Tyrosine kinase inhibitors and their pharmaceutical applications

Abstract

The present invention discloses a tyrosine kinase inhibitor and its pharmaceutical application. The tyrosine kinase inhibitor of the present invention is a quinazoline derivative with the following structural formula: #imgabs0# The present invention introduces a functional group containing XCH2(CH2)nC=O on the basis of the quinazoline structure, which can easily combine with the cysteine ​​thiol group through a nucleophilic reaction to form a covalent bond, thereby irreversibly inhibiting the activity of tyrosine kinase.

Description

Technical Field

[0001] The present invention relates to a tyrosine kinase inhibitor and application thereof in therapeutic drugs for inhibiting and treating diseases caused by overexpression of tyrosine kinase. Background Art

[0002] There are many signal pathway systems in cells, which interact to control cell proliferation, growth, metastasis and apoptosis. Tyrosine kinase plays a key role in signal transduction. The binding of human epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) can activate the activity of tyrosine kinase, leading to overexpression of EGF in various human solid tumors and causing uncontrollable cell division. Tyrosine kinase inhibitors (TKI) can block and control the activity of tyrosine kinase and control cell proliferation. Its mechanism of action is to compete with adenosine triphosphate (ATP) for the ATP binding site of kinase. Compared with traditional cytotoxic anticancer drugs, it has the characteristics of high selectivity and few adverse reactions. It has shown superiority in the treatment of chronic myeloid leukemia (CML), gastrointestinal stromal tumors (GIST), non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC).

[0003] At present, a variety of EGFR tyrosine kinase inhibitors have been developed as anti-tumor drugs. The first-class tyrosine kinase inhibitors, gefitinib and erlotinib, have good efficacy in treating recurrent and advanced non-small cell lung cancer. They belong to the aniline quinazoline class of compounds, which mainly act on EGFR. In order to improve the activity, the structure is generally modified in the following ways: adding lipophilic substituents to the 3-position substituent of aniline, such as introducing acetylene groups, or replacing H on aniline with F, Cl, Br or I atoms; connecting electronic groups at the 6th and 7th positions of the quinazoline ring; introducing N or O atoms at the 7th position of the quinazoline ring. The structures of various tyrosine kinase inhibitors containing quinazoline groups currently on the market all conform to the above structure-activity relationship.

[0004] The proportion of EGFR mutations in Chinese patients is relatively high and they are sensitive to EGFR-TKI treatment. However, the existence of drug resistance is a huge challenge faced clinically, and almost all patients who are effectively treated will experience disease progression after a period of remission.

[0005] EGFR mutations mostly occur in exons 18, 19, 20, and 21 of the gene, among which point mutations in exons 18 and 21 and deletion mutations in exon 19 are mainly associated with EGFR-TKI sensitivity. Clinical studies have shown that mutations in exons 19 and 21 account for about 90% of mutations in the tyrosine kinase region; gefitinib has a good therapeutic effect on patients with these mutations.

[0006] T790M is caused by the conversion of cytosine (C) at position 2369 of EGFR exon 20 to thymine (T), resulting in the substitution of threonine for methionine at position 790, which is associated with resistance to gefitinib or erlotinib. Threonine, as a "gatekeeper group", is located outside the core of the tyrosine kinase contact reaction and forms a highly affinity hydrogen bond with the aniline group of gefitinib, thereby ensuring that the drug is tightly bound to the tyrosine kinase and exerts an anti-tumor effect; once a mutation occurs, threonine is replaced by methionine, which introduces a larger amino acid side chain to the site to form a steric hindrance, thereby affecting the formation of hydrogen bonds between tyrosine kinase and gefitinib, and ultimately resulting in the inability of gefitinib to bind to it.

[0007] The first generation of tyrosine kinase inhibitors all work in a reversible inhibition (hydrogen bond formation) manner, so generally speaking, their selectivity is not good enough, their efficacy is not strong and lasting enough, and they are prone to drug resistance. The second generation (such as afatinib) and third generation (such as osimertinib) tyrosine kinase inhibitors are irreversible TKIs, which usually maintain the basic structural skeleton of reversible TKIs and connect an electrophilic functional group at the appropriate position, such as α, β-unsaturated aldehyde / ketone, acrylamide or alkyne. This electrophilic functional group can react electrophilically with the thiol group (electron-rich nucleophilic structure) on the cysteine ​​near the ATP binding domain to form a covalent bond. Compared with reversible TKIs, irreversible TKIs have many unique advantages. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a tyrosine kinase inhibitor which can irreversibly inhibit the activity of tyrosine kinase and inhibit and treat diseases caused by overexpression of tyrosine kinase.

[0009] The technical solution to achieve the first object of the present invention is a tyrosine kinase inhibitor, which is a quinazoline derivative, and has the following structural formula:

[0010]

[0011] wherein R1 is selected from an alkyl group containing from one to six carbon atoms, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group,

[0012] Phenyl, mono- and poly-substituted phenyl, trifluoromethyl, 2,2,2-trifluoroethyl, CH3O(CH2)n-, Wherein R is methyl, ethyl, isopropyl, trifluoromethyl, 2,2,2-trifluoroethyl, cyclopropyl, acetyl, acryloyl.

[0013] R2 and R3 are selected from halogen, hydrogen, amino, substituted amino, cyano, hydroxyl, sulfonic acid, sulfonamide, trifluoromethyl, 2,2,2-trifluoroethyl, methyl, methoxy, and ethynyl; the positions of R2 and R3 can be ortho, para, or meta.

[0014] Y is NH, O, S, ZN, wherein Z is selected from methyl, ethyl, isopropyl, trifluoromethyl, 2,2,2-trifluoroethyl, cyclopropyl.

[0015] X is selected from Cl, Br, F.

[0016] n represents an integer from 0 to 6.

[0017] Optionally, R1 is methyl, R2 is F, R3 is Cl, Y is NH, X is Br, and n=0; the tyrosine kinase inhibitor is 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-(N-bromoacetyl)aminoquinazoline.

[0018] The technical solution for achieving the second object of the present invention is the use of the above-mentioned tyrosine kinase inhibitor in the preparation of therapeutic drugs for inhibiting and treating diseases caused by overexpression of tyrosine kinase.

[0019] The active ingredient of the tyrosine kinase inhibitor of the present invention introduces a functional group containing XCH2(CH2)nC=O (X is a halogen atom, and n represents an integer of 0-6) on the basis of the quinazoline structure, which can easily combine with the cysteine ​​sulfhydryl group through a nucleophilic reaction to form a covalent bond, thereby irreversibly inhibiting the activity of tyrosine kinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the H NMR spectrum of the compound prepared in Example 1.

[0021] Figure 2 It is a statistical chart of the proliferation of mesothelioma 40L cells after pretreatment analyzed by plate cloning experiment. DETAILED DESCRIPTION

[0022] The basic reaction process for preparing the quinazoline derivatives of the present invention is as follows:

[0023]

[0024] The present invention will be further described below in conjunction with specific embodiments:

[0025] (Example 1)

[0026] Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-(N-bromoacetyl)aminoquinazoline, the steps are as follows:

[0027] (1) Preparation of 4-chloro-7-fluoro-6-nitroquinazoline.

[0028] 8.5 g of 4-hydroxy-7-fluoro-6-nitroquinazoline and 150 ml of phosphorus oxychloride were added to a 250 ml round-bottom flask, and the mixture was heated under reflux for 6 hours under stirring. Excess phosphorus oxychloride was removed by rotary evaporation, and then washed with 10:1 petroleum ether / ethyl acetate to obtain 8 g of solid 4-chloro-7-fluoro-6-nitroquinazoline, MS (m / z): 227.78 (M+1) + .

[0029] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline.

[0030] 2.3 g of 4-chloro-7-fluoro-6-nitroquinazoline, 1.6 g of 4-fluoro-3-chloroaniline, and 1.73 g of potassium carbonate were added to 30 ml of N, N-dimethylformamide, and the mixture was stirred and heated to 50 degrees. After 10 hours, the solvent was removed under reduced pressure distillation, 100 ml of ethyl acetate was added, and the mixture was washed with 30 ml of water, and the ethyl acetate was separated and dried over magnesium sulfate. Filtered and concentrated, the residue was separated by column separation with dichloromethane / methanol to obtain 3.0 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline, MS (m / z): 336.80 (M+1) + .

[0031] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-nitroquinazoline.

[0032] 3.3 g of 4-(4-fluoro-3-chlorophenylamino)7-fluoro-6-nitroquinazoline was dissolved in 100 ml of tetrahydrofuran, and 5 ml of 25% sodium methoxide methanol solution was added. The mixture was stirred at room temperature for 8 hours, and 50 ml of water was added. The mixture was extracted with 100 ml of ethyl acetate, and the ethyl acetate was separated and dried over magnesium sulfate. The mixture was filtered and concentrated, and the residue was separated by column chromatography with dichloromethane / methanol to obtain 2.5 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-nitroquinazoline. MS (m / z): 348.72 (M+1) + .

[0033] (4) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-aminoquinazoline.

[0034] 3.3 g of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-nitroquinazoline was dissolved in a mixed solvent of 150 ml of ethanol and 8 ml of water, and 1.6 g of ammonium chloride and 2.8 g of iron powder were added, and then heated under reflux for 5 hours under stirring. The filtrate was filtered and concentrated, and the obtained solid was washed with 15 ml of water to remove the ammonium chloride, and filtered and dried to obtain 2.6 g of the product, MS (m / z): 318.74 (M+1) + .

[0035] (5) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-(N-bromoacetyl)aminoquinazoline.

[0036] 160 mg of 4-(4-fluoro-3-chlorophenylamino)7-methoxy-6-aminoquinazoline and 129 mg of triethylamine were dissolved in 50 ml of dichloromethane, then cooled to 0 degrees, 120 mg of bromoacetyl bromide was slowly added dropwise, stirred for 3 hours, 10 ml of water was added, dichloromethane was separated and dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product, which was further purified by column to obtain 170 mg of the product.

[0037] The characterization of the prepared compound is as follows: MS (m / z): 438.80 (M+1) + .

[0038] H NMR spectrum Figure 1 .

[0039] H NMR (CD3OD / DMSO-D6): δ=8.92 (s, 1H), 8.55 (s, 1H), 8.12 (m, 1H), 7.79 (m, 1H), 7.38 (m, 1H), 7.30 (s, 1H), 4.28 (s, 2H), 4.06 (s, 3H).

[0040] In the figure, δ=4.04 is the peak of H2O, 3.13 is the peak of MeOH, and 2.60 is the peak of DMSO.

[0041] (Example 2)

[0042] Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-(N-chloroacetyl)aminoquinazoline, the steps are as follows:

[0043] 160 mg of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-aminoquinazoline and 129 mg of triethylamine were dissolved in 50 ml of dichloromethane, then cooled to 0 degrees, 68 mg of chloroacetyl chloride was slowly added dropwise, stirred for 5 hours, 10 ml of water was added, dichloromethane was separated and dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product, and further purified by column to obtain 120 mg of the product, MS (m / z): 394.68 (M+1) + .

[0044] (Example 3)

[0045] Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-(N-3'-chloropropionyl)aminoquinazoline, the steps are as follows:

[0046] 160 mg of 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-aminoquinazoline and 60 mg of p-dimethylaminopyridine were dissolved in 10 ml of N,N-dimethylformamide, 125 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 70 mg of 3-chloropropionic acid and 129 mg of N,N-diisopropylethylamine were added, stirred for 15 hours, 20 ml of water was added, extracted with 100 ml of ethyl acetate, the ethyl acetate was separated and dried with magnesium sulfate. Filtered and concentrated, the residue was separated by column separation with dichloromethane / methanol, and the product 4-(4-fluoro-3-chlorophenylamino)-7-methoxy-6-(N-3'-chloropropionyl)aminoquinazoline 55 mg was obtained, MS (m / z): 408.68 (M+1) + .

[0047] (Example 4)

[0048] Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-(N-bromoacetyl)aminoquinazoline, the steps are as follows:

[0049] (1) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-nitroquinazoline.

[0050] 6.0 g of propargyl alcohol was dissolved in 300 ml of tetrahydrofuran, 4.3 g of sodium hydride was slowly added in batches, and the mixture was stirred at room temperature for 1 hour, and then 13 g of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline was added. The mixture was stirred at room temperature for 5 hours, 100 ml of water was added to the reaction solution, and then extracted with 300 ml of ethyl acetate, the ethyl acetate was separated and dried with magnesium sulfate. Filtration and concentration were performed to obtain 2.5 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-nitroquinazoline, MS (m / z): 372.75 (M+1) + .

[0051] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-aminoquinazoline.

[0052] 10.0 g of 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-nitroquinazoline was dissolved in a mixed solvent of 250 ml of ethanol and 18 ml of water, and 4.5 g of ammonium chloride and 7.0 g of iron powder were added, followed by heating under reflux for 15 hours under stirring. The filtrate was filtered and concentrated, and the obtained solid was further purified by column to obtain 7.6 g of the product, MS (m / z): 342.76 (M+1) + .

[0053] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-(N-bromoacetyl)aminoquinazoline.

[0054] 343 mg of 4-(4-fluoro-3-chlorophenylamino)-7-propargyloxy-6-aminoquinazoline and 258 mg of triethylamine were dissolved in 100 ml of dichloromethane, then cooled to 0 degrees, 220 mg of bromoacetyl bromide was slowly added dropwise, stirred for 3 hours, 20 ml of water was added, dichloromethane was separated and dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product, which was further purified by column to obtain 300 mg of the product, MS (m / z): 462.69 (M+1) + .

[0055] (Example 5)

[0056] Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-phenol-6-(N-chloroacetyl)aminoquinazoline, the steps are as follows:

[0057] (1) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-phenyl-6-nitroquinazoline.

[0058] 2.0 g of 4-(4-fluoro-3-chlorophenylamino)-7-fluoro-6-nitroquinazoline was dissolved in 100 ml of tetrahydrofuran, and 2.2 g of sodium phenolate was added. The mixture was stirred at room temperature for 2 hours, and 50 ml of water was added. The mixture was extracted with 100 ml of ethyl acetate, and the ethyl acetate was separated and dried over magnesium sulfate. The mixture was filtered and concentrated, and the residue was separated by column chromatography with dichloromethane / methanol to obtain 1.5 g of the product 4-(4-fluoro-3-chlorophenylamino)-7-phenol-6-nitroquinazoline. MS (m / z): 410.79 (M+1) + .

[0059] (2) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-phenol-6-aminoquinazoline.

[0060] 1.5 g of 4-(4-fluoro-3-chlorophenylamino)-7-phenol-6-nitroquinazoline was dissolved in a mixed solvent of 100 ml of ethanol and 6 ml of water, and 2.0 g of ammonium chloride and 2.8 g of iron powder were added, and then heated under reflux for 5 hours under stirring. The filtrate was filtered and concentrated, and the obtained solid was washed with 15 ml of water and 20 ml of ether, and dried to obtain 1.0 g of the product, MS (m / z): 380.81 (M+1) + .

[0061] (3) Preparation of 4-(4-fluoro-3-chlorophenylamino)-7-phenolyl-6-(N-chloroacetyl)aminoquinazoline.

[0062] 160 mg of 4-(4-fluoro-3-chlorophenylamino)-7-phenol-6-aminoquinazoline and 129 mg of triethylamine were dissolved in 50 ml of dichloromethane, then cooled to 0 degrees, 120 mg of bromoacetyl bromide was slowly added dropwise, stirred for 3 hours, 10 ml of water was added, dichloromethane was separated and dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product, and further purified by column to obtain 170 mg of the product, MS (m / z): 456.74 (M+1) + .

[0063] (Example 6)

[0064] Preparation of 4-(3-ethynylphenylamino)-7-methoxy-6-(N-bromoacetyl)aminoquinazoline, the steps are as follows:

[0065] (1) Preparation of 4-(3-ethynylphenylamino)-7-fluoro-6-nitroquinazoline.

[0066] 2.3 g of 4-chloro-7-fluoro-6-nitroquinazoline, 1.3 g of 3-ethynylaniline, and 1.73 g of potassium carbonate were dissolved in 30 ml of N,N-dimethylformamide, and the mixture was stirred and heated to 50 degrees. After 10 hours, the solvent was removed under reduced pressure distillation, 100 ml of ethyl acetate was added, and the mixture was washed with 30 ml of water. The ethyl acetate was separated and dried over magnesium sulfate. The residue was filtered and concentrated, and the residue was separated by column separation with dichloromethane / methanol to obtain 2.1 g of the product 4-(3-ethynylphenylamino-7)-fluoro-6-nitroquinazoline, MS (m / z): 308.68 (M+1) + .

[0067] (2) Preparation of 4-(3-ethynylphenylamino)-7-methoxy-6-nitroquinazoline.

[0068] 1.3 g of 4-(3-ethynylphenylamino)7-fluoro-6-nitroquinazoline was dissolved in 100 ml of tetrahydrofuran, and 5 ml of 25% sodium methoxide methanol solution was added. The mixture was stirred at room temperature for 8 hours, and 50 ml of water was added. The mixture was extracted with 100 ml of ethyl acetate, and the ethyl acetate was separated and dried over magnesium sulfate. The mixture was filtered and concentrated, and the residue was separated by column chromatography with dichloromethane / methanol to obtain 0.8 g of the product 4-(3-ethynylphenylamino)7-methoxy-6-nitroquinazoline. MS (m / z): 320.70 (M+1) + .

[0069] (3) Preparation of 4-(3-ethynylphenylamino)-7-methoxy-6-aminoquinazoline.

[0070] 0.8 g of 4-(3-ethynylphenylamino)-7-methoxy-6-nitroquinazoline was dissolved in a mixed solvent of 50 ml of ethanol and 5 ml of water, and 0.6 g of ammonium chloride and 1.1 g of iron powder were added, and then heated under reflux for 5 hours under stirring. The filtrate was filtered and concentrated, and the obtained solid was washed with 10 ml of water to remove the ammonium chloride, and filtered and dried to obtain 0.5 g of the product, MS (m / z): 290.80 (M+1) + .

[0071] (4) Preparation of 4-(3-ethynylphenylamino)-7-methoxy-6-(N-bromoacetyl)aminoquinazoline.

[0072] 145 mg of 4-(3-ethynylphenylamino)-7-methoxy-6-aminoquinazoline and 129 mg of triethylamine were dissolved in 50 ml of dichloromethane, then cooled to 0 degrees, 120 mg of bromoacetyl bromide was slowly added dropwise, stirred for 3 hours, 10 ml of water was added, dichloromethane was separated and dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a crude product, and further purified by column to obtain 105 mg of the product, MS (m / z): 410.84 (M+1) + .

[0073] (Test Example 1)

[0074] The compound prepared in Example 1 was compared with gefitinib, and the results were as follows:

[0075] 1. Cell plate cloning experiment to detect cell proliferation

[0076] 1. Experimental steps:

[0077] (1) Mesothelioma cell line 40L was pretreated with 30 μM gefitinib and the drug of Example 1, respectively, and PBS was used as a blank control group;

[0078] (2) Collect 40L of cells from different treatment groups; count under a microscope, adjust the cell number to 200 cells / well (2 mL) with the corresponding complete culture medium, and seed them in a six-well plate and culture them in an incubator at 37°C with 5% CO2;

[0079] (3) Replace with fresh complete medium every 3 days;

[0080] (4) After continuous culture for about 7-10 days, the culture medium was discarded and the cells were carefully washed three times with 1× PBS. 1 mL of 4% paraformaldehyde was added to each culture dish to fix the cells for 30 minutes. The paraformaldehyde was discarded and the cells were washed three times with 1× PBS again. The cells were stained with crystal violet for 10 minutes at room temperature. The staining solution was discarded and the cells were washed three times with 1× PBS.

[0081] (5) Take photos with a camera and count and analyze the data.

[0082] 2. Experimental results

[0083] The effects of different drugs on the proliferation of 40L cells were analyzed by plate cloning experiments. After 10 days of experiments, the results were as follows: Figure 2 As shown in the figure, **P<0.01, ****P<0.0001, indicating that the drug of Example 1 has a significantly better ability to inhibit tumor cell proliferation than gefitinib.

Claims

1. A tyrosine kinase inhibitor having the following structural formula: .

2. Use of the tyrosine kinase inhibitor according to claim 1 in the preparation of a drug for treating mesothelioma.

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