CN106032359B - Indazole compounds and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of drug synthesis, and relates to a novel indazole compound, its preparation method and application, and a pharmaceutical composition containing the compound as an active ingredient, and it is used as a medicine to prepare and treat diseases related to tyrosine kinase FGFR , especially for use in medicines for FGFR-related cancers. The chemical formula of the indazole compound of the present invention is shown in the following general formula I, and the substituents in the formula are as defined in the claims specification; the results of biological experiments show that the compounds have good inhibitory activity on fibroblast growth factor receptor (FGFR), It can be used for preparing medicines for preventing and treating diseases related to FGFR, especially for preparing medicines for treating cancers related to FGFR.

Description

Indazole compound and its preparation method and use

technical field

The invention belongs to the field of drug synthesis, and relates to a novel indazole compound, its preparation method and application, and a pharmaceutical composition containing the compound as an active ingredient, and it is used as a medicine to prepare and treat diseases related to tyrosine kinase FGFR , especially for use in medicines for FGFR-related cancers.

technical background

According to reports, cancer is currently the number one killer threatening human life and health. According to incomplete statistics, there are about 20 million new cases in the world every year; in my country, there are about 1.6-2 million new cases and 1.3 million deaths each year. Because the tumor has the ability to metastasize in the early stage, about 50% of patients with clinically diagnosed primary tumors have developed distant metastases. Studies have shown that due to the rapid growth and mutation of tumor cells, multidrug resistance occurs, leading to the failure of chemotherapy. According to relevant statistics, more than 90% of chemotherapy failures are related to the multidrug resistance of tumor cells. Therefore, the current clinical antitumor drugs are far from meeting the treatment requirements.

In recent years, with the development of molecular oncology, molecular pharmacology and other disciplines, the nature of tumors is gradually being clarified, and people gradually realize that the essence of cell cancer is the unlimited proliferation of cells caused by the disorder of cell signal transduction pathways. Studies have shown that, as the most important member involved in cell signal transduction, protein tyrosine kinases (PTKs, referred to as tyrosine kinases) are the most common growth factor receptors, which are closely related to the occurrence and development of tumors; The activity of tyrosine kinase is too high, leading to the activation of its downstream signaling pathway, which leads to cell transformation, proliferation, anti-apoptosis, promotion of cell survival, and finally leads to the formation of tumors; Traditional cytotoxic drugs have turned to drugs targeting abnormal signal transduction in cells, and related drugs have been used clinically one after another. Compared with traditional cytotoxic antineoplastic drugs, this type of molecularly targeted drugs has strong curative effect and less toxic and side effects, and has gradually become a hot spot in the research and development of antineoplastic drugs.

Studies have shown that receptor tyrosine kinases play a key role in various aspects of tumor development, invasion and metastasis, and drug resistance due to their abnormal expression activation or gene mutation, and have become important targets for anti-tumor drug development. Fibroblast growth factor receptors (FGFRs) are important members of the receptor tyrosine kinase family, mainly including four subtypes, FGFR1, FGFR2, FGFR3 and FGFR4. (Fibroblast Growth Factors, FGFs). (Turner N., Grose R., Fibroblast growth factor signaling: from development to cancer, Nature Reviews Cancer. (2010) 10:116-129. Dieci M.V., Arnedos M., Andre F., Soria J.C., Fibroblast Growth Factor Receptor Inhibitors as a Cancer Treatment: From a Biologic Rationale to Medical Perspectives, Cancer Discovery. (2013) 3:264-279.) Due to gene amplification, mutation, fusion or ligand induction and other reasons, FGFR members are continuously activated to induce tumor cell proliferation, invasion, Migration, promote angiogenesis, and promote tumor development; FGFRs are highly expressed and abnormally activated in a variety of tumors, such as non-small cell lung cancer, breast cancer, gastric cancer, bladder cancer, endometrial cancer, prostate cancer, cervical cancer, colon cancer , esophageal cancer, keratinoblastoma, myeloma, rhabdomyosarcoma, etc. ; TurnerN, GroseR: Fibroblast growth factor signaling: from development to cancer. Nat. Rev. Cancer, 2010, 10, 116-29.), studies have shown that FGFR1 amplification accounts for 20% of non-small cell lung squamous cell carcinoma, and through FGFR1 Studies on the proliferation and signaling pathways of amplified lung cancer cell lines in vitro have shown that FGFR selective inhibitors can very effectively inhibit the activation of FGFR1 signaling pathways and cell proliferation (Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1 Dependency in Squamous Cell Lung Cancer (vol 3,66er5,2011 ), Sci TranslMed.(2010).I nhibitor-Sensitive FGFR1Amplification in HumanNon-Small CellLungCancer, PLoSONE.(2011)6:e20351.); In breast cancer, the amplification of the chromosome (8p11–12) region where FGFR1 is located accounts for about 10% of ER-positive patients, and its It is associated with high expression of FGFR1 mRNA and poor prognosis of patients (Dieci MV, Arnedos M, Andre F, Soria JC: Fibroblast growth factor receptor inhibitors as a cancer treatment: from abiologic rationale tomedical perspectives. Cancer discovery, 2013, 3, 264-79); FGFR2 gene amplification The abnormal activation of FGFR2 signaling pathway caused by amplification or mutation is mainly related to gastric cancer, triple-negative breast cancer, endometrial cancer, etc.; the amplification rate of FGFR2 in gastric cancer tissue is 5%-10% (Matsumoto K, Arao T, Hamaguchi T, Shimada Y, Kato K, OdaI, Taniguchi H, Koizumi F, Yanagihara K, Sasaki H, Nishio K, Yamada Y: FGFR2 gene amplification and clinicopathological features in gastric cancer. Br.J.Cancer, 2012, 106, 727-32.), there are studies on Analysis of 313 gastric cancer tissues showed that the amplification of FGFR2 was significantly correlated with tumor size, local invasion, lymph node metastasis and distant metastasis, and gastric cancer with FGFR2 amplification was generally a progressive tumor with a poor prognosis. The overall survival rate of patients is relatively low (JungEJ, JungEJ, Min SY, Kim MA, Kim WH: Fibroblast growth factor receptor 2gene amplification status and its clinicopathologic significance in gastric carcinoma. Humanpathology, 2012, 43, 1559-66.); FGFR2 amplification It accounts for 4% of refractory triple-negative breast cancer; endometrial cancer is a common gynecological reproductive tract tumor, and the mutation of FGFR2 accounts for about 12% of endometrial cancer; FGFR3 mutations account for 50%-60% of bladder cancer and 10%-15% of invasive bladder cancer; FGFR3t(4;14)(p16.3;q32) gene rearrangement in multiple myeloma It accounts for 15–20% (Dieci MV, Arnedos M, Andre F, Soria JC: Fibroblast growth factor receptor inhibitors as a cancer treatment: from abiologicrationale to medical perspectives. Cancer discovery, 2013, 3, 264-279). FGFR and its ligand FGFs have abnormal expression and activation, such as FGFR2, FGFR3, FGFR4, FGF19, FGF2, FGF5, FGF8, FGF9, etc. Many preclinical and clinical studies have shown that abnormal activation of the FGF/FGFR axis is important in liver cancer The importance in (ChengAL, Shen YC, Zhu AX: Targeting Fibroblast Growth Factor Receptor Signaling in Hepatocellular Carcinoma. Oncology-Basel, 2011, 81, 372-80.); It cannot be ignored that abnormal activation of the FGF/FGFR axis is associated with EGFR inhibitors , neovascularization inhibitors and endocrine therapy are closely related to drug resistance (Nicholas T, Richard G: Fibroblast growth factor signaling from development to cancer. Nature Reviews, 2010, 10, 116-129.). Therefore, the research and development of targeted FGFR inhibitors has become the frontier focus of anti-tumor drug research.

Based on the current status of research and development of molecularly targeted anti-tumor drugs, the inventors of the present application intend to provide new indazole compounds and their application in the preparation of anti-tumor drugs, especially in the preparation of drugs for the treatment of diseases related to tyrosine kinase FGFR Applications.

Contents of the invention

The object of the present invention is to provide a new indazole compound and its application in the preparation of anti-tumor drugs, especially in the preparation of drugs for treating diseases related to tyrosine kinase FGFR.

The present invention provides an indazole compound having a general formula I structure or a pharmaceutically acceptable salt thereof, and specifically relates to an indazole compound containing piperidine or piperazine, which can effectively inhibit the activity of FGFR, So as to play the role of preventing and treating tumors.

The second object of the present invention is to provide a preparation method of the compound represented by general formula I.

The third object of the present invention is to provide a pharmaceutical composition comprising the compound represented by general formula I or a pharmaceutically acceptable salt thereof.

The present invention provides a compound represented by the following general formula I or a pharmaceutically acceptable salt thereof:

In the formula, ring A represents a five-membered or six-membered aromatic ring, and a heteroaromatic ring containing nitrogen, oxygen, and sulfur atoms;

Ring B represents a five-membered or six-membered aromatic ring, and a heteroaromatic ring containing nitrogen, oxygen, and sulfur atoms;

R ₁ represents hydrogen, halogen, trifluoromethyl, methoxy, ethoxy, isopropoxy, isobutoxy, cyano, C1-C20 straight chain or branched hydrocarbon group;

R ₂ represents hydrogen,

The compound of the present invention represented by the following general formula I or a pharmaceutically acceptable salt thereof, wherein said Groups selected from the following structural formulas:

The compound of the present invention represented by the following general formula I or a pharmaceutically acceptable salt thereof, wherein said Groups selected from the following structural formulas:

The pharmaceutically acceptable salt of the compound of general formula I provided by the present invention comprises: hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, Lactic acid, malic acid, tartaric acid, citric acid, etc., or acidic amino acids such as aspartic acid and glutamic acid form esters and then form salts with inorganic bases, such as sodium, potassium, calcium, aluminum salts and ammonium salts; or with organic Salts formed by bases, such as methylamine salts, ethylamine salts, ethanolamine salts, etc.; or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. after forming esters with basic amino acids such as lysine, arginine, ornithine, etc. Acid salts, or salts with organic acids such as formic acid, acetic acid, picric acid, methanesulfonic acid, and ethanesulfonic acid.

The preparation method of the compound of general formula I of the present invention is according to the general formula shown below,

(1) Fragment C is prepared by stepwise condensation or fragment condensation;

(2) Fragments C and D are condensed to obtain intermediate F;

The condensation method used can be acid chloride method, acid fluoride method, mixed acid anhydride method or use the following condensing agents: N,N-dicyclohexylimine, 1-[3-dimethylamino]-propyl-3-ethylcarbodi imide hydrochloride or 7-azabenzoxazol-1-yl-oxy(tris-(dimethylamino)phosphine)hexafluorophosphate;

(3) Fragment F removes the tert-butyl ester protecting group. The reagents used are concentrated hydrochloric acid, ethyl hydrogen chloride acetate solution, hydrogen chloride dioxane solution, hydrogen bromide ethyl acetate solution, hydrogen bromide dioxane solution, bromide hydroacetic acid solution, hydrogen fluoride, trifluoroacetic acid or combinations thereof;

(4) Fragment F and E coupling reaction of removing tert-butyl ester protecting group obtains the compound shown in general formula I;

The coupling method used was suzuki coupling, or the following palladium catalysts were used: tetrakis(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, palladium acid palladium.

Among the present invention, preferred compound has following structure:

Taking compound 1-1 as an example, the preparation process of the compound of the present invention is as follows:

The present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the compound of the above general formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers; The composition may further contain odorant, flavoring agent, excipient or diluent, etc.

The present invention further provides the use of the compound represented by general formula I or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing and treating tumors.

The present invention has carried out the in vitro FGFR1 inhibitory activity test, and the results show that the compounds of the present invention show better FGFR1 inhibitory activity, wherein compounds 1-8, 1-13, 1-15, 1-16, and 1-17 have an inhibitory effect on FGFR1 Inhibitory activity IC ₅₀ value is less than 10nM;

The compound of the present invention has been tested for the inhibition of human gastric cancer cell SNU16 proliferation in vitro, and the results show that the compound has a better inhibitory effect on the proliferation of human gastric cancer cell SNU16.

In the present invention, the pharmacodynamic test method used is a method well known to those skilled in the art;

In the present invention, the FGFR1 kinase used is obtained by expressing and purifying the recombinant protein of the protein kinase region by the insect baculovirus expression system in our laboratory.

The compound of the present invention can be further developed to develop novel FGFR inhibitors and antitumor drugs.

The novel indazole compound containing piperazine or piperidine of the present invention can especially prepare FGFR inhibitors and antitumor drugs. In view of abnormal activation of FGFR signal transduction, it will lead to the occurrence of various tumors such as breast cancer, ovarian cancer, bladder cancer, melanoma, gastric cancer, endometrial cancer, etc., therefore, the malignant tumors described in the present invention include abnormal activation of FGFR signal pathway Related tumors, including breast cancer, ovarian cancer, bladder cancer, melanoma, gastric cancer, endometrial cancer and other tumors.

Detailed ways

Example 1: Synthetic compound 1-1,4-(4-ethylpiperazin-1-yl)-N-(6-phenyl-1H-indazol-3-yl)benzamide

Step 1. Synthesize 6-bromo-1H-indazol-3-amine according to the following reaction formula:

Under nitrogen protection, 4-bromo-2-fluoro-benzonitrile (5 g, 25.13 mmol) was dissolved in 20 mL of n-butanol, hydrazine hydrate (1.04 mL, 50.26 mmol) was added, and heated to reflux for 4 h. After cooling to room temperature, a large amount of solid precipitated out. After suction filtration and washing the filter cake with petroleum ether, 4.759 g of white solid was obtained after drying, with a yield of 85.7%. ¹ HNMR (400MHz, DMSO-d ₆ )δ11.52(brs,1H),7.64(d,1H,J=8.5Hz),7.37-7.52(m,1H),7.02(dd,1H,J=8.5, 1.6Hz), 5.47(brs,2H).ESI-MS(m/z): [M+H] ⁺ =213.0(calcd 212.05).

Step 2. Synthesize 1-tert-butoxycarbonyl-3-amino-6-bromo-1H-indazole according to the following reaction formula:

Under nitrogen protection, 6-bromo-1H-indazol-3-amine (4.759g, 22.43mmol) was dissolved in 20mL of tetrahydrofuran, and di-tert-butyl dicarbonate (5.385g, 24.67mmol) was added under ice-bath conditions, 4- Dimethylaminopyridine (1.0 g, 8.19 mmol). After reacting at room temperature for 3 h, TLC (petroleum ether/ethyl acetate=1/1) showed that the starting material disappeared, the reaction mixture was concentrated under reduced pressure, 1 mol/L hydrochloric acid was added to the residue to make it acidic, and extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (petroleum ether/ethyl acetate=2/1) to obtain 6.964 g of a yellow solid, with a yield of 88.8%. ¹ HNMR (400MHz, DMSO-d6) δ8.12(s, 1H), 7.80(d, J＝8.4Hz, 1H), 7.45(dd, J＝8.4, 1.7Hz, 1H), 6.44(s, 2H) ,1.57(s,9H).ESI-MS(m/z):[M+H] ⁺ ＝313.0(Calcd:312.16).

Step 3. Synthesize methyl 4-(4-ethylpiperazin-1-yl) methyl benzoate according to the following reaction formula:

Under nitrogen protection, methyl 4-fluorobenzoate (3.0mL, 20.69mmol) was dissolved in 20mL DMSO, and K ₂ CO ₃ (5.718g, 41.38mmol), N-ethylpiperazine (1.93mL, 24.83mmol) were added successively ). React overnight at 120°C. After the reaction is cooled to room temperature, add 50 mL each of ethyl acetate and saturated brine and shake to separate the layers. The aqueous layer is extracted with ethyl acetate (50 mL×3). The organic layers are combined and washed with saturated sodium chloride. The solution was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography (dichloromethane/methanol=10/1) to obtain 4.405 g of a white solid, with a yield of 85.8%. ESI-MS (m/z): [M+H] ⁺ = 249.2 (Calcd: 248.15).

Step 4. Synthesize methyl 4-(4-ethylpiperazin-1-yl) benzoic acid according to the following reaction formula:

Under nitrogen protection, methyl 4-(4-ethylpiperazin-1-yl)benzoate (4.405 g, 17.75 mmol) was dissolved in 15 mL of methanol, 15 mL of water, and NaOH solid (1.420 g, 35.5 mmol) was added. After heating to reflux for 2 hours, the reaction was cooled to room temperature, the reaction mixture was concentrated under reduced pressure, 1 mol/L hydrochloric acid was added to the residue to make it acidic, and ethyl acetate was extracted. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (dichloromethane/methanol=10/1) to obtain 3.009 g of a yellow solid, with a yield of 72.4%. ESI-MS (m/z): [M+H] ^- = 233.5 (Calcd: 234.14).

Step 5. Synthesize 1-tert-butoxycarbonyl-6-bromo-3-(4-ethylpiperazin-1-yl)-benzamide)-1H-indazolyl according to the following reaction formula:

Under nitrogen protection, methyl 4-(4-ethylpiperazin-1-yl)benzoic acid (3.009g, 12.85mmol) was dissolved in 15mL of dichloromethane, and oxalyl chloride (2.2mL, 25.70mmol ). After the reaction was refluxed for 2 h, the reaction mixture was concentrated under reduced pressure, added with 20 mL of dichloromethane and concentrated twice. The residue was added into a pyridine solution (20 mL) of 1-tert-butoxycarbonyl-3-amino-6-bromo-1H-indazole (4.011 g, 12.85 mmol) under ice-cooling conditions, and reacted overnight at room temperature. TLC (dichloromethane/methanol=5/1) showed that the starting material disappeared, the reaction mixture was concentrated under reduced pressure, 1 mol/L hydrochloric acid was added to the residue to make it acidic, and extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (dichloromethane/methanol=2/1) to obtain 3.136 g of a yellow solid, with a yield of 46.3%. ¹ HNMR (400MHz, DMSO-d6): δ12.86(brs, 1H), 10.56(brs, 1H), 7.94(d, J=8.7Hz, 2H), 7.61-7.70(m, 1H), 7.17(d ,J=9.9Hz,1H),6.99(d,J=9.0Hz,2H),3.29(t,J=7.4Hz,4H),2.51-2.56(m,4H),2.36(q,J=7.1Hz , 2H), 1.52(s, 9H), 1.02(t, J=7.2Hz, 3H).ESI-MS(m/z): [M+H] ⁺ =528.2(Calcd: 527.10).

Step 6. Synthesize 1-tert-butoxycarbonyl-3-(4-(4-ethylpiperazin-1-yl)-benzamide)-6-phenyl-1H-indazolyl according to the following reaction formula:

Under nitrogen protection, 1-tert-butoxycarbonyl-6-bromo-3-(4-ethylpiperazin-1-yl)-benzamide)-1H-indazolyl (100mg, 0.190mmol) was dissolved in 2mL di To the oxane, phenylboronic acid (46.3 mg, 0.380 mmol), cesium carbonate (185.7 mg, 0.570 mmol), and Pd(dppf)Cl ₂ (20 mg) were sequentially added. After reacting at 120°C for 2 hours, the reaction was lowered to room temperature, the reaction mixture was concentrated under reduced pressure, and 50ml of ethyl acetate and saturated brine were added to extract and separate layers. The aqueous layer was extracted with 50mL of ethyl acetate, and the organic layers were combined and washed with saturated sodium chloride. , dried over anhydrous sodium sulfate, evaporated the solvent under reduced pressure, and the residue was subjected to silica gel column chromatography (dichloromethane/methanol=10/1) to obtain 47.4 mg of white solid, yield 47.5%. ESI-MS (m/z): [M+H] ⁺ = 526.5 (Calcd: 526.22).

Step 7. Synthesize 4-(4-ethylpiperazin-1-yl)-N-(6-phenyl-1H-indazol-3-yl)benzamide according to the following reaction formula:

Under nitrogen protection, 1-tert-butoxycarbonyl-3-(4-(4-ethylpiperazin-1-yl)-benzamide)-6-phenyl-1H-indazolyl (47.4mg, 0.090mmol ) was dissolved in 2 mL of dichloromethane, and added to 1 mL of trifluoroacetic acid in an ice bath. After reacting in ice bath for 2 h, the reaction mixture was concentrated under reduced pressure, and 10 mL of dichloromethane was added and concentrated twice. Add 1mol/L sodium hydroxide aqueous solution to adjust the pH to alkaline, and extract with ethyl acetate, the organic layer is washed with saturated sodium chloride, dried over anhydrous sodium sulfate, the solvent is evaporated under reduced pressure, and the residue is subjected to silica gel column chromatography (dichloro Methane/methanol=10/1) to obtain 36.5 mg of white solid, yield 95.4%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.81(brs,1H),10.54(brs,1H),8.00(d,J=8.9Hz,2H),7.80(d,J=8.5Hz,1H), 7.75(d, J=7.3Hz, 2H), 7.66(s, 1H), 7.51(t, J=7.6Hz, 2H), 7.35-7.44(m, 2H), 7.03(d, J=8.9Hz, 2H ), 3.37(s, 4H), 2.49-2.59(m, 4H), 2.41(s, 2H), 1.06(t, J=7.1Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 426.6 (Calcd: 426.22)..

Example 2: Synthetic compound 1-2,4-(4-ethylpiperazin-1-yl)-N-(6-pyridyl-1H-indazol-3-yl)benzamide

Step 1. Synthesize N-(6-bromo-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl)benzamide according to the following reaction formula:

Under nitrogen protection, 1-tert-butoxycarbonyl-6-bromo-3-(4-ethylpiperazin-1-yl)-benzamide)-1H-indazolyl (300mg, 0.569mmol) was dissolved in 15mL di Chloromethane was added to 5 mL of trifluoroacetic acid in an ice bath. After reacting in ice bath for 2 h, the reaction mixture was concentrated under reduced pressure, and 10 mL of dichloromethane was added and concentrated twice. Add 1mol/L sodium hydroxide aqueous solution to adjust the pH to alkaline, and extract with ethyl acetate, the organic layer is washed with saturated sodium chloride, dried over anhydrous sodium sulfate, the solvent is evaporated under reduced pressure, and the residue is subjected to silica gel column chromatography (dichloro Methane/methanol=10/1) to obtain 220.9 mg of white solid, yield 90.9%. ¹ HNMR (400MHz, DMSO-d6): δ12.86(brs, 1H), 10.56(brs, 1H), 7.94(d, J=8.7Hz, 2H), 7.61-7.70(m, 1H), 7.17(d ,J=9.9Hz,1H),6.99(d,J=9.0Hz,2H),3.29(t,J=7.4Hz,4H),2.51-2.56(m,4H),2.36(q,J=7.1Hz , 2H), 1.02 (t, J=7.2Hz, 3H).ESI-MS (m/z): [M+H] ⁺ = 428.2 (Calcd: 427.10).

Step 2. Synthesize 4-(4-ethylpiperazin-1-yl)-N-(6-pyridyl-1H-indazol-3-yl)benzamide according to the following reaction formula:

Under nitrogen protection, N-(6-bromo-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl)benzamide (100 mg, 0.234 mmol) was dissolved in 2 mL of dioxane Into the ring, 4-pyridineboronic acid (57.5 mg, 0.468 mmol), cesium carbonate (228.7 mg, 0.792 mmol), and Pd(dppf)Cl ₂ (20 mg) were added sequentially. After reacting at 120°C for 2 hours, the reaction was lowered to room temperature, the reaction mixture was concentrated under reduced pressure, and 50ml of ethyl acetate and saturated brine were added to extract and separate layers. The aqueous layer was extracted with 50mL of ethyl acetate, and the organic layers were combined and washed with saturated sodium chloride. , dried over anhydrous sodium sulfate, evaporated the solvent under reduced pressure, and the residue was subjected to silica gel column chromatography (dichloromethane/methanol=10/1) to obtain 17.7 mg of white solid, yield 17.8%. ¹ H NMR (400MHz,DMSO-d ₆ )δ12.85(brs,1H),10.56(brs,1H),8.00(d,J=8.8Hz,2H),7.77(d,J=8.4Hz,1H) ,7.58-7.63(m,1H),7.41-7.54(m,4H),7.08-7.14(m,1H),7.04(d,J＝8.9Hz,2H),3.34(s,4H),2.51(s , 4H), 2.41(s, 2H), 1.06(t, J=7.1Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 427.4 (Calcd: 426.22)..

Example 3: Synthetic compound 1-3,4-(4-ethylpiperazin-1-yl)-N-(6-(1-methyl-1H-pyrazol-3-yl)-1H-indazole -3-yl)benzoyl

The synthesis method was similar to the preparation method of compound 1-2, and a total of 21.9 mg of white solid was obtained with a yield of 21.8%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.67(brs,1H),10.46(brs,1H),8.23(s,1H),7.98(d,J＝8.8Hz,2H),7.94(s,1H ),7.68(d,J=8.5Hz,1H),7.57(s,1H),7.30(d,J=8.5Hz,1H),7.02(d,J=8.8Hz,2H),3.89(s,3H ), 3.34(s, 4H), 2.51(s, 4H), 2.41(d, J=6.7Hz, 2H), 1.06(t, J=7.2Hz, 3H).ESI-MS(m/z):[ M+H] ⁺ = 430.4 (Calcd: 429.23).

Example 4: Synthetic compound 1-4,4-(4-ethylpiperazin-1-yl)-N-(6-(3-methoxyphenyl)-1H-indazol-3-yl)benzene Formamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 31.9 mg of white solid was obtained with a yield of 29.9%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.79(brs,1H),10.53(brs,1H),7.99(d,J=8.9Hz,2H),7.77(d,J=8.5Hz,1H), 7.67(s,1H),7.37(d,J=8.6Hz,1H),7.03(d,J=9.0Hz,2H),6.85(d,J=2.2Hz,2H),6.54(t,J=2.2 Hz, 1H), 3.84(s, 6H), 3.29-3.35(m, 4H), 2.50-2.52(m, 4H), 2.39(q, J=7.1Hz, 2H), 1.05(t, J=7.2Hz ,3H). ESI-MS (m/z): [M+H] ⁺ 456.0 (Calcd: 455.23).

Example 5: Synthesis of compound 1-5, N-(6-(4-cyanophenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl)benzyl Amide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 39.0 mg of white solid was obtained with a yield of 37.0%. ¹ HNMR (400MHz,DMSO-d ₆ )δ12.94(brs,1H),10.52(brs,1H),7.93-8.08(m,6H),7.85(d,J＝8.5Hz,1H),7.78(s ,1H),7.44(d,J=8.6Hz,1H),7.03(d,J=8.6Hz,2H),3.34(s,4H),2.51(s,4H),2.40(q,J=6.7Hz , 2H), 1.06 (t, J=7.1Hz, 3H). ESI-MS (m/z): [M+H] ⁺ 451.1 (Calcd: 450.22).

Example 6: Synthesis of compound 1-6, 4-(4-ethylpiperazin-1-yl)-N-(6-(4-trifluoromethylphenyl)-1H-indazol-3-yl) benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 45.2 mg of white solid was obtained with a yield of 39.2%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.92(brs,,1H),10.57(brs,1H),7.95-8.05(m,4H),7.85(d,J=8.4Hz,3H),7.77( s,1H),7.44(d,J=8.6Hz,1H),7.03(d,J=8.8Hz,2H),3.34(s,4H),2.51(s,4H),2.41(q,J=7.2 Hz, 2H), 1.06 (t, J=7.1Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 494.2 (Calcd: 493.21).

Example 7: Synthesis of compound 1-7, N-(6-(3-ethoxyphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl)- benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 45.2 mg of white solid was obtained with a yield of 39.2%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.80(brs,1H),10.54(brs,1H),8.00(d,J=8.9Hz,2H),7.78(d,J=8.5Hz,1H), 7.66(s,1H),7.35-7.43(m,2H),7.29(d,J=7.8Hz,1H),7.24(s,1H),7.03(d,J=8.9Hz,2H),6.96(dd ,J=8.1,1.9Hz,1H),4.13(q,J=6.9Hz,2H),3.33(s,4H),2.46-2.59(m,4H),2.41(s,2H),1.37(t, J=8.0Hz, 3H), 1.06(t, J=7.1Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 470.2 (Calcd: 469.25).

Example 8: Synthesis of Compound 1-8, N-(6-(3-isopropoxyphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl) -Benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 50.9 mg of white solid was obtained with a yield of 45.0%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.79(brs,1H),10.54(brs,1H),8.00(d,J=8.8Hz,2H),7.78(d,J=8.5Hz,1H), 7.65(s,1H),7.35-7.41(m,2H),7.27(d,J=7.9Hz,1H),7.22(s,1H),7.03(d,J=8.9Hz,2H),6.95(dd ,J=8.2,1.9Hz,1H),4.72-4.78(m,1H),3.34(s,4H),2.51(s,4H),2.40(s,2H),1.32(d,J=6.0Hz, 6H), 1.06 (t, J = 7.1 Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 484.2 (Calcd: 483.26).

Example 9: Synthesis of Compound 1-9, N-(6-(3-isobutoxyphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl) -Benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 29.4 mg of solid was obtained, with a yield of 25.3%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.79(brs,1H),10.54(brs,1H),8.00(d,J=8.9Hz,2H),7.78(d,J=8.5Hz,1H), 7.67(s,1H),7.37-7.40(m,2H),7.29(d,J=7.9Hz,1H),7.23-7.26(m,1H),7.03(d,J=9.0Hz,2H),6.96 (dd,J=8.1,1.8Hz,1H),3.85(d,J=6.5Hz,2H),3.34(s,4H),2.51(s,4H),2.40(s,2H),2.03-2.09( m, 1H), 1.06 (t, J = 7.2Hz, 3H), 1.02 (d, J = 6.7Hz, 6H). ESI-MS (m/z): [M+H] ⁺ = 498.3 (Calcd: 497.28).

Example 10: Synthesis of compound 1-10, N-(6-(thiophen-2-yl)-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl)-benzyl Amide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 36.6 mg of solid was obtained with a yield of 30.3%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.78(brs,1H),10.54(brs,1H),7.99(d,J=8.8Hz,2H),7.75(d,J=8.5Hz,1H), 7.67(s,1H),7.60(dd,J=8.6,4.3Hz,2H),7.41(d,J=8.5Hz,1H),7.18(dd,J=4.9,3.7Hz,1H),7.03(d , J=8.9Hz, 2H), 3.34(s, 4H), 2.51(s, 4H), 2.41(d, J=8.0Hz, 2H), 1.06(t, J=7.2Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 432.3 (Calcd: 431.18).

Example 11: Synthesis of compound 1-11, N-(6-(2,6-dimethylphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazin-1-yl )-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 20.4 mg of solid was obtained with a yield of 19.2%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.75(brs,1H),10.56(brs,1H),8.02(d,J=8.7Hz,2H),7.77(d,J=8.3Hz,1H), 7.18(d, J=8.4Hz, 2H), 7.14(d, J=8.2Hz, 2H), 7.07(d, J=8.6Hz, 2H), 6.82(d, J=8.3Hz, 1H), 3.37- 3.43 (m, 4H), 2.54-2.68 (m, 4H), 2.35 (q, J = 7.2Hz, 2H), 2.01 (s, 6H), 1.14 (t, J = 7.0Hz, 3H). ESI-MS (m/z): [M+H] ⁺ = 454.3 (Calcd: 453.25).

Example 12: Synthesis of compound 1-12, N-(6-(2-chloro-4-trifluoromethylphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazine- 1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 38.4 mg of solid was obtained with a yield of 21.1%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.94(brs,1H),10.59(brs,1H),7.99-8.03(m,3H),7.82(t,J=9.1Hz,2H),7.75(d ,J=8.0Hz,1H),7.55(s,1H),7.15(d,J=8.4Hz,1H),7.04(d,J=8.8Hz,2H),3.35(s,4H),2.58(s ,4H), 2.45(s,2H), 1.07(t,J=7.0Hz,4H). ESI-MS (m/z): [M+H] ⁺ = 528.1 (Calcd: 527.17)..

Example 13: Synthesis of compound 1-13, N-(6-(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazine-1 -yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 44.1 mg of solid was obtained with a yield of 36.3%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.86(brs,1H),10.59(brs,1H),8.00(d,J=8.9Hz,2H),7.80(d,J=8.5Hz,1H), 7.72(s,1H),7.40(dd,J=8.6,1.3Hz,1H),7.12-7.21(m,2H),7.03(d,J=9.0Hz,2H),6.87(d,J=10.9Hz ,1H), 3.87(s,3H), 3.32(s,4H), 2.51(s,4H), 2.39(q,J=7.1Hz,2H), 1.05(t,J=7.2Hz,3H). ESI-MS (m/z): [M+H] ⁺ = 473.8 (Calcd: 473.22)..

Example 14: Synthesis of compound 1-14, N-(6-(3-methoxyphenyl)-1H-indazol-3-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 71.4 mg of yellow solid was obtained with a yield of 65.0%. ¹ H NMR (400MHz, DMSO-d6) δ12.88 (brs, 1H), 10.86 (brs, 1H), 8.11 (d, J = 7.2Hz, 1H), 7.82 (d, J = 8.5Hz, 1H), 7.70 (s,1H),7.62(d,J=7.3Hz,1H),7.54-7.56(m,1H),7.40-7.42(m,1H),7.31(d,J=7.9Hz,1H),7.25- 7.29 (m, 1H), 6.98 (dd, J=8.1, 1.9Hz, 1H), 3.86 (s, 3H). ESI-MS (m/z): [M+H] ⁺ = 344.8 (Calcd: 343.13)..

Example 15: Synthesis of compound 1-15, N-(6-(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-(4-methylpiperazine-1 -yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 54.3 mg of solid was obtained with a yield of 16.9%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.86(brs,1H),10.56(brs,1H),7.99(d,J=8.8Hz,2H),7.79(d,J=8.5Hz,1H), 7.72(s,1H),7.40(d,J=8.6Hz,1H),7.12-7.20(m,2H),7.03(d,J=9.0Hz,2H),6.82-6.91(m,1H),3.87 (s,3H), 3.37(s,4H), 2.49(s,4H), 2.26(s,3H). ESI-MS (m/z): [M+H] ⁺ = 460.8 (Calcd: 459.21)..

Example 16: Synthesis of compound 1-16, N-(6-(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-(4-(dimethylamino)piper Pyridin-1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 74.3 mg of solid was obtained with a yield of 16.0%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.87(brs,1H),10.54(brs,1H),7.99(d,J=8.7Hz,2H),7.79(d,J=8.5Hz,1H), 7.72(s,1H),7.40(d,J=8.8Hz,1H),7.11-7.22(m,2H),7.04(d,J=8.8Hz,2H),6.87(d,J=10.9Hz,1H ), 3.98(d, J=12.2Hz, 2H), 3.87(s, 3H), 2.84(t, J=12.0Hz, 2H), 2.59(s, 1H), 2.35(s, 6H), 1.92(s , 2H), 1.51 (d, J=10.8Hz, 2H). ESI-MS (m/z): [M+H] ⁺ = 488.3 (Calcd: 487.24)..

Example 17: Synthesis of compound 1-17, N-(6-(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-(4-(3S,5R)- 3,5-Dimethyl-1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 66.5 mg of solid was obtained with a yield of 26.0%. HNMR(400MHz,DMSO-d ₆ )δ12.87(brs,1H),10.56(brs,1H),7.99(d,J=8.8Hz,2H),7.79(d,J=8.6Hz,1H),7.72 (s,1H),7.40(d,J=8.5Hz,1H),7.14-7.18(m,2H),7.04(d,J=9.0Hz,2H),6.87(d,J=10.9Hz,1H) ,4.47(brs,1H),3.87(s,3H),3.82(s,2H),2.96(s,2H),2.32-2.38(m,2H),1.11(s,3H),1.08(s,3H ). ESI-MS (m/z): [M+H] ⁺ = 473.8 (Calcd: 473.22)..

Example 18: Synthesis of compound 1-18, N-(6-(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-5-(4-methyl-piperazine- 1-yl)pyrazine-2-carboxamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 37.1 mg of solid was obtained with a yield of 37.4%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.95(brs,1H),10.42(brs,1H),8.80(s,1H),8.44(s,1H),7.94(d,J＝8.4Hz,1H ),7.74(s,1H),7.42(d,J=8.3Hz,1H),7.05-7.25(m,2H),6.87(d,J=10.9Hz,1H),3.87(s,7H),2.70 (s,4H),2.42(s,3H). ESI-MS (m/z): [M+H] ⁺ = 462.0 (Calcd: 461.20)..

Example 19: Synthesis of compound 1-19, N-(6-(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-(4-methyl-1,4 -homopiperazin-1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 30.1 mg of solid was obtained with a yield of 33.4%. ¹ HNMR (400MHz, DMSO-d ₆ )δ12.83(brs,1H),10.44(brs,1H),7.97(d,J=8.4Hz,2H),7.79(d,J=8.0Hz,1H), 7.71(s,1H),7.39(d,J=8.1Hz,1H),7.14-7.18(m,2H),6.85-6.89(m,1H),6.80(d,J=7.9Hz,2H),3.87 (s,7H),3.63-3.65(m,2H),3.53(t,J＝6.8Hz,2H),2.70-2.78(m,2H),2.55-2.62(m,2H),2.48-2.51(m ,4H), 2.36(s,3H). ESI-MS (m/z): [M+H] ⁺ = 474.0 (Calcd: 473.22)..

Example 20: Synthesis of compound 1-20, N-(6-(2-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazine-1 -yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 28.3 mg of solid was obtained with a yield of 30.8%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.96 (brs, 1H), 10.59 (brs, 1H), 8.01 (d, J = 8.5Hz, 2H), 7.79 (d, J = 8.5Hz, 1H), 7.63(s,1H),7.24-7.30(m,2H),7.09-7.11(m,1H),7.05(d,J＝8.0Hz,2H),6.97-7.00(m,1H),3.82(s, 3H),3.39-3.50(m,4H),2.68-2.80(m,4H),2.51-2.62(m,2H),1.11(t,J=7.2Hz,3H).ESI-MS(m/z) : [M+H] ⁺ = 474.0 (Calcd: 473.22).

Example 21: Synthesis of compound 1-21, N-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-4-(4-ethylpiperazine-1 -yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 29.0 mg of solid was obtained with a yield of 31.0%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.85 (brs, 1H), 10.57 (brs, 1H), 8.00 (d, J = 8.9Hz, 2H), 7.79 (d, J = 8.5Hz, 1H), 7.58(s,1H),7.19-7.28(m,3H),7.12-7.15(m,1H),7.03(d,J＝9.0Hz,2H),3.90(s,3H),3.25-3.41(m, 4H), 2.54-2.66(m, 4H), 2.41-2.48(m, 2H), 1.08(t, J=7.4Hz, 3H).ESI-MS(m/z): [M+H] ⁺ =474.0 (Calcd:473.22)..

Example 22: Synthesis of compound 1-22, N-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-4-(4-methyl-1,4 -homopiperazin-1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 32.1 mg of solid was obtained with a yield of 34.3%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.86 (brs, 1H), 10.56 (brs, 1H), 7.99 (d, J=8.9Hz, 2H), 7.79 (d, J=8.2Hz, 1H), 7.58(s,1H),7.19-7.21(m,3H),7.12-7.15(m,1H),7.04(d,J＝7.8Hz,2H),3.90(s,3H),3.82-3.85(m, 2H), 2.95(s,2H), 2.34-2.40(m,2H), 1.12(s,3H), 1.10(s,3H).ESI-MS(m/z):[M+H] ⁺ ＝474.0 (Calcd:473.22)..

Example 23: Synthesis of compound 1-23, N-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-4-(4-methyl-1,4 -homopiperazin-1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 22.1 mg of solid was obtained with a yield of 25.8%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.84 (brs, 1H), 10.47 (brs, 1H), 7.97 (d, J = 8.5Hz, 2H), 7.78 (d, J = 8.2Hz, 1H), 7.58(s,1H),7.20-7.26(m,3H),7.12-7.15(m,1H),6.81(d,J＝7.8Hz,2H),3.90(s,3H),3.63-3.66(m, 2H), 3.54(t, J=6.2Hz, 2H), 2.80(s, 2H), 2.65(s, 2H), 2.40(s, 3H), 1.96-2.03(m, 2H).ESI-MS(m /z): [M+H] ⁺ = 474.0 (Calcd: 473.22).

Example 24: Synthesis of compound 1-24, N-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-4-(4-methylpiperazine-1 -yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 19.2 mg of solid was obtained with a yield of 22.5%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.87 (brs, 1H), 10.57 (brs, 1H), 7.98 (d, J=8.2Hz, 2H), 7.76 (d, J=8.2Hz, 1H), 7.56(s,1H),7.19-7.24(m,3H),7.10-7.13(m,1H),7.02(d,J＝7.8Hz,2H),3.88(s,3H),3.24-3.39(s, 4H), 2.44-2.52 (s, 4H), 2.31 (s, 3H). ESI-MS (m/z): [M+H] ⁺ = 460.0 (Calcd: 459.20).

Example 25: Synthesis of compound 1-25, N-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-5-(4-methyl-piperazine- 1-yl)pyrazine-2-carboxamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 23.2 mg of solid was obtained, with a yield of 24.8%. ¹ H NMR (400MHz,DMSO-d ₆ )δ:12.93(brs,1H),10.40(brs,1H),8.78(s,1H),8.41(s,1H),7.94(d,J＝8.2Hz,1H ),7.59(s,1H),7.20-7.28(m,3H),7.12-7.15(m,1H),3.90(s,3H),3.72-3.83(m,4H),2.42-2.51(m,4H ), 2.26 (s, 3H). ESI-MS (m/z): [M+H] ⁺ = 462.1 (Calcd: 461.20).

Example 26: Synthesis of compound 1-26, N-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-4-(4-(dimethylamino)piper Pyridin-1-yl)-benzamide

The synthesis method was similar to the preparation method of compound 1-2, and a total of 35.4 mg of solid was obtained with a yield of 33.8%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.96 (brs, 1H), 10.55 (brs, 1H), 7.98 (d, J = 8.0Hz, 2H), 7.78 (d, J = 7.8Hz, 1H), 7.59(s,1H),7.20-7.26(m,3H),7.11-7.14(m,1H),7.02(d,J＝8.2Hz,2H),3.95(s,2H),3.90(s,3H) ,2.80-2.86(m,2H),2.19(s,6H),1.82-1.86(m,2H),1.43-1.48(m,2H).ESI-MS(m/z):[M+H] ⁺ = 488.3 (Calcd: 487.24)..

Example 27: In vitro FGFR1 inhibitory activity test

Dilute the enzyme reaction substrate Poly(Glu,Tyr) _4:1 with potassium ion-free PBS (10mM sodium phosphate buffer, 150mM NaCl, pH7.2-7.4) to 20μg/mL, 125μL/well to coat the microtiter plate, Place at 37°C for 12-16 hours. Wash the plate after discarding the liquid in the well, and wash the plate three times with 200 μL/well of T-PBS (PBS containing 0.1% Tween-20), 5 minutes each time. Dry the microtiter plate in an oven at 37°C for 1-2 hours;

Add 50 μL of ATP solution diluted with reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl ₂ , 0.5 mM MnCl ₂ , 0.2 mM Na ₃ VO _{4 ,} 1 mM DTT) into each well, with a final concentration of 5 μM. Dilute the compound with DMSO to an appropriate concentration, 1 μL/well or DMSO containing the corresponding concentration (negative control well), then add FGFR1 kinase domain recombinant protein diluted with 49 μL reaction buffer to start the reaction, and no ATP control is required for each experiment There are two holes. Put it on a shaker (100 rpm) at 37°C for 1 hour. Wash the plate three times with T-PBS. Add 100 μL/well of primary antibody PY99 dilution and react on a shaker at 37°C for 0.5 hours. Wash the plate three times with T-PBS. Add 100 μL/well of secondary antibody horseradish peroxidase-labeled goat anti-mouse IgG dilution, and react on a shaker at 37°C for 0.5 hours. Wash the plate three times with T-PBS. Add 100 μL/well of 2 mg/ml OPD chromogenic solution (diluted with 0.1M citric acid-sodium citrate buffer (pH=5.4) containing 0.03% H ₂ O ₂ ), and react in the dark at 25°C for 1-10 minutes. (Ultrasound is required for OPD dissolution, and the chromogenic solution must be prepared and used immediately). Add 50 μL/well of 2M H ₂ SO ₄ to stop the reaction, and read with a wavelength-tunable microplate microplate reader SPECTRAMAX 190 at a wavelength of 490 nm;

The inhibition rate of the sample was obtained by the following formula:

Inhibition rate (%)=[1-(OD value of compound-OD value of control well without ATP)/(OD value of negative control-OD value of control well without ATP)]×100%

The _IC50 value was obtained by regression with the four-parameter method using the accompanying software of the microplate reader;

The results show (as shown in Table 1), the compounds of the present invention show better FGFR1 inhibitory activity, wherein compounds 1-8, 1-13, 1-15, 1-16, 1-17 have inhibitory activity IC for FGFR1 _{The 50} value is less than 10 nM.

Table 1 is the results of the FGFR1 inhibitory activity of the compounds of the present invention in vitro.

Table 1

Example 28: Effect test of compounds on tumor cell proliferation ability mediated by FGFR2

CCK-8 cell counting kit (Dojindo) was used to detect the inhibitory effect of the compound of the present invention on the proliferation of SNU16 cells, and the specific steps included: SNU16 cells in the logarithmic growth phase were inoculated into a 96-well culture plate at an appropriate density, 90 μL per hole After culturing overnight, different concentrations of compounds were added to act for 72 hours, and a solvent control group (negative control) was set. After the compounds acted on the cells for 72 hours, the effect of the compounds on cell proliferation was detected by CCK-8 cell counting kit (Dojindo). Add 10 μL of CCK-8 reagent to each well, place it in a 37°C incubator for 2-4 hours, and read it with a full-wavelength microplate microplate reader SpectraMax 190, and the measurement wavelength is 450nm;

The inhibitory rate (%) of compound to tumor cell growth is calculated by following formula:

Inhibition rate (%)=[(OD control well-OD administration well)/OD control well]×100%

The IC ₅₀ value was obtained by regression with the four-parameter method using the accompanying software of the microplate reader.

The results show (as shown in Table 2) that the compound of the present invention has an in vitro proliferation inhibitory effect on human gastric cancer cell SNU16, and the results show that the compound shows a better proliferation inhibitory effect on human gastric cancer cell SNU16.

Table 2 is the in vitro proliferation inhibitory effect of the compounds of the present invention on human gastric cancer cell SNU16.

Table 2

Claims (8)

1. a kind of indazole compounds or its pharmaceutically acceptable salt, the compound is one kind in following compounds：

2. compound according to claim 1 or its pharmaceutically acceptable salt, the compound is in following compounds One kind：

3. a kind of method preparing claim 1~2 any one of them compound or its pharmaceutically acceptable salt, feature It is, by general formula I, to include the following steps：

(1) segment C is prepared using the gradually method of condensation or fragment condensation；

(2) intermediate F is made in segment C and D condensations,

The method of condensing is chloride method, acyl fluorides method or mixed anhydride method, or uses condensing agent：N, N- dicyclohexyl imines, 1- [3- dimethylaminos]-propyl -3- ethylcarbodiimides hydrochloride or 7- azepine benzothiazole -1- bases-oxygen (three-(dimethylamine Base) phosphine) hexafluorophosphate；

(3) segment F removes tert-butyl ester protecting group, and agents useful for same is concentrated hydrochloric acid, Hydrochloride/ethyl acetate, hydrogen chloride dioxy six Ring solution, hydrogen bromide ethyl acetate solution, hydrogen bromide dioxane solution, hydrogen bromide acetic acid solution, hydrogen fluoride, trifluoroacetic acid or A combination thereof；

(4) the segment F of removing tert-butyl ester protecting group obtains compound shown in claim 1~2 with E coupling reactions；Coupling side used Method is coupled for suzuki, or uses following catalyst：Tetrakis triphenylphosphine palladium, 1,1'- bis- (diphenyl phosphine) ferrocene dichloride Palladium or the sour palladium of acid；

Wherein R₁,R₂, A rings, B rings are claim 1, the substituent group of the corresponding position of compound shown in 2.

4. a kind of pharmaceutical composition, characterized in that the composition contains described in any one of claim 1~2 of therapeutically effective amount Compound or its pharmaceutically acceptable salt.

5. pharmaceutical composition according to claim 4, characterized in that the compound or its pharmaceutically acceptable salt Account for the 20%~99% of the pharmaceutical composition total weight.

6. pharmaceutical composition according to claim 4 or 5, characterized in that the composition further includes one or more Pharmaceutically acceptable carrier, odorant agent, flavouring agent, excipient or dilution.

7. claim 1~2 any one of them compound or its pharmaceutically acceptable salt are in preparing prevention tumour medicine Purposes, wherein the tumour is and the relevant tumours of tyrosine kinase FGFR.

8. purposes according to claim 7, characterized in that the tumour is breast cancer, oophoroma, carcinoma of urinary bladder, black Plain tumor, gastric cancer or carcinoma of endometrium.