WO2024022095A1

WO2024022095A1 - Axl＆c-met dual inhibitor, and preparation method and use

Info

Publication number: WO2024022095A1
Application number: PCT/CN2023/106523
Authority: WO
Inventors: 谢军; 姜春阳; 李惠; 裴欣宇; 舒海英
Original assignee: 上海博悦生物科技有限公司
Priority date: 2022-07-25
Filing date: 2023-07-10
Publication date: 2024-02-01
Also published as: CN117486860A

Abstract

Disclosed in the present invention are a class of Axl&c-Met dual inhibitors, and a preparation method therefor and the use thereof. The inhibitor is a compound as represented by formula (I) or a pharmaceutically acceptable salt, prodrug, hydrate or solvate thereof, wherein R1 is hydrogen, halogen, substituted or unsubstituted alkyl, alkoxy or haloalkoxy, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted aryl; R2 is halogen, or substituted or unsubstituted alkyl; and the value of n is 1 or 2. The Axl&c-Met dual inhibitor of the present invention has excellent pharmacokinetic properties and is expected to be developed into an anti-tumor drug. In particular, the series of inhibitors have an effective blood-brain barrier penetration ability, have good exposure in brain tissues, and can be used for developing drugs for brain tumors such as brain glioma and brain metastatic tumors.

Description

Axl&c-Met dual inhibitor, preparation method and use

Technical field

The invention relates to the field of medicinal chemistry, relates to a substituted pyridine amide compound, and specifically relates to a type of Axl&c-Met dual inhibitor and its preparation method and use.

Background technique

c-MET is a transmembrane receptor tyrosine kinase encoded by the MET gene and has high affinity to hepatocyte growth factor (HGF) structurally. Dysregulation of HGF/c-MET signaling leads to the activation of downstream pathways, including the RAS/MAPK, PI3K/AkT, and Rac/Rho pathways, which are involved in cell proliferation, survival, and metastasis. High-level MET gene amplification, protein overexpression or gene mutation are the main mechanisms leading to abnormal activation of the HGF/c-MET pathway. Increasing evidence shows that c-Met receptor tyrosine kinase plays a role in tumor development and metastasis progression. role. In addition, dysregulation of MET tyrosine kinases is associated with resistance to targeted therapies in cancer patients and often occurs in patients with non-small cell lung cancer (NSCLC) who are resistant to EGFR inhibitors. Drugs targeting MET signaling have the potential to improve treatment of patients with MET disorders. Currently, there are four drugs currently on the market targeting c-Met/HGF, namely Crizotinib, Cabozantinib, Capmatinib and Tepotinib. The indications are non-small cell lung cancer. host.

Receptor tyrosine kinases (RTKs) are transmembrane proteins that connect the extracellular and intracellular environments. As a mediator of signal transduction, it plays an important role in normal cellular processes, including differentiation, adhesion, migration, apoptosis, and metabolism. Axl (also known as Ufo, Ark or Tyro7) is a receptor tyrosine kinase, which together with Tyro3 and Mer constitute the TAM subfamily of receptor tyrosine kinases. Overexpression of Axl kinase was first discovered in chronic myelogenous leukemia and chronic myeloproliferation. Subsequently, Paccez et al. found that it also exists in various cancers such as breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, and liver cancer. Axl kinase overexpression. Abnormal expression of Axl activates and antagonizes tumor cell apoptosis, promotes tumor cell invasion and metastasis, promotes tumor angiogenesis, and promotes the occurrence and development of tumors in many aspects. What is particularly noteworthy is that recent studies have shown that the dimer produced by overexpression of Axl and binding to EGFR is an important reason for the acquired resistance of tumor cells to EGFR inhibitors; preclinical research on the combination of Axl inhibitors can Effectively overcome resistance to EGFR inhibitors. In addition, abnormal activation of Axl overexpression is also closely related to resistance to other targeted inhibitors and chemotherapy drugs, suggesting that Axl may have a wide range of application space for combination drugs. Unlike other kinases, Axl is highly expressed in macrophages and dendritic cells in the tumor microenvironment, and can interact with tumor cells and Other stromal cells interact and synergistically promote tumor progression. Therefore, in recent years, the development of Axl-targeted inhibitors has become the forefront and hot spot of anti-tumor drug research. Small molecule inhibitors developed for it have shown effects in tumor treatment.

At the same time, studies have shown that Axl and c-MET are overexpressed in a variety of malignant tumors such as glioma. One of the main obstacles in the treatment of brain tumors such as glioma is that drugs cannot effectively penetrate the blood-brain barrier. There is an urgent need to discover small molecule drugs with the ability to penetrate the blood-brain barrier.

The present invention provides a new class of Axl&c-Met dual inhibitors, which have excellent effects in treating various tumors. Moreover, this series of inhibitors has strong ability to penetrate the blood-brain barrier and is expected to be used to treat the brain. Tumor.

Contents of the invention

The purpose of the present invention is to develop a class of Axl&c-Met dual inhibitors, preparation methods and uses thereof. This new type of Axl&c-Met dual inhibitor has a completely new structure different from existing compounds and shows excellent anti-tumor activity.

The purpose of the present invention is achieved through the following technical solutions:

The invention provides a series of compounds, including compounds represented by formula (I) or pharmaceutically acceptable salts thereof, prodrugs thereof, hydrates or solvates thereof:

Wherein, R ₁ is hydrogen, halogen, substituted or unsubstituted alkyl, alkoxy or haloalkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl;

R ₂ is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkoxy, or hydroxyalkyl;

The value of n is 1 or 2.

As an embodiment of the present invention, the alkyl group is a C ₁ -C ₆ alkyl group, the cycloalkyl group is a C ₃ -C ₄ cycloalkyl group, and the alkoxy group is a C ₁ -C ₆ Alkoxy group, the hydroxyalkyl group is a C ₁ -C ₆ hydroxyalkyl group, and the substituent is fluorine or chlorine.

This compound has dual inhibitory effects on Axl&c-Met and is a new dual inhibitor of Axl&c-Met.

As an embodiment of the present invention, R ₁ is selected from hydrogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, fluorine or chlorine; R ₂ is selected from fluorine, chlorine, methyl.

As an embodiment of the invention, _R1 is selected from hydrogen, methyl, fluorine or chlorine, and _R2 is selected from fluorine, chlorine, methyl.

As an embodiment of the present invention, any one of the following compounds is selected:

If there is a discrepancy between the chemical nomenclature and the illustrated chemical structure, the illustrated chemical structure is preferred over the given chemical nomenclature.

The invention also provides a method for preparing an Axl&c-Met dual inhibitor compound, which method includes the following steps: compound and compounds The reaction produces a compound represented by formula (I), and the reaction is preferably carried out in the presence of an organic solvent, HATU and triethylamine. The organic solvent includes N,N-dimethylacetamide, N-methylpyrrolidone, dichloromethane, chloroform, tetrahydrofuran, methyl acetate, ethyl acetate, isopropyl acetate, 1,2-dichloro Ethane, acetonitrile, etc.

The reaction formula is as follows:

Wherein, R ₁ , R ₂ and n are as defined in the present invention.

compound The preparation includes the following steps:

A1. Compound React with ethyl chloroformyl acetate in the presence of dichloromethane and triethylamine to generate an intermediate compound

A2. Intermediate compounds It can be obtained by reacting with 4-methoxybut-3-en-2-one in the presence of ethanol and sodium ethoxide ethanol solution.

compound The preparation includes the following steps:

B1, compound with compounds In the presence of DMAc and potassium tert-butoxide, react at 80-90°C under nitrogen protection to generate an intermediate compound

B2, intermediate compounds It can be obtained by reacting with 1-methylpyrazole-4-boronic acid pinacol ester at 90-95℃ in the presence of dioxane, potassium carbonate and Pd(PPh ₃ ) ₄ .

The present invention also provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof for the preparation of a medicament for treating related diseases caused by Axl kinase and/or c-Met kinase. use in.

Wherein, the related diseases caused by the Axl kinase and/or c-Met kinase include brain tumors, gastric cancer, bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, and pancreatic cancer. Cancer/gallbladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, MFH/fibrosarcoma, glioblastoma/astrocytoma, melanoma or mesothelioma, psoriasis, cirrhosis, diabetes, angiogenesis, regeneration Stenosis, eye diseases, rheumatoid arthritis and other inflammatory diseases, immune diseases, cardiovascular diseases such as arteriosclerosis and kidney disease, etc.

Wherein, the brain tumors include brain gliomas, meningiomas, brain metastases, etc.

The present invention also provides the use of the compound represented by formula (I) or its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate in the preparation of Axl&c-Met inhibitors.

The invention also provides the use of a series of Axl&c-Met inhibitors in the preparation of drugs for treating tumors. The tumor Includes: brain tumors, stomach cancer, bladder cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic adenocarcinoma/gallbladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma , MFH/fibrosarcoma, malignant glioma/astrocytoma, melanoma or mesothelioma, etc.

As an embodiment of the present invention, the tumor drug is a brain tumor drug, including brain glioma drugs, meningioma drugs, and brain metastasis drugs.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

Figure 1 is a graph showing the changes in plasma concentration over time after intragastric administration of Compound 1 to mice (dose 10 mg/kg);

Figure 2 is a graph showing the changes in brain concentration over time after compound 1 was administered to mice by gavage (dose 10 mg/kg);

Figure 3 is a comparison chart of the effects of compounds on the body weight of nude mice;

Figure 4 is a comparison chart of the effects of compounds on tumor volume in the human gastric cancer cell MKN45 subcutaneous transplant tumor model;

Figure 5 is a comparison chart of the effects of compounds on the relative tumor volume of the human gastric cancer cell MKN45 subcutaneous transplant tumor model;

Figure 6 is a comparison chart of the effects of compounds on subcutaneous xenografts of human gastric cancer cells MKN45.

Detailed ways

The present invention will be described in detail below with reference to examples. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention. All publications mentioned are incorporated by reference in their entirety.

Example 1, synthesis of compound 1

1.1 Synthesis of intermediate B1

Add 20g of 4-fluoroaniline, 200mL of methylene chloride, and 23.6g of triethylamine into the 1000mL reaction bottle, stir and cool to 0°C. Dissolve 35.2g of ethyl chloroformyl acetate in 100 mL of methylene chloride, and add it dropwise into the reaction bottle at a temperature of 0 to 5°C. After dropping, raise to 20°C and react for 2 hours, add 200 mL of water, stir and separate. Wash the dichloromethane phase with 200 mL of saturated brine and concentrate to dryness. The residue was slurried with 90 mL of petroleum ether and 30 mL of ethyl acetate, filtered, and dried to obtain 30.0 g of intermediate B1A with a yield of 74.1%.

MS: m/z＝226.35[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ9.29 (s, 1H), 7.54 (m, 2H), 7.04 (m, 2H), 4.28 (q, J = 7.1Hz, 2H), 3.49 (s, 2H ),1.35(t,J＝7.1Hz,3H).

Add 15 g of intermediate B1A, 150 mL of ethanol, 10 g of 4-methoxybut-3-en-2-one, and 50 mL of sodium ethoxide ethanol solution into the 500 mL reaction bottle, and raise the temperature to reflux at 70-80°C and incubate the reaction for 6 hours. Cool to room temperature, add 150 mL each of 1 mol/L hydrochloric acid and methylene chloride, stir and separate the liquids. The dichloromethane phase was concentrated to dryness. The concentrated residue was beaten with 50 mL of ethyl acetate at 5°C, filtered, and dried to obtain 8.8 g of intermediate B1 with a yield of 53.9%.

MS: m/z＝248.33[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ13.87 (s, 1H), 8.53 (d, J = 7.5Hz, 1H), 7.36–7.30 (m, 2H), 7.28–7.23 (m, 2H), 6.57 (d,J＝7.5Hz,1H),2.18(s,3H).

1.2 Synthesis of intermediate A1

Add 3.07g of 4-amino-2-fluorophenol, 50mL of DMAc, and 2.71g of potassium tert-butoxide into the 500mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 5.5g of 2,3-dichloro-4-iodopyridine, raise the temperature to 85°C and react for 4 hours. After cooling to room temperature, the reaction solution was concentrated to dryness under reduced pressure. 150 mL of ethyl acetate was added to the concentrated residue, stirred, and filtered. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A1B.

MS: m/z＝273.21[M+H] ⁺

¹ H NMR(400MHz,Chloroform-d)δ8.09(d,J＝5.6Hz,1H),6.99(t,J＝8.6Hz,1H),6.59–6.52(m,2H),6.49(ddd,J =8.7,2.7,1.3Hz,1H),3.86(s,2H).

Add intermediate A1B, 100 mL of dioxane, 5.25 g of 1-methylpyrazole-4-boronic acid pinacol ester, 8.37 g of potassium carbonate, and 20 mL of water into a 250 mL reaction bottle, replace with nitrogen, and add Pd (PPh ₃ ) ₄ 1.5g, replace with nitrogen, raise the temperature to 90~95℃ and keep the reaction for 16 hours. Cool to room temperature, add 200 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 100 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 4.36 g of intermediate A1 with a yield of 67.6%.

MS: m/z＝319.37[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ8.29 (s, 1H), 8.27 (d, J = 5.5Hz, 1H), 8.21 (s, 1H), 7.01 (t, J = 8.7Hz, 1H), 6.55(dd,J＝11.9,2.7Hz,1H),6.49(ddd,J＝8.7,2.8,1.3Hz,1H),6.46(dd,J＝5.5,1.3Hz,1H),4.01(s,3H) ,3.83(s,2H).

1.3 Synthesis of Compound 1

Add 9g of intermediate A1, 6.98g of intermediate B1, 200mL of DMF, 12.88g of HATU, and 5.70g of triethylamine to the 500mL reaction bottle, and stir overnight for reaction. 400 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 10.53 g of crude compound 1, with a yield of 68.0%. Take 500 mg of the sample and perform TLC purification (the developing solvent is methylene chloride: ethanol 15:1) to obtain 289 mg of product with a purity of 98.18%.

MS: m/z＝548.45[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ11.94 (s, 1H), 8.66 (d, J = 7.5Hz, 1H), 8.28 (d, J = 5.5Hz, 2H), 8.21 (s, 1H), 7.97(dd,J＝12.4,2.5Hz,1H),7.38–7.31(m,3H),7.28–7.24(m,2H),7.15(t,J＝8.7Hz,1H),6.54(dd,J＝ 7.6,0.9Hz,1H),6.47(dd,J＝5.5,1.3Hz,1H),4.01(s,3H),2.15(s,3H).

Example 2, synthesis of compound 2

2.1 Synthesis of intermediate A2

Add 2.37g of 4-amino-2-fluorophenol, 50mL of DMAc, and 2.09g of potassium tert-butoxide into the 250mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 4g of 2-chloro-3-fluoro-4-iodopyridine, raise the temperature to 85°C and react for 4 hours. Cool to room temperature, add 200 mL each of water and ethyl acetate, stir, and let stand for liquid separation. The aqueous phase was extracted twice with 100 mL of ethyl acetate. Combine the ethyl acetate phases and concentrate to dryness under reduced pressure to obtain intermediate A2B.

Add the intermediate A2B obtained in the previous step, 100 mL of dioxane, 4.03 g of 1-methylpyrazole-4-boronic acid pinacol ester, 6.43 g of potassium carbonate, and 20 mL of water into a 250 mL reaction bottle. Replace with nitrogen and add Pd. (PPh ₃ ) ₄ 2.5g, replaced with nitrogen, heated to 90-95°C and kept for 20 hours. The temperature was cooled to room temperature, and the reaction solution was concentrated to dryness under reduced pressure. The concentrated residue was slurried with ethyl acetate and filtered through Celite. The filtrate was concentrated and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 2.5 g of intermediate A2 with a yield of 53.4%.

2.2 Synthesis of Compound 2

Add 0.6g of intermediate A2, 0.49g of intermediate B1, 15mL of DMF, 0.91g of HATU, and 0.40g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 0.70 g of crude compound 2, with a yield of 66.5%. Take 360 mg of the sample and perform TLC purification to obtain 118 mg of product with a purity of 95.28%.

MS: m/z＝532.47[M+H] ⁺

¹ H NMR(400MHz,Chloroform-d)δ11.94(s,1H),8.66(d,J＝7.6Hz,1H),8.19–8.16(m,2H),8.04(d,J＝2.0Hz,1H ),7.96(dd,J＝12.5,2.5Hz,1H),7.37–7.31(m,3H),7.27–7.24(m,2H),7.15(t,J＝8.7Hz,1H),6.56–6.51( m,2H),4.01(s,3H),2.15(s,3H).

Example 3, synthesis of compound 3

3.1 Synthesis of intermediate B2

Add 2g of 4-toluidine, 20mL of methylene chloride, and 2.64g of triethylamine into the 100mL reaction bottle, stir and cool down to 0°C. Dissolve 3.65g of ethyl chloroformyl acetate in 10 mL of methylene chloride, and add it dropwise into the reaction bottle while controlling the temperature at 0 to 5°C. After dropping, raise to 20°C and react for 2 hours, add 20 mL of water, stir and separate. Wash the dichloromethane phase with 20 mL of saturated brine and concentrate to dryness. The residue was beaten with 12 mL of petroleum ether and 3 mL of ethyl acetate, filtered, and dried to obtain 3.8 g of intermediate B2A with a yield of 92.0%.

Add 3.8g of intermediate B2A, 38mL of ethanol, 2.58g of 4-methoxybut-3-en-2-one, and 12.9mL of sodium ethoxide ethanol solution into the 100mL reaction bottle, and heat to reflux and incubate for 6 hours. Cool to room temperature, add 60 mL each of 1 mol/L hydrochloric acid and methylene chloride, stir and separate the liquids. The dichloromethane phase was concentrated to dryness. The concentrated residue was beaten with 20 mL of ethyl acetate at 5°C, filtered, and dried to obtain 1.73 g of intermediate B1 with a yield of 41.6%.

3.2 Synthesis of compound 3

Add 0.6g of intermediate A2, 0.48g of intermediate B2, 15mL of DMF, 0.91g of HATU, and 0.40g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. Add 30 mL of water dropwise, filter, and dry the filter cake to obtain 0.50 g of crude compound 3, with a yield of 48.2%. 360 mg of the sample was taken and purified by TLC to obtain 168 mg of product with a purity of 98.95%.

MS:m/z＝528.50[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ12.05 (s, 1H), 8.64 (d, J = 7.5Hz, 1H), 8.18 (d, J = 5.5Hz, 2H), 8.04 (s, 1H), 7.96(dd,J＝12.5,2.4Hz,1H),7.44(d,J＝8.0Hz,2H),7.35(d,J =8.9Hz,1H),7.18–7.10(m,3H),6.56–6.49(m,2H),4.01(s,3H),2.49(s,3H),2.15(s,3H).

Example 4, synthesis of compound 4

Add 1g of intermediate A1, 0.76g of intermediate B2, 25mL of DMF, 1.43g of HATU, and 0.63g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 50 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 0.98 g of crude compound 4, with a yield of 57.1%. 360 mg of the sample was taken and purified by TLC to obtain 214 mg of product with a purity of 97.86%.

MS: m/z＝544.47[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ12.06 (s, 1H), 8.64 (d, J = 7.5Hz, 1H), 8.28 (d, J = 5.5Hz, 2H), 8.21 (s, 1H), 7.97(dd,J＝12.5,2.4Hz,1H),7.44(d,J＝8.0Hz,2H),7.35(d,J＝9.4Hz,1H),7.17–7.11(m,3H),6.49(dd ,J＝19.6,6.2Hz,2H),4.01(s,3H),2.49(s,3H),2.15(s,3H).

Example 5, synthesis of compound 5

5.1 Synthesis of intermediate B3

Add 3g of aniline, 30mL of methylene chloride, and 4.56g of triethylamine into a 100mL reaction bottle, stir and cool down to 0°C. Dissolve 6.30 g of ethyl chloroformyl acetate in 15 mL of methylene chloride, and add it dropwise into the reaction bottle while controlling the temperature at 0 to 5°C. After dropping, raise to 20°C and react for 2 hours, add 30 mL of water, stir and separate. The dichloromethane phase was washed with 30 mL of saturated brine, and concentrated to dryness to obtain intermediate B3A.

Add the intermediate B3A obtained in the previous step, 30 mL of ethanol, 4.84 g of 4-methoxybut-3-en-2-one, and 24.1 mL of sodium ethoxide ethanol solution into the 100 mL reaction bottle, and heat to reflux and incubate for 7 hours. Cool to room temperature, add 60 mL each of 1 mol/L hydrochloric acid and methylene chloride, stir and separate the liquids. The dichloromethane phase was concentrated to dryness. The concentrated residue was beaten with 20 mL of ethyl acetate at 5°C, filtered, and dried to obtain 3.76 g of intermediate B3 with a yield of 50.9%.

5.2 Synthesis of compound 5

Add 0.6g of intermediate A2, 0.46g of intermediate B3, 15mL of DMF, 0.91g of HATU, and 0.40g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 0.52 g of crude compound 5, with a yield of 51.1%. 360 mg of the sample was taken and purified by TLC to obtain 210 mg of product with a purity of 99.01%.

MS: m/z＝514.47[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ12.00 (s, 1H), 8.66 (d, J = 7.5Hz, 1H), 8.18 (d, J = 5.5Hz, 2H), 8.04 (d, J = 1.6 Hz,1H),7.96(dd,J＝12.5,2.4Hz,1H),7.69–7.53(m,3H),7.35(dd,J＝8.8,1.4Hz,1H),7.27(s,1H),7.14 (t,J＝8.7Hz,1H),6.58–6.49(m,2H),4.01(s,3H),2.15(s,3H).

Example 6, synthesis of compound 6

Add 1g of intermediate A1, 0.72g of intermediate B3, 25mL of DMF, 1.43g of HATU, and 0.63g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 50 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 0.95 g of crude compound 6, with a yield of 56.9%. 360 mg of the sample was taken and purified by TLC to obtain 169 mg of product with a purity of 99.01%.

MS: m/z＝530.45[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ12.02 (s, 1H), 8.66 (d, J = 7.5Hz, 1H), 8.28 (d, J = 5.4Hz, 2H), 8.21 (s, 1H), 7.97(dd,J＝12.5,2.3Hz,1H),7.69–7.55(m,3H),7.36(d,J＝8.3Hz,1H),7.27(s,2H),7.14(t,J＝8.7Hz ,1H),6.50(dd,J＝25.7,6.5Hz,2H),4.01(s,3H),2.14(s,3H).

Example 7, synthesis of compound 7

7.1 Synthesis of intermediate B4

Add 3g of 4-chloroaniline, 30mL of methylene chloride, and 3.33g of triethylamine into a 100mL reaction bottle, stir and cool to 0°C. Dissolve 4.6 g of ethyl chloroformyl acetate in 15 mL of methylene chloride, and add it dropwise into the reaction bottle while controlling the temperature at 0 to 5°C. After dropping, raise to 20°C and react for 2 hours, add 30 mL of water, stir and separate. Wash the methylene chloride phase with 30 mL saturated brine. Concentrate to dryness, and the concentrated residue is slurried with 13.5 mL of petroleum ether and 4.5 mL of ethyl acetate, and filtered to obtain 4.52 g of intermediate B4A with a yield of 79.5%.

Add 4g of intermediate B4A, 40mL of ethanol, 2.48g of 4-methoxybut-3-en-2-one, and 12.5mL of sodium ethoxide ethanol solution into the 100mL reaction bottle, and heat to reflux and incubate for 7 hours. Cool to room temperature, add 60 mL each of 1 mol/L hydrochloric acid and methylene chloride, stir and separate the liquids. The dichloromethane phase was concentrated to dryness. The concentrated residue was beaten with 20 mL of ethyl acetate at 5°C, filtered, and dried to obtain 2.49 g of intermediate B4 with a yield of 57.1%.

7.2 Synthesis of Compound 7

Add 0.6g of intermediate A2, 0.52g of intermediate B4, 15mL of DMF, 0.91g of HATU, and 0.40g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 0.60 g of crude compound 7, with a yield of 55.3%. 360 mg of the sample was taken and purified by TLC to obtain 144 mg of product with a purity of 98.59%.

MS: m/z＝548.46[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ11.91 (s, 1H), 8.66 (d, J = 7.5Hz, 1H), 8.18 (d, J = 5.6Hz, 2H), 8.04 (s, 1H), 7.96(dd,J＝12.5,2.4Hz,1H),7.63(d,J＝8.6Hz,2H),7.34(d,J＝8.8Hz,1H),7.23(d,J＝8.6Hz,2H), 7.15(t,J＝8.7Hz,1H),6.56–6.51(m,2H),4.01(s,3H),2.16(s,3H).

Example 8, synthesis of compound 8

Add 1g of intermediate A1, 0.83g of intermediate B4, 25mL of DMF, 1.43g of HATU, and 0.63g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 50 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 1.18 g of crude compound 8, with a yield of 66.7%. Take 360 mg of the sample and perform TLC purification to obtain 160 mg of product with a purity of 97.92%.

MS:m/z＝564.39[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ11.92 (s, 1H), 8.66 (d, J = 7.6Hz, 1H), 8.28 (d, J = 5.4Hz, 2H), 8.21 (s, 1H), 7.97(dd,J＝12.5,2.4Hz,1H),7.63(d,J＝8.8Hz,2H),7.35(ddd,J＝8.7,2.4,1.3Hz,1H),7.25–7.20(m,2H) ,7.15(t,J＝8.7Hz,1H),6.57–6.43(m,2H),4.01(s,3H),2.16(d,J＝0.7Hz,3H).

Example 9, synthesis of compound 9

9.1 Synthesis of Intermediate A3

Add 0.84g of 4-amino-3-fluorophenol, 9mL of DMAc, and 0.74g of potassium tert-butoxide into the 50mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 1.5g of 2,3-dichloro-4-iodopyridine, raise the temperature to 85°C and react for 9 hours. After cooling to room temperature, the reaction solution was concentrated to dryness under reduced pressure. 60 mL of ethyl acetate was added to the concentrated residue, stirred, and filtered. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A3B.

Add intermediate A3B, 30 mL of dioxane, 1.43 g of 1-methylpyrazole-4-boronic acid pinacol ester, 2.27 g of potassium carbonate, 6 mL of water into a 100 mL reaction bottle, replace with nitrogen, and add Pd (PPh ₃ ) ₄ 0.5g, replace with nitrogen, raise the temperature to 90~95℃ and keep the reaction for 30 hours. Cool to room temperature, add 20 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 20 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 1.03 g of intermediate A3 with a yield of 59.2%.

9.1 Synthesis of Compound 9

Add 1.03g of intermediate A3, 0.80g of intermediate B1, 15mL of DMF, 1.47g of HATU, and 0.65g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 1.05 g of crude compound 9, with a yield of 59.3%. 360 mg of the sample was taken and purified by TLC to obtain 125 mg of product with a purity of 98.14%.

MS: m/z＝548.43[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ11.98 (s, 1H), 8.64 (d, J = 7.5Hz, 1H), 8.59 (t, J = 9.0Hz, 1H), 8.31 (d, J = 5.4 Hz,1H),8.29(s,1H),8.21(s,1H),7.31(dd,J＝9.8,7.2Hz,2H),7.28–7.23(m,2H),6.97–6.90(m,2H) ,6.58(d,J＝5.4Hz,1H),6.51(d,J＝7.9Hz,1H),4.01(s,3H),2.13(s,3H).

Example 10, synthesis of compound 10

10.1 Synthesis of Intermediate A4

Add 0.64g of 4-amino-2,3-difluorophenol, 6mL of DMAc, and 0.50g of potassium tert-butoxide into the 50mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 1.0g of 2,3-dichloro-4-iodopyridine, raise the temperature to 85°C and react for 4 hours. Cool to room temperature, add 30 mL each of ethyl acetate and water, stir, and separate the liquids. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A4B. Add the intermediate A4B obtained in the previous step, 30 mL of dioxane, 1.15 g of 1-methylpyrazole-4-boronic acid pinacol ester, 1.83 g of potassium carbonate, and 6 mL of water into a 100 mL reaction bottle. Replace with nitrogen and add Pd. (PPh ₃ ) ₄ 0.5g, replaced with nitrogen, heated to 90-95°C and kept for 20 hours. Cool to room temperature, add 20 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 20 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 1.30 g of intermediate A4 with a yield of 87.5%.

10.2 Synthesis of compound 10

Add 1.30g of intermediate A4, 0.95g of intermediate B1, 15mL of DMF, 1.76g of HATU, and 0.78g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 1.82 g of crude compound 10, with a yield of 83.3%. 400 mg of the sample was taken and purified by TLC to obtain 160 mg of product with a purity of 96.17%.

MS: m/z＝566.46[M+H] ⁺

¹ H NMR(400MHz,Chloroform-d)δ12.10(s,1H),8.64(d,J＝7.5Hz,1H),8.40–8.33(m,1H),8.31(d,J＝5.5Hz,1H ),8.30(d,J＝0.7Hz,1H),8.22(s,1H),7.35–7.30(m,2H),7.28–7.24(m,2H),7.03–6.97(m,1H),6.55– 6.48(m,2H),4.01(s,3H),2.15(s,3H).

Example 11, synthesis of compound 11

11.1 Synthesis of intermediate A5

Add 0.93g of 4-amino-2,5-difluorophenol, 9mL of DMAc, and 0.72g of potassium tert-butoxide into the 50mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 1.46g of 2,3-dichloro-4-iodopyridine, raise the temperature to 85°C and react for 4 hours. Cool to room temperature, add 30 mL each of ethyl acetate and water, stir, and separate the liquids. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A5B.

Add intermediate A5B, 30 mL of dioxane, 1.67 g of 1-methylpyrazole-4-boronic acid pinacol ester, 2.66 g of potassium carbonate, 6 mL of water into a 100 mL reaction bottle, replace with nitrogen, and add Pd (PPh ₃ ) ₄ 0.5g, nitrogen replacement, heating to 90~95℃ The reaction was kept warm for 20 hours. Cool to room temperature, add 20 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 20 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 0.97g of intermediate A5 with a yield of 44.9%.

11.2 Synthesis of compound 11

Add 0.97g of intermediate A5, 0.71g of intermediate B1, 15mL of DMF, 1.31g of HATU, and 0.58g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 1.13 g of crude compound 11, with a yield of 69.6%. 400 mg of the sample was taken and purified by TLC to obtain 143 mg of product with a purity of 99.00%.

MS: m/z＝566.46[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ12.13 (s, 1H), 8.67–8.62 (m, 2H), 8.32 (d, J = 5.4Hz, 1H), 8.29 (s, 1H), 8.21 (s ,1H),7.36–7.32(m,2H),7.28–7.22(m,2H),7.01(dd,J＝10.3,7.0Hz,1H),6.52(t,J＝6.9Hz,2H),4.01( s,3H),2.14(s,3H).

Example 12, synthesis of compound 12

12.1 Synthesis of intermediate A6

Add 1.25g of 4-amino-2,6-difluorophenol, 12mL of DMAc, and 0.97g of potassium tert-butoxide into the 50mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 1.96g of 2,3-dichloro-4-iodopyridine, raise the temperature to 85°C and react for 5 hours. Cool to room temperature, add 50 mL each of ethyl acetate and water, stir and separate the liquids. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A6B.

Add intermediate A6B, 30 mL of dioxane, 2.24 g of 1-methylpyrazole-4-boronic acid pinacol ester, 3.57 g of potassium carbonate, and 6 mL of water into a 100 mL reaction flask, replace with nitrogen, and add Pd (PPh ₃ ) ₄ 0.5g, replace with nitrogen, raise the temperature to 90~95℃ and keep the reaction for 20 hours. Cool to room temperature, add 20 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 20 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 2.05g of intermediate A6 with a yield of 70.7%.

12.2 Synthesis of compound 12

Add 2.05g of intermediate A6, 1.51g of intermediate B1, 15mL of DMF, 2.78g of HATU, and 1.23g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 2.40 g of crude compound 12, with a yield of 69.6%. 450 mg of the sample was taken for TLC purification to obtain 184 mg of product with a purity of 99.52%.

MS: m/z＝566.43[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ12.04 (s, 1H), 8.65 (d, J = 7.5Hz, 1H), 8.33–8.28 (m, 2H), 8.22 (s, 1H), 7.57–7.50 (m,2H),7.39–7.31(m,2H),7.28–7.23(m,2H),6.55(d,J＝7.9Hz,1H),6.48(d,J＝5.4Hz,1H),4.01( s,3H),2.16(s,3H).

Example 13, synthesis of compound 13

13.1 Synthesis of Intermediate A7

Add 0.50g of 4-amino-3,5-difluorophenol, 5mL of DMAc, and 0.39g of potassium tert-butoxide into the 50mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 0.79g of 2,3-dichloro-4-iodopyridine, raise the temperature to 85°C and react for 5 hours. Cool to room temperature, add 50 mL each of ethyl acetate and water, stir, and separate the liquids. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A7B.

Add the intermediate A7B obtained in the previous reaction, 30 mL of dioxane, 0.90 g of 1-methylpyrazole-4-boronic acid pinacol ester, 1.43 g of potassium carbonate, 6 mL of water, nitrogen replacement, and add to the 100 mL reaction bottle. Pd(PPh ₃ ) ₄ 0.5g, replaced with nitrogen, heated to 90-95°C and kept for 20 hours. Cool to room temperature, add 20 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 20 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 1.05 g of intermediate A7 with a yield of 90.2%.

13.2 Synthesis of compound 13

Add 1.05g of intermediate A7, 0.77g of intermediate B1, 15mL of DMF, and 1.42g of HATU into the 100mL reaction bottle. Triethylamine 0.63g, stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 1.06 g of crude compound 13, with a yield of 60.2%. Take 500 mg of the sample and perform TLC purification to obtain 151 mg of product with a purity of 96.61%.

MS: m/z＝566.45[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ11.17 (s, 1H), 8.65 (d, J = 7.5Hz, 1H), 8.39 (d, J = 5.4Hz, 1H), 8.29 (d, J = 0.7 Hz,1H),8.22(s,1H),7.35–7.30(m,3H),7.25(t,J＝5.0Hz,1H),6.77–6.71(m,3H),6.51(d,J＝8.3Hz ,1H),4.01(s,3H),2.15(s,3H).

Example 14, synthesis of compound 15

14.1 Synthesis of Intermediate A9

Add 0.90g of 4-amino-2-fluorophenol, 9mL of DMAc, and 0.80g of potassium tert-butoxide into a 50mL reaction bottle, and stir for 0.5 hours under nitrogen protection. Add 1.5g of 2-chloro-4-iodo-3-methylpyridine, raise the temperature to 85°C and react for 3 hours. Cool to room temperature, add 30 mL each of ethyl acetate and water, stir, and separate the liquids. The ethyl acetate phase was concentrated to dryness under reduced pressure to obtain intermediate A9B. Add intermediate A9B, 30 mL of dioxane, 1.54 g of 1-methylpyrazole-4-boronic acid pinacol ester, 2.45 g of potassium carbonate, 6 mL of water into a 100 mL reaction bottle, replace with nitrogen, and add Pd (PPh ₃ ) ₄ 0.5g, replace with nitrogen, raise the temperature to 90~95℃ and keep the reaction for 20 hours. Cool to room temperature, add 20 mL each of water and ethyl acetate, stir and separate the liquids. The aqueous phase was extracted twice with 20 mL of ethyl acetate. The ethyl acetate phases were combined, concentrated to dryness under reduced pressure, and separated by silica gel column chromatography (dichloromethane: ethanol = 100:1, v/v) to obtain 0.96 g of intermediate A9 with a yield of 54.4%.

14.2 Synthesis of compound 15

Add 0.96g of intermediate A9, 0.79g of intermediate B1, 15mL of DMF, 1.46g of HATU, and 0.65g of triethylamine to the 100mL reaction bottle, and stir overnight for reaction. 30 mL of water was added dropwise, filtered, and the filter cake was dried to obtain 1.25 g of crude compound 15, with a yield of 73.9%. 360 mg of the sample was taken and purified by TLC to obtain 191 mg of product with a purity of 99.16%.

MS:m/z＝528.50[M+H] ⁺

¹ H NMR (400MHz, Chloroform-d) δ11.91 (s, 1H), 8.66 (d, J = 7.5Hz, 1H), 8.27 (d, J = 5.7Hz, 1H), 7.97–7.90 (m, 2H ),7.86(s,1H),7.37–7.31(m,3H),7.26(dt,J＝6.8,2.3Hz, 2H),7.09(t,J＝8.7Hz,1H),6.54(dd,J＝7.5,0.9Hz,1H),6.43(d,J＝5.5Hz,1H),4.00(s,3H),2.51( s,3H).

Example 15 Kinase Activity Test

In this experiment, the inhibitory effect of the compound of the present invention on MET and AXL kinase was detected by using fluorescence microfluidic mobility detection technology (Mobility-Shift Assay). Kinase catalyzes the removal of a phosphate group from ATP to generate ADP, and transfers the phosphate group to the substrate peptide. The substrate peptide is fluorescently labeled, and the charge of the product changes due to the addition of a phosphate group. , during the electrophoresis process, the substrate and phosphorylated products are separated due to different mobilities and are detected respectively, and their amounts are proportional to the fluorescence signal. Use the Caliper instrument to measure the amounts of substrate and product, calculate the conversion rate of the product, and then calculate the inhibition rate.

The MET kinase reaction system is 25uL, which includes 5nM MET, concentration gradient small molecule inhibitor, 10mM MgCl2, 1M DTT, 26uM ATP (measured Km value), 3uM Peptide2 (5-FAM-EAIYAAPFAKKK-CONH2), 0.0015% Brij-35 and pH 7.5 50mM HEPES, 2mM DTT, 10mM MgCl2; AXL kinase reaction system is 25uL, including 6nM AXL, concentration gradient small molecule inhibitor, 10mM MgCl2, 1M DTT, 81uM ATP (measured Km value ), 3uM Peptide22 (5-FAM-EEPLYWSFPAKKK-CONH2), 0.0015% Brij-35 and 50mM HEPES, pH 7.5, 2mM DTT, 10mM MgCl2.

Add the enzyme and inhibitor to the 384-well plate and incubate at room temperature for 10 minutes, then add the substrate and ATP to start the reaction. After 30 minutes, add 25uL Stop Buffer to terminate the reaction. Caliper instrument data, with the Log concentration of the inhibitor as the X-axis, inhibition Draw a curve with the rate on the Y-axis, and get IC50 according to the formula Y=Bottom+(Top-Bottom)/(1+(IC50/X)^HillSlope).

The measured data are as follows in Table 1:

Table 1

Example 16 In vitro anti-tumor activity assay

The in vitro proliferation inhibitory effect of the compound of the present invention on human tumor cells derived from different tissues was detected.

experimental method

1) Cell culture and inoculation

Experimental tumor cell lines were cultured in RPMI-1640 and DMEM containing 10-20% Gibco serum at 37°C in a 5% CO2 incubator. According to the background data of the laboratory, 4000 tumor cells/well were seeded in a 96-well culture plate. The cells were in the logarithmic growth phase throughout the experiment.

2) Administration

After the cells were seeded in a 96-well plate and allowed to adhere overnight, 5 concentration gradients (0.625-10 μM/6.25-100 μM) were set according to different compounds, with two duplicate wells for each concentration.

3) Preparation of test products

Take the test substances separately, add DMSO to each tube to dissolve, aliquot and store at -20°C. Dilute to working concentration with fresh culture medium before use. See the experimental results section for specific concentration settings. 72 hours after administration, the cell proliferation inhibitory effect was measured.

4)SRB method

After the test substance acts on the cells for 72 hours, discard the culture medium, add pre-cooled 10% trichloroacetic acid (TCA) solution to each well to fix the cells, and place them in a 4°C refrigerator for 2 hours. Each well of the culture plate is washed with deionized water for 5 seconds. times to remove the trichloroacetic acid solution. After drying, add SRB solution (4mg/ml) prepared with 1% acetic acid to each well and leave it at room temperature for 20 minutes. Discard the liquid in each well and wash it 5 times with 1% acetic acid. For unbound SRB dye, add an appropriate volume of 10mM Tris-base (trishydroxymethylaminomethane) solution to each well after drying. After complete dissolution, measure the absorbance OD value with a microplate reader at a wavelength of 515nm.

5) Result processing

According to the OD value measured by the microplate reader, the inhibition rate is calculated according to the following formula:

Inhibition rate (%) = 1-OD administration/OD control × 100%. If the inhibition rate ≤ 0, it is recorded as 0.

Calculate IC50.

6)Experimental results

The tested compounds have varying degrees of inhibitory effects on U87-MG and MKN45, two human tumor cells derived from different tissues. Among them, compound 1, compound 2, compound 5, compound 6, compound 9, compound 11, compound 12 and compound 15 have inhibitory effects on U87 -MG cells have obvious inhibitory effects, especially compound 11 is more sensitive to U87-MG cells, with an IC50 value of 2.71 μM (see Table 2 for details). Compound 11 has a significant inhibitory effect on MKN45 cells, with an IC50 value of 8.71 μM, which is significantly better than the 19.37 μM of the control drug BMS777607.

Table 2: Inhibitory effects of compounds on tumor cell proliferation

Example 17 Evaluation of blood-brain barrier penetration ability

Compound 1 was dissolved in a solution of DMSO:PEG400:Water=1:6:3 (V/V/V) to prepare a preparation solution of 1 mg/mL, and administered to CD1 mice by gavage at a dose of 10 mg/kg. . Samples were taken from plasma and brain at 0.25h, 0.5h, 1h, 2h, 4h, 8h, and 24h, and then the amounts of chemical components in plasma and brain tissue were detected by LC-MS/MS to obtain a series of drugs. Kinetic parameters, the results are shown in Table 3 and Figures 1 and 2 below.

table 3

The results show that compound 1 shows excellent oral pharmacokinetic properties and can pass through the blood-brain barrier with a permeability of 0.24. The drug has high brain tissue exposure and can be developed to treat brain gliomas, meningiomas, and brain metastases. drugs for brain tumors such as tumors.

Example 18. Experimental therapeutic effect of compound 1 on subcutaneous transplantation of human gastric cancer cell MKN45 in nude mice.

Methods: 5×106 human gastric cancer MKN45 cells were injected into the left armpit of nude mice. Cut out the tumor pieces from MKN45 mice and place them in a glass dish filled with physiological saline. Peel off the surface blood vessels and remove the necrotic area. Cut the tumor pieces into 1-2 mm ³ and insert them into the nude mice with a trocar. After the tumor in the left armpit grew to an average volume of about 100 mm ³ , the animals were randomly divided into groups according to tumor volume and then administered. 32 mice were divided into 4 groups: G1 blank vehicle group (Control), G2BMS777607 30mg/kg group, G3 compound 1 15mg/kg group and G4 compound 1 30mg/kg group. There were 8 rats in each group, and each group was intragastrically administered the test substance of the corresponding concentration at a dosage volume of 10 mL/kg, once a day, with a 21-day dosing cycle.

Weigh and measure the tumor volume twice a week. Weigh the body weight on the 21st day. After measuring the tumor size, the mice are sacrificed and the tumor blocks are taken out and weighed. The relative tumor volume (RTV), relative tumor proliferation rate (T/C), and Tumor growth inhibition rate (TGI) and tumor inhibition percentage (IR), and SPSS was used for statistical analysis.

result:

At the end of the experiment, compared with the G1 blank vehicle group, there was no significant change in the body weight of mice in each administration group. See Table 4 and Figure 3 for details.

Table 4 Effect of compound 1 on body weight of nude mice

Compared with the G1 blank vehicle group, there was no significant difference between each group.

^Compared with the tumor volume of the G1 blank vehicle group of 2220±248mm3, the tumor volumes of the G2BMS777607 30mg/kg group, G3 compound 1 15mg/kg group and G4 compound 1 30mg/kg group were 1600±135 and 900±112 respectively (P<0.01 ) and 859±96mm3 (P<0.01). Compared with the RTV value of G1 blank solvent group of 24.25±3.24, the RTV of G2BMS777607 30mg/kg group, G3 compound 1 15mg/kg group and G4 compound 1 30mg/kg group The values were 16.92±1.17, 9.72±1.28 (P<0.05) and 8.94±0.72 (P<0.05) respectively; the T/C values were 69.78%, 40.07% and 36.87% respectively, and the TGI values were 30.22%, 59.93% and 63.13%. (See Figures 4 and 5 for details).

Compared with the tumor mass weight of the G1 blank vehicle group of 1.5907±0.2323g, the tumor mass weight of the G2BMS777607 30mg/kg group, G3 compound 1 15mg/kg group and G4 compound 1 30mg/kg group were 1.1525±0.1271 and 0.6556±0.0907 respectively (P< 0.05) and 0.5886±0.0840g (P<0.05), IR were 27.55%, 58.79% and 63.00% respectively. The effect of compound 1 on subcutaneous xenografts of human gastric cancer cells MKN45 (see Figure 6 for details).

Conclusion: Under the conditions of this experiment, Compound 1 administered intragastrically to nude mice at 15-30 mg/kg (q.d. for 21 days) can significantly inhibit the growth of human gastric cancer cell MKN45 subcutaneous transplanted tumors in nude mice in a dose-dependent manner, and the effect is much better than Positive control drug BMS777607.

Example 19 uses human liver microsomes to evaluate the inhibitory effect of Compound 1 of the present invention on 7 CYP450 isoforms (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4).

The inhibitors are CYP1A2: a-Naphthoflavone; CYP2C9: Sulfaphenazole; CYP2C19: Omeprazole; CYP3A4: Ketoconazole; CYP2D6: quinidine; CYP2C8: Nicardipine; CYP2B6: Clopidogrel;

The matrix is CYP1A2: Phenacetin at 30μM; CYP2C9: Diclofenac at 10μM; CYP2C19: S-Mephenytoin at 35μM; CYP3A4: Midazolam at 5μM; CYP2D6: Bufuralol at 10μM; CYP2C8: Paclitaxel at 10μM; CYP2B6: Bupropion at 70μM.

Detection system Human liver microsomes from Corning, incubation conditions CYP1A2, 2C9, 2D6, 2C8, 2B6: 10 minutes, 37℃; CYP2C19: 45 minutes, 37℃; CYP3A4: 5 minutes, 37℃. Sample size 2 copies (n=2 ), the bioanalytical method is LC-MS/MS.

Calculation method: Perform curve fitting using a Sigmoidal (nonlinear) dose-response model (GraphPad Prism 5.0 or Xlfit Model 205) to calculate IC50 based on data calculations using the following formula: Y=Bottom+(Top-Bottom)/(1+10 ^((LogIC50-X)*HillSlope)). where X is the logarithm of concentration. Y is the S-shaped response from bottom to top in response to inhibitor concentration from high to low. The results are shown in Table 5.

Table 5 Inhibitory effects of compound 1 of the present invention on 7 CYP450 subtypes

Compared with the prior art, the present invention has the following beneficial effects:

1) The Axl&c-Met dual inhibitor provided by the present invention has strong inhibitory activity against both AXL and c-Met. It can inhibit both GAS6/AXL and HGF/c-Met signaling pathways at the same time, and is expected to produce more significant effects. anti-tumor effect,

2) The inhibitory effect of the compound of the present invention on Axl and c-Met kinase is better than that of the positive control drug BMS777607. In the humanized gastric cancer MKN45 model, the Axl&c-Met dual inhibitor of the present invention showed significant anti-tumor activity, was significantly better than the positive control drug BMS777607, and was well tolerated by animals.

3) The Axl&c-Met dual inhibitor of the present invention has excellent pharmacokinetic properties and is expected to be developed into an anti-tumor drug; in particular, this series of inhibitors has a strong ability to penetrate the blood-brain barrier and in brain tissue With good exposure, it can be used to develop drugs for brain tumors such as glioma, brain metastases, and meningiomas.

4) The dual inhibitory effect of Axl&c-Met of the present invention is expected to overcome the resistance of tumor drugs.

5) The present invention also provides a simple preparation method for a series of compounds with dual Axl&c-Met inhibitory effects.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above. Those skilled in the art can make various variations or modifications within the scope of the claims, which does not affect the essence of the present invention.

Claims

For example, the compound represented by formula (I) or its pharmaceutically acceptable salt, its prodrug, its hydrate or solvate:

Wherein, R 1 is hydrogen, halogen, substituted or unsubstituted alkyl, alkoxy or haloalkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl;

R 2 is halogen, substituted or unsubstituted alkyl;

The value of n is 1 or 2.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof, wherein the alkyl group is a C1-C6 alkyl group, and the cycloalkyl group is C 3 -C 4 cycloalkyl group, the alkoxy group is a C 1 -C 6 alkoxy group, the hydroxyalkyl group is a C 1 -C 6 hydroxyalkyl group, and the substituent is fluorine or chlorine.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof, wherein R 1 is selected from hydrogen, methyl, methoxy, trifluoromethyl, Trifluoromethoxy, fluorine or chlorine; R 2 is selected from fluorine, chlorine, methyl.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof, wherein the compound is any one of the following compounds:
A pharmaceutical composition, characterized by comprising a pharmaceutically acceptable carrier and one or more therapeutically effective amounts of the compound of claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, or a hydrate thereof substance or solvate.
A method for preparing the compound of claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof, characterized in that the method includes the following steps:

The reaction produces a compound represented by formula (I).
The use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof in the preparation of anti-tumor drugs.
A compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate or a solvate thereof in the preparation of a medicament for treating related diseases caused by Axl kinase and/or c-Met kinase use.
The use according to claim 8, characterized in that the related diseases caused by the Axl kinase and/or c-Met kinase include brain tumors, gastric cancer, bladder cancer, breast cancer, colorectal cancer, head and neck cancer, and kidney cancer. , liver cancer, lung cancer, ovarian cancer, pancreatic adenocarcinoma/gallbladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, MFH/fibrosarcoma, glioblastoma/astrocytoma, melanoma or mesothelioma, psoriasis , liver cirrhosis, diabetes, angiogenesis, restenosis, ophthalmic diseases, rheumatoid arthritis and other inflammatory diseases, immune diseases, cardiovascular diseases such as arteriosclerosis and nephropathy.
The use according to claim 9, wherein the brain tumors include gliomas, meningiomas, and brain metastases.