Pharmaceutical compositions, uses thereof and methods of treating cancer

Abstract

The present application provides a pharmaceutical composition or kit comprising a therapeutically effective amount of a KRAS inhibitor and a therapeutically effective amount of another cancer therapeutic agent, and use of the KRAS inhibitor and the at least one other cancer therapeutic agent in the manufacture of a medicament for treating cancer, which have a good synergistic effect when used in combination in tumor cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a combination pharmaceutical composition comprising a KRAS inhibitor. BACKGROUND

[0002] Cancer is one of the major diseases threatening global human health. Lung cancer is the highest incidence of cancer in the world, and its incidence ranks first among all cancers in China. It is also the highest incidence and mortality cancer in China. According to the data published by the American Cancer Society in 2016, about 1.8 million people worldwide suffer from lung cancer every year, of which nearly 80% of lung cancer is non-small cell lung cancer (NSCLC).

[0003] RAS is a group of closely related monomeric globular proteins (21 kDa molecular weight) that have 188-189 amino acids and bind to either guanosine diphosphate (GDP) or guanosine triphosphate (GTP). RAS subfamily members include HRAS, KRAS, and NRAS. RAS functions as a molecular switch, when RAS contains bound GDP, it is in a dormant or off position and is "inactive." When cells are exposed to certain growth-promoting stimuli, RAS is induced to convert its bound GDP to GTP, when bound to GTP, RAS is "on" and is able to interact with and activate other downstream target proteins. The RAS protein has very little intrinsic ability to hydrolyze GTP back to GDP (thereby switching itself back to the off state). It requires an exogenous protein, GTPase-activating protein (GAP), to switch it back to the off state, GAP interacts with RAS to greatly accelerate the conversion of GTP to GDP. Any mutation in RAS that will affect the interaction of RAS with GAP, and the ability to convert GTP to GDP, will result in a prolonged period of protein activation, leading to prolonged cell signaling, which in turn leads to cells continuing to grow and divide. Since this signaling leads to cell growth and division, over-activated RAS signaling can ultimately lead to cancer. In lung cancer, mutations in the RAS gene are identified in about 32% of lung cancers, and a mutation in any of the three major isoforms of the RAS (HRAS, NRAS, or KRAS) gene can lead to the development of human tumors. It has been reported that the KRAS gene has the highest frequency of mutations in the RAS gene, and KRAS mutations are detected in 25-30% of tumors. In comparison, the rate of oncogenic mutations in the NRAS and HRAS family members is much lower (8% and 3%, respectively). The most common KRAS mutations are found at residues G12 and G13 in the P-loop and at residue Q61. The G12C mutation is a frequent mutation in the KRAS gene (glycine-12 mutated to cysteine). This mutation has been found in about 13% of cancers, about 43% of lung cancers, and almost 100% of MYH-associated polyposis (a familial colon cancer syndrome). It is therefore a good direction to develop inhibitors that selectively inhibit KRAS mutations, although KRAS inhibitors as monotherapy have efficacy in certain cancers, there is still a need for even more effective treatment regimens in cancer. SUMMARY

[0004] It is an object of the present application to provide pharmaceutical compositions or kits comprising a therapeutically effective amount of a KRAS inhibitor and a therapeutically effective amount of another cancer therapeutic agent, and the use of a KRAS inhibitor and at least one other cancer therapeutic agent in the manufacture of a medicament for treating cancer, which have a better synergistic effect when used in combination in tumor cells.

[0005] In one aspect of the present application, there is provided a pharmaceutical composition or kit comprising a therapeutically effective amount of a KRAS inhibitor and at least one therapeutically effective amount of another cancer therapeutic agent and a pharmaceutically acceptable carrier.

[0006] The KRAS inhibitor comprises a compound represented by Formula (I), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein R is CH3or CD3;

[0007]

[0008] In one embodiment, the another cancer therapeutic agent is selected from a PD-1 pathway inhibitor, a PD-L1 pathway inhibitor, an EGFR targeting agent, a CDK inhibitor and a PI3K inhibitor.

[0009] In one embodiment, the PD-1 pathway inhibitor is selected from a PD-1 antagonist, a PD-1 binding antagonist, a small molecule PD-1 antagonist, a PD-1 inhibitor and an anti-PD-1 biological product.

[0010] In one embodiment, the PD-L1 pathway inhibitor is selected from a PD-L1 antagonist, a PD-L1 binding antagonist, a small molecule PD-L1 antagonist, a PD-L1 inhibitor and an anti-PD-L1 biological product.

[0011] In one embodiment, the EGFR targeting agent is selected from an EGFR inhibitor and an EGFR antibody or antigen-binding fragment thereof.

[0012] In one embodiment, the CDK inhibitor is selected from a CDK4 / 6 inhibitor, a CDK2 / 4 / 6 inhibitor, a CDK7 inhibitor, a CDK8 inhibitor, a CDK9 inhibitor, a CDK10 inhibitor and a CDK11 inhibitor.

[0013] In one embodiment, the PI3K inhibitor is selected from a PI3K gamma inhibitor, a PI3K delta inhibitor, a PI3K beta inhibitor, a PI3K alpha inhibitor and a pan-PI3K inhibitor.

[0014] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and a PD-1 inhibitor.

[0015] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and a PD-L1 inhibitor.

[0016] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and an EGFR inhibitor.

[0017] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and an EGFR antibody or antigen-binding fragment thereof.

[0018] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and a CDK7 inhibitor.

[0019] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and a PI3K inhibitor.

[0020] In an embodiment, the PD-1 inhibitor is selected from AMG404, pembrolizumab, nivolumab (MDX 1106), pembrolizumab (MK-3475), pidilizumab (CT-011), cemiplimab (REGN2810), tislelizumab (BGB-A317), spartalizumab (PDR001), RN888, mAb15, MEDI-0680 (AMP-514), BGB-108, spartalizumab (AGEN-2034), IBI-308, mDX-400, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, and biosimilars, bioenhancers and bioequivalents of these inhibitors.

[0021] In an embodiment, the PD-L1 inhibitor is selected from BMS-936559 (MDX-1105), AMP-714, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab, avdilimumab, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, KY-1003, and biosimilars, bioenhancers and bioequivalents of these inhibitors.

[0022] In an embodiment, the EGFR inhibitor is selected from afatinib, erlotinib, lapatinib, gefitinib, osimertinib, icotinib, pyrotinib and olmutinib.

[0023] In an embodiment, the EGFR antibody or antigen-binding fragment thereof is selected from Cetuximab, nimotuzumab, panitumumab, zalutumumab, matuzumab and necitumumab; more preferably, Cetuximab.

[0024] In an embodiment, the CDK7 inhibitor is selected from the group consisting of SY-5609, SY- 1365, CT-7001, and BTX-A51.

[0025] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and SY-5609.

[0026] In an embodiment, the PI3K inhibitor is selected from the group consisting of BYL719, GDC0941, AMG511, LY294002, BEZ235, BKM120, and GSK-2636771.

[0027] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and BYL719.

[0028] In a preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (I) and GDC0941.

[0029] In an embodiment, the cancer is a KRAS G12C mutant cancer.

[0030] In an embodiment, the cancer is a solid or hematological tumor.

[0031] In an embodiment, the cancer is pancreatic ductal carcinoma, colorectal cancer, multiple myeloma, lung cancer, cutaneous melanoma, uterine endometrioid carcinoma, uterine carcinosarcoma, thyroid cancer, acute myeloid leukemia, bladder urothelial carcinoma, gastric cancer, cervical cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal cancer, chronic lymphocytic leukemia, lung squamous cell carcinoma, small cell lung cancer, renal papillary cell carcinoma, adenoid cystic carcinoma, chromophobe renal cell carcinoma, hepatocellular carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma, ovarian serous adenocarcinoma, adrenocortical carcinoma, prostate cancer, neuroblastoma, brain low grade glioma, glioblastoma, medulloblastoma, esophageal squamous cell carcinoma, renal clear cell carcinoma, osteosarcoma, ovarian small cell carcinoma, rhabdoid tumor, sarcoma, small intestine neuroendocrine tumor, T-cell prolymphocytic leukemia.

[0032] In an embodiment, the cancer is lung adenocarcinoma, colon cancer, rectal cancer, or lung cancer.

[0033] In an embodiment, the cancer is lung adenocarcinoma, rectal adenocarcinoma, or lung cancer.

[0034] In an embodiment, the lung cancer is small cell lung cancer or non-small cell lung cancer.

[0035] In an embodiment, the compound of Formula (I) is selected from the group consisting of:

[0036]

[0037] In one embodiment, the KRAS inhibitor is a compound of Formula (II) or a pharmaceutically acceptable salt thereof.

[0038]

[0039] In one embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and a PD-1 pathway inhibitor.

[0040] In one embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and a PD-L1 pathway inhibitor.

[0041] In one embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and an EGFR targeting agent.

[0042] In one preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and cetuximab.

[0043] In one embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and a CDK7 inhibitor.

[0044] In one preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and SY-5609.

[0045] In one embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and a PI3K inhibitor.

[0046] In one preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and BYL719.

[0047] In one preferred embodiment, the pharmaceutical composition or kit comprises a compound of Formula (II) and GDC0941.

[0048] In one embodiment, the pharmaceutical composition or kit comprises 0.1-1000 nM KRAS inhibitor and 1-10000 nM of another cancer therapeutic.

[0049] In one embodiment, the pharmaceutical composition or kit comprises 0.32-1000 nM KRAS inhibitor.

[0050] In one embodiment, the pharmaceutical composition or kit comprises 3-1000 nM KRAS inhibitor.

[0051] In one embodiment, the pharmaceutical composition or kit comprises 3.17-1000 nM KRAS inhibitor.

[0052] In an embodiment, the pharmaceutical composition or kit comprises 10-1000 nM of the KRAS inhibitor.

[0053] In an embodiment, the pharmaceutical composition or kit comprises 0.1-100 nM of the KRAS inhibitor.

[0054] In an embodiment, the pharmaceutical composition or kit comprises 3.17-1000 nM of the other cancer therapeutic agent.

[0055] In an embodiment, the pharmaceutical composition or kit comprises 31.74-10000 nM of the other cancer therapeutic agent.

[0056] In an embodiment, the pharmaceutical composition or kit comprises 100-10000 nM of the other cancer therapeutic agent.

[0057] In an embodiment, the pharmaceutical composition or kit comprises 316.91-10000 nM of the other cancer therapeutic agent.

[0058] In an embodiment, the pharmaceutical composition or kit comprises 10 mg / mL of the compound of formula (II) and 1 mg / mL of cetuximab.

[0059] In an embodiment, the pharmaceutical composition or kit comprises 1-3 mg / kg of the compound of formula (II) and 10 mg / kg of the PD-1 antibody.

[0060] In an embodiment, the pharmaceutical composition or kit comprises a pharmaceutical dosage equivalent to 10 mpk of the compound of formula (II) and 1 mpk of cetuximab.

[0061] In an embodiment, the pharmaceutical composition or kit comprises a pharmaceutical dosage equivalent to 1-3 mpk of the compound of formula (II) and 10 mpk of the PD-1 antibody.

[0062] In an embodiment, the pharmaceutical composition or kit comprises 0.1-100 nM of the compound of formula (II) and 1-1000 nM of SY-5609.

[0063] In an embodiment, the pharmaceutical composition or kit comprises 0.1-100 nM of the compound of formula (II) and 3.17-1000 nM of SY-5609.

[0064] In an embodiment, the pharmaceutical composition or kit comprises 1-1000 nM of the compound of formula (II) and 100-10000 nM of BYL719.

[0065] In one embodiment, the pharmaceutical composition or kit comprises 3-1000 nM of the compound of formula (II) and 100-10000 nM of BYL719.

[0066] In one embodiment, the pharmaceutical composition or kit comprises 10-1000 nM of the compound of formula (II) and 100-10000 nM of BYL719.

[0067] In one embodiment, the pharmaceutical composition or kit comprises 0.1-1000 nM of the compound of formula (II) and 10-10000 nM of GDC0941.

[0068] In one embodiment, the pharmaceutical composition or kit comprises 3.17-1000 nM of the compound of formula (II) and 31.74-10000 nM of GDC0941.

[0069] In one embodiment, the pharmaceutical composition or kit comprises 3.17-1000 nM of the compound of formula (II) and 100-10000 nM of GDC0941.

[0070] In one embodiment, the pharmaceutical composition or kit comprises 10-1000 nM of the compound of formula (II) and 31.74-10000 nM of GDC0941.

[0071] In one embodiment, the pharmaceutical composition or kit comprises 0.32-1000 nM of the compound of formula (II) and 316.91-10000 nM of GDC0941.

[0072] In one embodiment, the pharmaceutical composition or kit comprises the KRAS inhibitor and the other cancer therapeutic agent in a mass ratio of 0.00001-1000.

[0073] In one embodiment, the pharmaceutical composition or kit comprises the compound of formula (II) and cetuximab in a mass ratio of (1-10):1, for example 10:1.

[0074] In one embodiment, the pharmaceutical composition or kit comprises the compound of formula (II) and a PD-1 antibody in a mass ratio of (1-10):10, for example 1:10 or 3:10.

[0075] In one embodiment, the pharmaceutical composition or kit comprises the compound of formula (II) and SY-5609 in a mass ratio of 0.0001-31.6.

[0076] In one embodiment, the pharmaceutical composition or kit comprises a compound of formula (II) and BYL719 in a mass ratio of 0.0003-10, for example 0.001-10 or 0.0003-10.

[0077] In one embodiment, the pharmaceutical composition or kit comprises a compound of formula (II) and GDC0941 in a mass ratio of 0.000032-35, for example 0.001-31.5, 0.0003-10, 0.0003-31.5 or 0.00003-3.15.

[0078] In one embodiment, the pharmaceutical composition or kit comprises a pharmaceutical dose of a compound of formula (II) equivalent to 0.01-5000 mg per day, preferably 1-1500 mg per day, optionally 10 mg per day, 50 mg per day, 100 mg per day, 150 mg per day, 200 mg per day, 250 mg per day, 300 mg per day, 350 mg per day, 400 mg per day, 400 mg per day, 500 mg per day, 550 mg per day, 600 mg per day, 650 mg per day, 700 mg per day, 750 mg per day, 800 mg per day, 8500 mg per day, 900 mg per day, 950 mg per day, 1000 mg per day, 1200 mg per day, 1250 mg per day, 1300 mg per day, 1400 mg per day, 1500 mg per day.

[0079] In one embodiment, the pharmaceutical composition or kit comprises a pharmaceutical dose of a further cancer therapeutic agent equivalent to 0.01-1000 mg per day, preferably 1-500 mg per day, optionally 10 mg per day, 50 mg per day, 100 mg per day, 150 mg per day, 200 mg per day, 250 mg per day, 300 mg per day, 350 mg per day, 400 mg per day, 400 mg per day, 500 mg per day, 550 mg per day, 600 mg per day, 650 mg per day, 700 mg per day, 750 mg per day, 800 mg per day, 8500 mg per day, 900 mg per day, 950 mg per day, 1000 mg per day.

[0080] In one embodiment, the pharmaceutical composition or kit comprises 1-10000 mg of a KRAS inhibitor and 1-10000 mg of a further cancer therapeutic agent.

[0081] In one embodiment, the pharmaceutical composition or kit comprises 1-10000 mg of a compound of formula (II) and 1-10000 mg of cetuximab.

[0082] In one embodiment, the pharmaceutical composition or kit comprises 1-10000 mg of the compound of Formula (II) and 1-10000 mg of SY-5609.

[0083] In one embodiment, the pharmaceutical composition or kit comprises 1-10000 mg of the compound of Formula (II) and 1-10000 mg of BYL719.

[0084] In one embodiment, the pharmaceutical composition or kit comprises 1-10000 mg of the compound of Formula (II) and 1-10000 mg of GDC0941.

[0085] In another aspect of the present application, there is provided a method of treating cancer, comprising administering to a subject in need thereof a KRAS inhibitor as described above and another cancer therapeutic agent, wherein the therapeutically effective amount of the KRAS inhibitor and the therapeutically effective amount of the another cancer therapeutic agent can be administered simultaneously, separately or sequentially.

[0086] In one embodiment, the method of treating cancer further comprises administering to the subject another therapy selected from one or more of radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, phototherapy. The another therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0087] In another aspect of the present application, there is provided a use of the pharmaceutical composition as described above in the manufacture of a medicament for treating cancer. The therapeutically effective amount of the KRAS inhibitor and the therapeutically effective amount of the another cancer therapeutic agent can be administered simultaneously, separately or sequentially.

[0088] In one embodiment, the cancer in the use as described above is a solid tumor or a hematological tumor.

[0089] In one embodiment, the cancer in the use as described above is pancreatic ductal carcinoma, colorectal cancer, multiple myeloma, lung cancer, skin melanoma, uterine endometrioid carcinoma, uterine carcinosarcoma, thyroid cancer, acute myeloid leukemia, bladder urothelial carcinoma, gastric cancer, cervical cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal cancer, chronic lymphocytic leukemia, lung squamous cell carcinoma, small cell lung cancer, renal papillary cell carcinoma, adenoid cystic carcinoma, chromophobe renal cell carcinoma, liver cancer, breast invasive carcinoma, cervical squamous cell carcinoma, ovarian serous adenocarcinoma, adrenocortical carcinoma, prostate cancer, neuroblastoma, brain low-grade glioma, glioblastoma, medulloblastoma, esophageal squamous cell carcinoma, renal clear cell carcinoma, osteosarcoma, ovarian small cell carcinoma, rhabdoid tumor, sarcoma, small intestine neuroendocrine tumor, T-cell prolymphocytic leukemia.

[0090] In one embodiment, the cancer in the above use is lung adenocarcinoma, colon cancer, rectal cancer, or lung cancer.

[0091] In one embodiment, the cancer in the above use is lung adenocarcinoma, rectal adenocarcinoma, or lung cancer.

[0092] In one embodiment, the cancer in the above use is KRAS G12C mutant cancer.

[0093] In one embodiment, the lung cancer in the above use is small cell lung cancer or non-small cell lung cancer.

[0094] In another aspect, the present application provides use of a therapeutically effective amount of a KRAS inhibitor selected from one or more of the compounds of Formula (I), Formula (II)-Formula (V), or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, pharmaceutical combination, or kit for treating cancer. The KRAS inhibitor and the other cancer therapeutic agent, and the cancer in the use are as defined above.

[0095] In particular, in one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (I) and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, pharmaceutical combination, or kit for treating cancer.

[0096] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, pharmaceutical combination, or kit for treating cancer.

[0097] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (I) and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, pharmaceutical combination, or kit for treating lung cancer.

[0098] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (I) and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, pharmaceutical combination, or kit for treating small cell lung cancer or non-small cell lung cancer.

[0099] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, pharmaceutical combination, or kit for treating lung cancer.

[0100] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and at least one therapeutically effective amount of another cancer therapeutic agent in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer or non-small cell lung cancer.

[0101] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and a therapeutically effective amount of a PD-1 inhibitor in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colon cancer or rectal adenocarcinoma.

[0102] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and a therapeutically effective amount of a PD-L1 inhibitor in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colon cancer or rectal adenocarcinoma.

[0103] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and a therapeutically effective amount of an EGFR inhibitor in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colon cancer or rectal adenocarcinoma.

[0104] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and a therapeutically effective amount of an EGFR antibody or an antigen-binding fragment thereof in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colon cancer or rectal adenocarcinoma.

[0105] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and a therapeutically effective amount of a CDK7 inhibitor in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colon cancer or rectal adenocarcinoma.

[0106] In one aspect, the present application provides use of a therapeutically effective amount of a compound of Formula (II) and a therapeutically effective amount of a PI3K inhibitor in the manufacture of a medicament, a pharmaceutical combination or a kit for the treatment of small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, colon cancer or rectal adenocarcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0107] Figure 1 Matrix plot of the growth inhibition of the combination of the compound of Formula (II) and BYL719 on tumor cells LU99 in Example 1.

[0108] Figure 2A , Figure 2B , Figure 2C , Figure 2D Average synergy values calculated in four statistical models in Example 1.

[0109] Figure 3 Growth inhibition matrix for the combination of the compound of formula (II) and BYL719 on tumor cells SW837 in Example 2.

[0110] Figure 4A Figure 4B Figure 4C Figure 4D Average synergy values calculated in the four statistical models in Example 2.

[0111] Figure 5 Growth inhibition matrix for the combination of the compound of formula (II) and GDC0941 on tumor cells LU99 in Example 3.

[0112] Figure 6A Figure 6B Figure 6C Figure 6D Average synergy values calculated in the four statistical models in Example 4.

[0113] Figure 7 Growth inhibition matrix for the combination of the compound of formula (II) and GDC0941 on tumor cells SW837 in Example 4.

[0114] Figure 8A Figure 8B Figure 8C Figure 8D Average synergy values calculated in the four statistical models in Example 4.

[0115] Figure 9 Growth inhibition matrix for the combination of the compound of formula (II) and GDC0941 on tumor cells SW1463 in Example 5.

[0116] Figure 10A Figure 10B Figure 10C Figure 10D Average synergy values calculated in the four statistical models in Example 5.

[0117] Figure 11 Growth inhibition matrix for the combination of the compound of formula (II) and GDC0941 on tumor cells SW1573 in Example 6.

[0118] Figure 12A Figure 12B Figure 12C Figure 12D Average synergy values calculated in the four statistical models in Example 6.

[0119] Figure 13 ​​​​​​​​​​​​​​​The growth inhibition matrix of NCI-H2122 tumor cells is shown in Example 7, which is the combined action of compound (II) and SY-5609.

[0120] Figure 14A , Figure 14B , Figure 14C , Figure 14D This is the average synergistic value calculated from the four statistical models in Example 7.

[0121] Figure 15 The tumor growth curves (mean ± standard error) of each group of tumor-bearing mice in Example 8 are shown.

[0122] Figure 16 The figures show the tumor growth curves of individual tumor-bearing mice in each group in Example 8.

[0123] Figure 17 The tumor weight (mean ± standard error) of each group of tumor-bearing mice in Example 8.

[0124] Figure 18 The relative changes in body weight of tumor-bearing mice in Example 8 (mean ± standard error).

[0125] Figure 19 The tumor growth curves (mean ± standard error) of each group of tumor-bearing mice in Example 9 are shown.

[0126] Figure 20 The tumor growth curves are for each group of tumor-bearing mice in Example 9.

[0127] Figure 21 The tumor weight (mean ± standard error) of each group of tumor-bearing mice in Example 9.

[0128] Figure 22 The relative changes in body weight of tumor-bearing mice in Example 9 (mean ± standard error). Detailed Implementation

[0129] 1. Definitions and Explanations

[0130] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0131] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0132] As used herein, the use of the term "inhibition" is relative to a control. One of skill in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to the response in a subject or cell not treated with a compound. The disclosure of all ranges of the present application should be considered as a disclosure of all subranges and individual points within the ranges. For example: disclosure of a range of 1-1000 should be considered as a disclosure of a range of 1-200, 200-300, etc., as well as the individual points 200, 300, 400, 500, 600, 700, 800, 900, and 1000, etc.

[0133] The term "pharmaceutically acceptable" in reference to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0134] The compounds can exist in the pharmaceutical composition as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application that are within the scope of sound medical judgment, which are found to be suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. The salts can be prepared from the compounds of the present application by conventional chemical methods. The term "pharmaceutically acceptable salt" means a salt of a compound of this application which is suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, as well as the zwitterionic forms, where the zwitterions contain counterions such as sodium, potassium, ammonium, calcium, organic amino, or magnesium, and the like. When a compound of the present application contains relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base to provide the appropriate salt. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When a compound of the present application contains relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid to provide the appropriate salt. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric, hydrobromic, nitric, carbonic, monobasic, phosphoric, diphosphoric, sulfuric, hydrogen sulfate, hydroiodic, phosphorous, and the like; and the salts derived from organic acids, such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like; also salts derived from amino acids such as arginine, lysine, and the like. Certain specific compounds of the present application contain both basic and acidic functionalities as a result of which two salt forms can be formed. The compounds of the present application that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The compounds of the present application that are acidic in nature are capable of forming base salts with various cations. Pharmaceutically acceptable salts of the compounds of the present application include the following: acetate, benzenesulfonic, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, napsylate, nitrate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, sodium chloride, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, and the like.

[0135] The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by contacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid, in waters or in an organic solvent, or in a mixture of both, to provide the desired salt.

[0136] In addition to salt forms, the compounds provided herein can exist in a zwitterionic form. Some compounds disclosed herein can exist in different polymorphic or crystalline forms. In general, all physical forms are equivalent and within the scope of the present application. In addition to salt forms, the compounds provided by this application can exist in a zwitterionic form. The compounds described herein can be readily converted into the zwitterionic form by one skilled in the art. The compounds described herein can be converted into the zwitterionic form by mixing the compound with an appropriate acid or base, such as those described above. The compounds described herein can exist in different polymorphic or crystalline forms. In general, all physical forms are equivalent and within the scope of the present application.

[0137] Certain compounds of the present application can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are included in the scope of the present application. Certain compounds of the present application can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent and are intended to be within the scope of the present application.

[0138] The compounds of the present application can exist in particular geometric or stereoisomeric forms. The present application contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures and other mixtures thereof, as falling within the scope of the application. Additionally, it is understood that certain compounds of the present application can exist in different tautomeric forms. It is intended that all tautomeric forms are included within the scope of the present application.

[0139] The compounds disclosed herein can exist as atropisomers, which are conformational stereoisomers that arise when rotation about a single bond in the molecule is prevented or greatly slowed due to steric interactions with other parts of the molecule. The compounds disclosed herein include all atropisomers as pure individual atropisomer preparations, enriched preparations of each, or unspecified mixtures of each. If the barrier to rotation about the single bond is sufficiently high, and the interconversion between conformations is sufficiently slow, then separation and isolation of isomeric species can be permitted. Separation and isolation of isomeric species is suitably indicated by the well- known and generally accepted symbols "M" or "P".

[0140] The term "cancer" refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms include solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes precancerous lesions as well as malignant cancers and tumors.

[0141] The term "antigen-binding fragment" refers to any portion of a full-length antibody that is less than full-length, but that includes at least a portion of the variable region (e.g., one or more CDRs and / or one or more antibody combining sites) of the antibody that binds to an antigen, and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that includes an antigen-binding portion that binds to the same antigen as the antibody from which the fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments. The fragments can include multiple chains linked together, e.g., by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. An antigen-binding fragment includes any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of the corresponding region), results in an antibody that immunospecifically binds (i.e., exhibits a Ka of at least or at least about 107-108M"1) to an antigen. A "functional fragment" or "analog of an anti-GPC3 antibody" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as "antibody fragment," and with respect to an antibody, can refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, e.g., Fv, Fab, F(ab')2, and the like. An "Fv" fragment consists of the variable domain of a heavy chain, and the variable domain of a light chain, in a non-covalent association (VH-VL dimer). In this configuration, three CDRs from each of the variable domains interact to define an antigen- binding site on the surface of the VH-VL dimer, as in the case of whole antibodies. The six CDRs collectively impart target binding specificity of the whole antibody. However, even a single variable domain (or half of an Fv comprising only 3 target-specific CDRs) can have the ability to recognize and bind an antigen.

[0142] An "effective amount" or "therapeutically effective amount" as used herein includes an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disorder. An effective amount also means an amount that is sufficient to allow or promote diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0143] "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, sport animals, and pets.

[0144] The amount of compound administered can depend on the subject being treated, the age, health, sex, and weight of the subject, the kind of concurrent treatment, if any, the severity of the condition, the nature of the effect desired, the mode and frequency of administration, and the judgment of the prescribing physician. The frequency of administration can also depend on the pharmacodynamic effects on arterial partial pressure of oxygen. However, the optimal dose can be adjusted according to individual subject, as is understood by those skilled in the art and without undue experimentation. This will often include adjusting the standard dose (e.g., decreasing the dose if the patient is small).

[0145] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the application or salts thereof with a pharmaceutically acceptable carrier. The object of a pharmaceutical composition is to facilitate administration of a compound of the application to an organism. The inventive pharmaceutical composition can include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preserving, wetting, emulsifying, and dispersing agents.

[0146] The term "pharmaceutically acceptable carrier" refers to those auxiliaries which have no significant stimulating effect on the organism and do not impair the biological activity and properties of the active compounds. Suitable auxiliaries are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, liposomes, polymeric micelles or inorganic nanocarriers, etc.

[0147] The pharmaceutical composition of the present application can be prepared in any pharmaceutically acceptable dosage form for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration, for example, can be formulated into tablets, troches, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalants, or sprays.

[0148] The term "anti-PD-Ll biological product" refers to an antibody or antigen-binding fragment thereof that specifically binds to PD-1.

[0149] The term "synergism" refers to the phenomenon that the effect of two drugs used in combination is more effective than the sum of their individual effects, as opposed to antagonism.

[0150] The term "treatment" includes prevention and treatment, for example, treatment of KRAS G12C mutation-mediated diseases includes prevention and / or treatment of KRAS G12C mutation-mediated diseases.

[0151] The PD-1 pathway inhibitors, PD-L1 pathway inhibitors, EGFR targeting agents, CDK inhibitors, and PI3K inhibitors described herein, when the inhibitors are small molecule compounds, are intended to include pharmaceutically acceptable salts of the compounds. For example, in one embodiment, the pharmaceutical compositions described herein include a compound of Formula (II) and GDC0941, that is, a compound of Formula (II) and GDC0941 and pharmaceutically acceptable salts of GDC0941.

[0152] II, DETAILED DESCRIPTION

[0153] The KRAS inhibitor disclosed in the present application is a compound of Formula (II) as follows, which is named (4aR,8R)-3-acryloyl-11-chloro-10-(2-fluoro-6-hydroxyphenyl)-8-(2-isopropyl-4- methylpyridin-3-yl)-6-methyl-2,3,4,4a,6,8-hexahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3- c][1,8]naphthyridine-5,7-dione, and its preparation method is described in the step of Example 25 in the international patent application (PCT / CN2020 / 124226).

[0154]

[0155] Materials and reagents (Examples 1-7)

[0156] NCI-H2122 (ATCC, CRL-5985), LU99 (JCRB, JCRB0080), SW837 (ATCC, CRL-235), SW1463 (ATCC, CRL-234), SW1573 (ATCC, CRL-2170), L-15 (Gibco, 11415-064), RPMI1640 (Gibco, 11875-093), Trypsin-EDTA (Gibco, 25200-072), FBS (Gibco, 10099-141C), CellTiter Glo (Progema, G7573), cell counter (Count star, IC1000), cell counter (CHEMOMETEC, NC-200), microplate reader (PerkinElmer, Envison).

[0157] Method for calculating proportion of cell survival in combination group

[0158] The formula for calculating the cell survival rate (%) after the combination of two drugs is: cell survival rate = (1 - (DMSO control RLU - drug RLU) / (DMSO control RLU - blank control RLU)) x 100%. The DMSO control is a solvent control without drugs, and the blank control is a medium control without drugs and solvents.

[0159] Interpretation of results of calculation of proportion of cell survival

[0160] The graph of the calculation results of the cell survival rate is shown in FIG. Figure 1 , Figure 3 , Figure 5 , Figure 7 , Figure 9 , Figure 11 , Figure 13 ), the vertical coordinate is the concentration of the KRAS inhibitor compound of formula (II) of the present application (the effective concentration of the drug in the well solution), and the horizontal coordinate is the concentration of the combination drug (the effective concentration of the drug in the well solution). The vertical lines are drawn with the horizontal and vertical concentration scales as the vertical points, respectively, and the value in the grid at the intersection of the two vertical lines is the cell survival rate of the combination group using the two concentrations of drugs. For example, Figure 1 In FIG. 1, the horizontal coordinate reading is 0 nM, and the vertical coordinate reading is 10.03 nM. The vertical lines are drawn with the two concentrations as the vertical points, respectively, and the value in the grid at the intersection of the two vertical lines is 86.1. Therefore, the cell survival rate is 86.1%, which indicates that the cell survival rate is 86.1% when the concentration of BYL719 is 0 nM, i.e., when the compound of formula (II) is used alone.

[0161] Method for evaluating synergistic effect

[0162] The statistical method is used to evaluate the combined effect of the two drugs on tumor cells. The statistical software used is R systems, and the statistical model is Bliss, HSA, Loewe, ZIP. When the synergy value is >5, there is a synergistic effect, when the synergy value is >10, there is a significant synergistic effect, when the synergy value is <-5, there is an antagonistic effect, and when the synergy value is <-10, there is a significant antagonistic effect. The final efficacy of the combination of the two drugs is further determined by the synergy value.

[0163] Example 1 Effect of combination of compound of formula (II) and BYL719 (Alpelisib) on tumor cell LU99

[0164] Human non-small cell lung cancer cells LU99 were inoculated into 384-well plates at a density of 600 cells per well. After the cells adhered (24 h), the compound of formula (II) and BYL719 were diluted to a certain gradient concentration, and 2 replicate wells were added per concentration. After 120 h, the cell viability was detected by Cell Titer-Glo method, 30 μl of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 min, and then incubated for 10 min. The RLU value was read on the Envision instrument.

[0165] The cell survival rate calculation results are shown in Table 1. In human non-small cell lung cancer cells LU99, the cell survival rate of 100 nM compound of formula (II) was 83%, the cell survival rate of 1000 nM BYL719 was 62.6%, and the cell survival rate of 100 nM compound of formula (II) combined with 1000 nM BYL719 was 33.8%. Figure 1

[0166] The combined effect is shown in Figure 2. In human non-small cell lung cancer cells LU99, 10 nM-1000 nM of compound of formula (II) combined with 100 nM-10000 nM of BYL719, the average synergy value calculated by Bliss ( Figure 2A ), HSA ( Figure 2B ), Loewe ( Figure 2C ), and ZIP ( Figure 2D ) four statistical models are 8.622, 18.377, 17.27, and 8.959, respectively, all greater than 5. It shows that the combination of compound of formula (II) and BYL719 in tumor cells has a good synergistic effect.

[0167] Example 2 Effect of combination of compound of formula (II) and BYL719 on tumor cell SW837

[0168] ​Human rectal adenocarcinoma cells SW837 were seeded at a rate of 800 cells per well in 384-well plates. After cell adhesion (24 h), compound (II) and BYL719 were diluted to specific concentrations, with two replicates per concentration. Cell viability was assessed using the Cell Titer-Glo assay after 120 h. 30 μl of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 minutes, incubated for 10 minutes, and the RLU value was read on the Envision instrument.

[0169] The cell survival rate calculation results are as follows: Figure 3 As shown, in human rectal adenocarcinoma cells SW837, for example, the cell survival rate was 60.2% with 31.69 nM of compound (II), 62.9% with 10000 nM of BYL719, and 20.6% with the combination of 31.69 nM of compound (II) and 10000 nM of BYL719.

[0170] The combined effect is shown in Figure 4. In human rectal adenocarcinoma cells SW837, the combination of compound (II) at 3.17 nM–1000 nM with BYL719 at 100 nM–10000 nM, via Bliss ( Figure 4A ), HSA Figure 4B Loewe Figure 4C ZIP Figure 4D The average synergistic values ​​calculated in the four statistical models were 10.586, 18.221, 15.149, and 10.857, respectively, all greater than 5. This indicates that compound (II) and BYL719 have a good synergistic effect when used together in tumor cells.

[0171] Example 3: Compound of formula (II) and GDC0941 (Pictili s Effects of combined ib) and ib on LU99 tumor cells

[0172] Human non-small cell lung cancer cells LU99 were seeded at a rate of 600 cells per well in 384-well plates. After cell attachment (24 h), compound (II) and GDC0941 were diluted to a specific concentration gradient, with two replicates per concentration. Cell viability was assessed using the Cell Titer-Glo assay after 120 h. 30 μl of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 minutes, incubated for 10 minutes, and the RLU value was read on the Envision instrument.

[0173] The cell survival rate calculation results are as follows: Figure 5As shown, in human non-small cell lung cancer cells LU99, for example, the cell survival rate was 83.8% with 100 nM of compound (II), 85.4% with 100 nM of GDC0941, and 58% with the combination of 100 nM of compound (II) and 100 nM of GDC0941.

[0174] The combined effect is shown in Figure 6. In human non-small cell lung cancer cells LU99, the combination of 10 nM–1000 nM of compound (II) with 31.74 nM–10000 nM of GDC0941 via Bliss (… Figure 6A ), HSA Figure 6B Loewe Figure 6C ZIP Figure 6D The average synergistic values ​​calculated in the four statistical models were 8.876, 13.71, 8.626, and 7.761, respectively, all greater than 5. This indicates that compound (II) and GDC0941 have a good synergistic effect when used in combination in tumor cells.

[0175] Example 4: Effect of compound (II) in combination with GDC0941 on tumor cells SW837

[0176] Human rectal adenocarcinoma cells SW837 were seeded at a rate of 800 cells per well in 384-well plates. After cell attachment (24 h), compound (II) and drug GDC0941 were added at gradient concentrations, with two replicates per concentration. Cell viability was assessed using the Cell Titer-Glo assay after 120 h. 30 μL of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 minutes, incubated for 10 minutes, and the RLU value was read on the Envision instrument.

[0177] The cell survival rate calculation results are as follows: Figure 7 As shown, in human rectal adenocarcinoma cells SW837, for example, the cell survival rate was 56.2% with 31.69 nM of compound (II), 67.6% with 1000 nM of GDC0941, and 23.4% with the combination of 31.69 nM of compound (II) and 1000 nM of GDC0941.

[0178] The combined effect is shown in Figure 8. In human rectal adenocarcinoma cells SW837, the combination of compound (II) at 3.17 nM–1000 nM with GDC0941 at 100 nM–10000 nM, via Bliss ( Figure 8A ), HSA Figure 8B Loewe Figure 8C ZIP Figure 8DThe average synergistic values ​​calculated in the four statistical models were 8.733, 20.115, 18.31, and 9.181, respectively, all greater than 5. This indicates that compound (II) and GDC0941 have a good synergistic effect when used in combination in tumor cells.

[0179] Example 5: Effect of compound (11) in combination with GDC0941 on tumor cells SW1463

[0180] Human rectal adenocarcinoma cells SW1463 were seeded at a rate of 1200 cells per well in 384-well plates. After cell attachment (24 h), compound (II) and drug GDC0941 were added at gradient concentrations, with two replicates per concentration. Cell viability was assessed using the Cell Titer-Glo assay after 120 h. 30 μL of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 minutes, incubated for 10 minutes, and the RLU value was read on the Envision instrument.

[0181] The cell survival rate calculation results are as follows: Figure 9 As shown, in human rectal adenocarcinoma cells SW1463, for example, the cell survival rate was 43.2% with 31.69 nM of compound (II), 64.6% with 1000 nM GDC0941, and 8.9% with the combination of 31.69 nM of compound (II) and 1000 nM GDC0941.

[0182] The combined effect is shown in Figure 10. In human rectal adenocarcinoma cells SW1463, the combination of compound (II) at 3.17 nM–1000 nM and GDC0941 at 31.74 nM–10000 nM resulted in a significant improvement in efficacy via Bliss (…). Figure 10A ), HSA Figure 10B Loewe Figure 10C ZIP Figure 10D The average synergistic values ​​calculated in the four statistical models were 5.76, 12.967, 10.21, and 6.077, respectively, all greater than 5. This indicates that compound (II) and GDC0941 have a good synergistic effect when used in combination in tumor cells.

[0183] Example 6: Effect of compound (11) in combination with GDC0941 on tumor cells SW1573

[0184] Human non-small cell lung cancer cells SW1573 were seeded at a rate of 600 cells per well in 384-well plates. After cell attachment (24 h), compound (II) and GDC0941 were diluted to specific concentrations, with two replicates per concentration. Cell viability was assessed using the Cell Titer-Glo assay after 120 h. 30 μL of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 minutes, incubated for 10 minutes, and the RLU value was read on the Envision instrument.

[0185] The cell survival rate calculation results are as follows: Figure 11 As shown, in human non-small cell lung cancer cells SW1573, for example, the cell survival rate was 86.4% with 100 nM of compound (II), 46% with 3164 nM of GDC0941, and 23.6% with the combination of 100 nM of compound (II) and 3164 nM of GDC0941.

[0186] The combined effect is shown in Figure 12. In human non-small cell lung cancer cells SW1573, the combination of 0.32 nM–1000 nM of compound (II) with 316.91 nM–10000 nM of GDC0941 via Bliss (… Figure 12A ), HSA Figure 12B Loewe Figure 12C ZIP Figure 12D The average synergistic values ​​calculated in the four statistical models were 12.227, 19.12, 17.997, and 13.191, respectively, all greater than 5. This indicates that compound (II) and GDC0941 have a good synergistic effect when used in combination in tumor cells.

[0187] Example 7: Effect of compound (11) in combination with SY5609 (Synonyms) on NCI-H2122 tumor cells.

[0188] Human non-small cell lung cancer cells (NCI-H2122) were seeded at a rate of 600 cells per well in 384-well plates. After cell attachment (24 h), compound (II) and SY-5609 were diluted to specific concentrations, with two replicates per concentration. Cell viability was assessed using the Cell Titer-Glo assay after 120 h. 30 μL of Cell Titer-Glo reagent was added to each well, the plate was shaken for 2 minutes, incubated for 10 minutes, and the RLU value was read on the Envision instrument.

[0189] The cell survival rate calculation results are as follows: Figure 13As shown, in human non-small cell lung cancer cells NC-H2122, for example, the cell survival rate was 60.7% with 3.17 nM compound (II), 57.5% with 31.69 nM SY-5609, and 3.9% with the combination of 3.17 nM compound (II) and 31.69 nM SY-5609.

[0190] The combined effects are shown in Figure 14. In human non-small cell lung cancer cells NC-H2122, the combination of 0.1 nM–100 nM of compound (II) with 3.17 nM–1000 nM of SY-5609, via Bliss (… Figure 14A ), HSA Figure 14B Loewe Figure 14C ZIP Figure 14D The average synergistic values ​​calculated in the four statistical models were 8.249, 12.89, 10.518, and 8.805, respectively, all greater than 5. This indicates that compound (II) and SY-5609 have a good synergistic effect when used together in tumor cells.

[0191] Example 8: In vivo antitumor pharmacodynamic evaluation of compound (II) in combination with cetuximab in the NCI-H2122 human non-small cell lung cancer model.

[0192] Materials and reagents:

[0193] NCI-H2122 (ATCC, CRL-5807), Penicillin-Streptomycin (Gibco, 15140-122), RPMI1640 (Gibco, 11875-093), Trypsin-EDTA (Gibco, 25200-072), FBS (Gibco, 10099-141C), SolutolHS 15 (Sigma, 42966-1kg), HP-β-CD (Shaoyuan, SY004863100g).

[0194] During the experiment, the animals' health status was observed daily. If the animal's weight decreased by 10%, the dosage was halved; if the weight decreased by 15%, administration was stopped until the weight recovered; or if the animal's tumor volume exceeded 2,000 mm... 3 Immediate euthanasia should be performed. If the following health conditions occur, notify a veterinarian and perform euthanasia:

[0195] Significantly thin, with a weight loss of more than 20%.

[0196] They cannot freely obtain food and water.

[0197] Animals may develop infections, severely ruptured tumors, or hematomas.

[0198] Animals developed the following clinical signs and deteriorated: piloerection, hunched back, ear, nose eye or foot color whitening, rapid breathing, convulsions, diarrhea, pain, dehydration, moribund.

[0199] Experimental animals:

[0200] BALB / c nude mice, 6-8 weeks old, female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal qualification certificate number 20170011008676, SPF level feeding environment.

[0201] Experimental drugs:

[0202] Cetuximab was purchased from MERCK, batch number G00XED.

[0203] Preparation method: the compound of formula (II) was prepared using 5% DMSO + 10% Solutol HS 15 + 85% (6% HP-β-CD) solvent; Cetuximab was prepared using 0.9% sodium chloride injection.

[0204] Experimental methods and procedures

[0205] NCI-H2122 was cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Routine digestion was performed with 0.25% trypsin-EDTA for subculture. When the cells were in the exponential growth phase, the saturation degree was 80%-0%, the cells were collected and counted. The final concentration of the cells was adjusted to 2.0×10 6 / mL. The mice were inoculated subcutaneously on the right side of the back, the inoculation volume was 0.15 mL per animal, and the inoculation amount was 3×10 5 cells per animal. When the tumors grew to an average of about 242mm 3 , (D5 after inoculation), 32 tumor-bearing mice were selected, and were randomly divided into 4 groups according to the tumor volume and animal weight, 8 mice in each group, and the drug administration was started, and the administration method was carried out according to the experimental scheme in Table 1. The mice were administered at a dose of 10 mL / kg. The animals were weighed twice a week using an electronic balance, and the tumor volume was measured twice a week using a vernier caliper. D17 after inoculation was the end of the experiment, and the mice were euthanized after measuring the tumor volume in each group.

[0206] Table 1 Administration scheme of therapeutic drugs

[0207]

[0208] Note: p.o is oral, i.p is intraperitoneal injection, q.d is once a day, biw is twice a week.

[0209] Main evaluation index is :

[0210] Tumor volume: Tumor volume (TV) = (L × W) 2 ) / 2, where L is the long diameter of the tumor and W is the wide diameter of the tumor.

[0211] Tumor growth inhibition rate (TGI):

[0212] TGI(%) = [1-(avT)] i-0 / avC i-0 )]×100%; where avTi-0 is the average tumor volume of the treatment group on a specific day, minus the average tumor volume of the treatment group on the first day of administration; where avCi-0 is the average tumor volume of the solvent control group on a specific day, minus the average tumor volume of the solvent control group on the first day of administration.

[0213] Tumor weight inhibition (TWI):

[0214] TWI = (1-TW) treatment / Dx / TW c o ntr o l / Dx )×100%; of which TW c o ntr o l Mean tumor weight (g) in the control group, TW treatment Mean tumor weight (g) in the treatment group.

[0215] Relative change of body weight (RCBW):

[0216] RCBW(%) = (BW) i -BW0) / BW0×100%; where BW i BW0 is the animal's weight on a specific day, while BW0 is the animal's weight on the day the drug was first administered.

[0217] Data analysis, experimental results and conclusions

[0218] All experimental data analysis and graphing were done using GraphPad Prism software (GraphPad Software). Tumor volumes and animal body weights were statistically compared using two-way ANOVA (Dunnett's multiple comparisons test) for each group, and tumor weights at endpoint were statistically compared using one-way ANOVA (Dunnett's multiple comparisons test) for each group. Statistical significance was considered when P < 0.05. Tumor volumes, weights, and animal body weights were expressed as Mean ± SEM. The results are shown in Table 2 below.

[0219] Table 2 Experimental results

[0220]

[0221] Note: ***p < 0.001, compared with G1 vehicle control group, respectively.

[0222] The effects of each treatment group on the tumor growth of NCI-H2122 tumor-bearing mice (see Figure 15 and Figure 16 ); tumor weights of each tumor-bearing mouse in each treatment group at endpoint ( Figure 17 ); the animals were euthanized at endpoint, and the tumor tissues were collected and weighed, and photographed; the relative body weight changes of tumor-bearing mice in each group are shown in Figure 18 .

[0223] The results showed that, in the NCI-H2122 human non-small cell lung cancer model, the oral administration of the compound of formula (II) once a day and the intraperitoneal injection of Cetuximab twice a week could both significantly inhibit the growth of NCI-H2122 tumors, and had a certain joint strengthening effect; compared with the weight loss of the vehicle control group, the animals in the 10 mpk group of the compound of formula (II), the 1 mpk group of Cetuximab, and the combined administration group of the 10 mpk compound of formula (II) and the 1 mpk Cetuximab did not show significant weight loss; there was a statistically significant difference.

[0224] Example 9 In vivo anti-tumor pharmacodynamic evaluation of the combination of the compound of formula (II) and mPDl in a murine CT26 cell model

[0225] The experimental animals and main evaluation indexes were the same as in Example 8.

[0226] Experimental drugs:

[0227] Mouse PD1 antibody (mPD1) was purchased from BioXcell-BE0146, batch number 810421D1.

[0228] Configuration method: the compound of formula (II) was prepared using 5% DMSO + 10% Solutol HS 15 + 85% (6% HP-β-CD) solvent; mPD1 was prepared using PBS.

[0229] Experimental methods and procedures

[0230] The right dorsal of mice was subcutaneously inoculated with CT26 KRASG12C KI cells (KRASG12C mutant CT26 cells) with a volume of 0.1 mL per animal. On the 12th day after cell inoculation (D0), the average tumor volume reached 69 mm 3 The 32 tumor-bearing mice were selected from the middle and grouped for administration, and were divided into 4 groups according to the tumor volume and animal weight, with 8 mice in each group. The administration method was carried out according to the experimental scheme in Table 3. The administration volume of mice was 10 mL / kg. The animals were weighed twice a week using an electronic balance, and the tumor volume was measured twice a week using a vernier caliper.

[0231] Table 3 Administration scheme of therapeutic drugs

[0232]

[0233]

[0234] Note: p.o is oral, i.p is intraperitoneal injection, q.d is once a day, biw is twice a week.

[0235] The main evaluation indexes were tumor volume, TGI, TWI and RCBW, and the calculation formula and data analysis were the same as in Example 8. The results are shown in Table 4 below:

[0236] Table 4 Experimental results

[0237]

[0238] Note: *p<0.05, **p<0.01, compared with G1 solvent control group respectively.

[0239] The results showed that in the mouse CT26 KRASG12C KI cell model, the administration of the compound of formula (II) and mouse PD1 antibody (mPD1) could significantly inhibit tumor growth, and had a certain joint strengthening effect.

[0240] The effects of each treatment group on the tumor growth of CT26 KRAS G12C KI tumor-bearing mice (see Figure 19 and Figure 20); tumor weight of each tumor-bearing mouse in each treatment group at the end point (see Figure 21 ); at the end point, the animals were euthanized and the tumor tissues were collected and weighed; the relative body weight changes of tumor-bearing mice in each group are shown in Figure 22 .

[0241] The results show that, in the mouse colon cancer CT26 KRAS G12C KI subcutaneous tumor model, the single-agent treatment group of the compound of formula (II) and the single-agent treatment group of mPD1 both showed a mild tumor inhibition effect, with the average tumor volume being 1,065 mm 3 (T / C = 54.92%; TGI = 46.74%, p = 0.1640) and 968 mm 3 (T / C = 49.91%; TGI = 51.94%, p = 0.1094), respectively. The combination treatment group of the compound of formula (II) and mPD1 showed a significant tumor inhibition effect, with the average tumor volume being 632 mm 3 (T / C = 32.56%; TGI = 69.92%, p = 0.0223), and at the end of the experiment, one mouse in this group had a tumor volume less than 50 mm 3 , which was considered to have completely regressed. During the treatment, the tumor-bearing mice showed good tolerance to the tested drugs, and no obvious body weight loss or death of the mice due to the drugs was observed in all treatment groups.

[0242] As can be seen from the above experimental results, the KRAS inhibitor represented by formula (I) and the cancer treatment agents such as the PI3K inhibitor represented by BYL719 and GDC0941, the EGFR antibody represented by cetuximab, the CDK7 inhibitor represented by SY-5609, and the PD-1 antibody represented by mPD1 antibody all have obvious synergistic effects when used for the treatment of lung adenocarcinoma, rectal adenocarcinoma, or lung cancer.

[0243] The foregoing description of specific exemplary embodiments of the application is intended to illustrate and exemplify the application. These descriptions are not meant to limit the application to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application to thereby enable others skilled in the art to best utilize the application and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition or medicine box, characterized in that, It includes therapeutically effective amounts of KRAS inhibitors and therapeutically effective amounts of other cancer therapeutic agents and pharmaceutically acceptable carriers; The KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof; The other cancer treatment agent mentioned is BYL719; in, The pharmaceutical composition or kit comprises 3.17 nM-1000 nM of a compound of formula (II) and 100 nM-10000 nM of BYL719.

2. The pharmaceutical composition or kit according to claim 1, wherein, The pharmaceutical composition or kit comprises 10 nM-1000 nM of a compound of formula (II) and 100 nM-10000 nM of BYL719.

3. A pharmaceutical composition or medicine box, characterized in that, It includes therapeutically effective amounts of KRAS inhibitors and therapeutically effective amounts of other cancer therapeutic agents and pharmaceutically acceptable carriers; The KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof; The other cancer treatment agent mentioned is GDC0941; in, The pharmaceutical composition or kit comprises 3.17 nM–1000 nM of a compound of formula (II) and 31.74 nM–10000 nM of GDC0941.

4. The pharmaceutical composition or kit according to claim 3, wherein, The pharmaceutical composition or kit comprises 3.17 nM–1000 nM of a compound of formula (II) and 100 nM–10000 nM of GDC0941.

5. The pharmaceutical composition or kit according to claim 3, wherein, The pharmaceutical composition or kit comprises 10 nM–1000 nM of a compound of formula (II) and 31.74 nM–10000 nM of GDC0941.

6. A pharmaceutical composition or pillbox, characterized in that, It includes therapeutically effective amounts of KRAS inhibitors and therapeutically effective amounts of other cancer therapeutic agents and pharmaceutically acceptable carriers; The KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof; The other cancer treatment agent mentioned is GDC0941; in, The pharmaceutical composition or kit comprises 0.32 nM–1000 nM of a compound of formula (II) and 316.91 nM–10000 nM of GDC0941.

7. A pharmaceutical composition or pillbox, characterized in that, It includes therapeutically effective amounts of KRAS inhibitors and therapeutically effective amounts of other cancer therapeutic agents and pharmaceutically acceptable carriers; The KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof; The other cancer treatment agent mentioned is SY-5609; in, The pharmaceutical composition or kit comprises 0.1 nM–100 nM of a compound of formula (II) and 3.17 nM–1000 nM of SY-5609.

8. A pharmaceutical composition or pillbox, characterized in that, It includes therapeutically effective amounts of KRAS inhibitors and therapeutically effective amounts of other cancer therapeutic agents and pharmaceutically acceptable carriers; The KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof; The other cancer treatment agents mentioned are PD-1 antibodies; in, The pharmaceutical composition or kit contains 1-3 mpk of formula (II) compound and 10 mpk of PD-1 antibody.

9. The use of the pharmaceutical composition or kit according to any one of claims 2, 5 and 6 in the preparation of a medicament for treating non-small cell lung cancer; wherein the therapeutically effective amount of the KRAS inhibitor and the therapeutically effective amount of other cancer therapeutic agents may be administered simultaneously, separately or sequentially.

10. The use of the pharmaceutical composition or cassette according to any one of claims 1, 3 and 4 in the preparation of a medicament for treating rectal adenocarcinoma; wherein the therapeutically effective amount of the KRAS inhibitor and the therapeutically effective amount of other cancer treatment agents may be administered simultaneously, separately or sequentially.

11. The use of the pharmaceutical composition or kit of claim 7 in the preparation of a medicament for treating non-small cell lung cancer; wherein the therapeutically effective amount of the KRAS inhibitor and the therapeutically effective amount of other cancer therapeutic agents may be administered simultaneously, separately, or sequentially.

12. The use of the pharmaceutical composition or kit of claim 8 in the preparation of a medicament for treating KRAS G12C-mutant colon cancer; wherein the therapeutically effective amount of the KRAS inhibitor and the therapeutically effective amount of other cancer therapeutic agents may be administered simultaneously, separately, or sequentially.

13. The use of therapeutically effective amounts of KRAS inhibitors and other therapeutically effective amounts of other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of non-small cell lung cancer; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, in the range of 10 nM to 1000 nM. The other cancer treatment agent with an effective therapeutic amount is BYL719 at 100 nM to 10,000 nM.

14. The use of therapeutically effective amounts of KRAS inhibitors and other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of rectal adenocarcinoma; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, ranging from 3.17 nM to 1000 nM. The other cancer treatment agent with an effective therapeutic amount is BYL719 at 100 nM to 10,000 nM.

15. The use of therapeutically effective amounts of KRAS inhibitors and other therapeutically effective amounts of other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of non-small cell lung cancer; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, in the range of 10 nM to 1000 nM. Other cancer treatment agents with effective therapeutic amounts are GDC0941 at 31.74 nM–10000 nM.

16. The use of therapeutically effective amounts of KRAS inhibitors and other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of rectal adenocarcinoma; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, ranging from 3.17 nM to 1000 nM. Other cancer treatment agents with effective therapeutic amounts are GDC0941 at 100 nM–10000 nM.

17. The use of therapeutically effective amounts of KRAS inhibitors and other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of rectal adenocarcinoma; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, ranging from 3.17 nM to 1000 nM. Other cancer treatment agents with effective therapeutic amounts are GDC0941 at 31.74 nM–10000 nM.

18. The use of therapeutically effective amounts of KRAS inhibitors and other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of non-small cell lung cancer; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, ranging from 0.32 nM to 1000 nM. Other cancer treatment agents with therapeutically effective doses include GDC0941 at doses of 316.91 nM–10000 nM.

19. The use of therapeutically effective amounts of KRAS inhibitors and other therapeutically effective amounts of other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for the treatment of non-small cell lung cancer; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, ranging from 0.1 nM to 100 nM. The other cancer treatment agent with an effective therapeutic dose is SY-5609 at a dose of 3.17 nM–1000 nM.

20. The use of therapeutically effective amounts of KRAS inhibitors and other cancer therapeutic agents in the preparation of pharmaceutical compositions or kits for treating KRASG12C-mutant colon cancer; The therapeutically effective amount of the KRAS inhibitor is a compound of formula (II) at 1-3 mpk or a pharmaceutically acceptable salt thereof; The other cancer treatment agent in which the treatment is effective is a PD-1 antibody at 10 mpk.

Images