Use of macrocyclic compounds in combination with MEK inhibitors in drugs for treating diseases

Abstract

The present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, stereoisomer, in combination with a MEK inhibitor in the preparation of a medicament for treating a disease.

Description

Technical Field

[0001] This invention relates to the use of a combination of macrocyclic compounds, particularly fluorinated macrocyclic compounds, and MEK inhibitors in the treatment of KRAS-mutant cancers. Technical Background

[0002] Kirsten rat sarcoma virus oncogene homolog (KRAS) is one of the most common mutated oncogenes in human cancers (COSMIC database), indicating a poor prognosis. For many years, it was considered an untreatable target due to the lack of therapeutic options targeting this gene. However, with recent research advancements, Sotorasib (AMG510), a targeted drug against the KRAS G12C mutation, received accelerated approval from the FDA on May 29, 2021, for the treatment of KRAS G12C-mutant non-small cell lung cancer (NSCLC) that has undergone at least one prior systemic therapy.

[0003] KRAS protein is a cell membrane-bound guanylate triphosphatase (GTPase). It is activated when bound to GTP, and the GTPase activity of KRAS converts GTP to inactivated GDP. Guanylate exchange factors (GEFs), such as SOS1 protein, can promote the conversion of KRAS protein to the GTP-bound activated state. KRAS protein cycles between the GTP and GDP states, with a half-life of about 24 hours for resynthesis. KRAS acts like a "cell switch". When it is "turned on" by extracellular stimulation, it will activate a variety of downstream signaling pathways, including the oncology-related RAF-MEK-ERK, PI3K-AKT-mTOR, and Ral-GEF pathways, which are involved in cell proliferation, cell cycle regulation, metabolic changes, cell survival and cell differentiation (Liu P, Wang Y, Li X. Targeting the untargetable KRAS in cancer therapy[J]. Acta Pharmaceutica Sinica B, 2019, 9(5):871-879.).

[0004] Point mutations are a common form of KRAS mutation, resulting in KRAS being in a persistently GTP-binding activated state, activating downstream oncogenic signaling pathways. The three tumors with the highest frequency of KRAS mutations are pancreatic cancer (88%), colorectal cancer (45%-50%), and lung cancer (31%-35%). KRAS mutations are more common in lung adenocarcinoma (20%-40%) and relatively less common in lung squamous cell carcinoma (5%) (Jemal A, Siegel R, Ward E, et al: Cancer statistics, 2008. CA Cancer JClin 58:71-96, 2008). KRAS mutations are seen in up to 30% of NSCLC patients, primarily occurring at codons 12 and 13. Numerous studies have found that the most common KRAS mutation is G12C, accounting for 39%; followed by G12V (18-21%) and G12D (17-18%) (Clin. Cancer Res. 18 (2012) 6169–6177; J. Clin. Oncol. 35 (2017) 9021).

[0005] KRAS targeted therapy history

[0006] Drug development targeting KRAS has been conducted for nearly 40 years, including targeting the KRAS protein itself, post-translational modifications, membrane localization, protein-protein interactions, and downstream signaling pathways. However, most of these efforts have failed in clinical trials. This may be because the KRAS protein lacks small molecule drug binding regions in addition to the GTP / GDP binding pocket.

[0007] 1) Directly target KRAS

[0008] Direct targeting of KRAS is challenging due to the biochemical complexity of KRAS proteins, the high affinity of GTP for KRAS, and its limited active binding sites. While recent advances in computer modeling and structural crystallization studies have led to the discovery of small molecules that directly bind to specific RAS conformations, the binding affinity of these early compounds still needs improvement. Although Sotorasib (AMG510) received accelerated approval from the FDA on May 29, 2021, it only targets the G12C mutation.

[0009] 2) Indirect targeting of KRAS

[0010] This includes targeted post-translational modifications, membrane localization, protein-protein interactions, and inhibition of downstream signaling pathways. Tumor microenvironment reprogramming is a major characteristic of tumors; tumor cells can reprogram the tumor microenvironment through various pathways (such as immunosuppression, induction of angiogenesis, and metabolic alteration) (hanahan et al. Robert A. Weinberg, Hallmarks of Cancer: The Next Generation. 2011). Many studies have shown that the KRAS signaling pathway can induce the expression of numerous immunomodulatory factors, such as TGFβ, GM-CSF, CXCL8, IL-6, and IL-10. These immunomodulatory factors interact with the tumor microenvironment, leading to immunosuppression (Cavalho et al.). KRAS Oncogenic Signaling Extends beyond Cancer Cells to Orchestrate the Microenvironment. 2018 ;cullis et al. Jane Cullis, Kras and Tumor Immunity: Friend or Foe 2018 Maldegem et al. Mutant KRAS at the Heart of Tumor Immune Evasion. 2020 KRAS mutation-driven tumor cells can reprogram stromal cells into a state conducive to tumorigenesis (Cavalho et al.). Targeting the Tumor Microenvironment: An Unexplored Strategy for Mutant KRAS Tumors. 2019 They inhibit tumorigenesis by blocking autocrine cytokine signaling pathways (Zhu et al.). Inhibition of KRAS-Driven Tumorigenicity by Interrup tion of an Autocrine Cytokine Circuit. 2014 One example is that KRAS-mutant tumor cells can secrete IL-6. IL-6 is upregulated in lung cancer and mediates a signaling pathway that promotes KRAS-driven lung carcinogenesis (Brooks et al. IL6 Trans-signaling Promotes KRAS-Driven Lung Carcinogenesis. 2016). KRAS-mutant tumor cells promote angiogenesis by secreting vascular endothelial growth factor (VEGF) and other angiogenic factors (such as CXC chemokine), which is a paracrine process (Matsuo et al.). K-Ras Promotes Angiogenesis Mediated by Immortalized Human Pancreatic Epithelial Cells through Mitogen-Activated Protein Kinase Signaling Pathways. 2009Paracrine processes, besides influencing immune evasion and angiogenesis, can also alter tumor cell processes by remodeling the matrix. For example, KRAS-mutant cells can secrete insulin-like growth factor-1 (IGF1R), increasing mitochondrial volume in tumor cells via the IGF1R signaling pathway (Tape et al. Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation. 2016). In summary, for patients with KRAS-mutant tumors, effective treatment strategies need to target not only tumor cells but also the tumor microenvironment.

[0011] Farnesyltransferase inhibition: Farnesylation of the RAS protein is essential for its normal physiological function and its oncogenic mutational function. Farnesyltransferase inhibitors (FTIs) such as Tipifarnib and Salirasib have been clinically studied, but have not shown efficacy in KRAS-mutant NSCLC.

[0012] MEK Inhibition: MEK inhibitors have shown poor efficacy as monotherapy in clinical studies. Selumetinib and Trametinib are allosteric selective inhibitors of MEK1 / 2. Selumetinib has shown activity against KRAS-mutant tumors in preclinical studies. In a phase II clinical trial, among 87 previously treated patients with KRAS-mutant advanced NSCLC, there was no statistically significant difference in overall survival (OS) between Selumetinib plus Docetaxel and Docetaxel monotherapy (9.4 months vs. 5.2 months, p = 0.21). In another study, among 510 patients with KRAS-mutant NSCLC, there was no improvement in progression-free survival (PFS) between Selumetinib plus Docetaxel and Docetaxel monotherapy (HR = 0.93). In a phase II clinical trial comparing Trametinib and Docetaxel in patients with KRAS-mutant NSCLC, the trial was terminated early because the efficacy of Trametinib exceeded the nullipopulation threshold in the interim analysis. (Blumenschein et al 2015) Therefore, currently known MEK inhibitor monotherapy and MEK inhibitor combination chemotherapy are ineffective for patients with KRAS-mutant NSCLC.

[0013] Inherent and adaptive resistance are limiting factors for the clinical use of KRAS inhibitors as monotherapy. Resistance mechanisms include KRAS nucleotide cycling, negative feedback reactivation, and tumor cells bypassing KRAS dependence (Hallin et al., Cancer Discovery, 2020, 10(1), 54-71). Combining KRAS inhibitors with drugs that reactivate the MAPK feedback pathway, activate the RTK-induced PI3K pathway, increase apoptosis, or inhibit the pro-inflammatory tumor microenvironment may significantly improve clinical benefits. KRAS covalent inhibitors combined with immune checkpoint antibodies (such as PD-1 antibodies) have shown better efficacy (Canon et al., Nature 2019, 575, 217-223). Studies have found that the mechanisms by which KRAS-mutant tumors develop resistance to MEK inhibitors include compensatory upregulation of PI3K / AKT survival signaling and intrinsic or treatment-induced epithelial-mesenchymal transition (EMT) (Mohanty AJ, Sishc BJ, Falls KC, et al. GC4419 enhances the response of non-small cell lungcarcinoma cell lines to cisplatin and cisplatin plus radiation through a ROS-mediated pathway[J]. 2018.). The SRC / FAK signaling pathway regulates the PI3K / AKT survival signaling pathway and participates in integrin-mediated EMT signal transduction. Activation of the JAK2 / STAT3 pathway is a mechanism of resistance to MEK inhibitors.

[0014] SRC and FAK inhibition: Src kinases are associated with resistance to various cancer therapies, including radiotherapy, chemotherapy, and targeted therapy (Siyuan Zhang. Targeting Src family kinases in anti-cancertherapies: turning promise into triumph. 2012). The Src kinase family promotes mitotic signaling from growth factor receptors through multiple pathways, including signaling pathways required to initiate DNA synthesis, regulation of receptor expression, actin cytoskeleton rearrangement, migration, and survival (Bromann et al. The interplay between Src family kinases and receptor tyrosine kinases. 2004). It has been reported that KRAS drives pancreatic cancer cell metastasis and resistance by inducing a positive feedback amplification loop in Src / PEAK1 / ErbB2. Kelber et al. (Kelber et al. KRas Induces a Src / PEAK1 / ErbB2Kinase Amplification Loop ThatDrives Metastatic Growth and Therapy Resistance in Pancreatic Cancer. 2012) found that the Src inhibitor dasatinib enhances the antitumor activity of MEK inhibitors by inhibiting TAZ (Transcriptional coactivator with PDZ-binding motif) activity. The combination of dasatinib and trametinib is a potential therapeutic strategy for treating KRAS-driven tumors (Rao et al. Dasatinib sensitises KRAS-mutant cancer cells to mitogen-activated protein kinase kinase inhibitor via inhibition of TAZ activity. 2012).

[0015] FAK may be involved in inhibiting p53 expression to promote cell survival (Golubovskaya et al. Simultaneous Inhibition of Focal Adhesion Kinase and Src Enhances Detachment and Apoptosis in Colon Cancer Cell Lines. 2007). In preclinical studies, KRAS-mutant cell lines and xenografts with both TP53 and CDKN2A mutations were sensitive to FAK inhibitors. However, in a phase II clinical trial, the FAK inhibitor Defactinb monotherapy in patients with KRAS-mutant NSCLC who had received multiple lines of therapy showed weak clinical efficacy, with a progression-free survival (PFS) of only 45 days, and the efficacy was not correlated with TP53 and CDKN2A mutations.

[0016] FAK plays a crucial role in integrin, RTK, RAS, and TGFβ-mediated signaling pathways (Kanteti et al. FAK and paxillin, two potential targets in pancreatic cancer. 2012). Recent studies have found that integrins participate in the biological regulation of tumor stem cells and are involved in tumor development, metastasis, and drug resistance through the SRC / FAK signaling pathway (Seguin et al. Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance. 2015). SRC has been identified as a key mediator in the formation of thyroid proto-tumors and is a potential therapeutic target for thyroid cancer. However, inhibiting Src alone leads to activation of the IL-1β>FAK>p130Cas>c-Jun>MMP signaling axis, promoting tumor development toward an aggressive phenotype. In contrast, the combination of FAK and Src inhibitors may block the phenotypic switch induced by Src inhibitors and address drug resistance issues (Kessler et al. Resistance to Src inhibition alters the BRAF-mutant tumor secretome to promote an invasive phenotype and therapeutic escape through a FAK>p130Cas>c-Jun signaling axis. 2019).

[0017] Compensatory upregulation of PI3K / AKT signaling is one of the resistance mechanisms targeting KRAS mutations. FAK interacts directly with the SH2 domain of the PI3K regulatory subunit p85 by phosphorylating Y397, activating the PI3K pathway and inhibiting doxorubicin-induced apoptosis (van Nimwegen et al. Focal Adhesion Kinase and Protein Kinase BCooperate to Suppress Doxorubicin-Induced Apoptosis of Breast Tumor Cells. 2006). RhoA-FAK is an essential signaling axis for maintaining KRAS-driven lung adenocarcinoma. In vivo inhibition of FAK downregulates p-AKT but does not trigger PI3K / AKT-dependent compensatory mechanisms (Konstantinidou et al. RHOA-FAK Is a Required Signaling Axis for the Maintenance of KRAS-Driven Lung Adenocarcinomas. 2013).

[0018] Phosphorylation of FAK at Y925 forms a site that links to GRB2, which can activate the small GTP protein RAS and downstream ERK2 (MAPK) (Kanteti et al. FAK and paxillin, two potential targets inpancreatic cancer. 2016). Paxillins are major components of focal adhesion, connecting the extracellular matrix and the actin cytoskeleton. In tumor cells, Scr and FAK-mediated phosphorylation regulates paxillin function. Evidence such as increased cell detachment, inhibition of AKT / ERK1 / 2 and Src, and increased apoptosis suggests that dual inhibition of FAK and Src is more effective than inhibition of FAK alone (Golubovskaya et al. Simultaneous Inhibition of Focal Adhesion Kinase and Src Enhances Detachment and Apoptosis in Colon Cancer Cell Lines. 2003). Interferon and inflammation-related genes are enriched in KRAS-mutant colonic cell lines, exhibiting both intrinsic and acquired resistance to MEK inhibition (Wagner et al. Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer. 2019). Furthermore, Src and FAK can regulate STAT3, thereby controlling the expression of angiogenic factors such as VEGF and other cytokines (Niu et al. oncogene). Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. 2002; cavalho et al cancers Targeting the Tumor Microenvironment: An Unexplored Strategy for Mutant KRAS Tumors. 2019 ).

[0019] JAK2 inhibition: JAK2 provides signal transduction for inflammatory cytokines. Inhibiting JAK2 may reduce the secretion of interferon and inflammation-related genes, and sensitize KRAS mutant cell lines to MEK inhibitors. Studies have reported that KRAS mutations can activate p-STAT3 (Tyr705) in the absence of IL-6 secretion, and STAT3-mediated upregulation of BCL-XL contributes to apoptosis resistance in KRAS mutant colon cancer cells (zaanan et al.). The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism. 2015Therefore, inhibiting JAK2 can regulate STAT3 phosphorylation, thereby generating a synergistic apoptotic effect in KRAS-mutant tumors, including colorectal cancer. In preclinical studies, inhibition of MEK leads to autocrine activation of STAT3 via JAK and FGFR kinase activity, resulting in drug resistance. The MEK inhibitor Cobimetinib, in combination with the JAK1 / 2 inhibitor Ruxolitinib and the multi-target kinase inhibitor Ponatinib (including an FGFR inhibitor), showed enhanced efficacy in a mouse xenograft tumor model (Lee et al. Drug Resistance via Feedback Activation of Stat3 in Oncogene-Addicted Cancer Cells. 2019).

[0020] In summary, Src, FAK, and JAK2 play crucial roles in KRAS-mutant tumors by regulating angiogenesis in tumors, constructing pro-tumor immune responses within the tumor microenvironment, and modulating intracellular and extracellular signal transduction. However, drug development targeting the central downstream signaling effectors of the mutant RAS protein has remained challenging. The combination of SRC / FAK / JAK2 inhibitors with MEK inhibitors (especially Trametinib) represents a novel treatment approach, maximizing the anti-tumor activity and response time of MEK inhibitors, particularly highlighting the therapeutic benefit of Trametinib for KRAS-mutant patients.

[0021] Compound 1, a next-generation multi-target investigational drug, effectively inhibits SRC / FAK / JAK2 kinase activity at therapeutic concentrations. In a series of lung cancer, pancreatic cancer, and colorectal cancer cells with different types of KRAS mutations, the combination of Compound 1 and the MEK inhibitor Trametinib significantly increased antitumor proliferation activity and the proportion of tumor cells undergoing apoptosis compared to monotherapy. In some KRAS-mutant tumor subtypes, the combination of Trametinib and Compound 1 inhibited the rebound of Trametinib-induced AKP phosphorylation (pAKT) levels. However, the combination of SRC inhibitor Dasatinib, JAK1 / 2 inhibitor Ruxolitinib, or FAK inhibitor Defactinib with Trametinib did not inhibit Trametinib-induced AKT phosphorylation, indicating that the increased efficacy of the combination therapy requires simultaneous inhibition of SRC / FAK / JAK2. In xenograft experiments on KRAS-mutant mice, the in vivo antitumor effect of the combination therapy was stronger than that of Trametinib monotherapy. The combination of trametinib and compound 1 can exert a stronger and more durable antitumor effect by continuously inhibiting the mutated KRAS signaling pathway. Simultaneous inhibition of SRC, FAK, and JAK2 drugs in combination with MEK inhibitors may be a promising therapeutic approach for effectively targeting KRAS-mutated cancers. Furthermore, combined inhibition of SRC, FAK, and JAK2 may have potential roles in other inflammation-related diseases, such as asthma, inflammatory bowel disease, ulcerative colitis, Crohn's disease, and fibrosis. Summary of the Invention

[0022] This invention relates to the use of a compound of formula (I) shown or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, in combination with a MEK inhibitor, in the preparation of a medicament for treating diseases.

[0023]

[0024] Wherein R is hydrogen, halogen, methyl, or ethyl.

[0025] Further, in the uses described in this invention, R in the compound of formula (I) is hydrogen or F; more preferably R is F.

[0026] Further, in the uses described in this invention, the compound of formula (I) is an inhibitor of FAK, Src, or JAK2.

[0027] In some embodiments of the uses described herein, the disease is cancer or an inflammation-related disease, wherein the cancer is at least one genetically altered oncogene that has been previously identified in a patient, wherein the at least one genetically altered oncogene is genetically altered Kras or genetically altered MEK; and wherein the inflammation-related disease is asthma, inflammatory bowel disease, ulcerative colitis, Crohn's disease, or fibrosis.

[0028] In a further described use, the genetically altered Kras comprises at least one mutation selected from G12C, G12V, G12D, G12A, G13C, G12S, D12R, D12F, G13D, G13V, G13R, G13E, Q61H, Q61E, Q61L, and Q61R; or selected from G12D, G13D, and Q61H; or KRAS comprises at least one mutation that is not G12A, G12C, G12S, G12V, and Q61K.

[0029] Further, in the uses described in this invention, the disease is colorectal cancer, pancreatic cancer, lung cancer, or stomach cancer.

[0030] Further, in the uses described in this invention, the dosage of the compound of formula (I) is from about 1 mg to about 1000 mg.

[0031] Further, in the uses described in this invention, the dosage of the MEK inhibitor is from about 0.5 mg to about 100 mg.

[0032] Further, in the uses described in this invention, the MEK inhibitor is trametinib, slmumetinib, LY3214996, R05126766, TNO155 (SHP099), or midametinib, or a pharmaceutically acceptable salt thereof or a solvation thereof. Trametinib, or a pharmaceutically acceptable salt thereof or a solvation thereof, is preferred.

[0033] Further, in the use described in this invention, the compound of formula (I) is applied once or twice daily in an amount of about 1-500 mg.

[0034] Further, in the use described in this invention, the MEK inhibitor is administered in a dose of about 1-100 mg.

[0035] In some embodiments of the invention, the compound of formula (I) is administered simultaneously with a MEK inhibitor.

[0036] In some embodiments of the invention, the compound of formula (I) is administered before a MEK inhibitor.

[0037] In some embodiments of the invention, the compound of formula (I) is administered after a MEK inhibitor.

[0038] In some embodiments of the invention, the patient has not received prior treatment.

[0039] In some embodiments of the invention, the patient has received at least one prior treatment with one or more chemotherapeutic agents or immunotherapies.

[0040] In some embodiments of the invention, the patient has received at least one prior treatment with one or more chemotherapeutic agents or immunotherapies and has developed acquired resistance to said treatment, and / or has developed bypass resistance to said treatment.

[0041] On the other hand, the present invention provides a method for treating cancer in patients requiring such treatment by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof with a therapeutically effective amount of a MEK inhibitor.

[0042] On the other hand, the use of a compound of formula (I) shown in this invention, or a pharmaceutically acceptable salt, stereoisomer thereof, in the preparation of a medicament for treating FAK, SRC, or JAK2-related diseases.

[0043]

[0044] The R is hydrogen, halogen, methyl, or ethyl; preferably R is H or fluorine, and more preferably R is F.

[0045] Further, the stated uses are for cancer or autoimmune diseases.

[0046] Further, the cancers mentioned include: lung cancer, pancreatic cancer, and rectal cancer.

[0047] Further, the autoimmune diseases described herein are: asthma, inflammatory bowel disease, ulcerative colitis, Crohn's disease, and fibrosis.

[0048] The term "cancer" as used in this article includes, but is not limited to, lung cancer such as non-small cell lung cancer (NSCLC), adenocarcinoma, squamous cell carcinoma of the lung, large cell carcinoma, and large cell neuroendocrine tumors; small cell lung cancer (SCLC); neuroblastoma; inflammatory myofibroblastoma; adult renal cell carcinoma; pediatric renal cell carcinoma; breast cancer, such as triple-negative breast cancer; triple-positive breast cancer; colon adenocarcinoma; glioblastoma; glioblastoma multiforme; thyroid cancer, such as anaplastic thyroid cancer; bile duct cancer; ovarian cancer; gastric cancer, such as gastric adenocarcinoma; colorectal cancer (CRC); and inflammatory myofibroblastoma. Cytomas, angiosarcomas, epithelioid hemangioendotheliomas, intrahepatic cholangiocarcinomas, papillary thyroid carcinomas, pelvic sarcomas, astrocytomas, subclinical gliomas, secretory breast cancers, breast mimicry carcinomas, acute myeloid leukemia, congenital mesodermal nephroma, congenital fibrosarcoma, pH-like acute lymphoblastic leukemia, thyroid cancer, skin cancers such as cutaneous melanoma, head and neck squamous cell carcinoma (HNSC), pediatric glioma CML, prostate cancer, ovarian serous bladder cancer, cutaneous melanoma, castration-resistant prostate cancer, Hodgkin's lymphoma, serous and clear cell endometrial carcinoma. The term "cancer" can be understood to include both primary cancers or primary tumors and metastatic cancers or metastatic tumors. Examples include metastatic NSCLC, metastatic CRC, metastatic pancreatic cancer, metastatic colorectal cancer, metastatic HNSCC, etc. It is understood that the term "cancer" includes cancers involving the upregulation of certain genes or genetic mutations in certain genes that can lead to disease progression, such as the upregulation of the epidermal growth factor receptor.

[0049] In some embodiments of the various aspects described herein, the cancer is mediated by at least one genetically altered oncogene selected from genetically altered KRAS, of which genetically altered NRAS, genetically altered HRAS, genetically altered BRAF, genetically altered MEK, or genetically altered PI3K, or such genetically altered oncogenes, have been identified in patients. In some embodiments of the various aspects described herein, the cancer is non-small cell lung cancer mediated by genetically altered KRAS, which includes at least one mutation selected from G12C, G12V, G12D, G12A, G13C, G12S, D12R, D12F, G13D, G13V, G13R, G13E, Q61H, Q61E, Q61L, and Q61R. In some embodiments of the various aspects described herein, the cancer is non-small cell lung cancer mediated by a genetically altered KRAS containing at least one mutation selected from G12D, G13D, and Q61H; in some embodiments of the various aspects described herein, the cancer is non-small cell lung cancer mediated by a genetically altered KRAS containing at least one mutation that is not G12A, G12C, G12S, G12V, or Q61K.

[0050] In some embodiments of the various aspects described herein, the cancer is a genetically altered KRAS-mediated colorectal cancer, the KRAS comprising at least one mutation selected from G12D, G12V, G13D, A146T, G12C, G12A, G12S, K117N, Q61K, G12R, M72V, S17G, K5R, D69G, G13C, G13R, Q61H, K117E, Q61L, Q61R, K117R, A146V, A146P, K147N, and R97I. In some embodiments of the various aspects described herein, the cancer is a genetically altered KRAS-mediated pancreatic cancer, the KRAS comprising at least one mutation selected from G12D, G12V, G12R, Q61H, G12C, and G12S.

[0051] The term "KRAS" as used in this invention refers to the KRAS gene, the corresponding mRNA transcribed from the KRAS gene, or the protein encoded by the KRAS gene, referred to as K-ras, which is involved in the Ras / MAPK signaling pathway. The terms KRAS gene, K-ras, and Ras / MAPK signaling pathway will be known and understood by those skilled in the art. It is understood that KRAS mutations occur in approximately one-seventh of all metastatic cancers, and these mutations can occur at multiple locations within the KRAS gene coding sequence. KRAS mutations primarily occur at KRAS codons 12 and 13, but also at lower frequencies at codons 18, 61, 117, and 146, and play a significant role in codon-based and missense-based tumor cell signaling. Examples of KRAS mutations include, but are not limited to, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12S, KRAS G13C, KRAS G13D, KRAS A18D, KRASQ61H, KRAS K117N, etc.

[0052] The “MEK inhibitors” used in this invention include, but are not limited to, any compounds or reagents known in the art that inhibit the MAPK / ERK kinase-1 and -2 genes or inhibit proteins (MEK1 and MEK2; MAP2K1 and MAP2K2) encoded by the MAPK / ERK kinase-1 and -2 genes. Exemplary MEK inhibitors used in the methods and compositions described herein include, but are not limited to, trametinib, pimasatinib (AST03026); selemetinib (AZD6244); cobimetinib; midametinib (PD-0325901); refametinib (RDEA119); TAK733; MEK162; R05126766; WX-554; R04987655; GDC-0973; AZD8330; AZD6244; and CI-1040 (PD-184352); GDC-0623; HL-085.

[0053] The "trametinib" described in this invention relates to a compound having the following formula or a pharmaceutically acceptable salt thereof or a solvation thereof, also known as GSK1120212 or N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide. Trametinib is an orally bioavailable inhibitor of mitogen-activated protein kinase kinase (MEKMAPK / ERK kinase) with potential antitumor activity. Trametinib specifically binds to and inhibits MEK1 and 2, resulting in inhibition of growth factor-mediated cell signaling and cell proliferation in various cancers.

[0054] Pharmaceutically acceptable salts of trametinib are obtained by salting trametinib with acids, including but not limited to mesylate, maleate, tartrate, succinate, acetate, difluoroacetate, fumarate, citrate, benzenesulfonate, benzoate, naphthalenesulfonate, lactate, malate, hydrochloride, hydrobromide, sulfate, and phosphate.

[0055] "Pharmaceutically acceptable salts" refer to salts formed by base ions and free acids or salts formed by acid ions and free bases, such as hydrochloride, hydrobromide, sulfate, phosphate, formate, acetate, trifluoroacetate, fumarate, oxalate, etc.

[0056] An "effective dose" or "effective therapeutic dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicities.

[0057] The structure of compound 1 is as follows: It is reported in patent WO2021115401.

[0058] The structure of compound 2 is as follows: Prepared according to Example 6 of Patent WO2019210835.

[0059] Trametinib was purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., batch number 30987.

[0060] Experimental Example 1: In vitro proliferation inhibition experiment with combined drugs

[0061] Experimental objective: To detect the viability of tumor cells after treatment with the compound, using IC50 as the indicator. 50 The values ​​were used as indicators to evaluate the inhibitory effects of combination therapy and single therapy on the in vitro proliferation of HCT116 and other KRAS mutant cells.

[0062] Experimental materials: HCT116, KP-4, SW480, and NCI-H23 were all purchased from Nanjing Kebai, and CellCounting-Lite™ 2.0 was also used.

[0063] Experimental methods:

[0064] (1) Collect HCT116, KP-4, SW480 and NCI-H23 cells in the logarithmic growth phase, digest with trypsin, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, resuspend in culture medium, count, and divide according to 2-5*10. 3 Inoculate one per well into a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0065] (2) Dissolve the test compound in 100% DMSO to prepare a 10 mM stock solution and store at 4°C protected from light. Set up single-drug wells, combination wells, control wells, and blank wells. Single-drug wells contain gradient concentrations of compound 1 or compound 2 or trametinib (initial concentration 3 μM), diluted 3-fold, for a total of 9 concentrations; combination wells contain gradient concentrations of trametinib and 1 μM of compound 1 or compound 2; control wells contain cells and culture medium; blank wells contain only culture medium. The DMSO content of all wells is 0.5%. After drug addition, incubate the cells at 37°C in a 5% CO2 incubator for 72 h.

[0066] (3) Take out the cell culture plate to be tested, equilibrate at room temperature for 30 minutes, add an equal volume of CellCounting-Lite™ 2.0 to the cell culture to be tested, shake and mix for 10 minutes to fully lyse the cells, let stand for 5 minutes and then use a multi-functional microplate reader to detect the luminescence signal.

[0067] Data Analysis:

[0068] Calculation formula: Cell viability = (Ls - Lb) / (Lc - Lb) * 100%

[0069] Where: Ls represents the emission value of the experimental well, Lb represents the emission value of the blank well, and Lc represents the emission value of the control well.

[0070] Fitting dose-response curve

[0071] Plotting the concentration logarithmic value on the X-axis and cell viability on the Y-axis, dose-response curves were fitted using the log(inhibitor) vs. response–variable slope method in GraphPad Prism 5 to derive the IC50 values ​​for each compound. 50 value.

[0072]

[0073] Experimental conclusion: Trametinib in combination with compound 1 or compound 2 significantly enhanced the in vitro proliferation inhibition of tumor cells HCT116, NCI-H23, KP-4 and SW480 compared with the in vitro proliferation inhibition of each compound alone.

[0074] Experimental Example 2: JAK2 kinase activity assay

[0075] Experimental objective: To detect the inhibitory activity of the compound on JAK2 kinase.

[0076] Experimental materials:

[0077]

[0078] Experimental methods:

[0079] 1) Prepare 1X kinase buffer solution (prepared with ddH2O, containing 40mM Tris, 20mM MgCl2, 0.10% BSA, and 0.5mM MTT).

[0080] 2) Dilute the compound 3-fold with DMSO in a dilution plate. The initial concentration of the compound was 10 μM.

[0081] 3) Dilute the compound 50-fold in 1X kinase reaction buffer and shake on a shaker for 20 minutes.

[0082] 4) Prepare 2X kinase using 1X enzyme reaction buffer.

[0083] 5) Add 2 μL of kinase (prepared in step 4) to each well of the reaction plate.

[0084] 6) Add 1 μL of the compound diluted in buffer to each well, seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and incubate at room temperature for 10 minutes.

[0085] 7) Prepare a 4x MBP Protein and ATP mixture (final ATP concentration 10 μM) using 1X enzyme reaction buffer, and add 1 μL of the 4x MBP Protein / ATP mixture to the reaction plate.

[0086] 8) Seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and react at room temperature for 60 minutes.

[0087] 9) Transfer 4 μL of ADP-Glo ​​to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 40 min.

[0088] 10) Transfer 8 μL of Detection solution to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min.

[0089] 11) Use a Biotek multi-function plate reader to read the RLU (Relative Luminescence Unit) signal. The signal intensity is used to characterize the activity level of the kinase.

[0090] 12) Data processing:

[0091] a) Compound inhibition rate % = (1 - (RLU analyte - RLU positive control) / (RLU negative control - RLU positive control)) * 100%

[0092] b) Data analysis was performed using Graphpad 7.0 software. The IC50 (half-maximal inhibitory concentration) of the compound was obtained using the following nonlinear fitting formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hill Slope)). Where X is the log value of the compound concentration, and Y is the inhibition rate (%inhibition).

[0093] 13) Experimental results:

[0094]

[0095] Experimental conclusion: Compound 1 and Compound 2 have significant inhibitory effects on JAK2 kinase.

[0096] Experimental Example 3: FAK Kinase Activity Assay

[0097] Experimental objective: To detect the inhibitory activity of the compound on FAK kinase.

[0098] Experimental materials:

[0099]

[0100]

[0101] Experimental methods:

[0102] 1) Prepare 1X kinase buffer solution (prepared with ddH2O, containing 5mM MgCl2; 1mM DTT; 3.9nM SEB)

[0103] 2) Dilute the test compound 5-fold with 100% DMSO, and perform 3-fold serial dilutions in a 96-well dilution plate. Add 1 μL of the compound to 39 μL of kinase reaction buffer, and shake on a microplate shaker for 20 min.

[0104] 3) Transfer 2 μL of 2.5X kinase to a 384 reaction plate, add 1 μL of the test compound (prepared in step 2) to the 384 reaction plate (Greiner, 784075), centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min.

[0105] 4) Transfer 2 μL of the 2.5X substrate mixture to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min.

[0106] 5) Prepare a 2X Sa-XL 665 / TK-antibody-Cryptate mixture using HTRF detection buffer.

[0107] 6) Add 5 μL of Sa-XL 665 / TK-antibody-Cryptate to each well, centrifuge at 1000g for 30 seconds, and react at room temperature for 1 hour.

[0108] 7) Use Biotek to read the fluorescence signals at 615nm (Cryptate) and 665nm (XL665).

[0109] 8) Data Analysis

[0110] a) Calculate the ratio per pore (Ratio_665 / 615nm)

[0111] b) Substitute the ratio per well into the following formula to calculate the corresponding inhibition rate: Compound inhibition rate % = (1 - (compound - positive control) / (negative control - positive control)) * 100%

[0112] c) Using the logarithm of compound concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the analysis software GraphPadPrism 7.0 to obtain the IC50 of the compound. 50 The fitting formula is Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hill Slope)).

[0113] Experimental results:

[0114]

[0115] Experimental conclusion: Compound 1 and Compound 2 have significant inhibitory effects on FAK kinase.

[0116] Experiment Example 4: Src kinase activity assay

[0117] Experimental objective: To detect the inhibitory activity of the compound on Src kinase.

[0118] Experimental materials:

[0119] Materials and Reagents Manufacturer Catalog Number SRC Carna 08-173 Kinase substrate 4 GL 112395 DMSO Sigma D8418-1L 384-well plate Corning 3573 SRC Carna 08-173 Instruments and Equipment Manufacturer Catalog Number or Model Centrifuge Eppendorf 5430 Echo 550 Labcyte Echo 550 Microplate Reader Perkin Elmer Caliper EZ Reader

[0120] Experimental methods:

[0121] 1) Prepare 1X kinase buffer solution

[0122] 2) Preparation of compound concentration gradients: The test compound was initially tested at a concentration of 10 μM, diluted 3-fold, for a total of 10 concentrations, per well. It was diluted 100-fold to a final concentration of 100% DMSO in a 384-well plate. 250 nL of the 100-fold final concentration of the compound was transferred to the target 384-well plate using an Echo 550 dispenser. 250 nL of DMSO was added to the positive and negative control wells.

[0123] 3) Prepare a kinase solution with a final concentration of 2.5 times using 1X kinase buffer.

[0124] 4) Add 10 μL of kinase solution at 2.5 times the final concentration to the compound wells and the positive control wells, respectively; add 10 μL of 1x kinase buffer solution to the negative control wells.

[0125] 5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes.

[0126] 6) Prepare a mixed solution of ATP and kinase substrate 4 at a final concentration of 25 / 15 using 1x kinase buffer solution.

[0127] 7) Add 15 μL of a mixed solution of ATP and kinase substrate 4 at a final concentration of 25 / 15 to initiate the reaction.

[0128] 8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 30 minutes.

[0129] 9) Add 30 μL of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and vortex to mix.

[0130] 10) Use Caliper EZ Reader to read the conversion rate.

[0131] 11) Data Analysis:

[0132] a) Calculate the inhibition rate: Compound inhibition rate % = (Conversion%max - Conversion%sample) / (Conversion%max - Conversion%min) * 100%, where Conversion%sample is the conversion rate reading of the sample; Conversion%min is the average value of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Conversion%max is the average value of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

[0133] b) Using the logarithm of compound concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the analysis software GraphPadPrism 5.0 to obtain the IC50 of the compound. 50 The fitting formula is Y = Bottom + (Top - Bottom) / (1 + 10^(LogIC)). 50 -X)*Hill Slope)).

[0134] Experimental results:

[0135]

[0136] Experimental conclusion: Both compound 1 and compound 2 have inhibitory effects on Src kinase.

[0137] Trial Example 5: Combination Therapy in KRAS G12V Objective of the antitumor pharmacodynamic evaluation experiment on a mutant lung cancer MiniPDX model: To establish KRAS G12VA MiniPDX model of mutant lung cancer was used to detect the antitumor efficacy of compounds. The relative proliferation rate of tumor cells was used as an indicator to evaluate the effects of combination therapy and monotherapy on KRAS. G12V In vivo inhibition of mutation-induced lung cancer proliferation.

[0138] Experimental materials:

[0139] 1) Information on in vivo xenograft model:

[0140] Model Number Patient Gender Patient age Mutation type Pathological description LD1-0025-200616 female 75 <![CDATA[KRAS G12V ]]> Poorly to moderately differentiated adenocarcinoma

[0141] 2) Experimental animals: 18 female BALB / c-Nu mice, 6-8 weeks old, weighing 18-22g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0142] 3) Other reagents and instruments:

[0143]

[0144]

[0145] Experimental methods:

[0146] 1) MiniPDX fabrication:

[0147] a) The tumor model required for the resuscitation experiment, once the tumor grows to 500-800 mm 3 During the procedure, tumor tissue was aseptically removed and non-tumor tissue and necrotic tissue were removed in a biosafety cabinet.

[0148] b) Cut the tumor tissue into 1-3 cubic millimeter pieces, digest the tumor pieces with digestive solution at 37°C for 1-2 hours, then collect the cell suspension through a 70μm sieve, centrifuge at 1200rpm for 3 minutes to remove the supernatant, resuspend the cells in 10mL of PBS containing 1% FBS and count them with a hemocytometer.

[0149] c) After removing the mouse-derived cells, centrifuge at 1200 rpm for 3 minutes to remove the supernatant. Resuspend the cells in BIO-MPM-1 cell culture medium (serum-free), count the cells using a hemocytometer, and adjust the cell density. Fill the cell suspension into a MiniPDX device;

[0150] d) Mice were randomly divided into groups according to their body weight; two MiniPDX devices were implanted subcutaneously into the backs of the mice. The day of implantation was designated as day 0, and the entire experiment lasted for 7 days.

[0151] 2) Mouse grouping and administration:

[0152] Grouping and administration will begin on day 0 according to the table below.

[0153]

[0154]

[0155] Note: N: number of replicates; dosage volume is 10 μl / g; QD×7: once a day for a total of 7 times; po: oral administration.

[0156] Throughout the experiment, the use and observation of experimental animals were conducted in accordance with the relevant regulations for the use and management of animals by AAALAC. After being inoculated with the MiniPDX tumor device, the experimental animals were observed daily. Behavior, food intake, water intake, and fur were monitored in all experimental animals, and the weight of the mice was recorded.

[0157] The experiment ended 24 hours after the sixth day of drug administration (i.e., the seventh administration). All mice were euthanized, and the MiniPDX device was removed. Cell viability was assessed using the CellTiter-Glo method.

[0158] Data processing:

[0159] 1) Calculation formula:

[0160] a) Relative proliferation rate of tumor cells (%) = (Vdrug-treated group d7 - Vcontrol group d0) / (Vcontrol group d7 - Vcontrol group d0) × 100%. (Vdrug-treated group d7: fluorescence value of the treatment group on day 7; Vcontrol group d7: fluorescence value of the control group on day 7; Vcontrol group d0: fluorescence value of the control group on day 0).

[0161] b) The body weight of all tumor-bearing mice was measured daily. The percentage change in body weight after drug administration was also calculated: RCBW(%) = (BW) / (RCBW) / (RCBW) i –BW0) / BW0×100, where BWi is the average body weight after the start of administration and BW0 is the average body weight at the time of the first administration.

[0162] 2) Data Analysis:

[0163] All data are expressed as mean ± SEM. Student's t-test was used to compare the fluorescence values ​​after CTG detection between the treatment group and the control group to determine if there was a significant difference. All data were analyzed using GraphPad. A p-value < 0.05 was considered statistically significant.

[0164] Experimental results:

[0165] In LD1-0025-200616KRAS G12VIn the MiniPDX pharmacodynamic model of lung cancer, the relative proliferation rates of tumor cells on day 7 after administration of the following groups were 57%, 37%, 46%, 55%, and 28%, respectively: compound 1, 10 mg / kg; TPX-0005, 10 mg / kg; trametinib, 1 mg / kg + TPX-0005, 10 mg / kg; and trametinib, 1 mg / kg + compound 1, 10 mg / kg. Compared with the control group, the combination therapy group of trametinib, 1 mg / kg + compound 1, 10 mg / kg showed a significant reduction in fluorescence intensity (RLU) (p<0.05), demonstrating a significant antitumor effect. Furthermore, compared with the corresponding monotherapy groups, the combination therapy group of trametinib, 1 mg / kg + compound 1, 10 mg / kg showed a synergistic or additive effect. During the administration period, none of the mice in the administration groups showed significant weight loss (RCBW% < -15%), nor were any other abnormal behaviors or manifestations observed. All mice in this model showed good tolerance to the drug at the administered dose.

[0166]

[0167] Experimental conclusion: Trametinib combined with compound 1, in KRAS G12V In the mutant lung cancer miniPDX model, the inhibitory effect on tumor cell proliferation was significantly stronger than that of trametinib alone or compound 1 alone. The combination of compound 1 and trametinib was significantly better than the combination of TPX-0005 and trametinib.

[0168] Trial Example 6: Combination Therapy in KRAS G12D Objective of the antitumor pharmacodynamic evaluation experiment on a mutant lung cancer MiniPDX model: To establish KRAS G12D A MiniPDX model of mutant lung cancer was used to detect the antitumor efficacy of compounds. The relative proliferation rate of tumor cells was used as an indicator to evaluate the effects of combination therapy and monotherapy on KRAS. G12D In vivo inhibition of mutation-induced lung cancer proliferation.

[0169] Experimental materials:

[0170] 1) Information on in vivo xenograft model:

[0171] Model Number Patient gender Patient age Mutation type Pathological description LD1-0025-370740 male 70 <![CDATA[KRAS G12D ]]> Poorly differentiated adenocarcinoma

[0172] 2) Experimental animals: 18 female BALB / c-Nu mice, 6-8 weeks old, weighing 18-22g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0173] 3) Other reagents and instruments:

[0174]

[0175] Experimental methods:

[0176] 1) MiniPDX fabrication:

[0177] a) The tumor model required for the resuscitation experiment, once the tumor grows to 500-800 mm 3 During the procedure, tumor tissue was aseptically removed and non-tumor tissue and necrotic tissue were removed in a biosafety cabinet.

[0178] b) Cut the tumor tissue into 1-3 cubic millimeter pieces, digest the tumor pieces with digestive solution at 37°C for 1-2 hours, then collect the cell suspension through a 70μm sieve, centrifuge at 1200rpm for 3 minutes to remove the supernatant, resuspend the cells in 10mL of PBS containing 1% FBS and count them with a hemocytometer.

[0179] c) After removing the mouse-derived cells, centrifuge at 1200 rpm for 3 minutes to remove the supernatant. Resuspend the cells in BIO-MPM-1 cell culture medium (serum-free), count the cells using a hemocytometer, and adjust the cell density. Fill the cell suspension into a MiniPDX device;

[0180] d) Mice were randomly divided into groups according to their body weight; two MiniPDX devices were implanted subcutaneously into the backs of the mice. The day of implantation was designated as day 0, and the entire experiment lasted for 7 days.

[0181] 2) Mouse grouping and administration:

[0182] Grouping and administration will begin on day 0 according to the table below.

[0183]

[0184] Note: N: number of replicates; dosage volume is 10 μl / g; QD×7: once a day for a total of 7 times; po: oral administration.

[0185] Throughout the experiment, the use and observation of experimental animals were conducted in accordance with the relevant regulations for the use and management of animals by AAALAC. After being inoculated with the MiniPDX tumor device, the experimental animals were observed daily. Behavior, food intake, water intake, and fur were monitored in all experimental animals, and the weight of the mice was recorded.

[0186] The experiment ended 24 hours after the sixth day of drug administration (i.e., the seventh administration). All mice were euthanized, and the MiniPDX device was removed. Cell viability was assessed using the CellTiter-Glo method.

[0187] Data processing:

[0188] 1) Calculation formula:

[0189] a) Relative proliferation rate of tumor cells (%) = (Vdrug-treated group d7 - Vcontrol group d0) / (Vcontrol group d7 - Vcontrol group d0) × 100%. (Vdrug-treated group d7: fluorescence value of the treatment group on day 7; Vcontrol group d7: fluorescence value of the control group on day 7; Vcontrol group d0: fluorescence value of the control group on day 0).

[0190] b) The body weight of all tumor-bearing mice was measured daily. The percentage change in body weight after drug administration was also calculated: RCBW(%) = (BW) / (RCBW) / (RCBW) i –BW0) / BW0×100, where BWi is the average body weight after the start of administration and BW0 is the average body weight at the time of the first administration.

[0191] 2) Data Analysis:

[0192] All data are expressed as mean ± SEM. Student's t-test was used to compare the fluorescence values ​​after CTG detection between the treatment group and the control group to determine if there was a significant difference. All data were analyzed using GraphPad. A p-value < 0.05 was considered statistically significant.

[0193] Experimental results:

[0194] In LD1-0025-370740KRAS G12D In the MiniPDX pharmacodynamic model for lung cancer, the relative tumor cell proliferation rates on day 7 after administration of the following groups were 97%, 60%, 30%, 54%, and 19%, respectively: compound 1, 10 mg / kg; TPX-0005, 10 mg / kg; trametinib, 1 mg / kg + TPX-0005, 10 mg / kg; and trametinib, 1 mg / kg + compound 1, 10 mg / kg. Compared with the control group, all treatment groups showed varying degrees of reduction in fluorescence intensity (RLU). The trametinib, 1 mg / kg and trametinib, 1 mg / kg + compound 1, 10 mg / kg groups showed significantly reduced fluorescence intensity (p<0.05), demonstrating significant tumor-suppressive effects. Furthermore, compared with the corresponding monotherapy groups, the trametinib, 1 mg / kg + compound 1, 10 mg / kg combination therapy group showed a synergistic or additive effect. During the administration period, one mouse in the trametinib, 1 mg / kg + compound 1, 10 mg / kg group experienced a 15.14% weight loss on day 6, while the other two mice in this group showed normal weight changes. Meanwhile, no mice in any of the other administration groups exhibited significant weight loss (RCBW% < -15%), nor were any other abnormal behaviors or manifestations observed. This indicates that all tumor-bearing mice were able to tolerate the administered doses in this model, and the individual weight loss in some mice may be due to individual differences.

[0195]

[0196] Experimental conclusion: Trametinib combined with compound 1, in KRAS G12D In the mutant lung cancer miniPDX model, the inhibitory effect on tumor cell proliferation was significantly stronger than that of trametinib alone or compound 1 alone. The combination of compound 1 and trametinib was significantly better than the combination of TPX-0005 and trametinib.

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, stereoisomer, in combination with a MEK inhibitor, in the preparation of a medicament for treating a disease. Wherein R is hydrogen, halogen, methyl, or ethyl; The disease is cancer, specifically colorectal cancer, pancreatic cancer, and lung cancer. The MEK inhibitor is trametinib or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, wherein R in the compound of formula (I) is hydrogen or F.

3. The use according to claim 1, wherein the compound of formula (I) is an inhibitor of FAK, SRC or JAK2.

4. The use according to claim 1, wherein the cancer is at least one genetically altered oncogene previously identified in a patient, wherein the at least one genetically altered oncogene is genetically altered KRAS or genetically altered MEK.

5. The use according to claim 4, wherein the genetically altered KRAS comprises at least one mutation selected from G12C, G12V, G12D, G12A, G13C, G12S, D12R, D12F, G13D, G13V, G13R, G13E, Q61H, Q61E, Q61L, and Q61R; or the KRAS comprises at least one mutation that is not one of G12A, G12C, G12S, G12V, and Q61K.

6. The use according to any one of claims 1-5, wherein the dosage of the compound of formula (I) is from 1 mg to 1000 mg.

7. The use according to any one of claims 1-5, wherein the dosage of the MEK inhibitor is from 0.5 mg to 100 mg.

8. The use according to claim 6, wherein the compound of formula (I) is applied once or twice daily in an amount of 1-500 mg.

9. The use according to claim 7, wherein the MEK inhibitor is administered in an amount of 1-100 mg.

10. The use according to any one of claims 1-5, wherein the compound of formula (I) is administered simultaneously with a MEK inhibitor.

11. The use according to any one of claims 1-5, wherein the compound of formula (I) is administered before the MEK inhibitor.

12. The use according to any one of claims 1-5, wherein the compound of formula (I) is administered after the MEK inhibitor.

13. The use according to claim 4, wherein the patient has not received prior treatment.

14. The use according to claim 4, wherein the patient has received at least one prior treatment with one or more chemotherapeutic agents or immunotherapy.

15. The use according to claim 4, wherein the patient has received at least one prior treatment with one or more chemotherapeutic agents or immunotherapy and has developed acquired resistance to said treatment, and / or has developed bypass resistance to said treatment.

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