CN111303121A - 4-phenoxypyridine compound containing quinoxalinone and application thereof - Google Patents

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 compounds containing quinoxalinone and application thereof

technical field

The invention belongs to the field of medicine, and particularly relates to quinoxalinone-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof, their preparation methods and pharmaceutical compositions containing the compounds. The present invention also relates to the use of such compounds and their pharmaceutically acceptable salts in the preparation of medicines for the treatment of diseases caused by abnormally high expression of c-Met kinase, especially in the preparation of medicines for the treatment and/or prevention of colon cancer, lung cancer and other cancers Use in medicine.

Background technique

c-Met is a protein encoded by the proto-oncogene c-Met. Hepatocyte growth factor (HGF) binds to c-Met to initiate the HGF/c-Met signaling pathway, thereby exerting its biological effects (such as: embryonic development, tissue damage Repair, etc.); abnormal expression of c-Met is closely related to tumor occurrence, invasion, metastasis and drug resistance. HGF/Met has been internationally recognized as an anti-tumor target. The research of anti-tumor drugs based on c-Met signaling pathway has entered a new stage, especially the small molecule c-Met kinase inhibitor has become the focus and hotspot of the whole anti-tumor drug research. At present, most of the small-molecule c-Met kinase inhibitors that have been discovered are competitive inhibitors of ATP, and their mechanism of action is to inhibit the c-Met kinase by blocking the phosphorylation of tyrosine. 4-phenoxyquinoline compounds containing malonamide structure, among which Cabozantinib (XL-184) has relatively good inhibitory effect on c-Met/VEGFR-2, with IC ₅₀ values of 1.3nM respectively , 0.035nM, in addition, the compound also has a certain inhibitory effect on kinases such as Ret, Tie-2, Axl, Flt-3, Kit and Ron. In November 2012, the FDA approved cabozantinib for the treatment of unresectable malignant locally advanced or metastatic medullary thyroid cancer (MTC). In April 2016, the FDA approved cabozantinib as a second-line drug for the treatment of advanced renal cancer. In December 2017, the FDA approved the application of cabozantinib tablets for the expanded indication treatment of advanced renal cell carcinoma (RCC). Cabozantinib is currently the only marketed Type II small molecule c-Met kinase inhibitor. In the transformation of cabozantinib, scientists found that the cyclopropyl diamide side chain structure was retained, and the 6,7-dimethoxyquinoline core was replaced by a pyridine ring with a substituent at the 2-position to obtain a series of 4- Phenoxypyridine compounds, these compounds have strong inhibitory activity on c-Met kinase. Altiratinib, a c-Met kinase inhibitor developed by Deciphera Pharmaceuticals, is currently in Phase I clinical research. It has good anti-tumor research and development prospects. On the other hand, quinoxalinone compounds were discovered by scientists as early as 1989 to have obvious anti-tumor effects in vitro. In 1998, echinomycin containing quinoxalinone structure was approved as an anti-tumor drug and entered Phase II clinical trials. The two nitrogen atoms in the quinoxalinone structure are in the opposite position to each other, with small steric hindrance and strong modifiability. Because of its small molecular skeleton, it is often introduced into anticancer drugs as an antitumor pharmacophore. At present, the main problems in the research of c-Met inhibitors are as follows: large toxic and side effects in clinical application, unsatisfactory clinical therapeutic effect and pharmacokinetic parameters, and low oral bioavailability. Therefore, the development of novel, safe and effective c-Met kinase inhibitors with novel structure is still the key field of anti-tumor drug research at home and abroad.

SUMMARY OF THE INVENTION

The purpose of the present invention is to design and synthesize a series of new quinoxalinone-containing 4-phenoxypyridine compounds. After in vitro activity screening, it is shown that the compounds have anti-tumor activity and can be used for the development of anti-tumor drugs.

To achieve the above object, the present invention provides a quinoxalinone-containing 4-phenoxypyridine compound and a pharmaceutically acceptable salt thereof, the structural formula is shown in the general formula (I):

in:

R ₁ is selected from hydrogen, C ₁ -C ₁₀ alkyl, C ₃ -C ₇ cycloalkyl;

R ₂ is selected from 1-4 identical or different substituents of the following: hydrogen, halogen, C ₁ -C ₁₀ alkyl, or C ₁ -C ₄ alkoxy;

Ar is selected from C ₆ -C ₁₀ -membered aryl or 5-10-membered heteroaryl; the heteroaryl contains 1-3 heteroatoms selected from N, O or S, and Ar is optionally replaced by 1-5 replaced by the same or different R ₃ ;

The R ₃ is selected from hydrogen, halogen, hydroxyl, trifluoromethyl, nitro, amino, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₁ -C ₆ alkyl optionally substituted by hydroxy or amino or halogen.

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

Preferably, the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof,

R ₁ is selected from C ₁ -C ₁₀ alkyl or C ₃ -C ₇ cycloalkyl;

R ₂ is selected from 1-4 identical or different following substituents: hydrogen or halogen;

Ar is selected from C ₆ -C ₁₀ -membered aryl, C ₆ -C ₁₀ -membered aralkyl, C ₅ -C ₁₀ -membered heteroaryl; the heteroaryl contains 1-3 members selected from N, O or S of heteroatoms, and Ar is optionally substituted by 1-5 identical or different R ₃ ;

The R ₃ is selected from hydrogen, halogen, hydroxyl, trifluoromethyl, nitro, amino, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, C ₁ -C ₆ alkoxy.

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

Preferably, the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof,

R ₁ is selected from methyl, ethyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;

R ₂ is selected from 1-2 of the same or different following substituents: hydrogen or fluorine;

Ar is selected from phenyl, pyridyl, thiophene, furan, naphthyl, quinolinyl and indolyl, and Ar is optionally substituted by 1-5 identical or different R ₃ ;

The R ₃ is selected from hydrogen, halogen, hydroxyl, trifluoromethyl, nitro, amino, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, C ₁ -C ₆ alkoxy.

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

Preferably, the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof,

R ₁ is selected from methyl or cyclopropyl;

R ₂ is selected from 1-2 of the same or different following substituents: hydrogen or fluorine

Ar is selected from phenyl, and Ar is optionally substituted by 1-5 identical or different R ₃ ;

The R ₃ is hydrogen, halogen, methyl, trifluoromethyl, methoxy, etc.;

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

Preferably, the above-mentioned quinoxalinone structure-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof have the following structural formula:

A pharmaceutical composition, comprising the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and their pharmaceutically acceptable salts as active ingredients and pharmaceutically acceptable excipients.

Preferably, the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof or the above-mentioned pharmaceutical compositions are used in the preparation, treatment and/or prevention of abnormally high expression of c-Met kinase. The application of disease medicine.

Preferably, the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and pharmaceutically acceptable salts thereof or the above-mentioned pharmaceutical compositions are used in the preparation of medicines for the treatment and/or prevention of proliferative diseases.

Preferably, the above-mentioned quinoxalinone-containing 4-phenoxypyridine compounds and their pharmaceutically acceptable salts or the above-mentioned pharmaceutical compositions are used in the preparation of medicines for treating and/or preventing cancer.

Preferably, in the above application, the cancer is colon cancer and lung cancer.

Moreover, according to some common methods in the field to which the present invention belongs, the quinoxalinone-containing 4-phenoxypyridine compounds and configurational isomers represented by the general formula (I) in the present invention can form pharmaceutically acceptable compounds with acids. Accepted salt. Pharmaceutically acceptable addition salts include inorganic and organic acid addition salts, with 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, etc.

In the present invention, "halogen" refers to fluorine, chlorine, bromine or iodine; "alkyl" refers to straight-chain or branched alkyl; "alkylene" refers to straight-chain or branched alkylene; " "Cycloalkyl" refers to substituted or unsubstituted cycloalkyl; "Aryl" refers to unsubstituted or substituted phenyl, naphthyl; "Heteroaryl" refers to one or more selected from Monocyclic or polycyclic ring systems of N, O, S heteroatoms, the ring system is aromatic, such as imidazolyl, pyridyl, pyrazolyl, (1,2,3)- and (1,2 ,4)-triazolyl, furanyl, thienyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, naphthyl, quinolinyl, isoquinolinyl, benzimidazolyl and benzoxazolyl, etc.

The beneficial effects of the present invention are:

In the present invention, it is found by testing the c-Met enzyme activity that the compound of the present invention has significant c-Met kinase inhibitory activity, and can be used to prepare a c-Met inhibitor medicine, especially a medicine for treating and/or preventing cancer.

In the present invention, it is proved that the compounds of the present invention have significant inhibitory effects on lung cancer cells and human gastric cancer cells through in vitro inhibition of the activity of c-met high expression cell line human colon cancer cell line HT-29, human lung cancer cell H460 and human lung adenocarcinoma cell line A549. especially for the preparation of medicaments for the treatment and/or prevention of colon cancer and lung cancer.

Detailed ways

The examples and preparations provided hereinafter further illustrate and illustrate the compounds of the present invention and methods for their preparation. It should be understood that the scope of the following examples and preparations do not limit the scope of the invention in any way. The following examples are intended to illustrate rather than limit the scope of the invention. The following synthetic routes describe the preparation of derivatives of general formula (I) of the present invention, all starting materials were prepared by the following synthetic routes, by methods well known to those of ordinary skill in the art of organic chemistry, or are commercially available. All final derivatives of the present invention are prepared by the following synthetic routes 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 following synthetic routes are as defined below or as defined in the claims.

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

According to the compounds of general formula (I) of the present invention, intermediate M can be prepared by the method shown in Scheme 2, other substituents are as defined in the claims, wherein M-1 represents a substructure of intermediate M.

According to the compounds of general formula (I) of the present invention, intermediate M can also be prepared by the method shown in Scheme 3, with other substituents as defined in the claims, wherein M-2 represents another type of substructure of intermediate M.

According to the compounds of general formula (I) of the present invention, intermediate Q is prepared by the method shown in Scheme 4, with other substituents as defined in the claims.

Substituents R ₁ , R ₂ , D, E, Ar of all intermediates in the above four routes are as defined in the claims.

The examples are intended to illustrate rather than limit the scope of the invention. The hydrogen nuclear magnetic resonance spectrum of the compound was measured by BrukerARX-600, and the mass spectrum was measured by Agilent 6460QQQ; all reagents used were of analytical or chemical purity.

Preparation of Example 1 Compound 1

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

8.80 g of 2-amino-4-chloropyridine and 20.80 g of triethylamine were dissolved in 80 mL of dichloromethane, and 30 mL of a dichloromethane solution containing 9.30 g of cyclopropylcarbonyl chloride was added dropwise to the solution under ice bath conditions. After the addition is complete, warm to room temperature. Stir for 12h, after the reaction, the mixture was washed three times with 20% K ₂ CO ₃ solution and saturated brine, respectively, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness to obtain a crude product, which was separated by column chromatography to obtain a white Solid N-(4-chloropyridin-2-yl)cyclopropylcarboxamide. ¹ H NMR (600MHz, CDCl ₃ ) δ 8.79 (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

8.00 g of intermediate N-(4-chloropyridin-2-yl)cyclopropylcarboxamide and 15.98 g of 2-fluoro-4-nitrophenol were added to 100 mL of chlorobenzene, and reacted at 140°C for 40 h. Cooled to room temperature, concentrated under reduced pressure, the residue was dissolved in an appropriate amount of dichloromethane, washed three times with K ₂ CO ₃ solution and saturated brine respectively, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a brown color Solid, column chromatography gave N-[4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl]cyclopropylcarboxamide as a pale yellow solid product. ¹ HNMR (600MHz, DMSO-d ₆ )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]cyclopropylcarboxamide 6.00g, iron powder 5.28g, acetic acid 11.36g were added to 100mL of ethyl acetate 20 mL of water was added, heated to reflux for 2 h, the reaction was completed, filtered while hot, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain a white solid N-[4-(4-amino-fluorine -2-Fluorophenoxy)pyridin-2-yl]cyclopropylcarboxamide. ¹ H NMR (600MHz, DMSO-d ₆ )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).

Step 4: Preparation of 2-nitro-N-phenylaniline

Take 5.00 g of aniline, dissolve it in 40 mL of dry N,N-dimethylformamide, cool the solution to below 0 °C in an ice-salt bath, slowly add 3.21 g of 60% sodium hydride, keep stirring for 0.5 h, add to the above solution A solution of 9.10 g of 2-fluoronitrobenzene dissolved in 30 mL of N,N-dimethylformamide was added dropwise, the dropwise addition was completed, and the reaction was raised to room temperature for 20 h. The reaction solution was slowly poured into 300 mL of saturated aqueous ammonium chloride solution, a solid was precipitated, filtered and dried to obtain 9.2 g of a yellow product with a yield of 80%. MS (ESI) m/z (%): 213.3 [M-H]—..

Step 5: Preparation of N-phenyl-o-phenylenediamine

Take 8.0 g of intermediate 2-nitro-N-phenylaniline, dissolve it in 80 mL of ethyl acetate, add 30 mL of water, 10.43 g of iron powder and 22.4 g of glacial acetic acid to the solution, reflux for 6 h, filter while hot, The ethyl acetate layer was separated, washed twice with water, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain 5.2 g of a brown product. ESI-MS [M+H] (m/z): 185.3.

Step 5: Preparation of 3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-carboxylic acid ethyl ester

Take 5.60 g of intermediate N-phenyl-o-phenylenediamine and 6.4 g of diethyl ketomalonate monohydrate, add 90 mL of toluene, and reflux with a Dean-Stark trap for 12 h. After the reaction is completed, the solvent is removed by evaporation under reduced pressure. , the residue was dissolved in 150 mL of ethyl acetate, washed twice with water, dried over anhydrous sodium sulfate, and evaporated to dryness. 7.9 g of off-white product was isolated by column chromatography. MS(ESI) m/z(%): 295.0[M+H] ⁺ .

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

Take 5.00 g of ethyl 3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-carboxylate, dissolve it in 60 mL of tetrahydrofuran, add 15 mL of water, and add 1.44 g of lithium hydroxide monohydrate under stirring. g (34.00 mmol), the reaction was stirred at room temperature for 2 h, and the reaction was completed. The THF was evaporated under reduced pressure to remove THF, 30 mL of water was added, extracted twice with ethyl acetate, the aqueous phase was adjusted to pH 2 with 6 mol/L hydrochloric acid, an off-white solid was precipitated, suction filtered, the filter cake was washed with water, and dried to obtain 3-oxo-4- Phenyl-3,4-dihydroquinoxaline-2-carboxylic acid product 4.0 g. MS (ESI) m/z (%): 265.1 [MH] ^— .

Step 7: Preparation of Compound 1

2.11 g of intermediate 3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-carboxylic acid, intermediate N-[4-(4-amino-2-fluorophenoxy base)pyridin-2-yl]cyclopropylcarboxamide 1.90g, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (abbreviation : HATU) 3.02g, triethylamine 1.00g, N,N-dimethylformamide 10mL, stirring at room temperature for 12h, the reaction was completed. The reaction solution was poured into 150 mL of 20% potassium carbonate aqueous solution, extracted three times with 100 mL of dichloromethane respectively, the organic phases were combined, the organic phase was washed three times with 20% potassium carbonate aqueous solution, the organic layer was washed twice with saturated brine, and the organic layer was separated. Dry with anhydrous sodium sulfate. Filter, evaporate dichloromethane under reduced pressure, and recrystallize from methanol to obtain a white solid; ¹ H NMR (600MHz, DMSO-d ₆ )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] ⁺ .

Preparation of Example 2 Compound 2

Substitute 5.00 g of aniline in step 4 in Example 1 with 5.97 g of 4-fluoroaniline, and according to the preparation scheme of Example 1, Example 2 (Compound 2) was finally prepared. ¹ H NMR (600 MHz, CDCl ₃ ) δ 11.91 (s, 1H), 8.32 (d, J=7.0 Hz, 1H), 8.19-8.08 (m, 2H), 7.98 (dd, J=12.1, 2.3 Hz, 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] ⁺ .

Preparation of Example 3 Compound 3

Substitute 5.00 g of aniline in step 4 in Example 1 with 6.85 g of 4-chloroaniline, and follow the preparation scheme of Example 1 to finally obtain Example 3 (compound 3). ¹ H NMR (600 MHz, CDCl ₃ ) δ 11.88 (s, 1H), 8.30 (t, J=7.2 Hz, 1H), 8.25 (s, 1H), 8.11 (d, J=5.8 Hz, 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

Substitute 5.00 g of aniline in step 4 in Example 1 with 5.97 g of 2-fluoroaniline, and follow the preparation scheme of Example 1 to finally obtain Example 4 (compound 4). ¹ H NMR (600MHz, CDCl ₃ ) δ 11.84 (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

Substitute 5.00 g of aniline in step 4 in Example 1 with 6.85 g of 2-chloroaniline, and follow the preparation scheme of Example 1 to finally obtain Example 5 (compound 5). ¹ H NMR (600 MHz, CDCl ₃ ) δ 11.87 (s, 1H), 8.63 (s, 1H), 8.32 (d, J=8.0 Hz, 1H), 8.11 (d, J=5.8 Hz, 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

Substitute 5.00 g of aniline in step 4 in Example 1 with 5.97 g of 3-fluoroaniline, and according to the preparation scheme of Example 1, Example 6 (Compound 6) was finally prepared. ¹ H NMR (600 MHz, CDCl ₃ ) δ 11.86 (s, 1H), 8.46 (s, 1H), 8.31 (d, J=7.9 Hz, 1H), 8.11 (d, J=5.7 Hz, 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

Substitute 5.00 g of aniline in step 4 in Example 1 with 6.61 g of 4-methoxyaniline, and follow the preparation scheme of Example 1 to finally obtain Example 7 (compound 7). ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.29 (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

Substitute 5.00 g of aniline in step 4 in Example 1 with 8.22 g of 2,4-dimethoxyaniline, and follow the preparation scheme of Example 1 to finally obtain Example 8 (Compound 8). ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.28 (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

Substitute 5.00 g of aniline in step 4 in Example 1 with 9.24 g of 4-bromoaniline, and according to the preparation scheme of Example 1, Example 9 (compound 9) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.26 (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

Substitute 5.00 g of aniline in step 4 in Example 1 with 6.85 g of 3-chloroaniline, and follow the preparation scheme of Example 1 to finally obtain Example 10 (Compound 10). ¹ H NMR (600MHz, DMSO-d ₆ )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

5.00 g of aniline in step 4 in Example 1 was replaced with 5.75 g of 4-methylaniline, and according to the preparation scheme of Example 1, Example 11 (compound 11) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.28 (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

Substitute 5.00 g of aniline in step 4 in Example 1 with 8.70 g of 2,4-dichloroaniline, and follow the preparation scheme of Example 1 to finally obtain Example 12 (Compound 12). ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.26(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

Substitute 9.30 g of cyclopropylcarbonyl chloride in step 1 of Example 1 with 6.98 g of acetyl chloride, and according to the preparation scheme of Example 1, Example 13 (Compound 13) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ )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

Substitute 9.30 g of cyclopropylcarbonyl chloride in step 1 of Example 1 with 6.98 g of acetyl chloride, and replace 5.00 g of aniline in Step 4 of Example 1 with 5.97 g of 4-fluoroaniline, according to the preparation scheme of Example 1, Example 14 (compound 14) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.28(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

Substitute 9.30 g of cyclopropylcarbonyl chloride in step 1 of Example 1 with 6.98 g of acetyl chloride, and replace 5.00 g of aniline in Step 4 of Example 1 with 6.85 g of 2-chloroaniline, according to the preparation scheme of Example 1, Example 15 (compound 15) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.28(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

Substitute 9.30 g of cyclopropylcarbonyl chloride in step 1 of Example 1 with 6.98 g of acetyl chloride, and replace 5.00 g of aniline in Step 4 of Example 1 with 6.61 g of 4-methoxyaniline, according to the preparation of Example 1 scheme, and finally Example 16 (compound 16) was prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.29 (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

Substitute 15.98 g of 2-fluoro-4-nitrophenol in step 2 in Example 1 with 14.15 g of 4-nitrophenol, and according to the preparation scheme of Example 1, Example 17 (compound 17) was finally obtained. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.10 (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

Substitute 15.98 g of 2-fluoro-4-nitrophenol in step 2 in Example 1 with 14.15 g of 4-nitrophenol, and replace 5.00 g of aniline in Step 4 in Example 1 with 5.97 g of 4-fluoroaniline. According to the preparation scheme of Example 1, Example 18 (compound 18) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.09 (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

Replace 9.30 g of cyclopropylcarbonyl chloride in step 1 of Example 1 with 6.98 g of acetyl chloride, and replace 15.98 g of 2-fluoro-4-nitrophenol in step 2 of Example 1 with 14.15 g of 4-nitrophenol , according to the preparation scheme of Example 1, Example 19 (compound 19) was finally prepared. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.10 (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

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

Example 21 Preparation of Compound 21

Replace 9.30 g of cyclopropylcarbonyl chloride in step 1 of Example 1 with 6.98 g of acetyl chloride, and replace 15.98 g of 2-fluoro-4-nitrophenol in step 2 of Example 1 with 14.15 g of 4-nitrophenol , according to the preparation scheme of Example 1, Example 21 (compound 21) was finally obtained. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.09 (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=8.5 Hz,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-picolinecarboxamide

20g of 2-picolinic acid, 2.6g of sodium bromide and 30mL of chlorobenzene were placed in a 500m bottle. After the temperature was raised to 50°C, 48mL of thionyl chloride was slowly added dropwise. After the dropwise addition, stirring was continued for 30 minutes. Reflux for 20h. Most of the liquid was evaporated under reduced pressure, and 30 mL of toluene was added and stirred for 5 min. After the solvent was evaporated, 4-chloropicolinoyl chloride was obtained as a yellow oily liquid, and 100 mL of toluene was added, and the solution was directly used in the next step. Under ice-water bath conditions, the toluene solution of 4-chloropicolinoyl chloride prepared above was slowly added dropwise to 60 mL of 35% methylamine aqueous solution. The aqueous layer was extracted twice with 50 mL of toluene, all collected toluene layers were combined, and the solvent was evaporated to dryness under reduced pressure to obtain a brown oily liquid. The oily liquid was dissolved in 150 mL of tetrahydrofuran, and 50 mL of concentrated hydrochloric acid was slowly added at a temperature below 20 °C, and after the addition was completed, the mixture was stirred at the same temperature for 1 h, and filtered with suction. The filter cake was dissolved in 80 mL of water, adjusted to neutral pH with 20% NaOH solution, and the temperature was controlled at 15-20 °C, stirred at the same temperature for 1 h, and a light yellow solid was precipitated, which was washed with suction and dried to obtain 4-chloro-N-methyl Picolinamide. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.85(s, 1H), 8.63(d, J=5.2Hz, 1H), 8.03(s, 1H), 7.76(d, J=5.2Hz, 1H) , 2.84 (d, J=4.8 Hz, 3H).

Step 2: Preparation of 4-(4-aminophenoxy)-N-picolinecarboxamide

10.00 g of 4-chloro-N-picoline carboxamide and 8.30 g of p-hydroxyaniline obtained in the above steps were dissolved in 50 mL of DMF, and 13.20 g of potassium tert-butoxide was added with stirring at room temperature. After the addition, the temperature was raised to 90°C and refluxed for 5-10h. The reaction solution was cooled to 10-20°C, concentrated hydrochloric acid was added at the same temperature until a large amount of brown solid was precipitated, then stirred at the same temperature for 1 hour, suction filtered, the filter cake was placed in 200 mL of dichloromethane and stirred for 30 minutes, suction filtered, and the filter cake was Rinse with a small amount of dichloromethane. Dissolve the filter cake in water, adjust the pH to 7-8 with 20% NaOH aqueous solution at 10-20°C, and separate out a brown oily substance, stir at the same temperature until it solidifies, filter with suction after solidification, and place the filter cake in a 5% NaOH aqueous solution at room temperature. After stirring for 20 min, suction filtration, and washing with a large amount of water until the filtrate is colorless to obtain a gray solid, suction filtration, and drying to obtain 4-(4-aminophenoxy)-N-picolinecarboxamide. MS (ESI) m/z (%): 244.10 [M+H] ⁺ .

Step 3: Preparation of Compound 22

Substitute N-[4-(4-amino-2-fluorophenoxy)pyridine-2 in step 7 of Example 1 with 1.61 g of 4-(4-aminophenoxy)-N-picolinecarboxamide -yl]cyclopropylcarboxamide 1.90 g, according to the preparation scheme of step 7 in Example 1, Example 22 (compound 22) was finally obtained. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.14 (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

Substitute 3-oxo-4-phenyl-3 in step 3 in Example 22 with 3-oxo-4-(4-fluorophenyl)-3,4-dihydroquinoxaline-2-carboxylic acid 2.25 g , 2.11 g of 4-dihydroquinoxaline-2-carboxylic acid, according to the preparation scheme of Example 22, Example 23 (compound 23) was finally obtained. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.13 (s, 1H), 8.78 (d, J=4.7 Hz, 1H), 8.52 (d, J=5.6 Hz, 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

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

Example 25 Preparation of Compound 25

Substitute 3-oxo-4-phenyl-3 in step 3 of Example 22 with 3-oxo-4-(4-chlorophenyl)-3,4-dihydroquinoxaline-2-carboxylic acid 2.38 g , 2.11 g of 4-dihydroquinoxaline-2-carboxylic acid, according to the preparation scheme of Example 22, Example 25 (compound 25) was finally obtained. ¹ H NMR (600MHz, DMSO-d ₆ ) δ 11.12 (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

Replace 60 mL of 35% methylamine aqueous solution in step 1 in Example 22 with 60 mL of aqueous solution of 35.21 g of cyclopropylamine, and replace 8.30 g of p-hydroxyaniline in step 2 in Example 22 with 9.67 g of 4-amino-2-fluorophenol , 3-oxo-4-phenyl-3 in Example 22 was replaced with 3-oxo-4-(4-chlorophenyl)-3,4-dihydroquinoxaline-2-carboxylic acid 2.38g, 2.11 g of 4-dihydroquinoxaline-2-carboxylic acid, according to the preparation scheme of Example 22, Example 26 (compound 26) was finally obtained. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.33 (s, 1H), 8.76 (d, J=4.4 Hz, 1H), 8.52 (d, J=5.5 Hz, 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

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

Example 28 Preparation of Compound 28

Replace 60 mL of 35% methylamine aqueous solution in step 1 in Example 22 with 60 mL of aqueous solution of 35.21 g of cyclopropylamine, and replace 8.30 g of p-hydroxyaniline in step 2 in Example 22 with 9.67 g of 4-amino-2-fluorophenol , with 3-oxo-4-(4-fluorophenyl)-3,4-dihydroquinoxaline-2-carboxylic acid 2.25g replacing 3-oxo-4-phenyl- 2.11 g of 3,4-dihydroquinoxaline-2-carboxylic acid, according to the preparation scheme of Example 22, Example 28 (compound 28) was finally obtained. ¹ HNMR (600MHz, DMSO-d ₆ )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

Replace 60 mL of 35% methylamine aqueous solution in step 1 in Example 22 with 60 mL of aqueous solution of 35.21 g of cyclopropylamine, and replace 8.30 g of p-hydroxyaniline in step 2 in Example 22 with 9.67 g of 4-amino-2-fluorophenol , 3-oxo-4-benzene in step 3 in Example 22 was replaced with 2.35 g of 3-oxo-4-(4-methoxyphenyl)-3,4-dihydroquinoxaline-2-carboxylic acid 2.11 g of base-3,4-dihydroquinoxaline-2-carboxylic acid, according to the preparation scheme of Example 22, Example 29 (compound 29) was finally obtained. ¹ HNMR (600MHz, DMSO-d ₆ )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 anti-tumor cell activity of the quinoxalinone-containing 4-phenoxypyridine compounds of the present invention

Part of the quinoxalinone-containing 4-phenoxypyridine compounds of the present invention were screened for inhibiting c-met kinase activity in vitro and inhibiting human colon cancer cell HT-29, human lung cancer cell H460 and human lung adenocarcinoma cell line A549 Active screening.

Test of the inhibitory activity of the quinoxalinone-containing 4-phenoxypyridine compounds of the present invention to c-Met enzyme:

The assay used to measure c-Met kinase activity is based on an enzyme-linked immunosorbent assay (ELISA). The specific operation is: at room temperature, on a 0.25 mg/mL PGT-coated plate, the example compound, 50 pM c-Met (His-labeled recombinant human Met (amino acid 974-terminal), expressed by baculovirus) and 5 μM ATP in assay buffer (25 mM MOPS, pH 7.4, 5 mM MgCl ₂ , 0.5 raM MnCl ₂ , 100 μM sodium orthovanadate, 0.01% Triton X-100, 1 mM DTT, final DMSO concentration 1% (v/v)) Incubate for 20 minutes. The reaction mixture was removed by washing and the phosphorylated polymer substrate was detected with 0.2 μg/mL horseradish peroxidase (HRP)-conjugated phosphotyrosine-specific monoclonal antibody (PY20). The color of the developed substrate (TMB) was quantified spectrophotometrically at 450 nm after the addition of 1 M phosphoric acid to stop the development. The inhibitory data of example compounds and positive control drug (foretinib) on c-Met kinase are shown in Table 1.

Table 1:

Screening of the quinoxalinone-containing 4-phenoxypyridine compounds of the present invention for inhibiting tumor cell activity in vitro:

In vitro inhibition of human colon cancer cells HT-29, human lung cancer cells H460 and human lung adenocarcinoma cell lines A549 was carried out on some compounds of the quinoxalinone-containing 4-phenoxypyridine compounds of the general formula I according to the present invention. Screening for antitumor activity.

(1) After the cells were recovered and passaged 2-3 times to stabilize, they were digested from the bottom of the culture flask with trypsin solution (0.25%). The cell digestion solution was poured into a centrifuge tube, and then the culture solution was added to stop the digestion. Centrifuge the centrifuge tube at 800 r/min for 10 min, discard the supernatant, add 5 mL of culture medium, mix the cells by pipetting, pipette 10 μL of the cell suspension and add it to a cell counting plate for counting, and adjust the cell concentration to 10 ⁴ cells/well. In the 96-well plate, 100 μL of cell suspension was added to the rest of the 96-well plates, except that A1 was a blank well without cells. The 96-well plate was placed in an incubator for 24 h.

(2) Dissolve the test sample with 50 μL dimethyl sulfoxide, then add an appropriate amount of culture medium to dissolve the sample into 2.0 mg/mL liquid, and then dilute the sample to 20, 4, 0.8, 0.16 in a 24-well plate ,0.032μg/mL. Each concentration was added to 3 wells, and the growth of the cells in the surrounding two rows and two columns was greatly affected by the environment, and was only used for the blank cell wells. The 96-well plate was placed in an incubator for 48 h.

(3) Discard the medicated culture medium in the 96-well plate, wash the cells twice with phosphate buffered solution (PBS), add 100 μL of MTT (tetrazolium) (0.5 mg/mL) to each well and put it into the incubator After 4 h, the MTT solution was discarded, and 100 μL of dimethyl sulfoxide was added. Shake on a magnetic shaker to fully dissolve the surviving cells and the MTT reaction product formazan, and put it into a microplate reader to measure the results. The drug IC ₅₀ value can be obtained by the Bliss method. The compounds inhibited the activity of human colon cancer cell HT-29, human lung cancer cell H460 and human lung adenocarcinoma cell line A549, using Foreitinib as a positive control, the results are shown in Table 2:

Table 2

It can be clearly seen from Table 1 and Table 2 that the compounds of general formula (I) to be protected by the present invention have good in vitro inhibition of c-Met kinase activity and inhibition of human colon cancer cells HT-29, human lung cancer cells H460 and human Lung adenocarcinoma cell line A549 activity. Some compounds are comparable to or better than the control drug, Foreitinib. Such compounds have good application prospects for the development and application of antitumor drugs.

The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention. Necessary repetition, the present invention will not further describe various possible combinations. Any modifications, equivalent replacements or improvements made within the scope of the technical concept of the present invention are included within the protection scope of the present invention.

Claims (10)

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:

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).

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;

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.

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).

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

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;

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

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

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

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).

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.