the treatment group-tumor volume when the treatment group was grouped) / (tumor volume of the control group-tumor volume when the control group was grouped)) * 100%. The data were expressed as Mean ± SEM, and the significance between groups was compared by t-test.

[0158] Experimental results:

[0159] The results are shown in Figure 9 (A is the tumor growth curve, and B is the animal weight curve). The human lung cancer NCI-H358 xenograft tumor model grew well, and the tumor of the model group grew to 517.1 ± 77.7 mm 3 at the end of the experiment (Day 14). The tumor volumes of the SY-5933 (1 mg / kg) single drug, Conteltinib (100 mg / kg) single drug, and SY-5933 (1 mg / kg) + Conteltinib (100 mg / kg) groups were 345.5 ± 43.2 mm 3 , 415.4 ± 52.6 mm 3 , and 137.6 ± 14.7 mm 3, see Table 4. SY-5933 31 mg / kg showed a weak inhibitory effect on tumor growth, with TGI and T / C of 48.3% and 70.2% respectively at the end of the experiment (Day 14); Conteltinib 100 mg / kg showed no inhibitory effect on tumor growth, with TGI and T / C of 28.6% and 81.3% respectively at the end of the experiment (Day 14). Meanwhile, the combination of SY-5933 and Conteltinib showed a significantly better inhibitory effect on tumor growth than each single drug group (p<0.001), with TGI and T / C of 105.7% and 27.6% respectively at the end of the experiment (Day 14). No mouse death caused by the drugs was observed in the experiment, and the body weight of the mice in each drug group showed no significant change compared with the mice in the vehicle group, indicating good animal tolerance.

[0160] Table 4 Evaluation of the inhibitory effect of the combination of SY-5933 and Conteltinib on tumor growth in the NCI-H358 mouse subcutaneous xenograft model (based on data at Day 14 after grouping and administration)

[0161] Note: 1. Calculated according to the number of days after grouping and administration, data are mean ± standard error of the mean

[0162] 2. **: p<0.001, vs Vehicle group, T-test

[0163] 3. **: p<0.001, vs SY-5933+Conteltinib group, T-test

[0164] Example 8

[0165] In vivo anti-tumor efficacy study of the combination of SY-5933 and Conteltinib in the mouse subcutaneous xenograft model of rectal adenocarcinoma SW1463 cells

[0166] Experimental materials:

[0167] Zinc alloy digital caliper (MNT-200, resolution: 0.01 mm, Shanghai Meinaite Industry Co., Ltd.), JEB2002 electronic balance (resolution: 0.01 g, Shanghai Puchun Metrological Instrument Co., Ltd.), XS105 electronic balance (resolution: 0.01 mg, Mettler-Toledo Instrument Co., Ltd.), AL204 electronic balance (resolution: 0.1 mg, Mettler-Toledo Instrument Co., Ltd.), Haier biological safety cabinet (model: HR40-IIA2, Qingdao Haier Special Electrical Appliance Co., Ltd.).

[0168] Hydroxypropyl Methylcellulose (Batch No.: PDR527640, Shanghai Kalon Coating Technology Co., Ltd.); Polysorbate 80 (Batch No.: 20210902, Nanjing Well Pharmaceutical Group Co., Ltd.); Sodium Carboxymethylcellulose (Batch No.: 20210302, Shandong LiaoCheng Ahua Pharmaceutical Co., Ltd.)

[0169] SY-5933 (Batch No.: PCS0684-22001, content: 99.1%, Analysis and Preparation Technology Department of First Pharmaceutical Holding (Beijing) Co., Ltd.); Conteltinib (Batch No.: PCS0273-22003M1, content: 100.3%, Analysis and Preparation Technology Department of First Pharmaceutical Holding (Beijing) Co., Ltd.)

[0170] Female BALB / c-nude mice, 5 weeks old, 48, certificate No. SCXK(Jing)2021-0006, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were fed in IVC independent air supply isolation cages, and the cage size was 325*210*180mm. The barrier environment conditions were as follows: temperature: 20-26℃; humidity: 40-70%; light cycle: 12 hours of light and 12 hours of darkness per day. Feed (Co 60 sterilized feed, Beijing Kaoshehui Feed Co., Ltd.). Drinking water: purified water, sterilized by high-pressure steam. During the experiment, 5 mice were fed in one cage, and the animals were free to drink and eat. The cage, bedding, drinking water and feed were replaced twice a week, and the replaced cage and drinking bottle were sterilized by high-pressure steam after being washed. The cage rack, room wall and floor were cleaned with 84 disinfectant or new jieer disinfectant twice a week. The license number for the use of experimental animals was SYXK(Jing)2022-0050.

[0171] Experimental method:

[0172] Human rectal adenocarcinoma cells SW1463 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. The cells were cultured in monolayer in vitro, and the culture conditions were as follows: L15 medium with 10% fetal bovine serum, 37℃ 5% CO2 incubator. The cells were passaged by routine digestion with trypsin-EDTA 2-3 times a week. When the cells were in the exponential growth phase, the abundance was 80%-90%, the cells were collected, counted and inoculated. SW1463 cell suspension suspended in PBS 0.1mL (containing cell number 5.0*10 6 , 50% Matrigel) was subcutaneously injected into the subcutaneous part of the left and right sides of the back of each mouse.

[0173] When the average tumor volume was about 150mm 3Mice bearing tumors with regular shapes and uniform sizes were selected for the experiment. They were randomly stratified into four groups based on tumor volume and mouse weight: a model group, a SY-5933 (1.5 mpk) group, a Conteltinib (100 mpk) group, and a combination of SY-5933 / Conteltinib group, with 10 tumor-bearing mice in each group. Grouping information is shown in Table 5. Drug administration began on the day of grouping and was administered once daily. Tumor diameter and body weight were measured three times per week until day 12. At the end of the experiment, samples (plasma and tumor tissue) were collected at 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after drug administration, and the tumor-bearing mice and tumor masses were photographed using a digital camera. Blood samples were anticoagulated with heparin, centrifuged at 5200 rpm for 5 minutes, and plasma was separated and stored at -80℃ for the determination of SY-5933 and Conteltinib content. The tumor tissue was divided into three parts, flash-frozen in liquid nitrogen, and stored at -80°C for the determination of SY-5933 and Conteltinib content and Western blotting to detect protein expression.

[0174] Table 5. Dosing regimen of SY-5933 in combination with Conteltinib in subcutaneous xenograft tumors of SW1463 nude mice.

[0175] The primary indicators used were relative tumor proliferation rate (T / C) and tumor growth inhibition rate (TGI). The evaluation criteria were: T / C (%) > 40% was considered invalid; T / C (%) ≤ 40%, and statistically significant p < 0.05, was considered valid. The tumor volume was calculated using the formula V = 0.5 × a × b. 2 Where V is the tumor volume, and a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the tumor volume using the formula: RTV = V t / V0, where V0 is the tumor volume measured during group administration, V t This represents the tumor volume at each measurement. The relative tumor growth rate is calculated using the formula: T / C (%) = T RTV / C RTV *100%. (T) RTV Treatment group RTV; C RTV Negative control group (RTV). The tumor growth inhibition rate was calculated using the formula: TGI(%) = (1 - (tumor volume in the treatment group - tumor volume at the time of grouping in the treatment group) / (tumor volume in the control group - tumor volume at the time of grouping in the control group)) * 100%. Data are expressed as Mean ± SEM, and t-tests were used to compare the significance between groups.

[0176] Experimental results:

[0177] The results are shown in Figure 10 (A is the tumor growth curve, B is the animal weight curve). The human rectal adenocarcinoma cell line SW1463 xenograft tumor model grew well. At the end of the experiment (Day 12), the tumor in the model group grew to 461.5±49.1 mm. 3 The tumor volumes in the SY-5933 (1.5 mg / kg) monotherapy group, the Conteltinib (100 mg / kg) monotherapy group, and the SY-5933 (1.5 mg / kg) + Conteltinib (100 mg / kg) group were 292.1 ± 24.4 mm, respectively. 3 509.7±50.1mm 3 and 229.2±36.4mm 3 See Table 6 for details. At the end of the experiment (Day 12), SY-5933 1.5 mg / kg showed a TGI of 56.7% and a T / C of 64.1%, indicating a weak inhibitory effect on tumor growth. Conteltinib 100 mg / kg showed a TGI of -17.5% and a T / C of 115.1% at the end of the experiment (Day 12), showing no inhibitory effect on tumor growth. However, the SY-5933 combined with Conteltinib group showed a TGI of 75.9% and a T / C of 48.4% at the end of the experiment (Day 12), demonstrating superior tumor-inhibiting effects compared to the individual drug groups. Furthermore, no drug-induced mouse deaths were observed during the experiment, and the body weight of mice in each treatment group did not change significantly compared to the solvent group, indicating good animal tolerance.

[0178] Table 6 Evaluation of the tumor-suppressing effect of SY-5933 combined with Conteltinib in the SW1463 nude mouse subcutaneous xenograft tumor model (based on data on day 12 after group administration).

[0179] Note: 1. Calculated based on the number of days after group administration; data is the mean ± standard error of the mean.

[0180] 2.**: p<0.01; ***: p<0.001, vs. Vehicle group, T-test

[0181] 3.***: p<0.001, vs SY-5933+Conteltinib group, T-test

[0182] Example 9

[0183] In vivo antitumor efficacy study of SY-5933 combined with SY-4835 in a mouse subcutaneous xenograft model of pancreatic cancer MIA PaCa-2 cells.

[0184] Experimental materials:

[0185] Zinc alloy digital caliper (MNT-200, resolution: 0.01 mm, Shanghai Meinaite Industrial Co., Ltd.), JEB2002 electronic balance (resolution: 0.01 g, Shanghai Puchun Measuring Instruments Co., Ltd.), XS105 electronic balance (resolution: 0.01 mg, Mettler Toledo Instruments Co., Ltd.), AL204 electronic balance (resolution: 0.1 mg, Mettler Toledo Instruments Co., Ltd.), Haier biosafety cabinet (model: HR40-IIA2, Qingdao Haier Special Electric Appliance Co., Ltd.).

[0186] Hydroxypropyl methylcellulose (batch number: PDR527640, Shanghai Colorcon Coating Technology Co., Ltd.); Polysorbate 80 (batch number: 20210902, Nanjing Weier Pharmaceutical Group Co., Ltd.); Sulfobutyl beta-cyclodextrin sodium (batch number: SB20210917, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.)

[0187] SY-5933 (batch number: PCS0684-22001, content: 99.1%, Analysis and Formulation Technology Department of SYNCOM Pharmaceuticals (Beijing) Co., Ltd.); SY-4835 (batch number: CP5201-201201, purity: 99.71%, Analysis and Formulation Technology Department of SYNCOM Pharmaceuticals (Beijing) Co., Ltd.)

[0188] Female BALB / c-nude mice, 5 weeks old, certificate number: SCXK (Beijing) 2021-0006, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in IVC individually ventilated isolation cages with a cage size of 325*210*180 mm. Barrier environment conditions: Temperature: 20 - 26 °C; Humidity: 40 - 70%; Light cycle: 12 hours of light and 12 hours of darkness per day. Feed (Co 60 Sterilized feed, Beijing Keao Xieli Feed Co., Ltd.). Drinking water: Pure water, sterilized by high-pressure steam. During the experiment, 6 mice were housed in one cage, and the animals had free access to drinking water and food. The cages, bedding, drinking water, and feed were changed twice a week. The cages and drinking bottles to be replaced were washed and sterilized by high-pressure steam. The cage racks, room walls, and floors were cleaned twice a week with 84 disinfectant or bromogeramine. Experimental animal use license number: SYXK (Beijing) 2022-0050.

[0189] Experimental method:

[0190] Human pancreatic cancer cells MIAPaCa-2 were purchased from the China Center for Type Culture Collection, School of Life Sciences, Wuhan University. Cells were cultured in a monolayer in vitro under DMEM medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged using trypsin-EDTA digestion 2-3 times per week. When cells reached the exponential growth phase and abundance of 80%-90%, cells were harvested, counted, and seeded. Tumors were cultured until they reached 400-700 mm in size. 3 The tumors were passaged twice and then cryopreserved for later use. MIAPaCa-2 tumor masses were revived, passaged twice, and then inoculated subcutaneously into the left and right sides of the back of each mouse.

[0191] The average tumor volume is approximately 150 mm. 3 Mice bearing tumors with regular shapes and uniform sizes were selected for the experiment. They were randomly stratified into four groups based on tumor volume and mouse weight: a model group, a SY-5933 (0.5 mpk) group, a SY-4835 (40 mpk) group, and a combination of SY-5933 / SY-4835 group, with six tumor-bearing mice in each group. Grouping information is shown in Table 7. Drug administration began on the day of grouping. SY-5933 was administered once daily, and SY-4835 was administered for 5 days followed by a 2-day break. Tumor diameter and body weight were measured three times weekly during this period, continuing until day 11. At the end of the experiment, samples (plasma and tumor tissue) were collected at Tmax + 0.5 hours after drug administration. Blood samples were anticoagulated with heparin, centrifuged at 5200 rpm for 5 minutes, and plasma was separated and stored at -80℃ for the determination of SY-5933 and SY-4835 content. The tumor tissue was divided into two portions, flash-frozen in liquid nitrogen, and stored at -80°C for the determination of the content of SY-5933 and SY-4835 and the detection of protein expression by Western blotting.

[0192] Table 7. Dosing regimen of SY-5933 in combination with SY-4835 in subcutaneous xenograft tumors of MIAPaca-2 nude mice.

[0193] The primary indicators used were relative tumor proliferation rate (T / C) and tumor growth inhibition rate (TGI). The evaluation criteria were: T / C (%) > 40% was considered invalid; T / C (%) ≤ 40%, and statistically significant p < 0.05, was considered valid. The tumor volume was calculated using the formula V = 0.5 × a × b. 2 Where V is the tumor volume, and a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the tumor volume using the formula: RTV = V t / V0, where V0 is the tumor volume measured during group administration, V tThis represents the tumor volume at each measurement. The relative tumor growth rate is calculated using the formula: T / C (%) = T RTV / C RTV *100%. (T) RTV Treatment group RTV; C RTV Negative control group (RTV). The tumor growth inhibition rate was calculated using the formula: TGI(%) = (1 - (tumor volume in the treatment group - tumor volume at the time of grouping in the treatment group) / (tumor volume in the control group - tumor volume at the time of grouping in the control group)) * 100%. Data are expressed as Mean ± SEM, and t-tests were used to compare the significance between groups.

[0194] Experimental results:

[0195] The results are shown in Figure 11 (A is the tumor growth curve, B is the animal weight curve). The human pancreatic cancer MIAPaCa-2 xenograft model grew well. At the end of the experiment (Day 11), the tumor in the model group grew to 2110.2±160.5 mm. 3 The tumor volumes in the SY-5933 (0.5 mg / kg) monotherapy group, the SY-4835 (40 mg / kg) monotherapy group, and the SY-5933 (0.5 mg / kg) + SY-4835 (40 mg / kg) group were 1284.4 ± 147.8 mm. 3 1500.3±129.9mm 3 and 674.3±58.4mm 3 See Table 8 for details. At the end of the experiment (Day 11), SY-5933 (0.5 mg / kg) showed a TGI of 42.0% and a T / C of 62.3%, indicating a weak inhibitory effect on tumor growth. Similarly, SY-4835 (40 mg / kg) showed a TGI of 31.1% and a T / C of 73.5% at the end of the experiment (Day 11), also exhibiting a weak inhibitory effect on tumor growth. Meanwhile, the combination of SY-5933 and SY-4835 at the end of the experiment (Day 11) showed a TGI of 73.1% and a T / C of 33.6%, demonstrating significantly better tumor-inhibiting effects than the individual drug groups (p<0.01), indicating a significant synergistic inhibitory effect on tumor growth. Furthermore, no drug-induced mouse deaths were observed during the experiment, and the body weight of mice in each treatment group did not change significantly compared to the solvent group, indicating good animal tolerance.

[0196] Table 8 Evaluation of the tumor-suppressing effect of SY-5933 combined with SY-4835 in the MIAPaca-2 nude mouse subcutaneous xenograft tumor model (based on data from day 11 after group administration).

[0197] Note: 1. Calculated based on the number of days after group administration; data is the mean ± standard error of the mean.

[0198] 2. **: p < 0.01; ***: p < 0.001, vs Vehicle group, T-test

[0199] 3. **: p < 0.01; ***: p < 0.001, vs SY-5933+SY-4835 group, T-test

[0200] Example 10

[0201] In Vivo Antitumor Pharmacodynamic Study of SY-5933 in Combination with SY-4835 in a Subcutaneous Xenograft Tumor Model of Non-Small Cell Lung Cancer NCI-H358 Mice

[0202] Experimental Materials:

[0203] Zinc alloy digital caliper (MNT-200, resolution: 0.01 mm, Shanghai Meinaite Industrial Co., Ltd.), JEB2002 electronic balance (resolution: 0.01 g, Shanghai Puchun Measuring Instrument Co., Ltd.), XS105 electronic balance (resolution: 0.01 mg, Mettler Toledo Instruments Co., Ltd.), AL204 electronic balance (resolution: 0.1 mg, Mettler Toledo Instruments Co., Ltd.), Haier biological safety cabinet (model: HR40-IIA2, Qingdao Haier Special Electric Appliance Co., Ltd.).

[0204] Hypromellose (batch number: PDR527640, Shanghai Colorcon Coating Technology Co., Ltd.); Polysorbate 80 (batch number: 20210902, Nanjing Weier Pharmaceutical Group Co., Ltd.); Sulfobutyl ether β-cyclodextrin sodium (batch number: SB20210917, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.)

[0205] SY-5933 (batch number: PCS0684-22001, content: 99.1%, Analytical Preparation Technology Department of First Medicine Holdings (Beijing) Co., Ltd.); SY-4835 (batch number: CP5201-201201, purity: 99.71%, Analytical Preparation Technology Department of First Medicine Holdings (Beijing) Co., Ltd.)

[0206] Female NOD SCID mice, 5 weeks old, 25 in number, certificate number: SCXK (Beijing) 2021-0006, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in IVC individually ventilated isolation cages with a cage size of 325*210*180 mm. Barrier environment conditions: temperature: 20 - 26 °C; humidity: 40 - 70%; light cycle: 12 hours of light and 12 hours of darkness per day. Feed (Co 60Sterilized feed (Beijing Keao Xieli Feed Co., Ltd.). Drinking water: purified water, sterilized by high-pressure steam. During the experiment, every 5 mice were housed in one cage, and the animals had free access to drinking water and food. The cage, bedding, drinking water, and feed were changed twice a week. The replaced cages and water bottles were washed and sterilized by high-pressure steam. The cage rack, room walls, and floor were cleaned twice a week with 84 disinfectant or bromogeramine. Experimental animal use license number: SYXK(Beijing)2022 - 0050.

[0207] Experimental method:

[0208] Human non-small cell lung cancer cell line NCI-H358 was purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were cultured in monolayer in vitro under the condition of adding 10% fetal bovine serum to RPMI-1640 medium and cultured in a 37°C and 5% CO₂ incubator. Routine digestion and passage were carried out with trypsin-EDTA 2 - 3 times a week. When the cells were in the exponential growth phase with an abundance of 80% - 90%, the cells were harvested, counted, and then inoculated. A 0.1 mL cell suspension of NCI-H358 suspended in PBS (containing 5.0×10 6 , 30% Matrigel) was subcutaneously injected into the subcutaneous parts on the left and right sides of the back of each mouse.

[0209] When the average tumor volume was about 150 mm 3 , tumor-bearing mice with regular tumor shape and uniform size were selected for the experiment. They were randomly stratified into 4 groups according to tumor volume and mouse body weight, namely the model group, SY-5933 (1.5 mpk) group, SY-4835 (30 mpk) group, and SY-5933 / SY-4835 combination group, with 5 tumor-bearing mice in each group. The grouping information is shown in Table 9. Administration started on the day of grouping. SY-5933 was administered once a day, and SY-4835 was administered for 5 days and then discontinued for 2 days. During this period, the tumor diameter and body weight were measured 3 times a week, and continuous administration was carried out until the 16th day. At the end of the experiment, samples (plasma and tumor tissue) were collected at Tmax + 0.5 hours after administration. After the blood samples were anticoagulated with heparin, they were centrifuged at 5200 rpm for 5 minutes to separate plasma, which was stored at -80°C for the determination of the contents of SY-5933 and SY-4835. The tumor tissue was divided into two parts, quick-frozen in liquid nitrogen, and stored at -80°C for the determination of the contents of SY-5933 and SY-4835 and the detection of protein expression by Western Blotting.

[0210] Table 9 Administration scheme of SY-5933 combined with Conteltinib in subcutaneous xenograft tumors of NCI-H358 mice

[0211] The primary indicators used were relative tumor proliferation rate (T / C) and tumor growth inhibition rate (TGI). The evaluation criteria were: T / C (%) > 40% was considered invalid; T / C (%) ≤ 40%, and statistically significant p < 0.05, was considered valid. The tumor volume was calculated using the formula V = 0.5 × a × b. 2 Where V is the tumor volume, and a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the tumor volume using the formula: RTV = V t / V0, where V0 is the tumor volume measured during group administration, V t This represents the tumor volume at each measurement. The relative tumor growth rate is calculated using the formula: T / C (%) = T RTV / C RTV *100%. (T) RTV Treatment group RTV; C RTV Negative control group (RTV). The tumor growth inhibition rate was calculated using the formula: TGI(%) = (1 - (tumor volume in the treatment group - tumor volume at the time of grouping in the treatment group) / (tumor volume in the control group - tumor volume at the time of grouping in the control group)) * 100%. Data are expressed as Mean ± SEM, and t-tests were used to compare the significance between groups.

[0212] Experimental results:

[0213] The results are shown in Figure 12 (A is the tumor growth curve, B is the animal weight curve). The human lung cancer NCI-H358 xenograft model grew well, and at the end of the experiment (Day 16), the tumor in the model group grew to 582.4±65.7 mm. 3 The tumor volumes in the SY-5933 (1.5 mg / kg) monotherapy group, the SY-4835 (30 mg / kg) monotherapy group, and the SY-5933 (1.5 mg / kg) + SY-4835 (30 mg / kg) group were 412.1 ± 71.3 mm. 3 654.0±129.1mm 3 and 277.4±60.0mm 3See Table 10 for details. At the end of the experiment (Day 16), SY-5933 at 1.5 mg / kg showed a TGI of 37.8% and a T / C of 85.7%, indicating a weak inhibitory effect on tumor growth. SY-4835 at 30 mg / kg showed a TGI of -15.7% and a T / C of 108.1% at the end of the experiment (Day 16), showing no inhibitory effect on tumor growth. Meanwhile, the combination of SY-5933 and SY-4835 at the end of the experiment (Day 16) showed a TGI of 69.1% and a T / C of 46.3%, demonstrating superior tumor-inhibiting effects compared to the single-drug groups, and enhancing the inhibition of tumor growth. Furthermore, no drug-induced mouse deaths were observed during the experiment, and the body weight of mice in each treatment group did not change significantly compared to the solvent group, indicating good animal tolerance.

[0214] Table 10 Evaluation of the tumor-suppressive effect of SY-5933 combined with SY-4835 in the NCI-H358 mouse subcutaneous xenograft tumor model (based on data from day 16 after group administration).

[0215] Note: 1. Calculated based on the number of days after group administration; data is the mean ± standard error of the mean.

[0216] 2.**: p<0.01, vs. Vehicle group, T-test

[0217] 3.*: p<0.05, vs SY-5933+SY-4835 group, T-test

[0218] Example 11

[0219] In vivo antitumor efficacy study of SY-5933 combined with SY-4835 in a mouse subcutaneous xenograft tumor model of rectal adenocarcinoma SW1463 cells.

[0220] Experimental materials:

[0221] Zinc alloy digital caliper (MNT-200, resolution: 0.01mm, Shanghai Menet Industrial Co., Ltd.), JEB2002 electronic balance (resolution: 0.01g, Shanghai Puchun Measurement Instrument Co., Ltd.), XS105 electronic balance (resolution: 0.01mg, Mettler Toledo Instruments Ltd.), AL204 electronic balance (resolution: 0.1mg, Mettler Toledo Instruments Ltd.), Haier biosafety cabinet (model: HR40-ⅡA2, Qingdao Haier Special Electric Appliance Co., Ltd.).

[0222] Hypromellose (batch number: PDR527640, Shanghai Colorcon Coating Technology Co., Ltd.); Polysorbate 80 (batch number: 20210902, Nanjing Weier Pharmaceutical Group Co., Ltd.); Sulfobutyl ether β-cyclodextrin sodium (batch number: SB20210917, Shandong Binzhou Zhiyuan Biotechnology Co., Ltd.)

[0223] SY-5933 (batch number: PCS0684-22001, content: 99.1%, Analytical Preparation Technology Department of CSPC Pharmaceuticals (Beijing) Co., Ltd.); SY-4835 (batch number: CP5201-201201, purity: 99.71%, Analytical Preparation Technology Department of CSPC Pharmaceuticals (Beijing) Co., Ltd.)

[0224] Female BALB / c-nude mice, 5 weeks old, certificate number: SCXK (Beijing) 2021-0006, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in IVC individually ventilated isolation cages, and the cage size was 325*210*180 mm. Barrier environment conditions: temperature: 20-26 °C; humidity: 40-70%; light cycle: 12 hours of light and 12 hours of darkness per day. Feed (Co 60 Sterilized feed, Beijing Keao Xieli Feed Co., Ltd.). Drinking water: purified water, and sterilized by high-pressure steam. During the experiment, every 5 mice were housed in 1 cage, and the animals drank water and ate freely. The cages, bedding, drinking water and feed were changed twice a week. The cages and drinking water bottles to be replaced were washed and sterilized by high-pressure steam. The cage racks, room walls and floors were cleaned twice a week with 84 disinfectant or bromogeramine. Experimental animal use license number: SYXK (Beijing) 2022-0050.

[0225] Experimental method:

[0226] Human rectal adenocarcinoma cell line SW1463 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. The cells were cultured in vitro as a monolayer, and the culture conditions were adding 10% fetal bovine serum to L15 medium and culturing in a 37 °C 5% CO2 incubator. Routine digestion and passage were carried out with trypsin-EDTA 2-3 times a week. When the cells were in the exponential growth phase and the abundance was 80%-90%, the cells were harvested, counted and inoculated. 0.1 mL of the SW1463 cell suspension (containing 5.0×10 6 , 50% Matrigel) was subcutaneously injected into the left and right subcutaneous sites on the back of each mouse.

[0227] When the average tumor volume was about 150 mm 3Mice bearing tumors with regular shapes and uniform sizes were selected for the experiment. They were randomly stratified into four groups based on tumor volume and mouse weight: a model group, a SY-5933 (2 mpk) group, a SY-4835 (40 mpk) group, and a combination of SY-5933 / SY-4835 group, with five tumor-bearing mice in each group. Grouping information is shown in Table 11. Drug administration began on the day of grouping. SY-5933 was administered once daily, and SY-4835 was administered for 5 days followed by a 2-day break. Tumor diameter and body weight were measured three times weekly during this period, continuing until day 21. At the end of the experiment, samples (plasma and tumor tissue) were collected at Tmax + 0.5 hours after drug administration. Blood samples were anticoagulated with heparin, centrifuged at 5200 rpm for 5 minutes, and plasma was separated and stored at -80℃ for the determination of SY-5933 and SY-4835 content. The tumor tissue was divided into two portions, flash-frozen in liquid nitrogen, and stored at -80°C for the determination of the content of SY-5933 and SY-4835 and the detection of protein expression by Western blotting.

[0228] Table 11 Dosing regimen of SY-5933 in combination with SY-4835 in subcutaneous xenograft tumors of SW1463 nude mice

[0229] The primary indicators used were relative tumor proliferation rate (T / C) and tumor growth inhibition rate (TGI). The evaluation criteria were: T / C (%) > 40% was considered invalid; T / C (%) ≤ 40%, and statistically significant p < 0.05, was considered valid. The tumor volume was calculated using the formula V = 0.5 × a × b. 2 Where V is the tumor volume, and a and b are the length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the tumor volume using the formula: RTV = V t / V0, where V0 is the tumor volume measured during group administration, V t This represents the tumor volume at each measurement. The relative tumor growth rate is calculated using the formula: T / C (%) = T RTV / C RTV *100%. (T) RTV Treatment group RTV; C RTV Negative control group (RTV). The tumor growth inhibition rate was calculated using the formula: TGI(%) = (1 - (tumor volume in the treatment group - tumor volume at the time of grouping in the treatment group) / (tumor volume in the control group - tumor volume at the time of grouping in the control group)) * 100%. Data are expressed as Mean ± SEM, and t-tests were used to compare the significance between groups.

[0230] Experimental results:

[0231] The results are shown in Figure 13 (A is the tumor growth curve, B is the animal weight curve). The human rectal adenocarcinoma cell line SW1463 xenograft tumor model grew well. At the end of the experiment (Day 21), the tumor in the model group grew to 738.6±79.3 mm. 3 The tumor volumes in the SY-5933 (2 mg / kg) monotherapy group, the SY-4835 (40 mg / kg) monotherapy group, and the SY-5933 (2 mg / kg) + SY-4835 (40 mg / kg) group were 500.0 ± 79.9 mm. 3 804.7±40.5mm 3 and 407.1±25.3mm 3 See Table 12 for details. At the end of the experiment (Day 21), SY-59332 mg / kg showed a TGI of 43.9% and a T / C of 65.0%, indicating a weak inhibitory effect on tumor growth. SY-483540 mg / kg showed a TGI of -12.1% and a T / C of 111.6% at the end of the experiment (Day 21), showing no inhibitory effect on tumor growth. Meanwhile, the combination of SY-5933 and SY-4835 showed a TGI of 60.7% and a T / C of 55.5% at the end of the experiment (Day 21), demonstrating superior tumor-inhibiting effects compared to the individual drug groups, and enhancing the inhibition of tumor growth. Furthermore, no drug-induced mouse deaths were observed during the experiment, and the body weight of mice in each treatment group did not change significantly compared to the solvent group, indicating good animal tolerance.

[0232] Table 12 Evaluation of the tumor-suppressing effect of SY-5933 combined with SY-4835 in the SW1463 nude mouse subcutaneous xenograft tumor model (based on data from day 21 after group administration).

[0233] Note: 1. Calculated based on the number of days after group administration; data is the mean ± standard error of the mean.

[0234] 2.**: p<0.01, vs. Vehicle group, T-test

[0235] 3.***: p<0.001, vs SY-5933+SY-4835 group, T-test

[0236] Example 12

[0237] A study on the synergistic inhibition of the proliferation of KRAS(G12C)-mutant non-small cell lung cancer, pancreatic cancer, and colorectal cancer cells by combining SY-5933 and SY-3505.

[0238] Experimental plan:

[0239] Cell Titer-Glo reagent was used to detect the inhibitory effect of the combination of SY-5933 and SY-3505 on the proliferation of KRAS(G12C) mutant non-small cell lung cancer, pancreatic cancer and colorectal cancer cells, and the synergistic coefficient of the combination was calculated using Combenefit 2 software.

[0240] Experimental materials:

[0241] SY-5933 and SY-3505 were synthesized by the Pharmaceutical Chemistry Department of Shouyao Holdings (Beijing) Co., Ltd. The compounds were prepared into 10mM stock solutions using DMSO, and then aliquoted and stored at -20℃.

[0242] NCI-H358 cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; MIA PaCa-2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee; and SW1463 cells were purchased from ATCC.

[0243] DMEM medium was purchased from Biological Industries, catalog number 06-1055-57-1ACS. RPMI-1640 medium was purchased from Biological Industries, catalog number 01-100-1ACS. L-15 medium was purchased from VivaCell, catalog number C3070-0500. Fetal bovine serum (FBS) was purchased from Biological Industries, catalog number 04-001-1ACS. Penicillin-Streptomycin was purchased from GIBCO, catalog number 15070-063. 10×PBS buffer was purchased from Solarbio, pH 7.2-7.4, catalog number P1022.

[0244] Cell Titer-Glo was purchased from Promega, product number G7570.

[0245] Experimental methods:

[0246] NCI-H358 cells were cultured in RPMI-1640 medium, MIAPaCa-2 cells were cultured in DMEM medium, and SW1463 cells were cultured in L-15 medium. All the above media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0247] The cell proliferation inhibition assay was performed as follows: MIAPaCa-2, NCI-H358, and SW1463 cells were counted and seeded in 96-well cell culture plates, with 500 cells (MIAPaCa-2) and 2000 cells (NCI-H358 and SW1463) per well, respectively. 190 μL of culture medium was added to each well, and the plates were incubated overnight at 37°C. The next day, the combination compounds were added as follows: Compounds SY-3505 and SY-5933 were serially diluted with DMSO. 4 μL of each concentration was added to 96 μL of serum-free culture medium and vortexed. Then, 5 μL of the suspended compound was added to the test cells using a multipipe. The final concentrations of compound SY-3505 in MIAPaCa-2 cells were 2000, 600, 200, 60, 20, and 0 nM, and the final concentrations of compound SY-5933 were 6, 2, 0.6, 0.2, 0.06, and 0 nM. The final concentrations of compound SY-3505 in NCI-H358 cells were 250, 125, 62.5, 31.25, and 0 nM, while the final concentrations of compound SY-5933 were 5, 2.5, 1.25, 0.625, and 0 nM. For SW1463 cells, the final concentrations of compound SY-3505 were 10000, 3000, 1000, 300, 100, and 0 nM, while the final concentrations of compound SY-5933 were 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1, and 0 nM. After 5 days of compound treatment, the supernatant was aspirated, and 25 μL CellTiter-Glo reagent was added. The mixture was stirred in a shaker in the dark for 3 minutes, and the luminescence signal was read. Finally, Combenefit 2 was used to calculate the synergistic effect of the combined use of compound SY-5933 and SY-3505 on cell proliferation inhibition.

[0248] Experimental results:

[0249] After 5 days of combined treatment with SY-5933 and SY-3505, the proliferation of KRAS(G12C) mutant pancreatic cancer cells MIAPaCa-2, non-small cell lung cancer cells NCI-H358, and colorectal cancer cells SW1463 was significantly inhibited, and the combined use of the two showed a significant synergistic effect.

Claims

1. A pharmaceutical combination product, characterized in that, The product includes a first active pharmaceutical ingredient and a second active pharmaceutical ingredient, wherein the first active pharmaceutical ingredient is a KRAS inhibitor.

2. The product according to claim 1, characterized in that, The second active pharmaceutical ingredient is a FAK inhibitor and / or a WEE1 inhibitor.

3. The product according to claim 1 or 2, characterized in that, The KRAS inhibitor is a KRAS(G12C) inhibitor.

4. The product according to any one of claims 1-3, characterized in that, The product is used to treat abnormal cell growth, specifically cancer.

5. The product according to any one of claims 1-3, characterized in that, The product also includes one or more pharmaceutically acceptable excipients.

6. The product according to any one of claims 1-3, characterized in that, The first active pharmaceutical ingredient and the second active pharmaceutical ingredient are administered sequentially or simultaneously.

7. The product according to any one of claims 1-3, characterized in that, The KRAS inhibitor is selected from SY-5933 or a pharmaceutically acceptable salt thereof.

8. The product according to any one of claims 1-3, characterized in that, The FAK inhibitor is selected from Conteltinib, SY3505, or a pharmaceutically acceptable salt thereof.

9. The product according to any one of claims 1-3, characterized in that, The WEE1 inhibitor is selected from SY-4835, ZN-c3, Debio-0123, SC-0191, IMP-7068, APR-1051 or a pharmaceutically acceptable salt thereof.

10. Use of the product according to any one of claims 1-9 in the preparation of a medicament for treating tumors.

11. The use according to claim 10, characterized in that, The tumors mentioned are bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer, melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastoma, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct carcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastatic tumors caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or malignant hematological diseases.

12. The use according to claim 10, characterized in that, The tumors were selected from lung cancer, colorectal cancer, and pancreatic cancer.

13. The product according to any one of claims 1-9, and the use according to any one of claims 10-12, characterized in that, The KRAS inhibitor was administered in subjects at a dose range of 50 mg / day to 2000 mg / day.

14. The product according to any one of claims 1-9, and the use according to any one of claims 10-12, characterized in that, The FAK inhibitor was administered in subjects at a dose range of 50 mg / day to 2000 mg / day.

15. The product according to any one of claims 1-9, and the use according to any one of claims 10-12, characterized in that, The WEE1 inhibitor was administered in subjects at a dose range of 5 mg / day to 1000 mg / day.

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