CN111303121A

CN111303121A - 4-phenoxypyridine compound containing quinoxalinone and application thereof

Info

Publication number: CN111303121A
Application number: CN202010310205.5A
Authority: CN
Inventors: 刘举; 陈烨; 丁实; 史建涛; 宫益林; 李春艳; 朱翔龙; 赵雯雯; 王震
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2020-06-19

Abstract

The invention belongs to the field of medicines, and particularly relates to a 4-phenoxypyridine compound containing quinoxalinone and application thereof. The 4-phenoxypyridine compound containing the quinoxalinone has a structure shown in a general formula (I). The invention also relates to a compound with a general formula (I) which has a strong function of inhibiting c-Met kinase, provides a 4-phenoxypyridine compound containing quinoxalinone and pharmaceutically acceptable salts thereof, and application of the compound in preparing medicines for treating and/or preventing diseases caused by abnormal high expression of c-Met kinase, in particular application in preparing medicines for treating and/or preventing cancers.

Description

4-phenoxypyridine compound containing quinoxalinone and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a 4-phenoxypyridine compound containing quinoxalinone and pharmaceutically acceptable salts thereof, a preparation method thereof and a pharmaceutical composition containing the compound. The invention also relates to application of the compound and pharmaceutically acceptable salts thereof in preparing medicaments for treating diseases caused by abnormal high expression of c-Met kinase, in particular to application in preparing medicaments for treating and/or preventing cancers such as colon cancer, lung cancer and the like.

Background

The c-Met is a protein coded by the protooncogene c-Met, and after the Hepatocyte Growth Factor (HGF) is combined with the c-Met, an HGF/c-Met signal channel is started, so that the biological effects (such as embryonic development, tissue damage repair and the like) are exerted; the abnormal expression of c-Met is closely related to the occurrence, invasion, metastasis and drug resistance of tumors. HGF/Met has been internationally recognized as a target for anti-tumor. The research of the anti-tumor drugs based on the c-Met signal pathway enters a completely new stage, and particularly, the small-molecular c-Met kinase inhibitor becomes the key point and the hot point of the whole research of the anti-tumor drugs. At present, the discovered small molecule c-Met kinase inhibitor belongs to competitive inhibitors of ATP mostly, and the action mechanism of the inhibitor is to inhibit the c-Met kinase by blocking phosphorylation of tyrosine. 4-phenoxyquinoline compounds containing malonamide structures, wherein Cabozantinib (Cabozantinib, XL-184) has relatively good inhibition effect on c-Met/VEGFR-2, and IC₅₀The values are respectively 1.3nM and 0.035nM, and the compound also has certain inhibition effect on kinases such as Ret, Tie-2, Axl, Flt-3, Kit and Ron. FDA approval in us 11 months 2012 for cabozantinib to be marketed under the trade name Cometriq for the treatment of non-surgically resectable malignant locally advanced or metastatic Myeloid Thyroid Cancer (MTC). The FDA approved cabozantinib for marketing as a second line drug for treatment of advanced renal cancer at 4 months of 2016. 2017 application of FDA approval of cabozantinib tablets for advanced Renal Cell Carcinoma (RCC) expansion indication by month 12. Card with a detachable coverBotinib is currently the only Type II small molecule c-Met kinase inhibitor on the market. Scientists find in the reformation of cabozantinib that a cyclopropyl diamide side chain structure is reserved, a pyridine ring with a substituent at the 2-position is used for replacing a 6, 7-dimethoxy quinoline mother nucleus to obtain a series of 4-phenoxyl pyridine compounds, and the compounds have stronger inhibitory activity on c-Met kinase. Among them, Altiratinib is a c-Met kinase inhibitor developed by Deciphera pharmaceutical company, and is currently in the phase I clinical research stage. Has good research and development prospect for resisting tumors. On the other hand, the quinoxalinone compound is discovered by scientists as early as 1989 to have obvious in vitro anti-tumor effect, and echinomycin containing the quinoxalinone structure is approved as an anti-tumor drug to enter phase II clinical test in 1998. Two nitrogen atoms in the quinoxalinone structure are in mutual contraposition, the steric hindrance is small, the modifiability is strong, and the molecular skeleton is small, so the quinoxalinone structure is often used as an antitumor effect group and is introduced into an anticancer drug. At present, the following problems mainly exist in the research of c-Met inhibitors: the clinical application has large toxic and side effects, unsatisfactory clinical treatment effect and pharmacokinetic parameters, low oral bioavailability and the like. Therefore, the development of a novel c-Met kinase inhibitor with novel structure, safety and effectiveness is still the key field of research on antitumor drugs at home and abroad.

Disclosure of Invention

The invention aims to design and synthesize a series of novel 4-phenoxypyridine compounds containing quinoxalinone. In vitro activity screening shows that the compounds have antitumor activity and can be used for developing antitumor drugs.

In order to achieve the purpose, the invention provides a 4-phenoxypyridine compound containing quinoxalinone and pharmaceutically acceptable salts thereof, wherein the structural formula is shown as the general formula (I):

wherein:

R₁selected from hydrogen, C₁-C₁₀Alkyl radical, C₃-C₇A cycloalkyl group;

R₂is selected from 1 to 4 identical or different substituents from the group consisting of: hydrogen, halogen, C₁-C₁₀Alkyl, or C₁-C₄An alkoxy group;

ar is selected from C₆-C₁₀A membered aryl or 5-10 membered heteroaryl; said heteroaryl group containing 1 to 3 heteroatoms selected from N, O or S, and Ar is optionally substituted with 1 to 5 identical or different R₃Substituted;

said R₃Selected from hydrogen, halogen, hydroxyl, trifluoromethyl, nitro, amino, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkenyl radical, C₁-C₆Alkynyl, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio, C optionally substituted by hydroxy or amino or halogen₁-C₆An alkyl group.

D-E is a group containing an amide structure, and when D is an amino group (NH), E is a carbonyl group (C ═ O); when D is carbonyl (C ═ O), E is amino (NH).

Preferably, the aforementioned 4-phenoxypyridines containing quinoxalinones and pharmaceutically acceptable salts thereof,

R₁is selected from C₁-C₁₀Alkyl or C₃-C₇A cycloalkyl group;

R₂is selected from 1 to 4 identical or different substituents from the group consisting of: hydrogen or halogen;

ar is selected from C₆-C₁₀Aryl radicals, C₆-C₁₀A meta aralkyl radical, C₅-C₁₀A membered heteroaryl group; said heteroaryl group containing 1 to 3 heteroatoms selected from N, O or S, and Ar is optionally substituted with 1 to 5 identical or different R₃Substituted;

said R₃Selected from hydrogen, halogen, hydroxyl, trifluoromethyl, nitro, amino, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkenyl radical, C₁-C₆Alkynyl, C₁-C₆An alkoxy group.

R₁selected from methyl, ethyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;

R₂is selected from 1-2 identical or different substituents from the group consisting of: hydrogen or fluorine;

ar is selected from the group consisting of phenyl, pyridyl, thiophene, furan, naphthyl, quinolinyl, and indolyl, and Ar is optionally substituted with 1-5R, which may be the same or different₃Substituted;

R₁selected from methyl or cyclopropyl;

R₂is selected from 1-2 identical or different substituents from the group consisting of: hydrogen or fluorine

Ar is selected from phenyl, and Ar is optionally substituted with 1-5R, the same or different₃Substituted;

said R₃Hydrogen, halogen, methyl, trifluoromethyl, methoxy, etc.;

D-E is a group containing an amide structure, and when D is amino (NH), E is carbonyl (C ═ O); when D is carbonyl (C ═ O), E is amino (NH).

Preferably, the 4-phenoxypyridine compound containing the quinoxalinone structure and the pharmaceutically acceptable salt thereof have the following structural formula:

a pharmaceutical composition comprises the 4-phenoxypyridine compound containing the quinoxalinone and pharmaceutically acceptable salts thereof as active ingredients and pharmaceutically acceptable excipients.

Preferably, the 4-phenoxypyridine compound containing the quinoxalinone and the pharmaceutically acceptable salt thereof or the pharmaceutical composition are applied to the preparation of drugs for treating and/or preventing diseases caused by abnormal high expression of c-Met kinase.

Preferably, the 4-phenoxypyridine compound containing the quinoxalinone and the pharmaceutically acceptable salt thereof or the pharmaceutical composition are applied to the preparation of drugs for treating and/or preventing proliferative diseases.

Preferably, the 4-phenoxypyridine compound containing the quinoxalinone and the pharmaceutically acceptable salt thereof or the pharmaceutical composition are applied to the preparation of the drugs for treating and/or preventing cancers.

Preferably, for the above-mentioned use, the cancer is colon cancer and lung cancer.

Furthermore, the 4-phenoxypyridines containing quinoxalinones of the general formula (I) and configurational isomers of the present invention can form pharmaceutically acceptable salts with acids according to conventional methods in the art. Pharmaceutically acceptable addition salts include inorganic and organic acid addition salts, with the following acids being particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, succinic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, and the like.

"halogen" in the present invention means fluoro, chloro, bromo or iodo; "alkyl" refers to straight or branched chain alkyl; "alkylene" refers to straight or branched chain alkylene; "cycloalkyl" refers to a substituted or unsubstituted cycloalkyl; "aryl" refers to phenyl, naphthyl with no substituent or with a substituent attached; "heteroaryl" means a monocyclic or polycyclic ring system containing one or more heteroatoms selected from N, O, S, which ring system is aromatic, such as imidazolyl, pyridyl, pyrazolyl, (1,2,3) -and (1,2,4) -triazolyl, furyl, thienyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, naphthyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl and the like.

The invention has the beneficial effects that:

according to the invention, the c-Met enzyme activity test shows that the compound has significant c-Met kinase inhibition activity, and can be used for preparing c-Met inhibitor drugs, in particular for preparing drugs for treating and/or preventing cancers.

According to the invention, the in vitro inhibition activity tests of c-met high expression cell strains, namely human colon cancer cell HT-29, human lung cancer cell H460 and human lung adenocarcinoma cell strain A549 prove that the compound has a significant inhibition effect on lung cancer cells and human stomach cancer cells, and is particularly used for preparing medicines for treating and/or preventing colon cancer and lung cancer.

Detailed Description

The examples and preparations provided below further illustrate and exemplify the compounds of the present invention and their methods of preparation. It should be understood that the scope of the following examples and preparations are not intended to limit the scope of the invention in any way. The following examples are intended to illustrate, but not limit, the scope of the invention. The following synthetic schemes describe the preparation of the derivatives of formula (I) of the present invention, all starting materials are prepared by the following synthetic schemes, by methods well known to those of ordinary skill in the art of organic chemistry or are commercially available. All final derivatives of the invention are prepared by the synthetic routes described below or by methods analogous thereto, which are well known to those of ordinary skill in the art of organic chemistry. All variables used in the synthetic routes described below are as defined below or in the claims.

The derivatives of formula (I) according to the invention can be prepared according to the method of scheme 1 from the corresponding intermediates Q and the corresponding M by condensation;

according to the compounds of general formula (I) according to the present invention, intermediate M may be prepared by a process as shown in scheme 2, and the other substituents are as defined in the claims, wherein M-1 represents a class of substructures of intermediate M.

Compounds of general formula (I) according to the present invention, intermediate M, may also be prepared by a process as shown in scheme 3, the other substituents being as defined in the claims, wherein M-2 represents another class of substructures of intermediate M.

The compounds of general formula (I) according to the invention, intermediate Q, are prepared by a process as shown in scheme 4, the other substituents being as defined in the claims.

Substituents R of all intermediates in the four routes above₁、R₂D, E, Ar are as defined in the claims.

The examples are intended to illustrate, but not to limit, the scope of the invention. The nuclear magnetic resonance hydrogen spectrum of the compound is measured by BrukeraRx-600, and the mass spectrum is measured by Agilent 6460 QQQ; all reagents used were analytically or chemically pure.

EXAMPLE 1 preparation of Compound 1

Step 1 preparation of intermediate N- (4-chloropyridin-2-yl) cyclopropylcarboxamide

8.80g of 2-amino-4-chloropyridine and 20.80g of triethylamine were dissolved in 80mL of dichloromethane, and 30mL of a dichloromethane solution containing 9.30g of cyclopropylcarbonyl chloride was added dropwise to the solution under ice-bath conditions, and the temperature was raised to room temperature after completion of the addition. Stirring for 12h, and reacting, and mixing with 20% K₂CO₃Washing the solution and saturated saline solution for 3 times respectively, separating out an organic phase, drying by anhydrous sodium sulfate, filtering, evaporating the solvent to obtain a crude product, and separating by column chromatography to obtain a white solid N- (4-chloropyridine-2-yl) cyclopropylformamide.¹H NMR(600MHz,CDCl₃)δ8.79(s,1H),8.31(s,1H),8.16(d,J＝5.4Hz,1H),7.03(dd,J＝5.4,1.6Hz,1H),1.60–1.49(m,1H),1.17–1.09(m,2H),0.97–0.87(m,2H)；MS(ESI)m/z(％):197.1[M+H]⁺。

Step 2 preparation of intermediate N- [4- (2-fluoro-4-nitrophenoxy) pyridin-2-yl ] cyclopropanecarboxamide

8.00g of intermediate N- (4-chloropyridin-2-yl) cyclopropylcarboxamide and 15.98g of 2-fluoro-4-nitrophenol were added to 100mL of chlorobenzene and reacted at 140 ℃ for 40 hours. Cooling to room temperature, concentrating under reduced pressure, dissolving the residue in an appropriate amount of dichloromethane, adding₂CO₃Washing the solution and saturated saline solution for 3 times, separating organic phase, drying with anhydrous sodium sulfate, filtering, evaporating solvent to obtain brown solid, and performing column chromatography to obtain light yellow solid product N- [4- (2-fluoro-4-nitrophenoxy) pyridin-2-yl]Cyclopropyl carboxamide.¹HNMR(600MHz,DMSO-d₆)δ11.00(s,1H),8.43(m,1H),8.30(d,J＝5.7Hz,1H),8.19(m,1H),7.76(d,J＝2.2Hz,1H),7.61(t,J＝8.5Hz,1H),6.86(m,1H),2.04–1.95(m,1H),0.78(t,J＝6.3Hz,4H)。

Step 3 preparation of intermediate N- [4- (4-amino-2-fluorophenoxy) pyridin-2-yl ] cyclopropylcarboxamide

The intermediate N- [4- (2-fluoro-4-nitrophenoxy) pyridin-2-yl]6.00g of cyclopropyl formamide, 5.28g of iron powder and 11.36g of acetic acid are added into 100mL of ethyl acetate, 20mL of water is added, heating reflux is carried out for 2h, the reaction is finished, the hot reaction product is filtered, an organic phase is separated, anhydrous sodium sulfate is dried, the filtration is carried out, and the solvent is evaporated to dryness under reduced pressure to obtain a white solid N- [4- (4-amino-fluoro-2-fluorophenoxy) pyridin-2-yl]Cyclopropyl carboxamide.¹H NMR(600MHz,DMSO-d₆)δ10.79(s,1H),8.15(d,J＝5.7Hz,1H),7.59(s,1H),6.95(t,J＝9.0Hz,1H),6.67–6.61(m,1H),6.49(dd,J＝13.1,2.2Hz,1H),6.40(d,J＝8.7Hz,1H),5.44(s,2H),2.03–1.88(m,1H),0.76(br,4H)。

And 4, step 4: preparation of 2-nitro-N-phenylaniline

Dissolving 5.00g of aniline in 40mL of dry N, N-dimethylformamide, cooling the solution to below 0 ℃ in a ice salt bath, slowly adding 3.21g of 60% sodium hydride, keeping the temperature and stirring for 0.5h, dropwise adding 30mL of 2-fluoronitrobenzene solution dissolved in N, N-dimethylformamide into the solution, and after dropwise adding, heating to room temperature and reacting for 20 h. The reaction solution was slowly poured into 300mL of a saturated aqueous solution of ammonium chloride to precipitate a solid, which was then filtered and dried to obtain 9.2g of a yellow product with a yield of 80%. MS (ESI) M/z (%): 213.3[ M-H ] -.

And 5: preparation of N-phenyl o-phenylenediamine

Taking 8.0g of intermediate 2-nitro-N-phenylaniline, dissolving in 80mL of ethyl acetate, adding 30mL of water, 10.43g of iron powder and 22.4g of glacial acetic acid into the solution, carrying out reflux reaction for 6h, filtering while the solution is hot, separating an ethyl acetate layer, washing twice, drying with anhydrous sodium sulfate, and evaporating under reduced pressure to obtain 5.2g of brown product. ESI-MS [ M + H ] (M/z): 185.3.

And 5: preparation of ethyl 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylate

Taking 5.60g of intermediate N-phenyl o-phenylenediamine and 6.4g of ketomalonic acid diethyl ester monohydrate, adding 90mL of toluene, carrying out reflux reaction in a Dean-Stark water separator for 12h, removing the solvent by reduced pressure evaporation after the reaction is finished, dissolving the residue in 150mL of ethyl acetate, washing twice, drying with anhydrous sodium sulfate, and evaporating to dryness. Column chromatography separation to obtain 7.9g of off-white product. MS (ESI) M/z (%): 295.0[ M + H]⁺.

Step 6: preparation of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid

Taking 5.00g of intermediate 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-ethyl formate, dissolving in 60mL of tetrahydrofuran, adding 15mL of water, adding 1.44g (34.00mmol) of lithium hydroxide monohydrate under stirring, stirring at room temperature for reacting for 2h, and finishing the reaction. Removing THF by reduced pressure evaporation, adding 30mL of water, extracting with ethyl acetate twice, adjusting pH of the water phase with 6mol/L hydrochloric acid to 2, separating out a white solid, filtering, washing the filter cake with water, and drying to obtain 4.0g of a 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-formic acid product. MS (ESI) M/z (%): 265.1[ M-H]^—。

And 7: preparation of Compound 1

The intermediate 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid 2.11g, the intermediate N- [4- (4-amino-2-fluorophenoxy) pyridin-2-yl are added to a reaction flask]1.90g of cyclopropyl formamide, 3.02g of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU for short) and triethylamine1.00g, 10mL of N, N-dimethylformamide, stirring at room temperature for 12h, and completing the reaction. The reaction solution was poured into 150mL of 20% aqueous potassium carbonate solution, extracted three times with 100mL of dichloromethane, the organic phases were combined, the organic phase was washed three times with 20% aqueous potassium carbonate solution, the organic layer was washed twice with saturated brine, and the separated organic layer was dried over anhydrous sodium sulfate. Filtering, distilling under reduced pressure to remove dichloromethane, and recrystallizing with methanol to obtain white solid;¹H NMR(600MHz,DMSO-d₆)δ11.29(s,1H),10.87(s,1H),8.21(d,J＝5.7Hz,1H),8.03(d,J＝8.0Hz,1H),7.93(dd,J＝12.6,2.2Hz,1H),7.70(t,J＝7.6Hz,2H),7.68–7.57(m,3H),7.54(d,J＝8.9Hz,1H),7.48(t,J＝8.1Hz,3H),7.41(t,J＝8.9Hz,1H),6.74(dd,J＝5.7,2.4Hz,1H),6.68(d,J＝8.5Hz,1H),2.04–1.90(m,1H),0.89–0.65(m,4H)；MS(ESI)m/z(％):536.1[M+H]⁺,558.1[M+Na]⁺。

EXAMPLE 2 preparation of Compound 2

Example 2 (Compound 2) was finally obtained according to the preparation scheme of example 1 by substituting 5.97g of 4-fluoroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,CDCl₃)δ11.91(s,1H),8.32(d,J＝7.0Hz,1H),8.19–8.08(m,2H),7.98(dd,J＝12.1,2.3Hz,1H),7.79(s,1H),7.63–7.56(m,1H),7.55–7.49(m,1H),7.45–7.33(m,5H),7.15(t,J＝8.6Hz,1H),6.80(d,J＝8.3Hz,1H),6.60(dd,J＝5.7,2.2Hz,1H),1.52–1.47(m,1H),0.90–0.81(m,4H)；MS(ESI)m/z(％):554.1[M+H]⁺,576.1[M+Na]⁺.

EXAMPLE 3 preparation of Compound 3

Example 3 (Compound 3) was finally obtained according to the preparation scheme of example 1 by substituting 6.85g of 4-chloroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,CDCl₃)δ11.88(s,1H),8.30(t,J＝7.2Hz,1H),8.25(s,1H),8.11(d,J＝5.8Hz,1H),7.97(dd,J＝12.1,2.3Hz,1H),7.79(d,J＝1.6Hz,1H),7.69(d,J＝8.6Hz,2H),7.61–7.56(m,1H),7.55–7.49(m,1H),7.40(d,J＝8.8Hz,1H),7.32(d,J＝8.6Hz,2H),7.20–7.13(m,1H),6.81(d,J＝8.4Hz,1H),6.60(dd,J＝5.8,2.3Hz,1H),1.54–1.47(m,1H),1.09–1.03(m,2H),0.89–0.83(m,2H)；MS(ESI)m/z(％):569.3[M]⁺,592.1[M+Na]⁺.

EXAMPLE 4 preparation of Compound 4

Example 4 (Compound 4) was finally obtained according to the preparation scheme of example 1 by substituting 5.97g of 2-fluoroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,CDCl₃)δ11.84(s,1H),8.40–8.26(m,2H),8.11(d,J＝5.8Hz,1H),7.98(dd,J＝12.1,2.3Hz,1H),7.80(d,J＝1.7Hz,1H),7.72–7.64(m,1H),7.63–7.57(m,1H),7.56–7.51(m,1H),7.50–7.38(m,4H),7.15(t,J＝8.7Hz,1H),6.82(d,J＝8.4Hz,1H),6.59(dd,J＝5.8,2.3Hz,1H),1.55–1.46(m,1H),1.09–1.04(m,2H),0.89–0.82(m,2H)；MS(ESI)m/z(％):554.1[M+H]⁺,576.1[M+Na]⁺.

EXAMPLE 5 preparation of Compound 5

Example 5 (Compound 5) was finally obtained according to the preparation scheme of example 1 by substituting 6.85g of 2-chloroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,CDCl₃)δ11.87(s,1H),8.63(s,1H),8.32(d,J＝8.0Hz,1H),8.11(d,J＝5.8Hz,1H),7.98(dd,J＝12.1,2.1Hz,1H),7.81(s,1H),7.77–7.69(m,1H),7.62(s,3H),7.53(t,J＝7.5Hz,1H),7.42(dd,J＝10.3,5.0Hz,2H),7.15(t,J＝8.6Hz,1H),6.68(d,J＝8.3Hz,1H),6.59(dd,J＝5.7,2.1Hz,1H),1.57–1.47(m,1H),1.07(dd,J＝7.1,4.0Hz,2H),0.88–0.82(m,2H)；MS(ESI)m/z(％):570.1[M+H]⁺,592.1[M+Na]⁺.

EXAMPLE 6 preparation of Compound 6

Example 6 (Compound 6) was finally obtained according to the preparation scheme of example 1 by substituting 5.97g of 3-fluoroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,CDCl₃)δ11.86(s,1H),8.46(s,1H),8.31(d,J＝7.9Hz,1H),8.11(d,J＝5.7Hz,1H),7.97(dd,J＝12.1,2.1Hz,1H),7.80(s,1H),7.74–7.67(m,1H),7.59(t,J＝7.4Hz,1H),7.52(t,J＝7.5Hz,1H),7.43–7.35(m,2H),7.23–7.08(m,3H),6.80(d,J＝8.4Hz,1H),6.59(dd,J＝5.7,2.1Hz,1H),1.57–1.47(m,1H),1.09–1.02(m,2H),0.89–0.81(m,2H)；MS(ESI)m/z(％):554.1[M+H]⁺,576.1[M+Na]⁺.

EXAMPLE 7 preparation of Compound 7

Example 7 (Compound 7) was finally obtained according to the preparation scheme of example 1 by substituting 6.61g of 4-methoxyaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.29(s,1H),10.87(s,1H),8.21(d,J＝5.7Hz,1H),8.01(dd,J＝8.0,1.1Hz,1H),7.93(dd,J＝12.6,2.3Hz,1H),7.66(d,J＝2.2Hz,1H),7.63–7.57(m,1H),7.54(dd,J＝8.9,1.4Hz,1H),7.50–7.44(m,1H),7.44–7.34(m,3H),7.30–7.15(m,2H),6.79–6.68(m,2H),3.87(d,J＝9.4Hz,3H),2.14–1.71(m,1H),0.98–0.47(m,4H)；MS(ESI)m/z(％):566.1[M+H]⁺,588.1[M+Na]⁺.

EXAMPLE 8 preparation of Compound 8

Example 8 (Compound 8) was finally obtained according to the preparation scheme of example 1 by substituting 8.22g of 2, 4-dimethoxyaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.28(s,1H),10.87(s,1H),8.21(d,J＝5.7Hz,1H),8.00(d,J＝7.0Hz,1H),7.92(dd,J＝12.6,2.3Hz,1H),7.65(d,J＝2.1Hz,1H),7.62–7.58(m,1H),7.55(d,J＝10.1Hz,1H),7.46(t,J＝7.7Hz,1H),7.41(t,J＝8.9Hz,1H),7.29(d,J＝8.6Hz,1H),6.89(d,J＝2.5Hz,1H),6.80–6.71(m,3H),3.89(s,3H),3.72(s,3H),2.02–1.88(m,1H),0.84–0.70(m,4H)；MS(ESI)m/z(％):596.2[M+H]⁺,618.1[M+Na]⁺.

EXAMPLE 9 preparation of Compound 9

Example 9 (Compound 9) was finally obtained according to the preparation scheme for example 1 by substituting 9.24g of 4-bromoaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.26(s,1H),10.88(s,1H),8.21(d,J＝5.7Hz,1H),8.03(dd,J＝8.0,1.2Hz,1H),7.98–7.82(m,3H),7.65(d,J＝2.3Hz,1H),7.63–7.57(m,1H),7.55(dd,J＝8.9,1.4Hz,1H),7.51–7.44(m,3H),7.41(t,J＝8.9Hz,1H),6.84–6.63(m,2H),2.02–1.92(m,1H),0.81–0.74(m,4H)；MS(ESI)m/z(％):636.0[M+Na]⁺.

EXAMPLE 10 preparation of Compound 10

Example 10 (Compound 10) was finally obtained according to the preparation scheme of example 1 by substituting 6.85g of 3-chloroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.26(s,1H),10.88(s,1H),8.21(d,J＝5.7Hz,1H),8.08–7.99(m,1H),7.94(dd,J＝12.6,2.3Hz,1H),7.77–7.70(m,2H),7.69(s,1H),7.66(d,J＝2.2Hz,1H),7.64–7.59(m,1H),7.58–7.54(m,1H),7.51–7.46(m,2H),7.41(t,J＝8.9Hz,1H),6.97–6.49(m,2H),2.09–1.79(m,1H),0.98–0.52(m,4H)；MS(ESI)m/z(％):569.3[M]⁺,592.1[M+Na]⁺.

EXAMPLE 11 preparation of Compound 11

Example 11 (Compound 11) was finally obtained according to the preparation scheme of example 1 by substituting 5.75g of 4-methylaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.28(s,1H),10.88(s,1H),8.21(d,J＝5.7Hz,1H),8.02(dd,J＝8.0,1.2Hz,1H),7.93(dd,J＝12.6,2.3Hz,1H),7.66(d,J＝2.3Hz,1H),7.62–7.57(m,1H),7.54(dd,J＝8.8,1.4Hz,1H),7.52–7.45(m,3H),7.41(t,J＝8.9Hz,1H),7.33(d,J＝8.2Hz,2H),6.85–6.59(m,2H),2.45(s,3H),2.04–1.88(m,1H),0.82–0.72(m,4H)；MS(ESI)m/z(％):550.2[M+H]⁺,572.1[M+Na]⁺.

EXAMPLE 12 preparation of Compound 12

Example 12 (compound 12) was finally obtained according to the preparation scheme of example 1 by substituting 8.70g of 2, 4-dichloroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.26(s,1H),10.88(s,1H),8.21(d,J＝5.7Hz,1H),8.15–8.03(m,2H),7.93(dd,J＝12.6,2.3Hz,1H),7.79(dd,J＝8.5,2.3Hz,1H),7.76–7.69(m,1H),7.70–7.59(m,2H),7.61–7.54(m,1H),7.52(dd,J＝11.2,4.1Hz,1H),7.42(t,J＝8.9Hz,1H),6.79–6.68(m,2H),2.01–1.93(m,1H),0.82–0.72(m,4H)；MS(ESI)m/z(％):626.0[M+Na]⁺.

EXAMPLE 13 preparation of Compound 13

Example 13 (Compound 13) was finally obtained according to the preparation scheme of example 1 by substituting acetyl chloride 6.98g for cyclopropylcarbonyl chloride 9.30g in step 1 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.29(s,1H),10.57(s,1H),8.20(d,J＝5.7Hz,1H),8.09–7.99(m,1H),7.94(dd,J＝12.6,2.3Hz,1H),7.75–7.58(m,5H),7.55(dd,J＝8.8,1.3Hz,1H),7.52–7.45(m,3H),7.42(t,J＝8.9Hz,1H),6.77–6.58(m,2H),2.05(s,3H).

EXAMPLE 14 preparation of Compound 14

Example 14 (compound 14) was finally obtained according to the preparation scheme of example 1 by substituting 9.30g of cyclopropylcarbonyl chloride in step 1 of example 1 with 6.98g of acetyl chloride and 5.00g of aniline in step 4 of example 1 with 5.97g of 4-fluoroaniline.¹H NMR(600MHz,DMSO-d₆)δ11.28(s,1H),10.57(s,1H),8.20(d,J＝5.7Hz,1H),8.07–8.00(m,1H),7.94(dd,J＝12.6,2.3Hz,1H),7.67(s,1H),7.64–7.58(m,1H),7.55(dd,J＝8.8,3.9Hz,5H),7.48(t,J＝7.6Hz,1H),7.42(t,J＝8.9Hz,1H),6.77–6.67(m,2H),2.04(s,3H).

EXAMPLE 15 preparation of Compound 15

Example 15 (Compound 15) was finally obtained according to the preparation scheme of example 1 by substituting 9.30g of cyclopropylcarbonyl chloride in step 1 of example 1 with 6.98g of acetyl chloride and 5.00g of aniline in step 4 of example 1 with 6.85g of 2-chloroaniline.¹H NMR(600MHz,DMSO-d₆)δ11.28(s,1H),10.58(s,1H),8.20(d,J＝5.7Hz,1H),8.12–8.03(m,1H),7.94(dd,J＝12.6,2.3Hz,1H),7.89–7.82(m,1H),7.74–7.60(m,5H),7.57(dt,J＝18.2,9.0Hz,1H),7.51(t,J＝7.6Hz,1H),7.43(t,J＝8.9Hz,1H),6.71(dd,J＝5.7,2.4Hz,1H),6.62(d,J＝8.4Hz,1H),2.05(s,3H).

EXAMPLE 16 preparation of Compound 16

Example 16 (Compound 16) was finally obtained according to the preparation scheme of example 1 by substituting 9.30g of cyclopropylcarbonyl chloride in step 1 of example 1 with 6.98g of acetyl chloride and 5.00g of aniline in step 4 of example 1 with 6.61g of 4-methoxyaniline.¹H NMR(600MHz,DMSO-d₆)δ11.29(s,1H),10.57(s,1H),8.20(d,J＝5.7Hz,1H),8.02(dd,J＝8.0,1.1Hz,1H),7.94(dd,J＝12.7,2.3Hz,1H),7.68(s,1H),7.63–7.58(m,1H),7.57–7.52(m,1H),7.50–7.45(m,1H),7.44–7.35(m,3H),7.22(d,J＝8.9Hz,2H),6.78–6.65(m,2H),3.88(s,3H),2.05(s,3H)；MS(ESI)m/z(％):562.1[M+Na]⁺.

EXAMPLE 17 preparation of Compound 17

Example 17 (compound 17) was finally obtained according to the preparation scheme of example 1 by substituting 14.15g of 4-nitrophenol for 15.98g of 2-fluoro-4-nitrophenol in step 2 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.10(s,1H),10.82(s,1H),8.19(d,J＝5.7Hz,1H),8.02(dd,J＝8.0,1.2Hz,1H),7.81(d,J＝8.9Hz,2H),7.70(t,J＝7.6Hz,2H),7.68–7.61(m,2H),7.62–7.56(m,1H),7.51–7.42(m,3H),7.21(d,J＝8.9Hz,2H),6.76–6.61(m,2H),2.02–1.90(m,1H),0.86–0.68(m,4H)；MS(ESI)m/z(％):540.1[M+Na]⁺.

EXAMPLE 18 preparation of Compound 18

Example 18 (Compound 18) was finally obtained according to the preparation scheme for example 1 by substituting 14.15g of 4-nitrophenol for 15.98g of 2-fluoro-4-nitrophenol in step 2 of example 1 and 5.97g of 4-fluoroaniline for 5.00g of aniline in step 4 of example 1.¹H NMR(600MHz,DMSO-d₆)δ11.09(s,1H),10.82(s,1H),8.19(d,J＝5.7Hz,1H),8.02(dd,J＝8.0,1.1Hz,1H),7.81(d,J＝8.9Hz,2H),7.65(d,J＝2.3Hz,1H),7.64–7.57(m,1H),7.58–7.50(m,4H),7.48(t,J＝7.6Hz,1H),7.21(d,J＝8.9Hz,2H),6.76–6.64(m,2H),2.05–1.84(m,1H),0.85–0.65(m,4H)；MS(ESI)m/z(％):558.1[M+Na]⁺.

EXAMPLE 19 preparation of Compound 19

Example 19 (Compound 19) was finally obtained according to the preparation scheme of example 1 by substituting 9.30g of cyclopropylcarbonyl chloride in step 1 of example 1 with 6.98g of acetyl chloride and 15.98g of 2-fluoro-4-nitrophenol in step 2 of example 1 with 14.15g of 4-nitrophenol.¹H NMR(600MHz,DMSO-d₆)δ11.10(s,1H),10.52(s,1H),8.18(d,J＝5.7Hz,1H),8.02(d,J＝7.9Hz,1H),7.82(d,J＝8.9Hz,2H),7.74–7.56(m,5H),7.51–7.42(m,3H),7.21(d,J＝8.9Hz,2H),6.71–6.61(m,2H),2.04(s,3H)；MS(ESI)m/z(％):492.2[M+H]⁺,514.2[M+Na]⁺.

EXAMPLE 20 preparation of Compound 20

Example 20 (Compound 20) was finally prepared according to the preparation scheme of example 1 by substituting 9.30g of cyclopropylcarbonyl chloride in step 1 of example 1 with 6.98g of acetyl chloride, substituting 15.98g of 2-fluoro-4-nitrophenol in step 2 of example 1 with 14.15g of 4-nitrophenol, and substituting 5.00g of aniline in step 4 of example 1 with 6.85g of 2-chloroaniline. MS (ESI) M/z (%): 526.2[ M + H]⁺；548.2.2[M+Na]⁺.

EXAMPLE 21 preparation of Compound 21

Example 21 (Compound 21) was finally obtained according to the preparation scheme of example 1 by substituting 9.30g of cyclopropylcarbonyl chloride in step 1 of example 1 with 6.98g of acetyl chloride and 15.98g of 2-fluoro-4-nitrophenol in step 2 of example 1 with 14.15g of 4-nitrophenol.¹H NMR(600MHz,DMSO-d₆)δ11.09(s,1H),10.52(s,1H),8.18(d,J＝5.7Hz,1H),8.02(dd,J＝8.0,1.1Hz,1H),7.82(d,J＝8.9Hz,2H),7.67(s,1H),7.65–7.43(m,6H),7.21(d,J＝8.9Hz,2H),6.71(d,J＝8.5Hz,1H),6.66(dd,J＝5.7,2.4Hz,1H),2.04(s,3H)

EXAMPLE 22 preparation of Compound 22

Step 1: preparation of 4-chloro-N-methylpyridine formamide

20g of 2-picolinic acid, 2.6g of sodium bromide and 30mL of chlorobenzene are placed in a 500m bottle, after the temperature is raised to 50 ℃, 48mL of thionyl chloride is slowly dripped, stirring is continued for 30 minutes after the dripping is finished, the temperature is raised to 85 ℃, and reflux is carried out for 20 hours. Most of the liquid was distilled off under reduced pressure, 30mL of toluene was added and stirred for 5min, and after the solvent was distilled off, 4-chloropyridoformyl chloride was obtained as a yellow oily liquid, and 100mL of toluene was added and the solution was used directly in the next reaction. Slowly dripping the prepared toluene solution of 4-chloropyridoformyl chloride into 60mL of 35% methylamine water solution under the condition of ice-water bath, stirring at room temperature for 8h after dripping, carrying out suction filtration to separate toluene layers, extracting the water layers twice by using 50mL of toluene respectively, combining all the collected toluene layers, and evaporating the solvent to dryness under reduced pressure to obtain brown oily liquid. The oily liquid is dissolved in 150mL tetrahydrofuran, 50mL concentrated hydrochloric acid is slowly added under the temperature controlled below 20 ℃, and after the addition is finished, the mixture is stirred for 1 hour at the same temperature and filtered. Dissolving the filter cake in 80mL of water, adjusting the pH to be neutral by using a 20% NaOH solution, controlling the temperature to be 15-20 ℃, stirring for 1h at the same temperature, precipitating a light yellow solid, and performing suction filtration, washing and drying to obtain the 4-chloro-N-methylpyridine formamide.¹H NMR(400MHz,DMSO-d₆)δ8.85(s,1H),8.63(d,J＝5.2Hz,1H),8.03(s,1H),7.76(d,J＝5.2Hz,1H),2.84(d,J＝4.8Hz,3H)。

Step 2: preparation of 4- (4-aminophenoxy) -N-methylpyridine carboxamide

10.00g of 4-chloro-N-methylpyridine formamide and 8.30g of p-hydroxyaniline obtained in the above step were dissolved in 50mL of DMF, and potassium tert-butoxide 13 was added thereto with stirring at room temperature.20 g. After the addition, the temperature is raised to 90 ℃ and the reflux is carried out for 5 to 10 hours. And cooling the reaction liquid to 10-20 ℃, adding concentrated hydrochloric acid at the same temperature until a large amount of brown solid is separated out, then stirring at the same temperature for 1 hour, carrying out suction filtration, placing the filter cake in 200mL of dichloromethane, stirring for 30 minutes, carrying out suction filtration, and leaching the filter cake with a small amount of dichloromethane. Dissolving the filter cake in water, adjusting the pH value to 7-8 by using a 20% NaOH aqueous solution at 10-20 ℃, separating out brown oily substances, stirring at the same temperature until the oily substances are solidified, carrying out suction filtration after solidification, placing the filter cake in a 5% NaOH aqueous solution, stirring at room temperature for 20min, carrying out suction filtration, washing with a large amount of water until the filtrate is colorless, obtaining a gray solid, carrying out suction filtration, and drying to obtain the 4- (4-aminophenoxy) -N-methylpyridine formamide. MS (ESI) M/z (%): 244.10[ M + H]⁺。

And step 3: preparation of Compound 22

Replacement of N- [4- (4-amino-2-fluorophenoxy) pyridin-2-yl in step 7 of example 1 with 1.61g of 4- (4-aminophenoxy) -N-methylpyridine carboxamide]Cyclopropyl formamide 1.90g, following the procedure for step 7 in example 1, finally gave example 22 (compound 22).¹H NMR(600MHz,DMSO-d₆)δ11.14(s,1H),8.78(d,J＝4.8Hz,1H),8.52(d,J＝5.6Hz,1H),8.07–7.97(m,1H),7.85(d,J＝8.9Hz,2H),7.74–7.56(m,4H),7.51–7.41(m,4H),7.27(d,J＝8.9Hz,2H),7.16(dd,J＝5.6,2.6Hz,1H),2.79(d,J＝4.8Hz,3H).

EXAMPLE 23 preparation of Compound 23

Example 23 (Compound 23) was finally obtained according to the preparation scheme of example 22 by replacing 2.11g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid of step 3 in example 22 with 2.25g of 3-oxo-4- (4-fluorophenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid.¹H NMR(600MHz,DMSO-d₆)δ11.13(s,1H),8.78(d,J＝4.7Hz,1H),8.52(d,J＝5.6Hz,1H),8.03(d,J＝7.8Hz,1H),7.86(d,J＝8.8Hz,2H),7.71–7.36(m,7H),7.27(d,J＝8.8Hz,2H),7.16(dd,J＝5.5,2.5Hz,1H),6.72(d,J＝8.4Hz,1H),2.80(d,J＝4.8Hz,3H).

EXAMPLE 24 preparation of Compound 24

Example 24 (Compound 24) was finally obtained according to the preparation scheme of example 22 by replacing 2.11g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid of step 3 in example 22 with 2.25g of 3-oxo-4- (2-fluorophenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid. MS (ESI) M/z (%): 510.2[ M + H]⁺；532.2[M+Na]⁺.

EXAMPLE 25 preparation of Compound 25

Example 25 (Compound 25) was finally obtained according to the preparation scheme of example 22 by replacing 2.11g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid of step 3 in example 22 with 2.38g of 3-oxo-4- (4-chlorophenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid.¹H NMR(600MHz,DMSO-d₆)δ11.12(s,1H),8.78(d,J＝4.8Hz,1H),8.52(d,J＝5.6Hz,1H),8.11–7.96(m,1H),7.81(dd,J＝48.8,8.7Hz,4H),7.63–7.40(m,5H),7.27(d,J＝8.9Hz,

2H),7.21–7.11(m,1H),6.73(d,J＝8.3Hz,1H),2.80(d,J＝4.8Hz,3H).

EXAMPLE 26 preparation of Compound 26

Example 26 (Compound 26) was finally prepared according to the preparation scheme of example 22 by replacing 60mL of the 35% aqueous methylamine solution of step 1 in example 22 with 35.21g of cyclopropylamine in 60mL of an aqueous solution, replacing 8.30g of p-hydroxyaniline of step 2 in example 22 with 9.67g of 4-amino-2-fluorophenol, replacing 2.38g of 3-oxo-4- (4-chlorophenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid with 2.38g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid in example 22)。¹H NMR(600MHz,DMSO-d₆)δ11.33(s,1H),8.76(d,J＝4.4Hz,1H),8.52(d,J＝5.5Hz,1H),8.03(d,J＝7.8Hz,1H),7.98(d,J＝12.1Hz,1H),7.76–7.67(m,2H),7.66–7.56(m,3H),7.54–7.38(m,5H),7.22(d,J＝2.9Hz,1H),6.68(d,J＝8.4Hz,1H),2.97–2.79(m,1H),0.79–0.56(m,4H)；MS(ESI)m/z(％):536.1[M+H]⁺,558.1[M+Na]⁺.

EXAMPLE 27 preparation of Compound 27

Example 27 (Compound 27) was finally prepared according to the preparation scheme of example 22 by replacing 60mL of the 35% aqueous methylamine solution of step 1 in example 22 with 35.21g of cyclopropylamine in 60mL of an aqueous solution, replacing 8.30g of p-hydroxyaniline of step 2 in example 22 with 9.67g of 4-amino-2-fluorophenol, and replacing 2.11g of 3-oxo-4- (2-chlorophenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid of step 3 in example 22 with 2.38g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid. MS (ESI) M/z (%): 592.1[ M + Na [)]⁺；608.1[M+K]⁺.

EXAMPLE 28 preparation of Compound 28

Example 28 (Compound 28) was finally prepared according to the preparation scheme of example 22 by replacing 60mL of the 35% aqueous methylamine solution of step 1 in example 22 with 35.21g of cyclopropylamine in 60mL of an aqueous solution, replacing 8.30g of p-hydroxyaniline of step 2 in example 22 with 9.67g of 4-amino-2-fluorophenol, and replacing 2.11g of 3-oxo-4- (4-fluorophenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid of step 3 in example 22 with 2.25g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid.¹HNMR(600MHz,DMSO-d₆)δ11.32(s,1H),8.76(d,J＝5.0Hz,1H),8.52(d,J＝5.6Hz,1H),8.10–7.91(m,2H),7.68–7.37(m,9H),7.22(dd,J＝5.6,2.6Hz,1H),6.72(d,J＝8.2Hz,1H),3.00–2.77(m,1H),0.75–0.61(m,4H)；MS(ESI)m/z(％):554.2[M+H]⁺,576.2[M+Na]⁺.

EXAMPLE 29 preparation of Compound 29

Example 29 (Compound 29) was finally prepared according to the preparation scheme of example 22 by replacing 60mL of the 35% aqueous methylamine solution of step 1 in example 22 with 35.21g of cyclopropylamine in 60mL, replacing 8.30g of p-hydroxyaniline of step 2 in example 22 with 9.67g of 4-amino-2-fluorophenol, and replacing 2.11g of 3-oxo-4- (4-methoxyphenyl) -3, 4-dihydroquinoxaline-2-carboxylic acid of step 3 in example 22 with 2.35g of 3-oxo-4-phenyl-3, 4-dihydroquinoxaline-2-carboxylic acid.¹HNMR(600MHz,DMSO-d₆)δ11.33(s,1H),8.76(d,J＝4.9Hz,1H),8.52(d,J＝5.6Hz,1H),8.12–7.87(m,2H),7.60(dd,J＝15.0,7.8Hz,2H),7.44(m,5H),7.22(m,3H),6.74(d,J＝8.4Hz,1H),3.88(s,3H),3.02–2.75(m,1H),0.80–0.48(m,4H).

EXAMPLE 30 in vitro antitumor cell Activity of 4-phenoxypyridines containing quinoxalinones according to the invention

The 4-phenoxypyridine compounds containing part of quinoxalinone are subjected to in vitro inhibition c-met kinase activity screening and inhibition human colon cancer cell HT-29, human lung cancer cell H460 and human lung adenocarcinoma cell strain A549 activity screening.

The invention relates to a test of c-Met enzyme inhibition activity of a 4-phenoxypyridine compound containing quinoxalinone:

the assay used to measure c-Met kinase activity is based on an enzyme-linked immunosorbent assay (ELISA). The specific operation is as follows: the example compound, 50pM c-Met (His-tagged recombinant human Met (amino acid 974-terminus), expressed by baculovirus) and 5. mu.M ATP in assay buffer (25mM MOPS, pH 7.4, 5mM MgCl) were added to 0.25mg/mL PGT-coated plates at room temperature₂，0.5raM MnCl₂100 μ M sodium orthovanadate, 0.01% Triton X-100, 1mM DTT, and finally DMSO concentration 1% (v/v)) for 20 minutes. The reaction mixture was removed by washing and the phosphorylated polymer substrate was detected with 0.2. mu.g/mL of a phosphotyrosine-specific monoclonal antibody (PY20) conjugated with horseradish peroxidase (HRP). Adding intoAfter the color development was stopped with 1M phosphoric acid, the color of the developed substrate (TMB) was quantified spectrophotometrically at 450 nm. The data for the inhibition of c-Met kinase by the compounds of the examples and the positive control (foretinib) are shown in table 1.

Table 1:

the invention relates to a screening method for the in vitro inhibition tumor cell activity of 4-phenoxypyridine compounds containing quinoxalinone, which comprises the following steps:

the invention relates to a part of compounds of 4-phenoxypyridine compounds containing quinoxalinone of the general formula I, which are used for screening the antitumor activity of inhibiting human colon cancer cells HT-29, human lung cancer cells H460 and human lung adenocarcinoma cell strains A549 in vitro.

(1) After cells were thawed and passaged for 2-3 stabilities, they were digested from the bottom of the flask with trypsin solution (0.25%). The cell digest was poured into a centrifuge tube, after which culture broth was added to stop the digestion. Centrifuging the centrifuge tube at 800r/min for 10min, discarding supernatant, adding 5mL culture solution, blowing and beating the mixed cells, sucking 10 μ L cell suspension, adding into cell counting plate, counting, and adjusting cell concentration to 10⁴Per well. 100. mu.L of the cell suspension was added to the 96-well plate except that the A1 well was a blank well and no cells were added. The 96-well plate was placed in an incubator for 24 h.

(2) The test sample was dissolved in 50. mu.L of dimethyl sulfoxide, and then an appropriate amount of culture medium was added to dissolve the sample to 2.0mg/mL of the liquid, and then the sample was diluted to 20,4,0.8,0.16, 0.032. mu.g/mL in a 24-well plate. 3 wells were added for each concentration, two columns of cells surrounding each, which were greatly affected by the environment, and only used as blank wells. The 96-well plate was placed in an incubator for 48 h.

(3) The drug-containing culture medium in the 96-well plate was discarded, the cells were washed twice with Phosphate Buffered Saline (PBS), and 100. mu.l of MTT (tetrazolium) (0.5mg/mL) was added to each wellAfter L was placed in the incubator for 4 hours, the MTT solution was discarded, and 100. mu.L of dimethyl sulfoxide was added. And oscillating on a magnetic oscillator to fully dissolve the viable cells and the MTT reaction product formazan, and putting the formazan into an enzyme labeling instrument to measure the result. Determination of drug IC by Bliss method₅₀The value is obtained. The results of the activity of the compound in inhibiting human colon cancer cells HT-29, human lung cancer cells H460 and human lung adenocarcinoma cell line A549 are shown in Table 2, wherein the results of the results are shown in the following table, and the results of the results are shown in the following table:

TABLE 2

As is clear from tables 1 and 2, the compound of the general formula (I) to be protected in the invention has good in vitro c-Met kinase inhibition activity and human colon cancer cell HT-29, human lung cancer cell H460 and human lung adenocarcinoma cell strain A549 activity. Part of the compounds are equivalent to or better than a control drug, namely fluretinib (Foretinib). The compounds have good development and application prospects of antitumor drugs.

Although the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the details of the above embodiments, and various equivalent changes may be made to the technical solution of the present invention within the technical concept of the present invention, and various possible combinations of the present invention will not be described in detail in order to avoid unnecessary repetition. Any modification, equivalent replacement or improvement made within the technical spirit of the present invention is included in the scope of protection of the present invention.

Claims

1. A4-phenoxypyridine compound containing quinoxalinone and pharmaceutically acceptable salts thereof are characterized in that the structural formula is shown as a general formula (I):

wherein:

2. The quinoxalinone containing 4-phenoxypyridines and the pharmaceutically acceptable salts thereof according to claim 1, characterized in that:

R₁is selected from C₁-C₁₀Alkyl or C₃-C₇A cycloalkyl group;

3. The quinoxalinone containing 4-phenoxypyridines and the pharmaceutically acceptable salts thereof according to claim 2 characterized in that:

4. The quinoxalinone containing 4-phenoxypyridines and the pharmaceutically acceptable salts thereof according to claim 3 characterized in that:

R₁selected from methyl or cyclopropyl;

said R₃Selected from hydrogen, halogen, methyl, trifluoromethyl, methoxy;

5. The 4-phenoxypyridines containing a quinoxalinone structure and the pharmaceutically acceptable salts thereof according to claim 4, characterized by the following structural formula:

6. a pharmaceutical composition comprising the quinoxalinone containing 4-phenoxypyridine compound according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.

7. Use of the 4-phenoxypyridines containing quinoxalinones and the pharmaceutically acceptable salts thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 for the production of a medicament for the treatment and/or prevention of diseases caused by an abnormally high expression of c-Met kinase.

8. Use of the 4-phenoxypyridines containing quinoxalinones and the pharmaceutically acceptable salts thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 for the preparation of a medicament for the treatment and/or prevention of proliferative diseases.

9. Use of the 4-phenoxypyridines containing quinoxalinones and the pharmaceutically acceptable salts thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 for the production of a medicament for the treatment and/or prophylaxis of cancer.

10. The use of claim 9, wherein the cancer is colon cancer and lung cancer.