4-substituted aryl formyl-7-substituted 3, 4-dihydroquinoxaline-2 (1H)-ketone structure compound, and preparation method and medical application thereof

Abstract

The invention discloses a compound with a 4-substituted aryl formyl-7-substituted 3, 4-dihydroquinoxaline-2 (1H)-ketone structure as well as a preparation method and medical application thereof, and belongs to the technical field of medicines. The compound has a structural general formula (I): # imgabs0 #, wherein R1 represents ethyl, acetyl and 1-hydroxyethyl, # imgabs1 # imgabs2 # R2 represents hydrogen, fluorine, chlorine, bromine or methoxyl; r3 represents hydrogen, fluorine, chlorine, bromine or methoxyl; r4 represents hydrogen, fluorine, chlorine, bromine or methoxyl, # imgabs3 # imgabs4 # L is fluorine, chlorine, bromine, methyl, ethyl, hydroxyl, acetyl, methoxyl or cyano. The compound disclosed by the invention has tubulin polymerization inhibition activity, and the compound has good application in resisting tumors and other proliferative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a 4-substituted arylformyl-7-substituted 3,4-dihydroquinoxaline-2(1H)-one structural compound, a preparation method thereof and medical use thereof. Background Art

[0002] Microtubules are tubular polymers formed by the polymerization of α and β tubulin heterodimers. They have many functions in cells, especially participating in the mitosis process of cells in the form of spindles. Tumor cells have the ability to proliferate rapidly. If the formation of their spindles is inhibited, mitosis will be blocked, causing tumor cell growth to stagnate in the G2 / M phase and inhibiting cell proliferation.

[0003] Microtubules exist in two forms: cytoplasmic microtubules and spindle microtubules. They participate in cell movement, organelle positioning, intracellular material transport, and the mitotic process of eukaryotic cells. When a cell enters the division phase, its cytoplasmic microtubules disassemble into tubulin and then assemble into spindle microtubules. In the late stage of division, the spindle microtubules pull the sister chromatids from the equatorial plane to the two poles of the spindle. After the end of mitosis, the spindle microtubules disassemble and assemble into cytoplasmic microtubules. Therefore, this dynamic conversion (polymerization and depolymerization) between tubulin / microtubules is necessary for normal cell mitosis. If this cycle is disrupted, cells that have entered mitosis will stop dividing, thereby prolonging the cell cycle or inducing cell apoptosis.

[0004] Combretastatin A4 is a natural product isolated from Combretum caffrum, a South African willow, which can inhibit the polymerization of tubulin and has good anti-tumor activity. However, its poor water solubility and easy isomerization of double bonds to generate inactive trans isomers limit its clinical application.

[0005] The structural modification of Compruetin mainly includes: (1) introducing structures such as phosphoric acid and amino acids to improve its water solubility; (2) replacing double bonds with sulfonamide, carbonyl, five-membered aromatic heterocycle, etc. to solve the instability problem of its double bonds in the body. However, all the Compruetin derivatives reported above are different from the compounds involved in the present invention. Summary of the invention

[0006] The object of the present invention is to provide a 4-substituted arylformyl-7-substituted 3,4-dihydroquinoxaline-2(1H)-one structural compound having tubulin polymerization inhibitory effect; another object of the present invention is to provide a method for preparing the compound; the third object of the present invention is to provide the use of the compound or a pharmaceutical composition containing the compound in the preparation of a drug for preventing or treating tumors or other proliferative diseases.

[0007] Based on the above purpose, the specific technical solution adopted by the present invention is as follows:

[0008] As a first aspect of the present invention, the present invention provides a class of 4-substituted arylformyl-7-substituted 3,4-dihydroquinoxaline-2(1H)-one structural compounds and any combination thereof or pharmaceutically acceptable salts thereof, the general structural formula of which is shown in formula (I):

[0009]

[0010] R1 represents ethyl, acetyl, 1-hydroxyethyl, R2 represents hydrogen, fluorine, chlorine, bromine or methoxy; R3 represents hydrogen, fluorine, chlorine, bromine or methoxy; R4 represents hydrogen, fluorine, chlorine, bromine, methoxy,

[0011] L is fluorine, chlorine, bromine, methyl, ethyl, hydroxy, acetyl, methoxy or cyano.

[0012] Furthermore, R1 represents ethyl, acetyl, 1-hydroxyethyl, R2 represents fluorine, chlorine, bromine or methoxy; R3 represents hydrogen or methoxy; R4 represents fluorine, chlorine, bromine, methoxy,

[0013] L is fluorine, chlorine or cyano.

[0014] The compounds of the present invention can be prepared in the form of pharmaceutically acceptable salts according to conventional methods, including organic acid salts and inorganic acid salts thereof: organic acids include (but are not limited to) acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, oxalic acid, etc., and inorganic acids include (but are not limited to) hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, etc. Metal salts containing carboxylic acid compounds: include (but are not limited to) potassium salts, sodium salts, etc.

[0015] As a second aspect of the present invention, the compound of the present invention can be prepared by the following method, and its synthetic route is as follows:

[0016]

[0017] The specific steps are:

[0018] (1) Preparation of N-(4-methylsulfonyl-2-nitrophenyl)glycine methyl ester or N-(4-acetyl-2-nitrophenyl)glycine methyl ester (2a and 2b)

[0019] Compound 1a or 1b undergoes nucleophilic substitution reaction with glycine methyl ester hydrochloride in acetonitrile using potassium carbonate as a base to obtain compound 2a or 2b.

[0020] (2) Preparation of 7-methanesulfonyl-3,4-dihydroquinoxaline-2(1H)-one or 7-acetyl-3,4-dihydroquinoxaline-2(1H)-one (3a and 3b)

[0021] Compound 2a or 2b is prepared by using ethyl acetate as solvent and stannous chloride as catalyst through reduction and cyclization in one pot to obtain compound 3a or 3b.

[0022] (3) Preparation of 7-substituted-4-substituted benzoyl-3,4-dihydroquinoxaline-2(1H)-one (4a-4e)

[0023] Compound 3a or 3b is reacted with different substituted aryl acid chlorides in tetrahydrofuran (or dichloromethane) as solvent and 4-dimethylaminopyridine as catalyst to obtain compounds 4a-4e.

[0024] (4) Preparation of 7-substituted-4-(3,4,5-trimethoxybenzoyl)-3,4-dihydroquinoxaline-2(1H)-one (5a-5e)

[0025] Compound 4b is reduced with sodium borohydride in a mixture of tetrahydrofuran and methanol as solvent to obtain 5a; compound 4b is reacted with hydroxylamine hydrochloride or methoxyamine hydrochloride in ethanol as solvent and sodium acetate or potassium carbonate as base to obtain compound 5b or 5c; compound 4b is reacted with hydrazine hydrate in methanol as solvent to obtain compound 5d; compound 4b is reduced with triethylsilane in chloroform as solvent and indium tribromide as catalyst to obtain 5e.

[0026] (5) Preparation of 7-acetyl-4-(3-methoxy-5-substituted benzoyl)-3,4-dihydroquinoxaline-2(1H)-one (6a-6i)

[0027] Compound 4e is reacted with arylboronic acid via Suzuki reaction in a mixture of toluene, methanol and water as solvent, ferrocenepalladium dichloride as catalyst and sodium acetate as base to generate compounds 6a-6i.

[0028] (6) Preparation of 7-(1-hydroxyiminoethyl)-4-(3-methoxy-5-substituted benzoyl)-3,4-dihydroquinoxaline-2(1H)-one (7a-7c)

[0029] Compound 4e or 6a or 6c is reacted with hydroxylamine hydrochloride using ethanol as solvent and sodium acetate or potassium carbonate as base to generate compounds 7a-7c.

[0030] Further, in step (1), the molar ratio of compound 1a or 1b, glycine methyl ester hydrochloride and potassium carbonate is 1:1.1:2; in step (2), the molar ratio of compound 2a or 2b to stannous chloride is 1:6, and the reaction temperature is 40 to 60°C; in step (3), the molar ratio of 3a or 3b, aryl acid chloride and 4-dimethylaminopyridine is 1:1.5:0.2; in step (4), the volume ratio of tetrahydrofuran and methanol is 1:1, the molar ratio of compound 4b, sodium acetate or potassium carbonate and hydroxylamine hydrochloride or methoxyamine hydrochloride is 1:4:4, and the reaction temperature is 90 to 100°C; the molar ratio of compound 4b to hydrazine hydrate is 1: 4, the reaction temperature is 75-85°C; the molar ratio of compound 4b, indium tribromide and triethylsilane is 1:0.3:4, and the reaction temperature is 55-65°C; in step (5), the ferrocene palladium dichloride is 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, the volume ratio of toluene, methanol and water is 3:1:1, the molar ratio of compound 4e, arylboronic acid, ferrocene palladium dichloride and sodium acetate is 1:2.5:0.05:3, and the reaction temperature is 95-105°C; in step (6), the molar ratio of compound 4e or 6a or 6c, potassium carbonate or sodium acetate and hydroxylamine hydrochloride is 1:4:4, and the reaction temperature is 65-75°C.

[0031] As a third aspect of the present invention, there is also provided the use of any of the above compounds and any combination thereof or pharmaceutically acceptable salts thereof in the preparation of a drug for preventing or treating anti-tumor or other proliferative diseases. The tumor is breast cancer, ovarian cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, prostate cancer, melanoma or leukemia.

[0032] Use of any of the above compounds and any combination thereof or pharmaceutically acceptable salts thereof in the preparation of a microtubule protein polymerization inhibitor.

[0033] The present invention also provides a pharmaceutical composition. The compound of general formula (I), pharmaceutically acceptable salt or solvate can be added with a pharmaceutically acceptable carrier to prepare common pharmaceutical preparations, such as tablets, capsules, powders, syrups, liquids, suspensions, injections, and can be added with common pharmaceutical excipients such as flavorings, sweeteners, liquid or solid fillers or diluents.

[0034] In order to evaluate the anti-tumor activity of the synthetic compound of the present invention and the pharmaceutical composition thereof, the pharmacological activity tests at the cellular and molecular levels were performed on the compound of the present invention.

[0035] The compounds and pharmaceutical compositions of the present invention were subjected to in vitro cell growth inhibition experiments, and the results showed that the compounds generally had strong cell growth inhibition effects. The half inhibitory concentration (IC 50) reached several micromoles / liter (μM), and compounds 5b, 7b, and 7c reached nanomolar levels (nM). The cell growth inhibitory activity of these compounds was significantly better than that of doxorubicin.

[0036] The compounds and pharmaceutical compositions of the present invention were subjected to an in vitro tubulin polymerization inhibition experiment, and the results showed that compound 7c had a significant inhibitory effect on tubulin polymerization at 1 μM and 20 μM.

[0037] The compounds and pharmaceutical compositions of the present invention were subjected to in vitro cell growth cycle experiments, and the results showed that compound 7c at 30 nM could significantly arrest cells in the G2 / M phase.

[0038] The compounds and pharmaceutically acceptable carriers of the present invention can be administered orally or intravenously in clinical practice. They can be used as a single drug in clinical practice, or in combination with other clinically used chemotherapeutic drugs and radiotherapy for the treatment of the aforementioned cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a graph showing the inhibitory effect of 7c on tubulin polymerization in vitro.

[0040] Figure 2 The graph of the effect of compound 7c on the growth cycle of HeLa cells. A is a blank control, B, C, and D are the effects of compound 7c on the cell growth cycle at 10nM, 20nM, and 30nM concentrations, respectively; and B is a statistical graph of cells in different growth phases. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described in detail below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto. The ratios of the eluents used in the column chromatography in the following examples are all volume ratios.

[0042] Example 1

[0043] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-methylsulfonyl-3,4-dihydroquinoxalin-2(1H)-one (4a)

[0044]

[0045] (1) Preparation of N-(4-methylsulfonyl-2-nitrophenyl)glycine methyl ester (2a)

[0046]

[0047] 2-Fluoro-5-methylsulfonylnitrobenzene (i.e., compound 1a, 2.0 g, 9.1 mmol), glycine methyl ester hydrochloride (1.3 g, 10.0 mmol, 1.1 equiv), and potassium carbonate (2.5 g, 18.2 mmol, 2 equiv) were added to a 250 mL round-bottom flask in sequence, and 50 mL of anhydrous acetonitrile was added to dissolve the reactants, and stirred at room temperature for 24 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated under reduced pressure, and column chromatography (200-300 mesh silica gel, petroleum ether: ethyl acetate = 2:1) was performed to obtain 1.6 g of a light yellow solid with a yield of 60.9%. Melting point: 192.2-193.3°C. 1 H NMR (300MHz, DMSO-d6) δ8.85(t,J=5.9Hz,1H),8.54(d,J=2.2Hz,1H),7.94(dd,J =9.1,2.2Hz,1H),7.18(d,J=9.2Hz,1H),4.41(s,2H),3.72(s,3H),3.23(s,3H).

[0048] (2) Preparation of 7-methanesulfonyl-3,4-dihydroquinoxalin-2(1H)-one (3a)

[0049]

[0050] The intermediate 2a (1.0 g, 3.5 mmol) and stannous chloride (4.0 g, 20.8 mmol, 6 equiv) were added to a 250 mL round-bottom flask in sequence, and ethyl acetate (30 mL) was added to dissolve. After the reactant was dissolved, 1 mL of water was added, and the temperature was raised to 50 ° C and stirred for about 24 h. The reaction was monitored by TLC spot plate. After the reaction was complete, it was cooled to room temperature, and water was added to the reaction solution for extraction, dried over anhydrous sodium sulfate, and distilled under reduced pressure to distill off the solvent. Column chromatography (200-300 mesh silica gel, petroleum ether: ethyl acetate = 1:1) was performed to obtain 0.6 g of brown solid with a yield of 75.7%. Melting point: 253.4-254.1 ° C. 1 H NMR (300MHz, DMSO-d6) δ10.55(s,1H),7.28(dd,J=8.3,2.1Hz,1H),7.19(d,J=2. 0Hz,1H),6.90(s,1H),6.75(d,J=8.4Hz,1H),3.90(d,J=1.6Hz,2H),3.05(s,3H).

[0051] (3) Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-methylsulfonyl-3,4-dihydroquinoxalin-2(1H)-one (4a)

[0052]

[0053] Under nitrogen protection and 0°C, intermediate 3a (200.0 mg, 0.88 mmol) and DMAP (4-dimethylaminopyridine, 21.5 mg, 0.18 mmol, 0.20 equiv) were dissolved in 15 mL of anhydrous tetrahydrofuran. Then 3,4,5-trimethoxybenzoyl chloride (304 mg, 1.3 mmol, 1.5 equiv) was dissolved in 5 mL of anhydrous tetrahydrofuran and added dropwise to the above solution through a constant pressure dropping funnel and stirred for about 24 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure and column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 30:1) was performed to obtain 133 mg of a light-colored solid with a yield of 35.9%. Melting point: 199.3-201.5°C. 1 H NMR(300MHz,DMSO-d6)δ11.10(s,1H),7.55(d,J=1.8Hz,1H),7.44–7.36(m,1H),7.1 3(d,J=8.5Hz,1H),6.74(s,2H),4.43(s,2H),3.71(s,3H),3.67(s,6H),3.19(s,3H). 13 C NMR(125MHz,DMSO-d6)δ168.3,166.4,152.6,139.8,137.4,131.5,131.2,128.7,124.9,120.3,114.6,106.4,60.2,56.0,47.9,43.6.HRMS(ESI)cald.forC 19 H 20 N2O7SNa + [M+Na] + :443.08834,found:443.08875.

[0054] Example 2

[0055] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (4b)

[0056]

[0057] (1) Preparation of N-(4-acetyl-2-nitrophenyl)glycine methyl ester (2b)

[0058]

[0059] 4-Fluoro-3-nitroacetophenone (i.e., compound 1b, 5.0 g, 27.3 mmol), glycine methyl ester hydrochloride (3.8 g, 30.0 mmol, 1.1 equiv), and potassium carbonate (7.6 g, 54.6 mmol, 2.0 equiv) were added to a 250 mL round-bottom flask. Subsequently, 50 mL of anhydrous acetonitrile was added to dissolve, and the mixture was stirred at room temperature for 24 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was distilled under reduced pressure to remove the solvent, and column chromatography (200-300 mesh silica gel, petroleum ether: ethyl acetate = 2:1) was performed to obtain 5.6 g of a yellow solid with a yield of 81.3%. Melting point: 119.2-120.7°C. 1 H NMR (300MHz, CDCl3) δ8.83(d,J=2.1Hz,2H),8.13(d,J=9.0,2.1Hz,1H),6.78(d,J=9.0Hz,1H),4.22(s,2H),3.88(s,3H),2.60(s,3H).

[0060] (2) Preparation of 7-acetyl-3,4-dihydroquinoxaline-2(1H)-one (3b)

[0061]

[0062] The intermediate 2b (2.0 g, 7.9 mmol) and stannous chloride (9.0 g, 47.6 mmol, 6.0 equiv) were added to a 250 mL round-bottom flask in sequence, and ethyl acetate (30 mL) was added to dissolve. After the reactants were completely dissolved, 1 mL of water was added, and then the temperature was raised to 50 ° C and stirred for about 24 h. The reaction was monitored by TLC spot plate. After the reaction was complete, it was cooled to room temperature, and water was added to the reaction solution for extraction, dried over anhydrous sodium sulfate, and distilled under reduced pressure to evaporate the solvent. Column chromatography (200-300 mesh silica gel, petroleum ether: ethyl acetate = 2: 1) was performed to obtain 1.1 g of a yellow-brown solid with a yield of 73.2%. Melting point: 248.6-249.3 ° C. 1 H NMR (300MHz, DMSO-d6) δ10.42(s,1H),7.44(dd,J=8.3,1.9Hz,1H),7.30(d,J=1.8Hz,1H),6.83(s,1H),6.63(d,J=8.3Hz,1H),3.88(s,2H),2.39(s,3H).

[0063] (3) Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (4b)

[0064]

[0065] Under nitrogen protection and 0°C, intermediate 3b (1.0 g, 5.2 mmol) and 4-dimethylaminopyridine (DMAP) (127.1 mg, 1.0 mmol, 0.2 equiv) were dissolved in 15 mL of anhydrous tetrahydrofuran. Then 3,4,5-trimethoxybenzoyl chloride (1.8 g, 7.8 mmol, 1.5 equiv) was dissolved in 5 mL of anhydrous tetrahydrofuran and added dropwise to the above solution through a constant pressure dropping funnel and stirred for about 24 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure and column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 30:1) was performed to obtain 832 mg of a light yellow solid with a yield of 41.6%. Melting point: 214.9-216.2°C. 1 H NMR (300MHz, Methanol-d4) δ7.65 (d, J=2.0Hz, 1H), 7.49 (dd, J=8.5, 2.0Hz, 1H), 6.9 4(d,J=8.5Hz,1H),6.74(s,2H),4.52(s,2H),3.79(s,3H),3.70(s,6H),2.55(s,3H). 13 CNMR(75MHz,DMSO-d6)δ196.6,168.4,166.4,152.6,139.6,133.7,131.2,131.1 ,129.2,124.0,122.3,115.4,106.3,60.2,56.0,48.0,26.6.HRMS(ESI)cald.for C 20 H 20 N2O6Na + [M+Na] + :407.12136,found:407.12262.

[0066] Example 3

[0067] Preparation of 4-(3-fluoro-5-bromobenzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (4c)

[0068]

[0069] Except that 3-fluoro-5-bromo-benzoyl chloride was used instead of 3,4,5-trimethoxybenzoyl chloride in step (3), the other steps and purification method were the same as in Example 2 to obtain 256 mg of white solid with a yield of 38.7% and a HPLC purity of 98.6%. Melting point: 236.7-237.9°C. 1H NMR (300MHz, CDCl3) δ10.97(s,1H),7.88(s,1H),7.62(s,1H),7.56(d,J=9.0Hz,2H),7.46(d,J=12.0Hz,1H),6.99(s,1H),4.38(s,2H),2.50(s,3H). 13 C NMR (75MHz, DMSO-d6) δ197.97,166.52,166.34(d,J=3.0Hz,1C),161.94(d,J=248.25Hz,1C),138.71(d,J=8.3Hz,1C),134.61,131.88,130.56,1 28.07,124.53,122.74,122.48(d,J=9.8Hz,1C),121.61(d,J=24.8Hz,1C),115.99,115.53(d,J=0.75Hz,1C),48.08,27.03.HRMS(ESI)cald.for C17H12BrClN2O3Na + [M+Na] + :428.9612,found:428.96122.

[0070] Example 4

[0071] Preparation of 4-(3-chloro-5-bromobenzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (4d)

[0072]

[0073] Except that 3-chloro-5-bromo-benzoyl chloride was used instead of 3,4,5-trimethoxybenzoyl chloride in step (3), the other steps and purification method were the same as in Example 2 to obtain 0.12 g of yellow solid. The yield was 38.7%, HPLC purity: 95.5119%, melting point: 203.9-204.5°C. 1 H NMR (300MHz, DMSO-d6) δ8.40(s,1H),7.62-7.59(m,2H),7.48-7.42(m,2H),7.33(s,1H),6.84(d,J=10.7Hz,1H),4.56(s,2H),2.58(s,3H). 13C NMR(75MHz,DMSO-d6)δ196.00,166.52,166.23,138.53,134.57,134.55,133.44,131.84,130.51( 2C),128.03,124.61,122.76,122.61,115.94,48.11,27.01.HRMS(ESI)cald.forC17H12BrFN2O3Na +

[0074] [M+Na] + :412.99075,found:412.99083.

[0075] Example 5

[0076] Preparation of 4-(3-bromo-5-methoxybenzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (4e)

[0077]

[0078] Except that 3-bromo-5-methoxy-benzoyl chloride was used instead of 3,4,5-trimethoxybenzoyl chloride in step (3), the other steps and purification method were the same as in Example 2 to obtain 0.41 g of white solid. The yield was 38.7%, HPLC purity: 96.7166%, melting point: 203.9-204.5°C, HRMS (ESI) cald. for C18H15BrN2O4H + [M+H] + :403.0288,found:403.02886, 1 H NMR (300MHz, DMSO-d6) δ10.95(s,1H),7.57(s,1H),7.45(d,J=8.5Hz,1H),7. 28(s,1H),7.18(s,1H),6.98(s,2H),4.38(s,2H),3.72(s,3H),2.50(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.5,166.9,166.2,159.8,137.4,134.0,131.3,1 30.5,124.0,123.1,122.3,122.0,119.1,115.5,113.6,55.8,47.7,26.6.

[0079] Example 6

[0080] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-(1-hydroxyethyl)-3,4-dihydroquinoxalin-2(1H)-one (5a)

[0081]

[0082] The target compound 4b (200.0 mg, 0.52 mmol) was added to a 100 mL round-bottom flask, and 5 mL of a mixed solvent (volume ratio, MeOH:THF=1:1) was added to dissolve. After the reactants were dissolved, sodium borohydride (78.7 mg, 2.1 mmol, 4 equiv) was added and stirred at room temperature for 4-8 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure and column chromatography (200-300 mesh silica gel, dichloromethane:methanol=30:1) was performed to obtain 86.7 mg of a white solid with a yield of 43.3%. Melting point: 198.2-200.3°C. 1 H NMR (300MHz, DMSO-d6) δ10.75(s,1H),7.05(s,1H),6.73-6.81(m,2H),6.64(s,2H),5.19(d,J=4.1H z,1H),4.64(dt,J=10.3,5.1Hz,1H),4.37(s,2H),3.69(s,3H),3.64(s,6H),1.26(d,J=6.4Hz,3H). 13 C NMR(75MHz,DMSO-d6))δ167.8,166.9,152.4,145.4,139.4,130.9,129.4,125.3 ,123.8,118.7,112.8,106.3,67.4,60.1,55.9,47.9,25.9.HRMS(ESI)cald.for C20H22N2O6Na + [M+Na] + :409.13701,found:409.13886.

[0083] Example 7

[0084] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-((1-hydroxyimino)ethyl)-3,4-dihydroquinoxalin-2(1H)-one (5b)

[0085]

[0086] The target compound 4b (200.0 mg, 0.52 mmol), hydroxylamine hydrochloride (145 mg, 2.1 mmol, 4 equiv), and sodium acetate (171 mg, 2.1 mmol, 4 equiv) were added to a 100 mL round-bottom flask. Subsequently, 10 mL of ethanol was added. After the reactants were dissolved, 1 mL of water was added and the temperature was raised to 95 ° C and stirred for about 12 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure and column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 30: 1) was performed to obtain 91 mg of white solid with a yield of 43.8%. Melting point: 232.6-234.3 ° C. 1 H NMR (300MHz, DMSO-d6) δ11.26(s,1H),10.86(s,1H),7.38(d,J=1.9Hz,1H),7.10(d,J=8.2 Hz,1H),6.87(brs,1H),6.70(s,2H),4.39(s,2H),3.69(s,3H),3.65(s,6H),2.08(s,3H). 13 C NMR(75MHz,DMSO-d6)δ168.0,166.7,152.5,152.0,134.3,131.1,129.4,124.0,119.2,112.8,106.2,99.5,60.2,55.9,48.2,11.4.HRMS(ESI)cald.for C20H21N3O6Na + [M+Na] + :422.13226,found:422.13380.

[0087] Example 8

[0088] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-((1-methoxyimino)ethyl)-3,4-dihydroquinoxalin-2(1H)-one (5c)

[0089]

[0090] The target compound 4b (200.0 mg, 0.52 mmol), methoxyamine hydrochloride (145 mg, 2.1 mmol, 4 equiv), and sodium acetate (171 mg, 2.1 mmol, 4 equiv) were added to a 100 mL round-bottom flask in sequence, and 10 mL of ethanol was added to dissolve. After the reactants were dissolved, 1 mL of water was added, and the temperature was raised to 95°C and stirred for 12 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure, and column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 30:1) was performed to obtain 88.2 mg of a white solid with a yield of 40.9%. Melting point: 206.1-208.5°C. 1 H NMR (300MHz, DMSO-d6) δ10.81(s,1H),7.38(d,J=1.9Hz,1H),7.12(dd,J=9.0,1.8Hz,1H),6. 89(brs,1H),6.70(s,2H),4.38(s,2H),3.90(s,3H),3.69(s,3H),3.66(s,6H),2.11(s,3H). 13 C NMR(75MHz,DMSO-d6)δ168.1,166.5,153.1,152.5,139.4,133.0,131.1,129.4,12 7.7,124.1,119.5,113.1,106.2,61.7,60.2,55.9,48.1,12.1.HRMS(ESI)cald.for C21H23N3O6Na + [M+Na] + :436.14791,found:436.14974.

[0091] Example 9

[0092] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-((1-hydrazino)ethyl)-3,4-dihydroquinoxalin-2(1H)-one (5d)

[0093]

[0094] The target compound 4b (200.0 mg, 0.52 mmol) and hydrazine hydrate (104 mg, 2.1 mmol, 4 equiv) were added to a 100 mL round-bottom flask in sequence, and 10 mL of methanol was added to dissolve. After the reactants were dissolved, the temperature was raised to 80°C and reacted for 12 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure and column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 30:1) was performed to obtain 93.1 mg of a pale white solid with a yield of 44.9%. Melting point: 188.7-190.3°C.1 HNMR(300MHz,DMSO-d6)δ10.78(s,1H),7.36(d,J=1.9Hz,1H),7.04(d,J=8.5Hz,1H),6.79 (brs,1H),6.68(s,2H),6.42(s,2H),4.37(s,2H),3.68(s,3H),3.65(s,6H),1.94(s,3H). 13 CNMR(125MHz,DMSO-d6)δ167.9,166.7,152.5,140.8,139.4,137.4,130.9,12 9.5,125.8,123.7,118.3,112.1,106.2,60.2,55.9,11.2.HRMS(ESI)cald.for C20H22N4O5Na + [M+Na] + :421.14824,found:421.14981.

[0095] Example 10

[0096] Preparation of 4-(3,4,5-trimethoxybenzoyl)-7-ethyl-3,4-dihydroquinoxalin-2(1H)-one (5e)

[0097]

[0098] The target compound 4b (200.0 mg, 0.52 mmol), indium (III) tribromide (55 mg, 0.16 mmol, 0.3 equiv), and triethylsilane (241.9 mg, 2.1 mmol, 4 equiv) were added to a 100 mL round-bottom flask in sequence, and 10 mL of chloroform was added to dissolve the mixture. The mixture was heated to 60°C and reacted for 8 h. The reaction was monitored by TLC spot plate. After the reaction was complete, the solvent was distilled off under reduced pressure and column chromatography (200-300 mesh silica gel, DCM:MeOH=50:1) was performed to obtain 97 mg of white powder with a yield of 50.3%. Melting point: 126.1-128.7°C. 1 H NMR(300MHz, CDCl3)δ9.80(s,1H),6.82(s,1H),6.69-6.63(m,2H),6.61(s,2H),4. 60(s,2H),3.85(s,3H),3.68(s,6H),2.57(q,J=7.6Hz,2H),1.17(t,J=7.6Hz,3H). 13CNMR (75MHz, CDCl3) δ169.4,168.7,152.9,143.1,140.5,129.9,128.8,125.3, 124.7,122.8,115.8,106.7,61.1,56.2,47.9,28.5,15.8.HRMS(ESI)cald.for C20H22N2O5Na + [M+Na] + :393.14209,found:393.14364.

[0099] Embodiment 11

[0100] Preparation of 4-(3-(methyloxy)-5-(furan-2-yl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6a)

[0101]

[0102] Intermediate 4e (0.37 g, 0.92 μmol, 1.0 equiv), 2-furanboronic acid (0.26 g, 2.30 μmol, 2.5 equiv), 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (PdCl2(dppf)) (0.034 g, 0.046 μmol, 0.05 equiv), and sodium acetate (0.293 g, 2.76 μmol, 3.0 equiv) were sequentially added into a 100 mL round-bottom flask, and dry toluene:methanol:water (9 mL:3 mL:3 mL) was added to dissolve, and nitrogen was replaced three times at room temperature. The reaction system was stirred for 3 hours. Place in an oil bath and slowly heat to 100°C, and react at 100°C for about 16 hours. Monitor the reaction by TLC and observe under UV light. After the reaction is complete, cool to room temperature, add dichloromethane and water, extract with dichloromethane (3*50mL), dry the organic layer with anhydrous sodium sulfate, filter, distill under reduced pressure, evaporate the solvent, column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 500: 1 to 50: 1), and obtain 0.195g of white solid. The yield is 54.3%, HPLC purity: 97.2216%, melting point: 186.4-188.5°C, HRMS (ESI) cald.for C22H18N2O5H + [M+H] + :391.12885,found:391.12893, 1H NMR (300MHz, DMSO-d6) δ10.97(s,1H),7.76(s,1H),7.57(s,1H),7.44(d,J=9.1Hz,1H),7.35 (s,2H),7.07-7.01(m,2H),6.88(s,1H),6.60(s,1H),4.42(s,2H),3.75(s,3H),2.49(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.5,168.2,166.4,159.4,151.8,143.5,136.5,133.9,131.8,1 31.3,130.8,124.0,122.4,115.6,115.5,112.9,112.2,111.0,107.3,55.5,47.8,26.6.

[0103] Example 12

[0104] Preparation of 4-(3-methoxy-5-(furan-3-yl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6b)

[0105]

[0106] Except that 3-furanboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.046 g of a white solid. The yield was 47.5%, HPLC purity: 98.7354%, melting point: 196.5-197.8°C, HRMS (ESI) cald. for C22H18N2O5Na + [M+Na] + :413.11079,found:413.11089, 1 H NMR (300MHz, DMSO-d6) δ10.96(s,1H),8.22(s,1H),7.72(s,1H),7.57(d,J=1.6Hz,1H),7.45(d,J=8.5Hz,1H), 7.28(d,J=6.1Hz,2H),7.05(d,J=7.5Hz,1H),6.94(s,1H),6.83(s,1H),4.40(s,2H),3.73(s,3H),2.48(s,3H). 13C NMR(75MHz,DMSO-d6)δ196.5,168.4,166.3,159.4,144.4,140.2,136.2,133.9,133.7,1 31.3,130.8,125.0,123.9,122.3,117.7,115.5,113.6,112.1,108.7,55.4,47.9,26.5.

[0107] Embodiment 13

[0108] Preparation of 4-(3-methoxy-5-(thiophen-2-yl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6c)

[0109]

[0110] Except that 2-thiopheneboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.077 g of white solid. Yield: 51.6%, HPLC purity: 99.4978%, melting point: 199.3-200.6°C, HRMS (ESI) cald. for C22H18N2O4SNa + [M+Na] + :429.08795,found:429.08894, 1 H NMR (300MHz, CDCl3) δ9.64(s,1H),7.66(d,J=1.5Hz,1H),7.41(dd,J=8.4,1.8Hz,1H),7.28(s,1H),7.23(t,J=1.6Hz,1H),7.21 -7.19(m,2H),7.04(dd,J=5.1,3.6Hz,1H),6.93(d,J=8.4Hz,1H),6.86(t,J=1.6Hz,1H),4.62(s,2H),3.77(s,3H),2.55(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.5,168.0,166.4,159.5,142.0,136.4,135.2,134.0,131.3,1 30.8,128.5,126.5,124.8,124.1,122.3,117.8,115.4,113.2,112.9,55.5,47.7,26.5.

[0111] Embodiment 14

[0112] Preparation of 4-(3-methoxy-5-(thiophen-3-yl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6d)

[0113]

[0114] Except that 3-thiopheneboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.077 g of white solid. The yield was 38.2%, HPLC purity: 98.9647%, melting point: 200.7-201.8°C, HRMS (ESI) cald. for C22H18N2O4SH + [M+Na] + :429.08795,found:429.08900, 1 H NMR (300MHz, DMSO-d6) δ10.99(s,1H),7.92(s,1H),7.67-7.58(m,2H),7.52(s,1H),7.45(d,J=8.7Hz, 1H),7.39(d,J=8.8Hz,2H),7.08(d,J=5.4Hz,1H),6.91(s,1H),4.44(s,2H),3.77(s,3H),2.50(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.5,168.4,166.4,159.5,140.3,136.7,136.2,133.9,131.3,1 30.9,127.3,126.2,124.0,122.4,122.2,118.3,115.5,114.2,112.3,55.5,47.9,26.5.

[0115] Embodiment 15

[0116] Preparation of 4-(3-methoxy-5-phenylbenzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6e)

[0117]

[0118] Except that phenylboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.065 g of a white solid. The yield was 50.3%, HPLC purity: 99.6080%, melting point: 181.7-182.8°C, HRMS (ESI) cald.for C24H20N2O4H+[M+H]+:401.14958, found:401.14975, 1HNMR(300MHz,DMSO-d6)δ10.99(s,1H),7.65-7.53(m,3H),7.51–7.37(m,4H),7.3 2(s,1H),7.23(s,1H),7.11-6.97(m,2H),4.44(s,2H),3.80(s,3H),2.50(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.5,168.4,166.4,159.5,141.7,139.1,136.1,133.9,131.3,131.0 ,129.0(2C),128.1,126.8(2C),124.2,122.3,119.0,115.5,114.8,113.0,55.5,47.9,26.6.

[0119] Example 16

[0120] Preparation of 4-(3-methoxy-5-(4-fluorophenyl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6f)

[0121]

[0122] Except that 4-fluorophenylboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.055 g of white solid. The yield was 53.0%, HPLC purity: 99.3643%, melting point: 230.5-231.6°C, HRMS (ESI) cald. for C24H19FN2O4H + [M+H] + :419.14016,found:419.14018, 1 H NMR (300MHz, CDCl3) δ8.35 (s, 1H), 7.57 (d, J = 1.5, 1H), 7.46-7.34 (m, 3H), 7.16-7.13 (m ,2H),7.03(t,J=8.7Hz,2H),7.01-6.93(m,2H),4.60(s,2H),3.80(s,3H),2.55(s,3H). 13C NMR (75MHz, DMSO-d6) δ196.6,168.3,166.4,162.2(d,J=243.8Hz),160.5,159.5,140.7,136.2,135.5(d,J=3.0Hz),133 .9,131.3,130.9,128.9(d,J=7.5Hz),124.1,122.3,118.9,115.9(d,J=21.0Hz),115.5,114.8,112.9,55.5,47.8,26.6.

[0123] Embodiment 17

[0124] Preparation of 4-(3-methoxy-5-(4-chlorophenyl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6 g)

[0125]

[0126] Except that 4-chlorophenylboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.097 g of gray solid. The yield was 90.0%, HPLC purity: 97.9813%, melting point: 245.7-246.5°C, HRMS (ESI) cald. for C24H19ClN2O4H + [M+H] + :435.11061,found:435.11069, 1 H NMR (300MHz, DMSO-d6) δ10.97(s,1H),7.62(d,J=8.4Hz,2H),7.58(s,1H),7.49(d,J=8.4Hz,2H),7.44(d,J=8. 7Hz,1H),7.32(s,1H),7.27(s,1H),7.06(d,J=6.5Hz,1H),7.00(s,1H),4.43(s,2H),3.78(s,3H),2.49(s,3H). 13 C NMR(75MHz,DMSO-d6)196.5,168.2,166.4,159.5,140.4,137.8,136.3,133.9,132.9,1 31.3,130.8,128.9,128.6,124.1,122.3,118.9,115.5,114.7,113.3,55.5,47.8,26.5.

[0127] Embodiment 18

[0128] Preparation of 4-(3-methoxy-5-(4-cyano)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6h)

[0129]

[0130] Except that 4-cyanophenylboronic acid was used instead of 2-furanboronic acid, the other steps and purification method were the same as in Example 11 to obtain 0.068 g of brown solid. Yield: 64.5%, HPLC purity: 99.4879%, melting point: 234.8-236.0°C, HRMS (ESI) cald. for C25H19N3O4 H + [M+H] + :426.14483,found:426.14871, 1 H NMR(300MHz,DMSO-d6)δ11.04(s,1H),7.96-7.75(m,4H),7.61(s,1H),7.4 9-7.34(m,3H),7.16-6.98(m,2H),4.45(s,2H),3.81(s,3H),2.50(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.5,168.1,166.3,159.6,143.5,139.8,136.5,133.9,132.8(2C),131. 3,130.8,127.8(2C),124.1,122.3,119.3,118.7,115.6,115.1,114.2,110.6,55.6,47.9,26.6.

[0131] Embodiment 19

[0132] Preparation of 4-(3-methoxy-5-(naphthalen-2-yl)benzoyl)-7-acetyl-3,4-dihydroquinoxalin-2(1H)-one (6i)

[0133]

[0134] Except that 2-naphthaleneboric acid was used instead of 2-furanboric acid, the other steps and purification method were the same as in Example 11 to obtain 0.108 g of a white solid. The yield was 48.3%, HPLC purity: 100.0000%, melting point: 29.8-211.9°C, HRMS (ESI) cald. for C28H22N2O4H + [M+H] + :451.16523,found:451.16526, 1H NMR(300MHz,DMSO-d6)δ11.00(s,1H),8.11(s,1H),8.05–7.87(m,3H),7.74(d,J=8.4Hz,1H),7.63(s,1H),7 .59-7.51(m,2H),7.51-7.44(m,2H),7.39(s,1H),7.16-7.00(m,2H),4.48(s,2H),3.84(s,3H),2.51(s,3H). 13 CNMR(75MHz,DMSO-d6)δ196.5,168.3,166.4,159.6,141.5,136.4,136.2,134.0,133.1,132.5,131.4,131.0, 128.5,128.1,127.5,126.6,126.4,125.7,125.0,124.2,122.4,119.4,115.5,115.1,113.1,55.5,47.8,26.6.

[0135] Embodiment 20

[0136] Preparation of 4-(3-bromo-5-methoxybenzoyl)-7-(1-hydroxyiminoethyl)-3,4-dihydroquinoxalin-2(1H)-one (7a)

[0137]

[0138] Compound 4e (0.10 g, 0.25 μmol, 1.0 equiv), hydroxylamine hydrochloride (0.07 g, 1.0 μmol, 4.0 equiv), potassium carbonate (0.106 g, 1.0 μmol, 4.0 equiv) were added to a 100 mL round-bottom flask, and 30 mL of anhydrous ethanol was added. The reaction system was refluxed at 70 ° C for about 10 hours. The reaction was monitored by TLC and observed under ultraviolet light. After the reaction was complete, it was cooled to room temperature and distilled under reduced pressure to evaporate the solvent. Column chromatography (200-300 mesh silica gel, dichloromethane: methanol = 200: 1 to 50: 1) was performed to obtain 0.065 g of a yellow solid. The yield was 62.7%, HPLC purity: 98.7876%, melting point: 271.3-272.6 ° C, HRMS (ESI) cald. for C18H16BrN3O4H + [M+H] + :418.0397,found:418.03974, 1H NMR(300MHz,DMSO-d6)δ11.32(s,1H),10.96(s,1H),7.42(s,1H),7.26(s,1H),7.13(s ,1H),7.08(d,J=8.1Hz,1H),7.01-6.87(m,2H),4.36(s,2H),3.71(s,3H),2.07(s,3H). 13 C NMR(300MHz,DMSO-d6)δ166.6,166.4,159.8,151.8,137.6,134.6,131.2, 126.6,124.0,123.0,121.9,119.1,118.9,113.5,113.0,55.8,47.6,11.3.

[0139] Embodiment 21

[0140] Preparation of 4-(3-methoxy-5-(furan-2-yl)benzoyl)-7-(1-hydroxyiminoethyl)-3,4-dihydroquinoxalin-2(1H)-one (7b)

[0141]

[0142] Except for using 6a to replace 4e, the other steps and purification methods were the same as in Example 20 to obtain 0.089 g of a white solid. The yield was 57.1%, HPLC purity: 99.8585%, melting point: 262.4-263.5°C, HRMS (ESI) cald. for C18H15BrN2O4H + [M+H] + :406.13975, found:406.13942, 428.12129, 1 H NMR(300MHz,DMSO-d6)δ11.28(s,1H),10.94(s,1H),7.75(s,1H),7.46 -7.29(m,3H),7.15-6.97(m,3H),6.82(s,1H),6.59(s,1H),4.39(s,2H),3.74(s,3H),2.05(s,3H). 13 C NMR(75MHz,DMSO-d6)δ167.9,166.6,159.3,151.9(2C),143.4,136.7,134.4,131.8,13 1.2,126.9,124.0,119.2,115.5,113.0,112.8,112.2,110.8,107.3,55.5,47.8,11.3.

[0143] Embodiment 22

[0144] Preparation of 4-(3-methoxy-5-(thiophen-2-yl)benzoyl)-7-(1-hydroxyiminoethyl)-3,4-dihydroquinoxalin-2(1H)-one (7c)

[0145]

[0146] Except for using 6c to replace 4e, the other steps and purification methods were the same as those in Example 20 to obtain 0.095 g of a white solid with a yield of 66.1%, HPLC purity: 99.1123%, melting point: 250.3-251.6°C, HRMS (ESI) cald. for C18H15BrN2O4Na + [M+Na] + :444.09885,found:444.09968, 1 H NMR (300MHz, DMSO-d6) δ11.26(s,1H),10.88(s,1H),7.55(d,J=4.9Hz,1H),7.50(d,J=3.0Hz,1H),7.40(s,1H),7.28(s,1H),7.19(s,1H),7.14 -7.01(m,3H),6.95-6.84(m,2H),4.41(s,2H),3.75(s,3H),2.06(s,3H). 13 C NMR(75MHz,DMSO-d6)δ168.2,167.1,159.9,152.4,142.5,137.0,135.6,135.0,131.6,1 29.9,129.0,127.4,126.9,125.2,124.6,119.7,118.2,113.5,113.3,55.9,48.1,11.8.

[0147] Below are some pharmacological experiments and results described in the present invention:

[0148] Example 1: Experiment on inhibition of tumor cell proliferation (MTT experiment)

[0149] (1) Experimental methods

[0150] The cell proliferation inhibitory activity of the compounds of the present invention on human liver cancer cell lines (HepG-2) and human cervical cancer cell lines (HeLa) was tested. The above cell lines were frozen and passaged by the laboratory. Methods: For HepG-2 and HeLa tumor cells, the cells were inoculated in a 96-well flat-bottom plate (about 4000-6000 cells per well), incubated at 37°C, 5% CO2 for 24 hours, and then different concentrations of the test compound or positive control drug were added to each well. After culturing for another 72 hours, 50 μL of MTT PBS buffer (concentration of 1 mg / mL) was added to each well, incubated at 37°C for 4 hours, MTT and culture medium were removed, and 100 μL of DMSO was added to each well, and detected at 490nm (using a plate reader). Compretin (CA-4) and doxorubicin were selected as positive control drugs. The data were statistically analyzed.

[0151] (2) Experimental results

[0152] The experimental results are shown in Table 1. The results show that this class of compounds generally have a strong cell growth inhibitory effect. The half inhibitory concentration (IC 50 ) reached the nanomolar level (nM). The cell growth inhibitory activity of these compounds was significantly better than that of doxorubicin.

[0153] Table 1 MTT test results of compounds

[0154]

[0155]

[0156] Note: Compound CA-4 in the table is a positive control drug and is not included in the scope of patent protection.

[0157] 2. Tubulin polymerization inhibition experiment

[0158] (1) Experimental methods

[0159] The inhibitory effect of the compounds of the present invention on tubulin polymerization was tested using the BK011P kit of cytoskeleton. A 96-well plate was placed on a multifunctional microplate reader (CLARIOstar, BMG LABTECH, Germany) and preheated at 37°C for 10 minutes. According to the instructions, the test compound solution (10×), buffer 1, GTP mother solution, tubulin glycerol buffer, and tubulin mother solution were prepared for standby use. According to the experiment, a suitable ratio was selected, and buffer 1, GTP mother solution, tubulin glycerol buffer, and tubulin mother solution were mixed to prepare a microtubule reaction solution, which was placed on an ice bath. 5 μL of control buffer, Colchicine solution (5 μM), and a test compound solution (set 1 replicate) were added to the 96-well plate, preheated at 37°C for 1 minute on the microplate reader, and the prepared microtubule reaction solution was quickly added, and the change in fluorescence intensity within 60 minutes was recorded.

[0160] Preparation of buffer 1: ① Dissolve the substances in each bottle of buffer 1 with 10 mL of Milli-Q ultrapure water (two bottles in total); ② Mix the substances in the two bottles; ③ Divide the above buffer into 13 cryovials, with 1.5 mL in each tube, and store at -70°C.

[0161] Tubulin glycerol buffer, stored at 4°C.

[0162] Preparation of GTP mother solution: ① Dissolve the substance in each tube of GTP mother solution with 100 μL of ice-cold sterile distilled water (3 tubes in total); ② Place it on ice; ③ Mix the substances in the three tubes; ④ Divide the above solution into 13 cryotubes, 20 μL in each tube, and store at -70°C.

[0163] Preparation of tubulin solution: ① Place the vial containing freeze-dried tubulin on ice and place liquid nitrogen in a Dewar flask; ② Label 12 cryovials and place them on ice for use; ③ Defrost a 20μL GTP stock solution; ④ Mix 1.5mL of ice-cold buffer 1 with 15μL GTP stock solution; ⑤ Use 1.1mL of the above mixture to dissolve the freeze-dried tubulin powder; ⑥ Place on ice for 2 minutes until the powder is completely dissolved and suspended; ⑦ Divide the above tubulin solution into 12 cell cryovials, 88μL per tube, and quickly freeze in liquid nitrogen; ⑧ Store at -70℃.

[0164] (2) Experimental results

[0165] like Figure 1 As shown, the results showed that compound 7c had a significant inhibitory effect on microtubule polymerization at 1 μM and 20 μM.

[0166] 3. Cell cycle experiment

[0167] (1) Experimental methods

[0168] The effect of the compound of the present invention on the cancer cell cycle (human cervical cancer cell line HeLa was selected) was tested by PI (propidium iodide) staining. Method: The cells were inoculated in a 6-well flat-bottom plate (about 100,000 cells per well), incubated at 37°C, 5% CO2 for 24 hours, and different concentrations of 7c were added to each well (the final concentrations were 10nM, 20nM, and 30nM, respectively), and a blank control group was set up (the blank control group did not add compound 7c), and incubated at 37°C, 5% CO2 for 24 hours. The cells were collected, washed with precooled PBS (4°C, 0.01mol / L), and the cells were added to precooled (4°C) 70% ethanol, and the fixed cells were placed at -20°C overnight. The cells were centrifuged, ethanol was removed, and the cells were washed with pre-cooled PBS (4°C, 0.01 mol / L). PI dye containing RNase was added and the cells were stained at 4°C in the dark for 1 hour. The cells were centrifuged, the dye was removed, and the cells were washed with PBS. The cells were filtered through a 300-mesh filter and tested on a flow cytometer (BD FACS, USA).

[0169] (2) Experimental results

[0170] like Figure 2 As shown, the results showed that compound 7c at 30 nM could significantly arrest cells in the G2 / M growth phase.

Claims

1. A 4-substituted arylformyl-7-substituted 3,4-dihydroquinoxaline-2(1H)-one structural compound and a pharmaceutically acceptable salt thereof, characterized in that: The compound has the structure shown in formula (I): Wherein, R1 represents ethyl, acetyl, 1-hydroxyethyl, R2 represents hydrogen, fluorine, chlorine, bromine or methoxy; R3 represents hydrogen, fluorine, chlorine, bromine or methoxy; R4 represents hydrogen, fluorine, chlorine, bromine, methoxy, L is fluorine, chlorine, bromine, methyl, ethyl, hydroxy, acetyl, methoxy or cyano.

2. The 4-substituted arylformyl-7-substituted 3,4-dihydroquinoxaline-2(1H)-one structural compound and its pharmaceutically acceptable salt according to claim 1, characterized in that: R1 represents ethyl, acetyl, 1-hydroxyethyl, R2 represents fluorine, chlorine, bromine or methoxy; R3 represents hydrogen or methoxy; R4 represents fluorine, chlorine, bromine, methoxy, L is fluorine, chlorine or cyano.

3. The method for preparing the 4-substituted arylformyl-7-substituted 3,4-dihydroquinoxaline-2(1H)-one structure compound according to claim 1 or 2, characterized in that: The synthetic route of this type of compound is as follows: The specific synthesis steps are: (1) Compound 1a or 1b is reacted with glycine methyl ester hydrochloride in acetonitrile using potassium carbonate as a base to undergo nucleophilic substitution reaction to obtain compound 2a or 2b; (2) Compound 2a or 2b is respectively treated with ethyl acetate as solvent and stannous chloride as catalyst, and subjected to reduction and cyclization in one pot to obtain compound 3a or 3b; (3) Compound 3a or 3b is reacted with different substituted aryl acid chlorides in tetrahydrofuran or dichloromethane as solvent and 4-dimethylaminopyridine as catalyst to obtain compounds 4a-4e; (4) Compound 4b is subjected to reduction reaction with sodium borohydride in a mixture of tetrahydrofuran and methanol as solvent to obtain 5a; Compound 4b is subjected to reaction with hydroxylamine hydrochloride or methoxyamine hydrochloride in ethanol as solvent and sodium acetate or potassium carbonate as base to obtain compound 5b or 5c; Compound 4b is subjected to reaction with hydrazine hydrate in methanol as solvent to obtain compound 5d; Compound 4b is subjected to reduction reaction with triethylsilane in chloroform as solvent and indium tribromide as catalyst to obtain 5e; (5) Compound 4e was reacted with arylboronic acid via Suzuki reaction in a mixture of toluene, methanol and water as solvent, ferrocene palladium dichloride as catalyst and sodium acetate as base to generate compounds 6a-6i; (6) Compound 4e or 6a or 6c is reacted with hydroxylamine hydrochloride using ethanol as solvent and sodium acetate or potassium carbonate as base to produce compounds 7a-7c.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of compound 1a or 1b, glycine methyl ester hydrochloride and potassium carbonate is 1:1.1:2; in step (2), the molar ratio of compound 2a or 2b to stannous chloride is 1:6, and the reaction temperature is 40 to 60°C; in step (3), the molar ratio of 3a or 3b, aryl acid chloride and 4-dimethylaminopyridine is 1:1.5:0.2; in step (4), the volume ratio of tetrahydrofuran and methanol is 1:1, and the molar ratio of compound 4b to sodium borohydride is 1:4; the molar ratio of compound 4b, sodium acetate or potassium carbonate and hydroxylamine hydrochloride or methoxyamine hydrochloride is 1:4:4, and the reaction temperature is 90 to 100°C; compound 4b and hydrazine hydrate The molar ratio of compound 4b, indium tribromide and triethylsilane is 1:0.3:4, and the reaction temperature is 55-65°C; in step (5), the ferrocene palladium dichloride is 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride, the volume ratio of toluene, methanol and water is 3:1:1, the molar ratio of compound 4e, arylboronic acid, ferrocene palladium dichloride and sodium acetate is 1:2.5:0.05:3, and the reaction temperature is 95-105°C; in step (6), the molar ratio of compound 4e or 6a or 6c, sodium acetate or potassium carbonate and hydroxylamine hydrochloride is 1:4:4, and the reaction temperature is 65-75°C.

5. A pharmaceutical composition, characterized in that Containing the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

6. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating tumors or proliferative diseases other than tumors.

7. The use according to claim 6, characterized in that The tumor is breast cancer, ovarian cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, prostate cancer, melanoma or leukemia.

8. Use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a microtubule polymerization inhibitor.

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