WO2023061434A1 - Use of tricyclic compound - Google Patents

Abstract

The present invention provides a use of a compound represented by formula (A) or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating cancer mediated by EGFR, FGFR2, KIT, ALK and/or ROS1 mutations. The compound has obvious inhibitory activity against cancer mediated by these mutation types.

Description

A kind of purposes of tricyclic compound

This application claims the priority of the Chinese patent application 202111200131.0 with the filing date of October 14, 2021, the Chinese patent application 202210330403.7 with the filing date of March 31, 2022, and the priority of the Chinese patent application 202211221824.2 submitted on October 8, 2022 Right, this application cites the full text of the above-mentioned Chinese patent application.

technical field

The present invention provides a use of a tricyclic compound or a pharmaceutically acceptable salt thereof in treating cancer mediated by EGFR, FGFR2, KIT, ALK and/or ROS1 mutations.

Background technique

EGFR, the epidermal growth factor receptor, is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells and other cells. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation. EGFR mutation is also the most common type of mutation in NSCLC patients, especially in Asian populations, accounting for 40% to 50%. Therefore, EGFR has always been one of the hottest targets in the field of drug development.

At present, the EGFR inhibitors on the market are divided into the first, second and third generations. The first generation is reversible targeted drugs, targeting L858R mutation and Del19 mutation, such as gefitinib, erlotinib, and icotinib. The second generation is irreversible targeted drugs, such as afatinib and dacomitinib. Although the first and second generation targeted drugs are effective, most patients will develop drug resistance after 1-2 years of drug use. Among patients with EGFR inhibitor resistance, 50% of drug resistance is related to T790M mutation. The third-generation EGFR-targeted drug osimertinib can overcome the tumor resistance caused by the T790M mutation and bring better survival benefits to more lung cancer patients. However, the third-generation targeted drugs will inevitably produce drug resistance, and the reasons for drug resistance include further C797S mutation, G724S mutation, L792H mutation, E709K mutation, and EGFR amplification. At present, there is no mature treatment for osimertinib resistance clinically, and the clinical needs are imminent.

Fibroblast growth factor and its receptor (FGFR) drive important developmental signaling pathways affecting cell proliferation, migration and survival. Aberrant FGF signaling plays a role in many cancers. The FGFR family consists of FGFR1, FGFR2, FGFR3 and FGFR4. FGFRs are tyrosine kinases that are activated in a subset of tumors by gene amplification, mutation, or chromosomal translocations or rearrangements. Amplification of FGFR1 occurs in squamous cell lung cancer and estrogen receptor-positive breast cancer. FGFR2 is also amplified in gastric and breast cancers. FGFR mutations have been observed in endometrial cancer and FGFR3 mutations in bladder cancer.

The encoded product of c-KIT is a transmembrane receptor protein with tyrosine kinase activity and a molecular weight of 145 kilodaltons. It has five immunoglobulin G-like domains in the extracellular region, so it belongs to Member of the type III tyrosine kinase superfamily. Under physiological conditions, a small amount of c-KIT is expressed in mast cells, stem cells, sperm cells and intestinal Cajal cells. Under physiological conditions, when stem cell factor (a ligand of c-KIT) binds to the immunoglobulin G-like domain of c-KIT, the c-KIT molecule undergoes homodimerization, resulting in the Y568 and Y570 tyrosine Autophosphorylation of acid residues, which in turn leads to phosphorylation of tyrosine residues in many substrate proteins in cells, and activation of multiple signal transduction pathways related to cell proliferation, including Jak-Stat3/Stat5 pathway, Src kinase, Ras-MEK-Erk1/2 and PI3K-AKT pathways, thereby enabling cell proliferation. Gain-of-function point mutations in the tyrosine kinase domain of c-KIT cause ligand-independent and persistent activation, leading to uncontrolled cell growth and resistance to apoptosis. It has been clear that c-KIT mutation is the cause of gastrointestinal stromal tumor (GIST), systemic mastocytosis, and is closely related to small cell lung cancer.

Gene fusion is a chimeric gene formed by connecting the coding regions of two or more genes end to end and placed under the control of the same set of regulatory sequences (including promoters, enhancers, ribosome binding sequences, terminators, etc.). A fusion of the echinoderm microtubule-binding protein 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene has been found in NSCLC. EML4-ALK fusion gene is a cancer-promoting gene mutation that occurs in non-small cell lung cancer, accounting for 4-5% of the incidence of non-small cell lung cancer. EML4-ALK leads to abnormal expression of tyrosine kinases, causing malignant transformation of cells. The incidence of SLC34A2-ROS1 fusion gene in NSCLC is about 1.0%-3.4%, and the incidence in EGFR/KRAS/ALK negative population can reach 5.7%. The pathological type is mainly adenocarcinoma. When the SLC34A2-ROS1 gene fusion occurs, the extracellular region is lost, and the transmembrane and intracellular tyrosine kinase regions are retained. The fusion sites mainly occur in exons 32, 34, 35, and 36 of the ROS1 gene. ROS1 receptor tyrosine kinase is involved in the activation of multiple downstream signal transduction pathways, including RAS-MAPK/ERK, PI3K/AKT/mTOR, JAK/STAT3, PLC/IP3 and SHP2/VAV3 pathways, thereby regulating the growth and proliferation of tumor cells , cell cycle, differentiation, metastasis and migration. There is 49% homology between ROS1 gene and ALK gene in the sequence of tyrosine kinase region, and the homology between them is as high as 77% in the ATP binding site of the kinase catalytic region. ROS1 fusion gene provides a new method for individualized treatment of lung cancer It is of great significance for clinical practice to clarify the positive rate of ROS1 fusion gene in lung adenocarcinoma.

The applicant disclosed a small molecule EGFR inhibitor against C797S mutation in the patent PCT/CN2021/086941, the structure of which is shown in formula (A). The Del19/T790M/C797S mutation has good kinase inhibitory activity and cell anti-proliferation activity, and the molecule has good anti-tumor activity and tolerance in mouse models. In order to improve the clinical value of this compound, it is of great significance to develop more uses of it.

Contents of the invention

The invention provides a use of a tricyclic compound or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR, FGFR2, KIT, ALK and/or ROS1 mutations.

specific,

The present invention provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19 mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is T790M mutation without C797S mutation.

In some solutions of the present invention, the T790M mutation without the C797S mutation described in the above uses is selected from one or a combination of the following: L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation Mutation, E709K/T790M/L858R triple mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19/C797S double mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R/C797S double mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer mediated by EGFR amplification.

In some schemes of the present invention, the above-mentioned EGFR amplification is the amplification of Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation and V674L/E746_A750del/T790M triple mutation EGFR amplification.

In some schemes of the present invention, the above-mentioned EGFR amplification is EGFR amplification accompanied by Del19/T790M/C797S triple mutation, EGFR amplification accompanied by L858R/T790M/D537H triple mutation or accompanied by V674L/E746_A750del/T790M triple mutation EGFR amplification.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is exon 20 insertion mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating cancer mediated by EGFR mutation or amplification, and the type of EGFR mutation is selected from one or any combination of the following: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, 20 outside Exon mutation; EGFR amplification selected from Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation, V674L/E746_A750del/T790M triple mutation EGFR amplification.

The present invention also provides the use of a compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EGFR mutation or amplification, wherein the type of EGFR mutation is selected from one or any combination of the following : Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, 20 Exon mutation; the EGFR amplification is selected from one or any combination of the following: EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation .

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer with high expression of FGFR2.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer with C-KIT mutation, and the type of C-KIT mutation is V560G mutation and/or D816Y mutation and/or D816H mutation and/or V559 and V560 amino acid deletion mutation and/or D816V mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EML4-ALK fusion protein.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EML4-ALK fusion protein L1196M mutation and/or F1174L mutation and/or L1196M/L1198F double mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by SLC34A2-ROS1 fusion protein.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by the D2033N mutation of the SLC34A2-ROS1 fusion protein.

In some aspects of the present invention, the pharmaceutically acceptable salt of the compound (A) in any of the above uses is hydrochloride.

In some aspects of the present invention, the pharmaceutically acceptable salt of the compound (A) in any of the above uses is monohydrochloride.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19 mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is T790M mutation without C797S mutation.

In some solutions of the present invention, the T790M mutation without the C797S mutation described in the above uses is selected from one or a combination of the following: L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation Mutation, E709K/T790M/L858R triple mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19/C797S double mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R/C797S double mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR amplification.

In some schemes of the present invention, the EGFR amplification described in the above uses is the amplification of Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation and V674L/E746_A750del/T790M triple mutation EGFR amplification.

In some schemes of the present invention, the EGFR amplification in the above use is EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is exon 20 insertion mutation.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation or amplification, and the type of EGFR mutation is selected from one or any combination of the following: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, exon 20 mutation ; EGFR amplification is selected from the amplification of Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation, V674L/E746_A750del/T790M triple mutation EGFR.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation or amplification, wherein the type of EGFR mutation is selected from one or any combination of the following: Del19 mutation , L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, exon 20 Mutation; the EGFR amplification is selected from one or any combination of the following: EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation.

The present invention also provides a method for treating cancer mediated by EGFR mutation or amplification, which comprises administering a compound of formula (A) or a pharmaceutically acceptable salt thereof to a patient, wherein the type of EGFR mutation is selected from one or any of the following Combination: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, Exon 20 mutation; the EGFR amplification is selected from one or any combination of the following: EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation increase.

The present invention also provides the compound of formula (A) for preparing the above-mentioned Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19 Application in EGFR mutation regulators of /C797S double mutation, L858R/C797S double mutation and exon 20 mutation.

In some aspects of the present invention, the above-mentioned EGFR mutation regulator is used as an inhibitor of the above-mentioned mutation of EGFR.

The present invention also provides the use of the compound of formula (A) in preparing regulators for EGFR amplification of the above-mentioned Del19/T790M/C797S triple mutation, the above-mentioned L858R/T790M/D537H triple mutation or the above-mentioned V674L/E746_A750del/T790M triple mutation.

In some aspects of the present invention, the cancer in any of the above uses is lung cancer.

In some aspects of the present invention, the cancer in any of the above uses is non-small cell lung cancer.

In some aspects of the present invention, the cancer in any of the above uses is treatment-naive non-small cell lung cancer.

In some aspects of the present invention, the cancer in any of the above uses is non-small cell lung cancer that develops drug resistance after receiving EGFR inhibitor therapy in the past.

In some aspects of the present invention, the aforementioned EGFR inhibitors include first-generation EGFR inhibitors, second-generation or third-generation EGFR inhibitors.

In some aspects of the present invention, the above-mentioned first-generation EGFR inhibitors include gefitinib, icotinib, and erlotinib.

In some aspects of the present invention, the above-mentioned second-generation EGFR inhibitors include afatinib and dacomitinib.

In some aspects of the present invention, the aforementioned third-generation EGFR inhibitor includes osimertinib.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancers with high FGFR2 expression.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer with C-KIT mutation, and the type of C-KIT mutation is V560G mutation and/or D816Y mutation and/or D816H mutation and /or 559 and 560 amino acid deletion mutations and/or D816V mutations.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EML-ALK fusion protein.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancers mediated by L1196M mutation and/or F1174L mutation and/or L1196M/L1198F double mutation of EML4-ALK fusion protein.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by SLC34A2-ROS1 fusion protein.

The present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by the D2033N mutation of the SLC34A2-ROS1 fusion protein.

The present invention also provides a method for treating the above-mentioned cancer with high FGFR2 expression, the above-mentioned cancer with C-KIT mutation, the above-mentioned cancer mediated by EML-ALK fusion protein, and the above-mentioned cancer mediated by SLC34A2-ROS1 fusion protein, which comprises administering to patients A compound of formula (A) or a pharmaceutically acceptable salt thereof.

The present invention also provides the use of the compound of formula (A) in preparing regulators of the above-mentioned FGFR2, the above-mentioned C-KIT mutation, the above-mentioned EML-ALK fusion protein, and the above-mentioned SLC34A2-ROS1.

In some aspects of the present invention, the aforementioned modulators are inhibitors.

In some aspects of the present invention, the pharmaceutically acceptable salt of the compound of formula (A) in any of the above uses is hydrochloride.

In some aspects of the present invention, the pharmaceutically acceptable salt of the compound of formula (A) in any of the above uses is monohydrochloride.

technical effect

The compound of formula (A) of the present invention not only has good activity against L858R/T790M/C797S triple mutation and Del19/T790M/C797S triple mutation, but also has good activity against L858R or Del19 single mutation, exon 20 insertion mutation, L858R/T790M or L858R/C797S or Del19/C797S double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation and Del19/T790M/C797S triple mutation accompanied by EGFR amplification, L858R/ The T790M/D537H triple mutation and the V674L/E746_A750del/T790M triple mutation also have good in vitro kinase or cell anti-proliferation activities, and the compound has shown a strong anti-proliferative activity in the mouse models of the Del19 single mutation, the L858R single mutation, and the Del19/C797S double mutation. Good antitumor activity and well tolerated by mice.

In addition, the compound of formula (A) of the present invention is effective against high expression of FGFR2, C-KIT V560G mutation, C-KIT D816Y mutation, C-KIT D816H mutation, C-KIT V559 and V560 amino acid deletion mutation, C-KIT D816V mutation , EML4-ALK fusion protein mutation, EML4-ALK fusion protein L1196M or F1174L mutation or L1196M/L1198F double mutation, SLC34A2-ROS1 fusion protein mutation, SLC34A2-ROS1 fusion protein D2033N mutation cell lines all have good anti-proliferation activity.

Definition and Description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered indeterminate or unclear if it is not specifically defined, but should be understood according to its ordinary meaning.

The term "pharmaceutically acceptable salt" refers to derivatives prepared from the compounds of the present invention with relatively non-toxic acids or bases. These salts can be prepared during compound synthesis, isolation, purification, or alone by reacting the free form of the purified compound with an appropriate acid or base. When the compound contains relatively acidic functional groups, it can react with alkali metal, alkaline earth metal hydroxide or organic amine to obtain base addition salts, including cations based on alkali metals and alkaline earth metals and non-toxic ammonium, quaternary ammonium and amine cations, Salts of amino acids and the like are also contemplated. When the compound contains a relatively basic functional group, it reacts with an organic acid or an inorganic acid to form an acid addition salt. In the present invention, the EGFR mutation-mediated tumor or cancer refers to the cancer driver mutation (driver mutation) of EGFR that can be detected in these tumors or cancer patients, including but not limited to Del19 mutation, L858R mutation, T790M mutation , 20 exon insertion mutation (Exon 20ins), C797S and other mutations. Among them, the Del19 mutation refers to the deletion of some bases in exon 19, resulting in a non-frameshift partial amino acid deletion; L858R refers to the change of amino acid 858 from L to R due to a missense mutation of the base; T790M refers to the change of amino acid 790 from T to M due to the missense mutation of the base in the gene; the exon 20 insertion (Exon 20ins) mutation refers to the in-frame duplication/ Insertion mutation; C797S mutation refers to the mutation of cysteine residue at position 797 to serine. In the present invention, the EGFR mutations include not only the above-mentioned single mutants of EGFR, but also compound mutants of T790M, Del19, L858R, Exon 20ins, C797S and other sites freely combined, including but not limited to the L858R/T790M double mutation , Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, etc.

In the present invention, the EGFR amplification refers to the increase of the copy number of EGFR gene or the high-level expression of protein. It can occur on mutant cells as well as on EGFR receptor cells without the mutation (wild type).

Description of drawings

Figure 1 is the animal tumor growth curve in the in vivo pharmacodynamic study of EGFR Del19/C797S mutation.

Fig. 2 is a graph of animal body weight in the in vivo pharmacodynamic study of EGFR Del19/C797S mutation.

Figure 3 is the animal tumor growth curve in the in vivo pharmacodynamic study of EGFR L858R mutation.

Fig. 4 is the animal body weight curve in the in vivo pharmacodynamic study of EGFR L858R mutation.

Figure 5 is a graph of animal tumor growth curves in the in vivo pharmacodynamic study of EGFR Del19 mutation.

Figure 6 is a graph of animal body weights in the in vivo pharmacodynamic study of EGFR Del19 mutation.

Figure 7 is the animal tumor growth curve in the PDX model study of Osimertinib-resistant human lung cancer.

Fig. 8 is a graph of animal body weight in the PDX model study of Osimertinib-resistant human lung cancer.

Figure 9 is a graph of animal tumor growth curves in the in vivo pharmacodynamic study of EGFR L858R/C797S mutation.

Fig. 10 is a curve diagram of animal body weight in the in vivo pharmacodynamic study of EGFR L858R/C797S mutation.

Figure 11 is the animal tumor growth curve in the in vivo pharmacodynamic study of EGFR L858R/T790M mutation.

Figure 12 is a graph of animal body weights in the in vivo pharmacodynamic study of the EGFR L858R/T790M mutation.

Detailed ways

The present invention will be described in detail through examples below, but it does not imply any unfavorable limitation to the present invention. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

Embodiment 1, the preparation of formula (A) compound

1.1 Preparation of Intermediate 6A

Compound 6A-1:

Compound 1C-4 (3.5 g, 15.5 mmol) was dissolved in acetonitrile (40 mL), and N-iodosuccinimide (4.9 g, 21.7 mmol) was added at 0°C. The reaction was stirred at room temperature for 5 hours. After LCMS monitoring showed that the raw materials disappeared, it was concentrated under reduced pressure, added water (30mL), extracted with dichloromethane (45mL×3 times), and combined the organic phases. The organic phases were first washed with saturated brine (60mL×2 times), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=1/1) to obtain 3.57 g of compound 6A-1.

MS (ESI, m/z): 352.0 [M+H] ⁺ .

Compound 6A-2:

Compound 6A-1 (3.4g, 9.7mmol) and 1A (3.7g, 12.5mmol) were dissolved in 1,4-dioxane (30mL) and water (6mL), and potassium carbonate ( 2.7 g, 19.4 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (790 mg, 1.0 mmol). Under the protection of nitrogen, the reaction system was heated to 80°C and stirring was continued for 2 hours. After LCMS monitoring showed that the raw materials disappeared, the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=2/1) to obtain 2.8 g of compound 6A-2 .

MS (ESI, m/z): 395.3 [M+H] ⁺ .

Compound 6A-3:

Compound 6A-2 (2.7 g, 6.8 mmol) was dissolved in N,N-dimethylformamide (28 mL). Subsequently, potassium carbonate (1.9 g, 13.5 mmol) was added to the above reaction liquid. The reaction system was heated to 100°C and stirring was continued for 24 hours. After LCMS monitoring showed disappearance of starting material, the reaction solution was cooled to room temperature and quenched by adding water (50 mL). The mixture was extracted with ethyl acetate (60 mL×4 times), and the organic phases were combined. The organic phase was washed with saturated brine (50 mL×3 times), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate=15/1) to obtain 1.2 g of Compound 6A-3.

MS (ESI, m/z): 375.2 [M+H] ⁺ .

Compound 6A-4:

Compound 6A-3 (1.2 g, 3.3 mmol) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (4M, 15 mL). Stirring at 30° C. for 6 hours, LCMS monitoring showed that the starting material disappeared, the reaction solution was concentrated, water (40 mL) was added, and the pH was adjusted to 9 with saturated aqueous sodium bicarbonate solution. The mixture was extracted with chloroform/isopropanol=3/1 (50 mL×3 times), and the organic phases were combined, then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol = 30/1) to obtain 842 mg of compound 6A-4.

MS (ESI, m/z): 275.0 [M+H] ⁺ .

Compound 6A-5:

Compound 6A-4 (300 mg, 1.1 mmol) and cesium carbonate (1.07 g, 3.3 mmol) were dissolved in N,N-dimethylformamide (6 mL). Subsequently, iodoisopropane (1.86 g, 10.9 mmol) was added to the above reaction liquid. The reaction system was heated to 80°C and stirring was continued for 16 hours. After LCMS monitoring showed disappearance of the starting material, the reaction was cooled to room temperature and quenched by adding water (30 mL). The mixture was extracted with ethyl acetate (50 mL×3 times), and the organic phases were combined. The organic phase was washed with saturated brine (50 mL×3 times), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=10/1) to obtain 75 mg of compound 6A-5.

MS (ESI, m/z): 317.2 [M+H] ⁺ .

Intermediate 6A:

Compound 6A-5 (75 mg, 0.2 mmol) was dissolved in ethanol (8 mL) and water (1.6 mL). Subsequently, ammonium chloride (50.7 mg, 0.9 mmol) and reduced iron powder (132.4 mg, 2.4 mmol) were added to the above reaction liquid. The reaction system was heated to 80°C and stirring was continued for 5 hours. After LCMS monitoring showed disappearance of starting material, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=10/1) to obtain 48 mg of Compound 6A.

MS (ESI, m/z): 287.2 [M+H] ⁺ .

Preparation of Intermediate 35A

化 ^合物35A-l，

^Compound 35A-l,

6-Aminoquinoxaline (10 g, 68.89 mmol) was dissolved in concentrated sulfuric acid (20 mL). At 0° C., potassium nitrate (9.054 g, 89.55 mmol) was added in portions to the reaction solution and stirring was continued at this temperature for 30 minutes. After LCMS monitoring showed that the starting material disappeared, the reaction solution was poured into ice water (100 g). Its pH was adjusted to 8 with 1M aqueous sodium hydroxide solution. The mixture was extracted with ethyl acetate (200 mL x 2 times), and the organic phases were combined. The organic phase was washed with saturated brine (100 mL x 3 times), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=10/1) to obtain 2 g of Compound 35A-1.

MS (ESI) M/Z: 191.2 [M+H] ⁺ .

1.2 Intermediate 35A:

Compound 35A-1 (2 g, 10.5 mmol) was dissolved in N,N-dimethylformamide (20 mL). The reaction solution was lowered to 0°C, under nitrogen protection, sodium hydride (60wt, 1.3g, 31.5mmol) was added in batches and stirring was continued for 20 minutes. Subsequently, 2,4-dichloro-5-bromopyrimidine (4.8 g, 21.0 mmol) was added to the above reaction solution, and the reaction was warmed to room temperature and stirred for 1 hour. After LCMS monitoring showed that the raw materials disappeared, the reaction solution was cooled to 0° C. and quenched by adding saturated aqueous ammonium chloride solution (80 mL). The mixture was extracted with ethyl acetate (100 mL×3 times), and the organic phases were combined. The organic phase was washed with saturated brine (80 mL×3 times), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=1/2) to obtain 2.7 g of compound 35A.

MS (ESI, m/z): 381.0, 383.0 [M+H] ⁺ .

Compound 53A:

Compound 6A (2.7 g, 9.43 mmol) and 35A (3.6 g, 9.43 mmol) were dissolved in N-methylpyrrolidone (30 mL). Subsequently, methanesulfonic acid (2.72 g, 28.28 mmol) was added to the above reaction liquid. The reaction system was heated to 95°C and stirring was continued for 3 hours. After LCMS monitoring showed that the starting material disappeared, the reaction solution was cooled to room temperature and purified by a reverse phase C18 column. Purification conditions: chromatographic column 330g C18 reverse phase column; mobile phase water (containing 0.1% formic acid) and acetonitrile; flow rate 70mL/min; gradient within 20 minutes, acetonitrile rises from 10% to 50%; detection wavelength 254nm. The collected products were concentrated under reduced pressure to obtain 3.4 g of compound 53A.

MS (ESI, m/z): 631.2, 633.2 [M+H] ⁺ .

Compound 53B:

Compound 53A (3.4 g, 5.38 mmol) was dissolved in a mixed solvent of ethanol (40 mL) and water (8 mL). Subsequently, iron powder (1.50 g, 26.92 mmol) and ammonium chloride (0.86 g, 16.15 mmol) were added to the above reaction solution, and the reaction system was heated to 80° C. and continued to stir for 2 hours. After LCMS monitoring showed disappearance of starting material, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=10/1) to obtain 2.8 g of Compound 53B.

MS (ESI, m/z): 601.2, 603.2 [M+H] ⁺ .

1.3 Compound A:

Compound 53B (5 g, 8.31 mmol) was dissolved in pyridine (50 mL). Methanesulfonyl chloride (1.9 g, 16.62 mmol) was then added dropwise to the reaction solution. The reaction system was warmed to 50 °C and stirring was continued for 2 hours. After LCMS monitoring showed disappearance of starting material, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in a mixed solvent of methanol/tetrahydrofuran (1/1, 50 mL), and an aqueous solution of sodium hydroxide (2M, 5 mL) was added to the reaction solution at 0°C. The reaction system was warmed up to room temperature and stirred for 1 hour, then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=10/1), the crude product was slurried with dichloromethane/methanol (20/1, 30mL), and washed with acetonitrile/water (50mL) After lyophilization, 3 g of compound A was obtained.

MS (ESI, m/z): 679.0, 681.0 [M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ9.88(br s,1H),8.94(d,J=2.0Hz,1H),8.85(d,J=2.0Hz,1H),8.76(s,1H ),8.67(br s,1H),8.35(s,1H),8.27(s,1H),7.73(s,1H),7.49(s,1H),7.38(s,1H),6.58(s,1H ),3.99-3.91(m,1H),3.76(s,3H),3.71(s,3H),3.21(t,J＝5.6Hz,2H),3.00(s,3H),2.94(t,J＝ 5.6Hz, 2H), 1.29 (d, J = 6.4Hz, 6H).

1.4 Compound A hydrochloride:

Compound A (67 g, 98.59 mmol) was dissolved in a mixed solvent of dichloromethane (880 mL) and methanol (440 mL) and stirring was continued at room temperature for 1 hour. Then, methanol solution of hydrogen chloride (4M, 24.65 mL, 98.59 mmol) was added dropwise to the reaction solution at room temperature. After the reaction system continued to stir at room temperature for 4 hours, the reaction solution was concentrated to 70 mL under reduced pressure. Methyl tert-butyl ether (880 mL) was added to the above mixture and stirring was continued at room temperature for 2 hours. The precipitated solid was filtered and lyophilized with acetonitrile/water (500 mL) to obtain 60.2 g of compound A hydrochloride.

MS (ESI) M/Z: 679.0, 681.0 [M+H] ⁺ .

¹ H NMR (300MHz,DMSO-d ₆ )δ9.97(s,1H),9.30-9.16(m,2H),8.99(s,1H),8.91(s,1H),8.43(s,2H), 7.75-7.30(m,3H),6.57(s,1H),3.95-3.86(m,1H),3.79(s,3H),3.69(s,3H),3.28-3.14(m,2H),3.03( s, 3H), 2.98-2.88 (m, 2H), 1.27 (brs, 6H).

Embodiment 2: biological test evaluation:

(1) In vitro enzyme experiment

In this experiment, the fluorescence resonance energy transfer (TR-FRET) method was used to test the inhibitory effect of compound A on the kinase activity of EGFR WT, EGFR Del19, EGFR L858R, EGFR L858R/T790M, EGFR L858R/C797S and EGFR ex19del/C797S, and concluded that The half inhibitory concentration IC ₅₀ of compound A to EGFR kinase activity.

2. Experimental materials

EGFR, EGFR Del19, EGFR L858R, EGFR L858R/T790M, EGFR L858R/C797S, EGFR ex19del/C797S recombinases were purchased from Signalchem.

HTRF KinEASE-TK kit was purchased from Cisbio.

DTT, MnCl2, and MgCl2 were purchased from Sigma.

ATP was purchased from Promega.

3. Experimental method

1) Prepare 1× working solution: 5 mM MgCl ₂ ; 1 mM DTT; 1 mM MnCl ₂ and 1× kinase buffer (in the kit), wherein SEB is added to the buffer of EGFR L858R/T790M.

2) Use Echo 550 (Labcyte) to transfer 10 nL (or 1 μL) of the serially diluted compound to a 384-well experimental plate.

3) Add 5 μL (or 2 μL) of 2× recombinase solution to the 384-well experimental plate, and incubate at room temperature for 10 minutes.

4) Add 5 μL (or 2 μL) of 2×TK-substrate-biotin substrate solution (including ATP) to the 384-well experimental plate, and incubate at room temperature for 40 minutes (or 1 h).

5) Add 5 μL of detection solution containing Sa-XL 665HTRF, and 5 μL of TK-antibody-Cryptate, and incubate at room temperature for 1 hour.

6) A microplate reader detects the 615nm and 665nm fluorescence signal values of each well.

7) Calculate the ratio of the fluorescence signal 665nm/615nm of each well.

8) Use GraphPad Prism software for data analysis to obtain the IC ₅₀ of Compound A.

The results of kinase activity inhibition are shown in Table 1.

4. Experimental results and conclusions

From Table 1, we can see that Compound A has a good inhibitory effect on Del19 single mutant and L858R single mutant kinases.

Table 1. Enzyme inhibition results

(2) Cell Proliferation Inhibition Experiment

I. A431 cell proliferation inhibition experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of A431 cells, and the concentration IC ₅₀ at which the compound inhibited half of the cell growth was obtained.

1. Experimental materials

A431 cells were purchased from ATCC.

DMEM medium, fetal bovine serum (FBS), and Penicillin-Streptomycin were purchased from GIBCO.

Brigatinib was purchased from Selleck Company.

CellTiter-Glo reagent was purchased from Promega Company.

2. Experimental method

1) A431 cells were seeded in a 384-well culture plate at a density of 800 cells per well, 30 μl per well, and placed in a cell culture incubator for 24 hours (37° C., 5% CO ₂ ).

2) Day 0: Add 30 nL of the compound to be tested in a gradient dilution to the culture plate cells using Echo, the final concentration of DMSO is 0.1%, and place the culture plate in a cell culture incubator for 72 hours (37° C., 5% CO ₂ ). For the blank control, 30 nL of DMSO was added to each well.

3) Day 3: Add 30 μL Cell Titer-Glo reagent to each well, and keep it in the dark for 30 minutes at room temperature.

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

II.NCI-H3255 cell proliferation inhibition experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of NCI-H3255 (EGFR L858R mutation) cells, and the concentration IC ₅₀ of compound A inhibiting half of the cell growth was obtained.

2. Experimental materials

NCI-H3255 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin, purchased from GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Inoculate NCI-H3255 cells in a 384-well culture plate, 30 μL per well.

2) Day0: Add 30 nL of the compound to be tested in a gradient dilution to the culture plate cells using Echo, the final concentration of DMSO is 0.1%, and place the culture plate in a cell culture incubator for 72 hours (37° C., 5% CO ₂ ). For the blank control, 30 nL of DMSO was added to each well.

3) Day3: Add 30 μL Cell Titer-Glo reagent to each well, and keep it in the dark for 30 minutes at room temperature.

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

III. PC-9 Cell Proliferation Inhibition Experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on PC-9 (EGFR Del19 mutation) cell proliferation, and the concentration IC ₅₀ of the compound inhibiting half of the cell growth was obtained.

2. Experimental materials

PC-9 cells were purchased from European Collection of Authenticated Cell Cultures.

1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin, purchased from GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Inoculate PC-9 cells in a 384-well culture plate, 30 μL per well.

2) Day0: Add 30 nL of the compound to be tested in a gradient dilution to the culture plate cells using Echo, the final concentration of DMSO is 0.1%, and place the culture plate in a cell culture incubator for 72 hours (37° C., 5% CO ₂ ). For the blank control, 30 nL of DMSO was added to each well.

3) Day3: Add 30 μL Cell Titer-Glo reagent to each well, and keep it in the dark for 30 minutes at room temperature.

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

IV. NCI-H1975 Cell Proliferation Inhibition Experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of NCI-H1975 (EGFR L858R/T790M mutation) cells, and the concentration IC ₅₀ of compound A inhibiting half of the cell growth was obtained.

2. Experimental materials

NCI-H1975 cells were from ATCC.

1640 medium, fetal bovine serum (FBS), and Penicillin-Streptomycin were purchased from GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Inoculate NCI-H1975 cells in a 384-well culture plate, 30 μL per well.

2) Day0: Add 30 nL of the compound to be tested in a gradient dilution to the culture plate cells using Echo, the final concentration of DMSO is 0.1%, and place the culture plate in a cell culture incubator for 72 hours (37° C., 5% CO ₂ ). For the blank control, 30 nL of DMSO was added to each well.

3) Day3: Add 30 μL Cell Titer-Glo reagent to each well, and keep it in the dark for 30 minutes at room temperature

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

V. HCC827 cell proliferation inhibition experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of HCC827 (EGFR Del mutation) cells, and the concentration IC ₅₀ of compound A inhibiting half of the cell growth was obtained.

2. Experimental materials

HCC827 cells were purchased from ATCC.

1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin, purchased from GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Inoculate HCC827 cells in a 384-well culture plate, 30 μL per well.

2) Day0: Add 30 nL of the compound to be tested in a gradient dilution to the culture plate cells using Echo, the final concentration of DMSO is 0.1%, and place the culture plate in a cell culture incubator for 72 hours (37° C., 5% CO ₂ ). For the blank control, 30 nL of DMSO was added to each well.

3) Day3: Add 30 μL Cell Titer-Glo reagent to each well, and keep it in the dark for 30 minutes at room temperature.

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

VI.Ba/F3 EGFR-Del19/G724S/T790M, Ba/F3 EGFR-E709K/T790M/L858R, Ba/F3 EGFR-L858R/T790M/L792H cell proliferation inhibition experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of Ba/F3 EGFR-Del19/G724S/T790M and Ba/F3 EGFR-E709K/T790M/L858R and Ba/F3 EGFR-L858R/T790M/L792H cells. And the concentration IC ₅₀ of compound A inhibiting half of the cell growth was obtained.

2. Experimental materials

Ba/F3 EGFR-Del19/G724S/T790M cells were from Kangyuan Biotech (Beijing) Co., Ltd.

Ba/F3 EGFR-E709K/T790M/L858R cells were from Kangyuan Biotech (Beijing) Co., Ltd.

Ba/F3 EGFR-L858R/T790M/L792H cells were from Kangyuan Biotech (Beijing) Co., Ltd.

1640 medium, fetal bovine serum (FBS) was purchased from Hyclone and GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Harvest the cells in the logarithmic growth phase for cell counting. The cell viability was detected by the trypan blue exclusion method to ensure that the cell viability was above 90%.

2) The cell density was adjusted using complete medium, and then inoculated in a 96-well cell culture plate with 90 μL per well, totaling 3000 cells.

3) Culture the cells in the 96-well plate at 37°C and 5% CO2.

4) Prepare a 10-fold drug solution, transfer 10 μL of each serially diluted compound to the corresponding experimental well of a 96-well cell plate, so that the detection concentration of the compound starts from 1 μM, 9 concentrations, 3-fold dilution, and then set three for each drug concentration Repeat hole.

5) The drug-dosed 96-well plate was placed at 37°C and 5% CO2 to continue culturing for 72 hours, and then CTG analysis was performed.

6) Thaw the CTG reagent and equilibrate the cell plate to room temperature for 30 minutes.

7) Add an equal volume of CTG solution to each well.

8) Lyse the cells by shaking on an orbital shaker for 5 minutes.

9) Place the cell plate at room temperature for 20 minutes to stabilize the luminescence signal.

10) Read the luminescence value and collect data.

11) Use GraphPad Prism software for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

VII. Ba/F3 (EGFR-Del19/C797S), Ba/F3 (EGFR-L858R/C797S) cell proliferation inhibition experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of Ba/F3 (EGFR-Del19/C797S) and Ba/F3 (EGFR-L858R/C797S) cells, and the concentration of compound A that inhibited half of the cell growth was obtained _IC50 .

2. Experimental materials

Ba/F3 EGFR-Del19/C797S cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3 EGFR-L858R/C797S cells were from Kangyuan Biotech (Beijing) Co., Ltd.

1640 medium, fetal bovine serum (FBS) was purchased from GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Inoculate Ba/F3 (EGFR Del19/C797S) and Ba/F3 (EGFR L858R/C797S) cells in a 96-well culture plate at a density of 3000 cells per well, 90 μL per well.

2) Day 0: Add 10 μL of the compound to be tested in serial dilution to the cells on the culture plate, the final concentration of DMSO is 0.2%, and place the culture plate in a cell culture incubator and incubate for 72 hours (37° C., 5% CO ₂ ). For the blank control, 10 μL of DMSO was added to each well.

3) Day3: Add 100 μL CellTiter-Glo reagent to each well, and shake at room temperature for 10 minutes in the dark

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) Use GraphPad Prism software for data analysis to obtain the IC ₅₀ of the compound.

The cell viability inhibition results are shown in Table 2.

VIII. NCI-H716 Cell Proliferation Inhibition Experiment

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of NCI-H716 (highly expressed FGFR2) cells, and the concentration IC ₅₀ of the compound inhibiting half of the cell growth was obtained.

2. Experimental materials

NCI-H716 cells were purchased from ATCC.

1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin, purchased from GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Inoculate NCI-H716 cells in a 96-well culture plate, 100 μL per well.

2) Day0: Add 92 μL medium and 8 μL serially diluted compound to be tested to the cells in the culture plate, the final concentration of DMSO is 0.1%, and place the culture plate in a cell culture incubator for 72 hours (37°C, 5% CO ₂ ) . For the blank control, 92 μL of medium and 8 μL of DMSO were added to each well.

3) Day3: Add 100 μL of Cell Titer-Glo reagent to each well, and keep in the dark for 30 minutes at room temperature.

4) Envision microplate reader (PerkinElmer) detects chemiluminescent signal.

5) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

IX.Ba/F3 C-KIT-V560G, Ba/F3 C-KIT-D816Y, Ba/F3 C-KIT-D816H, Ba/F3 C-KIT-Del(V559V560), Ba/F3 C-KIT-D816V, NCI-H3122(EML4-ALK), Ba/F3-EML4-ALK-L1196M, Ba/F3 EML4-ALK-F1174L, Ba/F3-EML4-ALK-L1196M/L1198F, Ba/F3 SLC34A2/ROS1, BaF3 SLC34A2- ROS1-D2033N cell proliferation inhibition experiment

1. Purpose of the experiment

In this experiment, the method of CellTiter-Glo was used to test the effect of compound A on Ba/F3 C-KIT-V560G, Ba/F3 C-KIT-D816Y, Ba/F3 C-KIT-D816H, Ba/F3 C-KIT-Del(V559- V560), Ba/F3 C-KIT-D816V, NCI-H3122(EML4-ALK), Ba/F3-EML4-ALK-L1196M, Ba/F3 EML4-ALK-F1174L, Ba/F3-EML4-ALK-L1196M/ Inhibitory effect of L1198F, Ba/F3 SLC34A2/ROS1, Ba/F3 SLC34A2-ROS1-D2033N cell proliferation, and the concentration IC ₅₀ of compound A inhibiting half of cell growth was obtained.

2. Experimental materials

Ba/F3 C-KIT-V560G cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3 C-KIT-D816Y cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3 C-KIT-D816H cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3 C-KIT-Del (V559V560) cells were from Kangyuan Biotech (Beijing) Co., Ltd.

Ba/F3 C-KIT-D816V cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

NCI-H3122 (EML4-ALK) cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3-EML4-ALK-L1196M cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3 EML4-ALK-F1174L cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3-EML4-ALK-L1196M/L1198F cells were from Kangyuan Biotech (Beijing) Co., Ltd.

Ba/F3 SLC34A2/ROS1 cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

Ba/F3 SLC34A2-ROS1-D2033N cells were from Kangyuan Biotech (Beijing) Co., Ltd.

1640 medium, fetal bovine serum (FBS) was purchased from Hyclone and GIBCO.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) Harvest the cells in the logarithmic growth phase for cell counting. The cell viability was detected by the trypan blue exclusion method to ensure that the cell viability was above 90%.

2) The cell density was adjusted using complete medium, and then inoculated in a 96-well cell culture plate with 90 μL per well, totaling 3000 cells.

3) Culture the cells in the 96-well plate at 37°C and 5% CO2.

4) Prepare a 10-fold drug solution, transfer 10 μL of each serially diluted compound to the corresponding experimental well of a 96-well cell plate, and set three replicate wells for each drug concentration.

5) The drug-dosed 96-well plate was placed at 37°C and 5% CO2 to continue culturing for 72 hours, and then CTG analysis was performed.

6) Thaw the CTG reagent and equilibrate the cell plate to room temperature for 30 minutes.

7) Add an equal volume of CTG solution to each well.

8) Lyse the cells by shaking on an orbital shaker for 5 minutes.

9) Place the cell plate at room temperature for 20 minutes to stabilize the luminescence signal.

10) Read the luminescence value and collect data.

11) Use GraphPad Prism software for data analysis to obtain the IC ₅₀ of Compound A.

The cell viability inhibition results are shown in Table 2.

4. Experimental results and conclusions

As can be seen from the experimental results in Table 2, compound A of the present invention is effective against NCI-H3255 L858R EGFR mutation, PC9 Del19 EGFR mutation, HCC827 Del19 EGFR mutation, NCI-H1975 L858R/T790M EGFR mutation, Ba/F3 (Del19/G724S/ T790M) EGFR triple mutant cell line, Ba/F3 (L858R/T790M/L792H) EGFR triple mutant cell line, Ba/F3 (E709K/T790M/L858R) EGFR triple mutant cell line, Ba/F3 (Del19/C797S) EGFR double mutant cell line Mutant cell lines and Ba/F3 (L858R/C797S) EGFR double mutant cell lines, NCI-H716 (FGFR2), Ba/F3 C-KIT-V560G, Ba/F3 C-KIT-D816Y, Ba/F3 C-KIT- D816H, Ba/F3 C-KIT-Del(V559V560), Ba/F3 C-KIT-D816V, NCI-H3122(EML4-ALK), Ba/F3-EML4-ALK-L1196M, Ba/F3 EML4-ALK-F1174L , Ba/F3-EML4-ALK-L1196M/L1198F, Ba/F3 SLC34A2/ROS1, Ba/F3 SLC34A2-ROS1-D2033N have good inhibitory effect on cell proliferation; the inhibitory effect on EGFR wild-type cell line A431 is weak , with better selectivity.

Table 2. Results of Cell Proliferation Inhibition Test Data

(3) PDO proliferation inhibition test

1. Purpose of the experiment

In this experiment, the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of osimertinib-resistant PDO (Patient Derived Tumor Organoids), and the concentration IC ₅₀ of compound A inhibiting half of the cell growth was obtained.

2. Experimental materials

PDO, from Beijing Ketu Medical Technology Co., Ltd.

Accutase was purchased from Sigma.

K2 Oncology, from Beijing Ketu Medical Technology Co., Ltd.

CellTiter-Glo reagent was purchased from Promega Company.

3. Experimental method

1) After PDO grows to a diameter of 200 μm in a 6-well cell culture plate, digest it with cell digestion solution at 37°C for 10-30 minutes, and pipette it several times with a burnt blunted Baker tube until the cells are single cells or State of the oligocellular mass.

2) After cell counting, adjust the concentration to 100-160 cells/μL with pre-cooled complete medium, add an equal volume of 20% Matrigel, mix completely, and add 50 μL/well to the bottom of the low-adsorption cell culture plate.

3) After incubating in a 37° C. incubator for 30 minutes, add 50 μL/well complete medium to the upper layer, and continue culturing in a 5% carbon dioxide incubator at 37° C. for 2 days.

4) Add serial dilutions of the compound to be tested to the cells on the culture plate, the final concentration of DMSO is 0.2%, and place the culture plate in a cell incubator and incubate for 5 days (37° C., 5% CO ₂ ).

5) Add 70 μL of chemiluminescent cell lysate, shake for 5 minutes, gently pipette until the cells are completely lysed, and transfer 100 μL from the cell culture plate to a white low-permeability enzyme-labeled detection plate.

6) Chemiluminescent signals were detected in a chemiluminescence microplate reader (FLUOstar Omega).

7) GraphPad Prism software was used for data analysis to obtain the IC ₅₀ of Compound A.

4. Experimental results and conclusions

From the results in Table 3, compound A has a better inhibitory effect on the proliferation of osimertinib-resistant PDO.

Table 3. Results of PDO Proliferation Inhibition Test Data

(4) In vivo pharmacodynamic study of EGFR Dellg/C797S mutation

1. Purpose of the experiment

Evaluation of compound A (the hydrochloride of compound A used in this experiment) was administered orally for 14 consecutive days, and its antitumor activity and toxic and side effects on Ba/F3 EGFR Del19/C797S were evaluated.

2. Experimental materials

NU/NU mice, female, SPF grade, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Ba/F3 EGFR Del19/C797S cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

3. Experimental steps

3.1 Cell culture

Ba/F3 EGFR Del19/C797S cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator, and cells in the exponential growth phase were collected for inoculation.

3.2 Cell inoculation

Under sterile conditions, take the Ba/F3 EGFR Del19/C797S cell suspension cultured in vitro, adjust the cell concentration to 3× ¹⁰⁷ cells/mL after centrifugation, and inoculate it subcutaneously in the right axilla of mice (0.1 mL/mouse). The day of inoculation was set as day 0.

3.3 Tumor grouping, drug administration and measurement

a, When the average tumor volume is 100-200mm ³ , 32 mice with moderate tumor volume were selected and randomly divided into 4 groups according to the tumor volume: G1: vehicle control group, G2: compound A (15mg/kg), G3 : Compound A (30 mg/kg) and G4: Compound A (65 mg/kg), 8 rats/group.

b, after the animals were grouped, the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was weighed once a day, and the drug was administered continuously for 14 days; the tumor diameter was measured twice a week.

c. Tumor volume (Tumor volume, TV): The tumor volume was measured twice a week to observe the volume change and growth rate of the tumor mass. Tumor volume V=1/2×a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The growth inhibitory effect of compounds on tumor tissue was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[1-(the average tumor volume of a certain administration group-the average tumor volume of the administration group grouping day)/(the average tumor volume of the negative control group-the average tumor volume of the negative control group grouping day)] ×100%. The data of the administration group and the negative control group were collected on the same day.

d, During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.

e. After the end point of the experiment, the mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

4. Experimental results

Table 4. Experimental data

a, mean ± standard error;

b, P value for statistical analysis of tumor volume, compared with G1 group, *P﹤0.05; **P﹤0.01.

5. Experimental conclusion

It can be seen from the above results that compound A doses of 15, 30, and 65 mg/kg can significantly inhibit tumor growth (Figure 1), and there is an obvious dose-effect relationship, and the mice are well tolerated (Figure 2).

(5) In vivo drug efficacy study of L858R mutation

1. Purpose of the experiment

The antitumor activity and side effects of compound A on NCI-H3255(L858R) were evaluated by oral administration for 22 consecutive days.

2. Experimental materials

NOD SCID mice, female, SPF grade, were purchased from Beijing Huafukang Biotechnology Co., Ltd.

NCI-H3255 (L858R) cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

3. Experimental steps

3.1 Cell Culture

NCI-H3255 tumor cells were cultured in RPMI-1640 medium containing inactivated 10% fetal bovine serum in an incubator at 37°C and 5% CO ₂ . Tumor cells in logarithmic growth phase were used for inoculation of tumors in vivo.

3.2 Cell inoculation

NCI-H3255 tumor cells resuspended in serum-free RPMI-1640 culture medium at a concentration of 1×10 ⁷ /100 μL were inoculated subcutaneously on the right flank of experimental animals, and the day of inoculation was set as day 0.

3.3 Tumor grouping, drug administration and measurement

a, When the average tumor volume was about 236mm ³ , 24 mice with moderate tumor volume were selected and randomly divided into 3 groups according to the tumor volume: G1: vehicle control group, G2: compound A (15mg/kg) and G3: Compound A (60mg/kg), 8 rats/group.

b, after the animals were grouped, the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was administered once a day by weight, and the drug was administered continuously for 22 days; the tumor diameter was measured twice a week.

c. Tumor volume (Tumor volume, TV): The tumor volume was measured twice a week to observe the volume change and growth rate of the tumor mass. Tumor volume V=1/2×a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The growth inhibitory effect of compounds on tumor tissue was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[1-(the average tumor volume of a certain administration group-the average tumor volume of the administration group grouping day)/(the average tumor volume of the negative control group-the average tumor volume of the negative control group grouping day)] ×100%. The data of the administration group and the negative control group were collected on the same day.

d, During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.

e. After the end point of the experiment, the mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

4. Experimental results

Table 5. Experimental data

a, mean ± standard error;

b, P value for statistical analysis of tumor volume, compared with G1 group, *P﹤0.05; **P﹤0.01.

5. Experimental conclusion

It can be seen from the above results that compound A doses of 15 and 60 mg/kg can significantly inhibit tumor growth ( FIG. 3 ), and there is a certain dose-effect relationship. Mice tolerated it well (Figure 4).

(6) Drug efficacy study of Del19 mutation in vivo

1. Purpose of the experiment

The antitumor activity and side effects of compound A against PC-9 (Del19) were evaluated after oral administration for 21 consecutive days.

2. Experimental materials

CB-17 SCID mice, female, SPF grade, were purchased from Beijing Huafukang Biotechnology Co., Ltd.

PC-9 (Del19) cells were purchased from European Collection of Authenticated Cell Cultures.

3. Experimental steps

3.1 Cell culture

Culture PC-9 (Del19) tumor cells in RPMI-1640 medium containing inactivated 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator at 37 °C and 5% CO2 , after the cells were congested, they were divided into flasks for passage. Tumor cells in logarithmic growth phase were used for inoculation of tumors in vivo.

3.2 Cell inoculation

PC-9 (Del19) tumor cells resuspended in serum-free RPMI-1640 culture medium at a concentration of 5×10 ⁶ /100 uL were inoculated subcutaneously on the right flank of the experimental animal, and the day of inoculation was set as day 0.

3.3 Tumor grouping, drug administration and measurement

a, When the average tumor volume was about 181 mm ³ , 20 mice with moderate tumor volume were selected and randomly divided into 4 groups according to the tumor volume: G1: vehicle control group, G2: Gefitinib (gefitinib, 100 mg/kg ), G3: Compound A (15 mg/kg) and G4: Compound A (45/60 mg/kg), 5 rats/group.

b, Animals were grouped and started to be dosed with a volume of 10 mL/kg, administered orally (po); the dose was weighed once a day for 21 consecutive days; the tumor diameter was measured twice a week.

c. Tumor volume (Tumor volume, TV): The tumor volume was measured twice a week to observe the volume change and growth rate of the tumor mass. Tumor volume V=1/2×a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The growth inhibitory effect of compounds on tumor tissue was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[1-(the average tumor volume of a certain administration group-the average tumor volume of the administration group grouping day)/(the average tumor volume of the negative control group-the average tumor volume of the negative control group grouping day)] ×100%. The data of the administration group and the negative control group were collected on the same day.

d, During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.

e. After the end point of the experiment, the mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

4. Experimental results

Table 6. Experimental data

a, mean ± standard error;

b, P value for statistical analysis of tumor volume, compared with G1 group, *P﹤0.05; **P﹤0.01.

5. Experimental conclusion

It can be seen from the above results that compound A doses of 15, 45/60 mg/kg and Gefitinib dose of 100 mg/kg can significantly inhibit tumor growth, and compound A has an obvious dose-effect relationship (Figure 5). Compound A 45/60mg/kg and Gefitinib 100mg/kg have similar curative effect and no statistical difference. Mice tolerated it well (Figure 6).

(7) Osimertinib (Osimertinib) drug-resistant human lung cancer PDX model study

1. Purpose of the experiment

To evaluate the anti-tumor activity and toxic and side effects of Compound A administered orally for 21 consecutive days against Osimertinib-resistant human lung cancer PDX model LD1-0025-200717.

2. Experimental materials

NU/NU mice, female, SPF grade, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.

LD1-0025-200717, human lung cancer tissue, 54-year-old male patient, clinical diagnosis: left upper lung primary bronchial lung cancer, adenocarcinoma; EGFR triple mutation, 19del&T790M&C797S; Osimertinib resistance; PDX pathological diagnosis: poorly-moderately differentiated adenocarcinoma cancer. Passed to FP2+5 generation for this efficacy test.

3. Experimental steps

3.1 In vivo inoculation of human lung cancer xenografts

The LD1-0025-200717 tumor tissue was evenly cut into a tumor mass of about 3mm×3mm×3mm (about 50-90mg) and inoculated subcutaneously on the right side of NU/NU mice. Post-inoculation mice were then observed and tumor growth monitored.

3.2 Tumor grouping, drug administration and measurement

a, when the average volume of the tumor reached 143.53mm ³ , they were randomly divided into 3 groups according to the tumor volume: G1: vehicle control group, G2: compound A (15 mg/kg) and G3: compound A (60 mg/kg), each group 8 only. The day of grouping is Day 0.

b, Animals were grouped into groups and administered on the same day, with a volume of 10 mL/kg, administered orally (po); administered once a day by weight, for 21 consecutive days; tumor diameter was measured twice a week.

c. Tumor volume (Tumor volume, TV): The tumor volume was measured twice a week to observe the volume change and growth rate of the tumor mass. Tumor volume V=1/2×a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The growth inhibitory effect of compounds on tumor tissue was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[1-(the average tumor volume of a certain administration group-the average tumor volume of the administration group grouping day)/(the average tumor volume of the negative control group-the average tumor volume of the negative control group grouping day)] ×100%. The data of the administration group and the negative control group were collected on the same day.

d, During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.

e. After the end point of the experiment, the mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

4. Experimental results

Table 7. Experimental data

a, mean ± standard error;

b, P value for statistical analysis of tumor volume, compared with G1 group, *P﹤0.05; **P﹤0.01.

5. Experimental conclusion

From the above results, it can be seen that in the Osimertinib-resistant human lung cancer PDX model LD1-0025-200717, compound A doses of 15 and 60 mg/kg can significantly inhibit tumor growth, and there is an obvious dose-effect relationship (Figure 7 ). Mice tolerated it well (Figure 8).

(8) In vivo drug efficacy study of L858R/C797S mutation

1. Purpose of the experiment

To evaluate the antitumor activity and side effects of compound A on Ba/F3 EGFR L858R/C797S after oral administration for 14 consecutive days.

2. Experimental materials

NU/NU mice, female, SPF grade, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Ba/F3 EGFR L858R/C797S cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.

3. Experimental steps

3.1 Cell culture

Cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator. The initial concentration of cell culture is 5×10 ⁶ , and the cells are divided into flasks for passage every 2-3 days after the cells are full. Tumor cells in logarithmic growth phase were used for inoculation of tumors in vivo.

3.2 Cell inoculation

Ba/F3 EGFR L858R/C797S cells were inoculated subcutaneously in the right axilla of NU/NU mice at 2×10 ⁶ cells/0.1 mL, and the day of inoculation was set as day 0.

3.3 Tumor grouping, drug administration and measurement

a, when the average tumor volume was about 120mm ³ , they were randomly divided into 5 groups according to the tumor volume: G1: Vehicle, G2: Osimertinib (10mg/kg), G3: Compound A (15mg/kg), G4: Compound A (30mg /kg) and G5: Compound A (65mg/kg), 7 rats/group.

b, after the animals were grouped, the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was weighed once a day, and the drug was administered continuously for 14 days; the tumor diameter was measured twice a week.

c. Tumor volume (Tumor volume, TV): The tumor volume was measured twice a week to observe the volume change and growth rate of the tumor mass. Tumor volume V=1/2×a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The growth inhibitory effect of compounds on tumor tissue was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[1-(the average tumor volume of a certain administration group-the average tumor volume of the administration group grouping day)/(the average tumor volume of the negative control group-the average tumor volume of the negative control group grouping day)] ×100%. The data of the administration group and the negative control group were collected on the same day.

d, During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.

e. After the end point of the experiment, the mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

4. Experimental results

Table 8. Experimental data

a, mean ± standard error;

b, P value for statistical analysis of tumor volume, compared with G1 group, *P﹤0.05; **P﹤0.01.

5. Experimental conclusion

It can be seen from the above results that compound A doses of 15, 30, and 65 mg/kg can significantly inhibit tumor growth, and there is an obvious dose-effect relationship, and Osimertinib has almost no inhibitory effect on the L858R/C797S double mutation model (Figure 9 ), confirming its drug resistance. Mice tolerated it well (Figure 10).

(9) In vivo drug efficacy study of L858R/T790M mutation

1. Purpose of the experiment

The antitumor activity and side effects of compound A against H1975 (L858R/T790M) were evaluated by oral administration for 21 consecutive days.

2. Experimental materials

BALB/c nude mice, female, SPF grade, Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

H1975 cells were purchased from ATCC.

3. Experimental steps

3.1 Cell culture

Add 10% fetal calf serum, 1% double antibody (penicillin/streptomycin solution) to the medium containing RPMI1640, and culture at 37° C. with 5% CO ₂ . Routine centrifugation with 0.25% trypsin twice a week for passage. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.

3.2 Cell inoculation

PBS containing 5× ¹⁰⁶ H1975 cells (final volume: 100uL) was inoculated subcutaneously in the axilla of the right forelimb of each mouse. When the average tumor volume of the enrolled animals reached ^127mm3 , group administration began.

3.3 Tumor grouping, drug administration and measurement

a, When the average tumor volume reached 127mm ³ , they were randomly divided into 4 groups according to the tumor volume: G1: vehicle control group, G2: compound A (15 mg/kg), G3: compound A (30 mg/kg) and G4: compound A (65mg/kg), 8/group. The day of grouping is Day 0.

b, after the animals were grouped, the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was weighed once a day, and the drug was administered continuously for 21 days; the diameter of the tumor was measured twice a week.

c. Tumor volume (Tumor volume, TV): The tumor volume was measured twice a week to observe the volume change and growth rate of the tumor mass. Tumor volume V=1/2×a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The growth inhibitory effect of compounds on tumor tissue was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[1-(the average tumor volume of a certain administration group-the average tumor volume of the administration group grouping day)/(the average tumor volume of the negative control group-the average tumor volume of the negative control group grouping day)] ×100%. The data of the administration group and the negative control group were collected on the same day.

d, Closely observe the living conditions of the mice during the experiment, including appearance signs, general behavioral activities, mental state, feeding situation, respiratory state, feces and urine properties, injection site and other toxic manifestations.

e. After the end point of the experiment, the mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

4. Experimental results

Table 9. Experimental data

a, mean ± standard error;

b, P value for statistical analysis of tumor volume, compared with G1 group, *P﹤0.05; **P﹤0.01.

5. Experimental conclusion

It can be seen from the above results that compound A doses of 15, 30, and 65 mg/kg can significantly inhibit tumor growth, and compound A has a certain dose-effect relationship (Figure 11). Mice tolerated it well (Figure 12).

Claims (17)

The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation,

It is characterized in that the EGFR mutation type is Del19 mutation. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation,

It is characterized in that the EGFR mutation type is L858R mutation. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation,

It is characterized in that, the EGFR mutation type is T790M mutation without C797S mutation; Preferably, the T790M mutation not accompanied by the C797S mutation is selected from one of the following L858R/T790M double mutations, Del19/G724S/T790M triple mutations, L858R/T790M/L792H triple mutations, and E709K/T790M/L858R triple mutations. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation,

It is characterized in that the EGFR mutation type is Del19/C797S double mutation. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation,

It is characterized in that the EGFR mutation type is L858R/C797S double mutation. Use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of cancer mediated by EGFR amplification,

Preferably, the EGFR amplification is EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation,

It is characterized in that, the EGFR mutation type is exon 20 insertion mutation; Preferably, the exon 20 insertion mutation is an insertion mutation accompanied by a L747_P753 mutation. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EGFR mutation or amplification,

It is characterized in that the EGFR mutation type is selected from one or any combination of the following: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/ T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, exon 20 mutation; EGFR amplification can be selected from one or any combination of the following: accompanied by Del19/T790M/C797S triple mutation, L858R/T790M EGFR amplification of /D537H triple mutation or V674L/E746_A750del/T790M triple mutation. The use according to any one of claims 1-8, wherein the cancer is lung cancer; Preferably, the cancer is non-small cell lung cancer; Preferably, the cancer is treatment-naive non-small cell lung cancer; Preferably, the cancer is drug-resistant non-small cell lung cancer that has been previously treated with an EGFR inhibitor. The use according to claim 9, wherein the EGFR inhibitor comprises a first-generation EGFR inhibitor, a second-generation EGFR inhibitor or a third-generation EGFR inhibitor; Preferably, the first-generation EGFR inhibitors include gefitinib, icotinib, and erlotinib; Preferably, the second-generation EGFR inhibitors include afatinib and dacomitinib; Preferably, the third-generation EGFR inhibitors include osimertinib, alimertinib, vomitinib, and befutinib. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer with high expression of FGFR2,

The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of C-KIT mutation cancer,

It is characterized in that the C-KIT mutation type is V560G mutation and/or D816Y mutation and/or D816H mutation and/or V559 and V560 amino acid deletion mutation and/or D816V mutation. The purposes of the compound of formula (A) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of cancer mediated by EML4-ALK fusion protein,

The use of a compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating EML4-ALK fusion protein L1196M mutation and/or F1174L mutation and/or L1196M/L1198F double mutation-mediated cancer,

Use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer mediated by SLC34A2-ROS1 fusion protein,

Use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of cancer mediated by the D2033N mutation of the SLC34A2-ROS1 fusion protein,

The use according to any one of claims 1-16, characterized in that the pharmaceutically acceptable salt of compound (A) is hydrochloride.

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