Compound with diarylamine structure as well as preparation method and medical application thereof

Abstract

The invention discloses a diarylamine structure compound and a preparation method and medical application thereof. The compound has a structural general formula shown in the specification, wherein R1 represents hydrogen, halogen, methyl, cyano, methoxyl, acetyl, methoxycarbonyl and methyl oximido; r2 represents hydrogen, halogen, methyl, methoxyl or cyano; r < 3 > represents hydrogen or halogen, R < 4 > represents hydrogen, halogen or methyl, R < 5 > represents hydrogen, halogen, methyl, methoxyl, N-methyl piperazinyl and N-ethyl piperazinyl, W represents a carbon atom or a nitrogen atom, X represents an oxygen atom or NH, and Y represents methylene or carbonyl. The compounds have good application in resisting tumors and proliferative diseases except tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a diarylamine structure compound, a preparation method thereof and medical use thereof. Background Art

[0002] Microtubules are tubular polymers composed of α- and β-tubulin heterodimers. They have numerous functions in cells, particularly participating in mitosis as spindles. Tumor cells have the ability to proliferate rapidly. Inhibiting spindle formation will inevitably lead to a blockage of mitosis, causing tumor cell growth to arrest 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 transport, and the mitotic process of eukaryotic cells. When cells enter mitosis, cytoplasmic microtubules disaggregate into tubulin and then assemble into spindle microtubules. During anaphase, spindle microtubules pull sister chromatids from the equatorial plane toward the spindle poles. After mitosis, spindle microtubules disaggregate and reassemble into cytoplasmic microtubules. Therefore, this dynamic conversion of tubulin / microtubules (polymerization and depolymerization) is essential for normal mitosis. Disruption of this cycle can cause cells entering mitosis to cease division, thereby prolonging the cell cycle or inducing apoptosis. Therefore, tubulin has become an important target for anti-tumor drugs.

[0004] CDKs belong to the serine / threonine protein kinase family and form heterodimers with their respective regulatory cell cycle protein subunits to exert their effects. They can be divided into three groups according to their functions. The first group is mitosis-related CDKs (CDK 1, CDK2, CDK4 and CDK6), which directly promote cell cycle progression, although other CDKs also play a role in mammalian cell cycle regulation; the second group is transcription-related CDKs (CDK 7, CDK8 and CDK 9); the third group is atypical CDKs (CDK 5, CDK14, CDK15, CDK18), etc. Among them, CDK4 / 6 controls the transition from the G1 phase to the S phase of the cell cycle. The classic CDK4 / 6 pathway in cell proliferation involves CDK4 / 6 binding to cyclin D1, phosphorylating retinoblastoma protein (RB1) and RB-like proteins (RBL1 and RBL2). This leads to the dissociation of RB1 from members of the E2F transcription family. The released E2F enters the cell nucleus and regulates the transcription of substances necessary for entry into the S phase. Conversely, E2F promotes the transcription of cyclin E bound to CDK2. This complex further phosphorylates RB1, ultimately prompting cells to enter the S phase. Abnormalities in the CDK4 / 6-RB-E2F axis have been observed in various cancers. Therefore, CDK4 / 6 inhibitors have become important anti-cancer drug targets. To date, six CDK4 / 6 small molecule drugs have been approved for marketing.

[0005] Multi-target drugs have become an effective strategy to overcome mechanism-based drug resistance. In addition, G1 Therapeutics recently developed a short-acting CDK4 / 6 inhibitor, Trilaciclib, which has an inhibitory activity IC of CDK4 / 6. 50 The values ​​are 1nM and 4nM, respectively. It is used in combination with cytotoxic chemotherapy drugs to reduce the toxic side effects of chemotherapy drugs. Its mechanism of action is that patients are first administered Trilaciclib, which induces hematopoietic stem cells in the bone marrow to temporarily arrest in the G1 phase, followed by the administration of chemotherapy drugs to protect myeloid cells from damage by cytotoxic chemotherapy drugs. In clinical practice, Trilaciclib is combined with platinum / etoposide or topotecan-containing regimens for the treatment of extensive-stage small cell lung cancer (ES-SCLC), showing good myeloid cell protection efficacy. Therefore, the research and development of dual-target inhibitors of tubulin and CDK4 / 6 is an effective strategy to effectively reduce resistance to tubulin inhibitors and reduce their toxicity. Summary of the Invention

[0006] The present invention aims to provide a diarylamine structural compound; another object of the present invention is to provide a method for preparing the compound; and a 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 anti-tumor or proliferative diseases other than tumors.

[0007] Based on the above objectives, the specific technical solutions adopted by the present invention are as follows:

[0008] As the first aspect of the present invention, the bicyclic acetamide structural compound and any combination thereof or pharmaceutically acceptable salts thereof have the following general structural formula:

[0009]

[0010] where R 1 represents hydrogen, halogen, methyl, cyano, methoxy, acetyl, methoxycarbonyl, or oxime; R 2 represents hydrogen, halogen, methyl, methoxy or cyano; R 3 represents hydrogen or halogen, R 4 represents hydrogen, halogen or methyl, R 5 represents hydrogen, halogen, methyl, methoxy, N-methylpiperazinyl, N-ethylpiperazinyl, W represents a carbon atom or a nitrogen atom, X represents an oxygen atom or NH, and Y represents a methylene group or a carbonyl group.

[0011] A biologically acceptable salt formed by the above-mentioned diarylamine structural compound and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

[0012] 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:

[0013] Synthesis route:

[0014]

[0015] R 1 represents hydrogen, F, Cl, Br, methyl, cyano, methoxy, acetyl, methoxycarbonyl; R 2 represents hydrogen, F, Cl, Br, methyl or cyano; R 3 Represents hydrogen, F, Cl, Br, R 4 represents hydrogen, F, Cl, Br or methyl, R 5 represents hydrogen, F, Cl, Br, methyl, methoxy, N-methylpiperazinyl or N-ethylpiperazinyl, W represents a carbon atom or a nitrogen atom, Het is The specific steps are:

[0016] The specific steps are:

[0017] (a) Using substituted o-fluoronitrobenzene as the starting material, a nucleophilic substitution reaction with glycine methyl ester hydrochloride under alkaline conditions was performed to obtain compound 2;

[0018] (b) Compound 2 is reduced with hydrogen gas under the catalysis of palladium on carbon to obtain compound 3;

[0019] (c) Compound 4 reacts with a substituted aromatic amine under acidic conditions to give compound 5 through nucleophilic substitution reaction;

[0020] (d) Compound 5 is reacted with phosphorus oxychloride to obtain compound 6;

[0021] (e) Compound 3 reacts with compound 6 under acidic conditions to obtain compound 7 through nucleophilic substitution reaction;

[0022] (f)R 1 Compound 7 with an acetyl group is subjected to dehydration condensation with hydroxylamine hydrochloride under alkaline conditions to obtain compound 8;

[0023] (g)R 4 or R 5 Compound 7 containing F, Cl, or B is reacted with N-methylpiperazine or N-ethylpiperazine via Bach-Wald coupling to obtain compound 9;

[0024] (h) Compound 7 is reduced by borane to obtain compound 10;

[0025] (i) Compound 6 reacts with benzomorpholine or 2,3-dihydro-1H-quinolin-4-one to obtain compound 11 through nucleophilic substitution reaction.

[0026] Specifically, the present application synthesized the following compounds:

[0027]

[0028]

[0029] Furthermore, in step (a), the base is potassium carbonate, the molar ratio of substituted o-fluoronitrobenzene, glycine methyl ester hydrochloride and base is 1:(1-1.5):(1.5-2.5), and the reaction temperature is room temperature.

[0030] Furthermore, in step (b), the molar ratio of compound 2 to Pd / C is 1:(0.05-0.1), and the reaction is carried out in an ice bath.

[0031] Furthermore, in step (c), the reaction is carried out in pivalic acid, and the molar ratio of compound 4 to the substituted aromatic amine is 1:(1-3).

[0032] Furthermore, in step (d), compound 5 reacts with phosphorus oxychloride in the presence of pyridine at 95-105° C., and the molar ratio of compound 5, phosphorus oxychloride and pyridine is 1:(5-7):(1-3).

[0033] Furthermore, in step (e), the acid is hydrochloric acid, the reaction solvent used is 1,4-dioxane, the hydrochloric acid is prepared into a hydrochloric acid-1,4-dioxane solution with a concentration of 0.5 to 2 mol / L, the molar ratio of compound 3, compound 6 and hydrochloric acid is 1:(0.5 to 1):(10 to 20), and the reaction temperature is 95 to 105°C.

[0034] Furthermore, in step (f), the base is sodium acetate, R 1 The molar ratio of the acetyl compound 7, hydroxylamine hydrochloride and sodium acetate is 1:(1.5-2):(1-3), the reaction temperature is 90-100°C, and the reaction solvent used is a mixed solvent of anhydrous ethanol and water, with the volume ratio of ethanol in the mixed solvent being 50-75%.

[0035] Furthermore, in step (g), R 4 or R 5 Compound 7 containing F, Cl, or Br reacts with N-methylpiperazine or N-ethylpiperazine in the presence of lithium bis(trimethylsilyl)amide, x-phos, and Pd2(dba)3 at a temperature of 65 to 75°C. 4 or R 5 The molar ratio of compound 7 containing F, Cl, and Br to N-methylpiperazine or N-ethylpiperazine, lithium bis(trimethylsilyl)amide, x-phos, and Pd2(dba)3 is 1:(1.5-2.5):(4.5-5.5):(0.1-0.3):(0.03-0.07).

[0036] Furthermore, in step (h), the molar ratio of compound 7 to borane is 1:(9-11), and the reaction temperature is -5°C to 5°C.

[0037] Furthermore, in step (i), during the nucleophilic substitution reaction, the acid used is hydrochloric acid, the reaction solvent used is 1,4-dioxane, the hydrochloric acid is prepared into a hydrochloric acid-1,4-dioxane solution with a concentration of 0.5 to 2 mol / L, the molar ratio of benzomorpholine or 2,3-dihydro-1H-quinolin-4-one, compound 6 and hydrochloric acid is 1:(0.5 to 1):(10 to 20), and the reaction temperature is 95 to 105°C.

[0038] A pharmaceutical composition comprising the above compound or the above biologically acceptable salt.

[0039] As a third aspect of the present invention, there is also provided use of any of the above compounds, any combination thereof, or biologically acceptable salts thereof in the preparation of a medicament for preventing and / or treating tumors or proliferative diseases other than tumors.

[0040] The tumor disease is breast cancer, ovarian cancer, cervical cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, prostate cancer, melanoma or leukemia.

[0041] Use of the above compound or the above biologically acceptable salt in the preparation of tubulin polymerization inhibitors and / or CDK4 / 6 inhibitors.

[0042] The present invention also provides a pharmaceutical composition. The compound of the general formula, pharmaceutically acceptable salts or solvates can be added with pharmaceutically acceptable carriers to prepare common pharmaceutical preparations, such as tablets, capsules, powders, syrups, liquids, suspensions, and injections. Common pharmaceutical excipients such as flavorings, sweeteners, liquid or solid fillers or diluents can be added.

[0043] Use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a microtubule polymerization inhibitor and / or a CDK4 / 6 inhibitor.

[0044] In order to evaluate the anti-tumor activity of the synthetic compounds of the present invention and the pharmaceutical compositions thereof, pharmacological activity tests at the cellular and molecular levels were performed on the compounds of the present invention.

[0045] In vitro cell growth inhibition experiments conducted on the compounds and pharmaceutical compositions of the present invention have shown that these compounds generally have strong cell growth inhibition effects. The cell growth inhibition activity of some compounds is comparable to that of the positive control drug, palbociclib.

[0046] 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 9a had a significant inhibitory effect on tubulin polymerization.

[0047] The compounds of the present invention and the pharmaceutical composition thereof were subjected to in vitro cell growth cycle experiments, and the results showed that compound 9a at 2 μM could significantly arrest cells in the G2 / M phase.

[0048] The compounds and pharmaceutically acceptable carriers of the present invention can be administered clinically by oral administration, intravenous injection, etc. They can be used clinically as monotherapy or in combination with other clinically used chemotherapeutic drugs and radiotherapy for the treatment of the aforementioned cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Figure 2 is the in vitro inhibition of tubulin polymerization. Control is the blank control, colchicine (5 μM), and compound 9a (5 μM) on tubulin polymerization.

[0050] Figure 2 This is a graph showing the effect of compound 9a on the growth cycle of HepG2 cells;

[0051] Figure 3 The positive drugs palbociclib and compound 9a showed inhibitory effects on CDK activity. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described in detail below with reference to specific examples, but the scope of protection of the present invention is not limited thereto. The ratios of the eluents used in the column chromatography in the following examples are all by volume.

[0053] Example 1

[0054] Preparation of 4-((2-phenylamino)pyrimidin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (7aa)

[0055]

[0056] (1) Preparation of N-(2-nitrophenyl)glycine methyl ester (2)

[0057] 2-Fluoronitrobenzene (5.0 g, 35.4 mmol, 1.0 equiv), glycine methyl ester hydrochloride (4.9 g, 38.9 mmol, 1.1 equiv), and potassium carbonate (9.8 g, 70.8 mmol, 2.0 equiv) were added sequentially to a 250 mL round-bottom flask. Dissolved in 50 mL of anhydrous acetonitrile, the mixture was stirred at room temperature for 24 hours, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was extracted with dichloromethane and water. The organic phase was evaporated under reduced pressure and purified by column chromatography (200-300 mesh silica gel, PE:EA = 2:1) to yield 5.4 g of a yellow solid with a yield of 72.5%. 1 H NMR (300MHz, Chloroform-d) δ8.43(brs,1H),8.23(d,J=8.4Hz,1H),7.50(t,J=7.5Hz,1H),6.85–6.62(m,2H),4.15(d,J=5.0Hz,2H),3.86(s,3H).

[0058] (2) Preparation of 3,4-dihydroquinoxaline-2(1H)-one (3)

[0059] Intermediate 2 (1.0 g, 4.7 mmol, 1.0 equiv) and 0.3 mmol of 10% Pd / C were added sequentially to a 100 mL round-bottom flask. Dissolved with 30 mL of methanol, the hydrogen atmosphere was replaced three times, and the mixture was stirred in an ice bath at 0°C for 4 h. The reaction was monitored by TLC. After completion of the reaction, the mixture was filtered, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Column chromatography (200-300 mesh silica gel, DCM:MeOH = 10:1) afforded 0.42 g of a yellow-brown solid in a 59.6% yield. 1 H NMR (300MHz, DMSO-d6) δ10.22(brs,1H),6.80-6.70(m,2H),6.65(d,J=6.5Hz,1H),6.62-6.55(m,1H),5.94(brs,1H),3.69(s,2H).

[0060] (3) Preparation of 2-phenylaminopyrimidine-4-ol (5)

[0061] 4-Methylthio-2-hydroxypyrimidine (2.0 g, 14.1 mmol, 1.0 equiv) and aniline (2.6 mL, 28.1 mmol, 2.0 equiv) were added sequentially to a 50 mL round-bottom flask. 15 mL of pivalic acid was added for dissolution. The mixture was stirred at 130°C in a heating mantle for 10 h. The reaction was monitored by TLC. Upon completion, a large amount of n-hexane was added to the reaction mixture, and the mixture was stirred at room temperature for 0.5 h. The mixture was then filtered to obtain 2.3 g of a white solid with a yield of 88.3%. 1 H NMR (300MHz, DMSO-d6) δ11.44(brs,1H),8.85(brs,1H),7.76(d,J=6.0Hz,1H),7.61( d,J=8.0Hz,2H),7.31(t,J=7.8Hz,2H),7.03(t,J=7.3Hz,1H),5.81(d,J=6.0Hz,1H).

[0062] (4) Preparation of 4-chloro-N-phenylpyrimidin-2-amine (6)

[0063] Intermediate 5 (2.0 g, 10.8 mmol, 1.0 equiv), phosphorus oxychloride (6.1 mL, 64.8 mmol, 6.0 equiv), and pyridine (1.7 mL, 21.6 mmol, 2.0 equiv) were added sequentially to a 25 mL round-bottom flask and then placed in a 100°C oil bath for 2 h. The reaction was monitored by TLC. After completion, saturated sodium bicarbonate was added to adjust the pH to approximately 6-7. The product was then extracted with dichloromethane and water. The organic layer was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by column chromatography (PE:EA = 2:1) to yield 1.2 g of a white solid in a 54.6% yield.1 H NMR (300MHz, DMSO-d6) δ10.04(brs,1H),8.43(d,J=5.2Hz,1H),7.70(d,J=7.8Hz,2H),7.30(t,J=7.8Hz,2H),7.00-6.97(m,1H),6.95(d,J=5.2Hz,1H).

[0064] (5) Preparation of 4-((2-phenylamino)pyrimidin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (7aa)

[0065] Intermediate 3 (0.30 g, 2.1 mmol, 1.0 equiv) and intermediate 6 (0.34 g, 1.6 mmol, 0.8 equiv) were added sequentially to a 25 mL round-bottom flask. 3.0 mL of 1,4-dioxane was added for dissolution. A hydrochloric acid-1,4-dioxane solution (1 mol / L, 31.5 mL, 15 equiv) was then added. The atmosphere was purged with nitrogen three times and the mixture was reacted in an oil bath at 100°C for 8 h. The reaction was monitored by TLC. Upon completion, the mixture was extracted with saturated sodium bicarbonate and ethyl acetate. The organic phase was evaporated under reduced pressure and purified by column chromatography (200-300 mesh silica gel, DCM:MeOH = 10:1) to afford 0.138 g of a white solid in a 21.5% yield. HPLC purity: 92.7%. Melting point: 270.8-271.4°C. 1 H NMR (300MHz, DMSO-d6) δ10.80(brs,1H),9.93(brs,1H),8.09(d,J=5.8Hz,1H),7.65(d,J=7.5Hz,2H),7.5 3(d,J=7.6Hz,1H),7.39-7.27(m,2H),7.27-7.20(m,1H),7.06(m,3H),6.58(d,J=5.8Hz,1H),4.64(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.6,160.5,159.6,157.6,140.6,132.1,128.4,127.2,125.5,122.9,122.3,121.2,118.9,116.8,96.8,46.9.HRMS(ESI)cald.for C 18 H 15 N5OH + [M+H] + :318.13494,found:318.13572.

[0066] Examples 2 to 8

[0067] Except that 2-fluoronitrobenzene was replaced by 2,5-difluoronitrobenzene, 2-fluoro-5-chloronitrobenzene, 2-fluoro-5-bromonitrobenzene, 3-nitro-4-fluorobenzonitrile, 3-nitro-4-fluoroanisole, 3-nitro-4-fluoroacetophenone, and 3-nitro-4-fluorobenzoic acid methyl ester in step (1), the other steps and purification methods were the same as those in Example 1, and 7ab, 7ac, 7ad, 7ae, 7af, 7ag, and 7ah were prepared, respectively.

[0068]

[0069] The experimental data of 7ab are as follows: yield: 23.9%. HPLC purity: 100%. Melting point: 267.8-268.1℃. 1 HNMR(300MHz,DMSO-d6)δ10.82(brs,1H),9.38(brs,1H),8.10(d,J=5.0Hz,1H),7.70(d,J =6.7Hz,2H),7.25(t,J=7.3Hz,2H),6.98-6.43(m,4H),6.43(d,J=5.0Hz,1H),4.62(s,2H). 13 CNMR(75MHz,DMSO-d6)δ167.6,160.7,159.4,159.3( 1 J C-F =240Hz),157.5,140.5,133.7( 6 J C-F =11.25Hz),128.4,124.9( 5 J C-F =10.50Hz),123.6( 4 J C-F =2.25Hz),121.2,119.0,108.7( 3 J C-F =23.25Hz),103.4( 2 J C-F =25.50Hz),96.5,46.9.HRMS(ESI)cald.forC 18 H 14 FN5OH + [M+H] + :336.12551,found:336.12684.

[0070] The experimental data of 7ac are as follows: yield: 27.3%. HPLC purity: 100%. Melting point: 254.9-255.8℃. 1HNMR (300MHz, DMSO-d6) δ10.86(brs,1H),9.42(brs,1H),8.12(d,J=5.5Hz,1H),7.70(d,J=7.8Hz,2H),7. 54(d,J=8.4Hz,1H),7.26(t,J=7.4Hz,2H),7.15(m,2H),6.98(m,1H),6.49(d,J=5.5Hz,1H),4.61(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.4,160.4,159.5,157.8,140.5,133.4,128.8,128.4,126.3,124.4,121.9,121.2,118.9,116.1,96.9,46.9.HRMS(ESI)cald.for C 18 H 14 ClN5OH + [M+H] + :352.09596,found:352.09586.

[0071] The experimental data of 7ad are as follows: yield: 26.8%. HPLC purity: 100%. Melting point: 260.9-261.7℃. 1 HNMR(300MHz,DMSO-d6)δ10.81(brs,1H),9.39(brs,1H),8.12(d,J=5.7Hz,1H),7.70(d,J=7.7Hz,2H) ,7.48(d,J=11.4Hz,1H),7.32-7.18(m,4H),6.93(t,J=7.0Hz,1H),6.49(d,J=5.7Hz,1H),4.60(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.4,160.3,159.5,157.8,140.5,133.6,128.4,126.7,124.7,124.7,121.2,118.9,116.7,97.0,46.9.HRMS(ESI)cald.forC 18 H 14 BrN5OH + [M+H] + :396.04545,found:396.04598.

[0072] The experimental data of 7ae are as follows: yield: 28.9%. HPLC purity: 99.3%. Melting point: 291.2-291.9℃. 1HNMR (300MHz, DMSO-d6) δ10.99(brs,1H),9.44(brs,1H),8.20(d,J=5.8Hz,1H),7.70(d,J=8.1Hz,3H),7.47(d ,J=8.4Hz,1H),7.36(s,1H),7.25(t,J=7.7Hz,2H),6.94(t,J=7.2Hz,1H),6.58(d,J=5.8Hz,1H),4.58(s,2H). 13 C NMR(75MHz,DMSO-d6)δ166.7,159.9,159.6,158.1,140.4,132.0,131.9,128.4,1 26.2,122.6,121.3,119.4,119.0,118.6,106.1,98.1,47.3.HRMS(ESI)cald.for C 19 H 14 N6OH + [M+H] + :396.04545,found:396.04598.

[0073] The experimental data of 7af are as follows: yield: 21.6%. HPLC purity: 96.2%. Melting point: 286.7-287.4℃. 1 HNMR (300MHz, DMSO-d6) δ10.66(brs,1H),9.37(brs,1H),8.06(d,J=6.8Hz,1H),7.72(d,J=8.2Hz,2H),7.43(s,1H),7. 26(t,J=8.5Hz,2H),6.99(m,1H),6.69(d,J=6.8Hz,1H),6.63(s,1H),6.39(d,J=6.6Hz,1H),4.62(s,2H),3.76(s,3H). 13 C NMR(75MHz,DMSO-d6)δ167.7,160.8,159.5,157.4,157.1,140.7,133.4,128.3, 124.3,121.1,120.3,118.8,107.7,102.1,96.3,55.4,47.0.HRMS(ESI)cald.for C 19 H 17 N5O2H + [M+H] + :348.14550,found:348.14539.

[0074] The experimental data of 7ag are as follows: yield: 31.1%. HPLC purity: 90.3%. Melting point: 288.3-289.1℃. 1 HNMR(300MHz,DMSO-d6)δ10.90(brs,1H),9.46(brs,1H),8.18(d,J=5.4Hz,1H),7.68(m,6 H),7.38–7.21(m,2H),7.03–6.88(m,1H),6.59(d,J=5.4Hz,1H),4.60(s,2H),2.55(s,3H). 13 C NMR (75MHz, DMSO-d6) δ196.2,166.9,160.0,159.6,157.9,140.5,133.1,131.7,131. 5,128.4,123.0,121.6,121.3,119.0,115.9,97.9,47.2,26.5.HRMS(ESI)cald.forC 20 H 18 N5O2H + [M+H] + :360.14550,found:360.14594.

[0075] The experimental data of 7ah are as follows: yield: 31.7%. HPLC purity: 93.5%. Melting point: 278.8-279.2℃. 1 HNMR(300MHz,DMSO-d6)δ10.91(brs,1H),9.45(brs,1H),8.18(d,J=5.8Hz,1H),7.68(m,6H), 7.25(t,J=7.6Hz,2H),6.93(t,J=7.2Hz,1H),6.58(d,J=5.8Hz,1H),4.60(s,2H),3.86(s,3H). 13 C NMR(75MHz,DMSO-d6)δ166.9,165.5,160.0,159.6,157.8,140.4,131.7,131.5,128 .3,125.5,123.3,121.7,121.3,119.0,117.2,97.9,52.1,47.1.HRMS(ESI)cald.for C 20 H 18 N5O3H + [M+H] + :376.14042found:376.14318.

[0076] Examples 9 to 14

[0077] Except that 2-fluoronitrobenzene was replaced by 2,4-difluoronitrobenzene, 2-fluoro-4-chloronitrobenzene, 2-fluoro-4-bromonitrobenzene, 3-fluoro-4-nitrotoluene, 3-fluoro-4-nitrobenzenenitrile and 2-fluoro-3-chloronitrobenzene in step (1), the other steps and purification method were the same as those in Example 1, and 7ai, 7aj, 7ak, 7al, 7am and 7an were prepared respectively.

[0078]

[0079] The experimental data of 7ai are as follows: yield: 34.5%. HPLC purity: 100%. Melting point: 266.5-267.4℃. 1 HNMR (300MHz, DMSO-d6) δ10.74(brs,1H),9.41(brs,1H),8.16(d,J=6.8Hz,1H),7.70(d,J=7.3Hz,2H),7.42(d,J=9 .8Hz,1H),7.24(t,J=6.8Hz,2H),7.04(d,J=5.9Hz,2H),6.92(t,J=7.0Hz,1H),6.56(d,J=6.8Hz,1H),4.56(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.0,160.3,159.5,157.9,157.1( 1 J C-F =236.25Hz),140.5,128.4( 6 J C-F =2.25Hz),128.3,128.2( 5 J C-F =8.25Hz),121.3,118.9,117.5( 4 J C-F =9.3Hz),111.9( 3 J C-F =23.2Hz),109.9( 2 J C-F =26.9Hz),97.65,47.49.HRMS(ESI)cald.for C 18 H 14 FN5OH + [M+H] + :336.12551,found:336.12619.

[0080] The experimental data of 7aj are as follows: yield: 32.4%. HPLC purity: 91.2%. Melting point: 252.9-253.3℃. 1HNMR(300MHz,DMSO-d6)δ10.72(brs,1H),9.38(brs,1H),8.10(d,J=5.3Hz,1H),7.72(d,J=5.5Hz,2H), 7.51(d,J=3.8Hz,1H),7.26(d,J=3.5Hz,1H),7.17(d,J=7.3Hz,2H),6.94(d,J=5.3Hz,1H),4.62(s,2H). 13 C NMR(75MHz,DMSO-d6)δ168.0,160.9,158.0,141.1,132.6,128.8,126.0,123.4,122.7,121.6,119.4,117.3,97.3,47.4.HRMS(ESI)cald.forC 18 H 14 ClN5OH + [M+H] + :352.09596,found:352.09699.

[0081] The experimental data of 7ak are as follows: yield: 35.9%. HPLC purity: 100%. Melting point: 271.8-271.1℃. 1 HNMR(300MHz,DMSO-d6)δ10.83(brs,1H),9.42(brs,1H),8.18(d,J=9.4Hz,1H),7.87– 7.62(m,3H),7.46–7.18(m,3H),7.11–6.87(m,2H),6.53(d,J=9.4Hz,1H),4.56(s,2H). 13 CNMR(75MHz,DMSO-d6)δ167.1,160.3,159.5,158.0,140.5,131.3,128.8,128 .4,127.8,125.1,121.2,118.8,118.3,113.4,97.0,47.1.HRMS(ESI)cald.for C 18 H 14 BrN5OH + [M+H] + :396.04545,found:396.04521.

[0082] The experimental data of 7al are as follows: yield: 35.2%. HPLC purity: 100%. Melting point: 273.4-274.1℃. 1HNMR (300MHz, DMSO-d6) δ10.62(brs,1H),9.36(brs,1H),8.10(d,J=5.3Hz,1H),7.72(d,J=7.4Hz,2H),7. 32(s,1H),7.25(t,J=7.1Hz,2H),6.96(p,J=7.5Hz,3H),6.50(d,J=5.3Hz,1H),4.58(s,2H),2.29(s,3H). 13 C NMR(75MHz,DMSO-d6)δ167.4,160.5,159.5,157.6,140.6,131.5,129.6,128.4, 127.1,126.1,123.1,121.1,118.8,116.6,96.9,47.0,20.5.HRMS(ESI)cald.for C 19 H 18 N5OH + [M+H] + :332.15059,found:332.15139.

[0083] The experimental data of 7am are as follows: yield: 32.6%. HPLC purity: 94.5%. Melting point: 285.3-282.1℃. 1 HNMR(300MHz,DMSO-d6)δ11.13(brs,1H),9.43(brs,1H),8.26–8.12(m,1H),8.0 1(s,1H),7.64(m,3H),7.22(m,3H),6.94(s,1H),6.63–6.53(m,1H),4.59(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.3,160.4,159.4,158.2,140.4,136.0,129.2,128.3,1 27.7,126.7,121.3,118.9,118.8,117.3,104.3,97.1,47.1.HRMS(ESI)cald.for C 19 H 18 N5OH + [M+H] + :332.15059,found:332.15139.

[0084] The experimental data of 7an are as follows: yield: 35.8%. HPLC purity: 100%. Melting point: 236.2-237.1℃. 1HNMR(300MHz,DMSO-d6)δ10.85(brs,1H),9.40(brs,1H),8.14(dd,J=6.0Hz 1H),7.62(d,J=7.9Hz,2H),7.27(m,5H),7.07(d,J=7.6Hz,2H),6.91(dd,J=6.0Hz,1H),4.65(s,2H). 13 CNMR(75MHz,DMSO-d6)δ168.3,161.1,159.3,157.8,140.5,135.5,129.4,128.3, 127.9,125.5,123.6,121.1,118.7,115.5,97.9,54.9,48.2.HRMS(ESI)cald.for C 18 H 15 ClN5OH + [M+H] + :352.09596,found:352.09927.

[0085] Examples 15 to 22

[0086] Except that 3-chloroaniline, 3-methylaniline, 4-fluoroaniline, 4-chloroaniline, 4-bromoaniline, 4-methylaniline, 4-methoxyaniline, and 2-amino-5-bromopyridine were used in place of aniline in step (3), the other steps and purification methods were the same as those in Example 1, to prepare 7ao, 7ap, 7aq, 7ar, 7as, 7at, 7au, and 7av, respectively.

[0087]

[0088]

[0089] The experimental data of 7ao are as follows: yield: 33.2%. HPLC purity: 98.1%. Melting point: 278.8-279.2℃. 1 HNMR (300MHz, DMSO-d6) δ10.72(brs,1H),9.59(brs,1H),8.13(d,J=5.9Hz,1H),7.96(s,1H),7.62(d,J=8.3Hz,1H),7.50(d,J=7.8 Hz,1H),7.27(t,J=8.1Hz,1H),7.23–7.16(m,2H),7.08(t,J=5.5Hz,2H),6.95(d,J=7.9Hz,1H),6.53(d,J=5.9Hz,1H),4.62(s,2H). 13CNMR(75MHz,DMSO-d6)δ167.4,160.5,159.3,157.5,142.2,132.9,132.1,129.9,127. 1,125.6,122.7,122.3,120.5,117.9,117.0,116.8,97.5,46.9.HRMS(ESI)cald.forC 18 H 15 ClN5OH + [M+H] + :352.09596,found:352.09679.

[0090] The experimental data of 7ap are as follows: yield: 27.6%. HPLC purity: 98.7%. Melting point: 280.7-281.3℃. 1 HNMR(300MHz,DMSO-d6)δ10.72(brs,1H),9.30(brs,1H),8.08(d,J=5.1Hz,1H),7.59(s,1H),7.49(d,J =4.8Hz,2H),7.25–7.02(m,5H),6.75(d,J=6.8Hz,1H),6.46(d,J=5.1Hz,1H),4.62(s,2H),2.27(s,3H). 13 C NMR(75MHz,DMSO-d6)167.6,160.4,159.6,157.6,140.5,137.4,132.1,128.2,127.3,1 25.5,122.8,122.3,121.9,119.4,116.8,116.1,96.6,46.9,21.3.HRMS(ESI)cald.for C 19 H 18 N5OH + [M+H] + :332.15059,found:332.15009.

[0091] The experimental data of 7aq are as follows: yield: 35.1%. HPLC purity: 99.6%. Melting point: 264.1-264.3℃. 1 HNMR(300MHz,DMSO-d6)δ10.70(brs,1H),9.39(brs,1H),8.22–8.00(m,1H),7.8 7–7.64(m,2H),7.60–7.42(m,1H),7.12(m,6H),6.61–6.41(m,1H),4.60(s,2H). 13C NMR(75MHz,DMSO-d6)δ167.5,160.5,159.5,157.5,157.0( 1 J C-F =234.75Hz),137.0,132.1,127.2,125.5,122.4( 4 J C-F =46.5Hz),120.5( 3 J C-F =7.5Hz),116.7,114.8( 2 J C-F =21.75Hz),96.9,54.8,46.9.HRMS(ESI)cald.for C 18 H 15 FN5OH + [M+H] + :336.12551,found:336.12639.

[0092] The experimental data of 7ar are as follows: yield: 34.2%. HPLC purity: 100%. Melting point: 264.5-265.0℃. 1HNMR (300MHz, DMSO-d6) δ10.72 (brs, 1H), 9.52 (brs, 1H), 8.20–8.03 (m, 1H), 7.76 (d, J=7.0 Hz, 2H), 7.50 (d, J=7.6 Hz, 1H), 7.29 (d, J=6.3 Hz, 2H), 7.18 (d, J=7.8 Hz, 2H), 7.14–6.99 (m, 2H), 6.60–6.36 (m, 1H), 4.60 (s, 2H). 13C NMR(75MHz,DMSO-d6)δ167.5,160.5,159.3,157.5,139.6,132.1,128.2,127.1,125.6,124.5,122.9,122.2,120.2,116.7,97.2,47.0.HRMS(ESI)cald.for C18H15ClN5OH+[M+H] + :352.09596,found:352.09548.

[0093] The experimental data of 7as are as follows: yield: 30.9%. HPLC purity: 90.1%. Melting point: 280.1-280.9°C. 1HNMR (300MHz, DMSO-d6) δ10.73(brs,1H),9.54(brs,1H),8.11(d,J=5.8Hz,1H),7.71(d,J=8.6Hz,2H),7.50(d,J =7.5Hz,1H),7.41(d,J=8.6Hz,2H),7.28–7.14(m,1H),7.08(t,J=7.4Hz,2H),6.51(d,J=5.8Hz,1H),4.60(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.5,160.5,159.3,157.5,140.1,132.1,131.1,127.1,125.6,122.9,122.3,120.7,116.8,112.4,97.3,47.0.HRMS(ESI)cald.for C 18 H 15 BrN5OH + [M+H] + :396.04545,found:396.04529.

[0094] The experimental data of 7at are as follows: yield: 39.3%. HPLC purity: 95.6%. Melting point: 281.2-281.5℃. 1 HNMR (300MHz, DMSO-d6) δ10.72(brs,1H),9.27(brs,1H),8.07(d,J=5.8Hz,1H),7.59(d,J=8.1Hz,2H),7.49(d ,J=8.0Hz,1H),7.19(d,J=7.0Hz,1H),7.06(d,J=7.6Hz,4H),6.44(d,J=5.8Hz,1H),4.60(s,2H),2.24(s,3H). 13 C NMR(75MHz,DMSO-d6)δ167.6,160.4,159.7,157.6,138.0,132.1,129.9,128.8, 127.3,125.5,122.8,122.2,119.0,116.7,96.5,46.9,20.3.HRMS(ESI)cald.for C 19 H 18 N5OH + [M+H] + :332.15059,found:332.14994.

[0095] The experimental data of 7au are as follows: yield: 36.2%. HPLC purity: 100%. Melting point: 269.6-270.4℃. 1 HNMR (300MHz, DMSO-d6) δ10.71(brs,1H),9.19(brs,1H),8.04(d,J=5.6Hz,1H),7.60(d,J=8.4Hz,2H),7.49(d,J=8.1Hz ,1H),7.23–7.14(m,1H),7.07(d,J=5.0Hz,2H),6.86(d,J=8.3Hz,2H),6.42(d,J=5.6Hz,1H),4.60(s,2H),3.72(s,3H). 13 C NMR(75MHz,DMSO-d6)δ167.5,160.4,159.6,157.4,154.1,133.7,132.1,127.3, 125.4,122.8,122.2,120.7,116.7,113.6,96.2,55.1,46.8.HRMS(ESI)cald.for C 19 H 18 N5O2H + [M+H] + :348.14550,found:348.14499.

[0096] The experimental data of 7av are as follows: yield: 28.9%. HPLC purity: 93.1%. Melting point: 281.5-281.9℃. 1 HNMR (300MHz, DMSO-d6) δ10.73(brs,1H),9.54(brs,1H),8.11(d,J=5.8Hz,1H),7.72(d,J=8.7Hz,2H),7.51(d,J =7.8Hz,1H),7.42(d,J=8.6Hz,2H),7.24–7.15(m,1H),7.09(t,J=7.5Hz,2H),6.52(d,J=5.8Hz,1H),4.61(s,2H). 13 C NMR(75MHz,DMSO-d6)δ167.5,160.5,159.3,157.5,140.1,132.1,131.1,127.1,125.6,122.9,122.2,120.6,116.8,112.4,97.3,47.0.HRMS(ESI)cald.for C 17 H 14 BrN6OH + [M+H] +:397.04070,found:397.04182.

[0097] Examples 23 to 29

[0098] Except that 3-nitro-4-fluoroacetophenone was used instead of 2-fluoronitrobenzene in step (1), and 3-chloroaniline, 3-methylaniline, 4-fluoroaniline, 4-chloroaniline, 4-bromoaniline, 4-methylaniline, and 4-methoxyaniline were used instead of aniline in step (3), the other steps and purification methods were the same as those in Example 1, and 7aw, 7ax, 7ay, 7az, 7ba, 7bb, and 7bc were prepared, respectively.

[0099]

[0100] The experimental data of 7aw are as follows: yield: 31.7%. HPLC purity: 100%. Melting point: 281.1-281.7℃. 1 HNMR(300MHz,DMSO-d6)δ10.90(brs,1H),9.67(brs,1H),8.23(d,J=5.6Hz,1H),7.96(s,1H),7.73–7.5 2(m,5H),7.26(t,J=8.0Hz,1H),6.95(d,J=7.8Hz,1H),6.64(d,J=5.6Hz,1H),4.59(s,2H),2.55(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.2,166.8,160.1,159.2,157.9,142.1,133.1,132.9,131.5,131 .5,130.0,123.1,121.6,120.7,117.9,117.1,115.9,98.6,47.3,26.5.HRMS(ESI)cald.for C 20 H 17 ClN5O2H + [M+H] + :394.10653,found:394.10763.

[0101] The experimental data for 7ax are as follows: yield: 34.9%. HPLC purity: 97.4%. Melting point: 284.6-285.2°C. 1HNMR (300MHz, DMSO-d6) δ10.87(brs,1H),9.35(brs,1H),8.18(d,J=5.8Hz,1H),7.74–7.57(m,5H),7.47(d,J=8.1H z,1H),7.13(t,J=7.8Hz,1H),6.75(d,J=7.4Hz,1H),6.56(d,J=5.8Hz,1H),4.59(s,2H),2.55(s,3H),2.25(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.2,166.9,160.0,159.6,158.0,140.4,137.4,133.0,131.7,131.5, 128.2,123.1,122.0,121.5,119.5,116.2,115.9,97.8,47.3,26.4,21.3.HRMS(ESI)cald.for C 21 H 20 N5O2H + [M+H] + :374.16115,found:374.16162.

[0102] The experimental data of 7ay are as follows: yield: 32.4%. HPLC purity: 96.3%. Melting point: 282.8-283.2℃. 1 HNMR(300MHz,DMSO-d6)δ10.89(brs,1H),9.47(brs,1H),8.17(d,J=5.7Hz,1H),7.72(d,J=8.1Hz,2H),7.67 -7.60(m,4H),7.09(t,J=8.6Hz,2H),6.58(d,J=5.7Hz,1H),4.58(s,2H),2.55(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.2,166.8,160.1,159.5,157.9,157.0( 1 J C-F =237Hz),136.8( 4 J C-F =2.25Hz),133.1,131.7,131.5,123.0,121.5,120.6( 3 J C-F =7.5Hz),115.9,114.8( 2 J C-F=21.75Hz),98.0,47.2,26.4.HRMS(ESI)cald.for C 20 H 17 FN5O2H + [M+H] + :378.13608,found:378.13858.

[0103] The experimental data of 7az are as follows: yield: 36.6%. HPLC purity: 96.1%. Melting point: 274.8-275.3℃. 1 HNMR(300MHz,DMSO-d6)δ10.90(brs,1H),9.60(brs,1H),8.19(d,J=5.8Hz,1H),7.75(d,J=8.6Hz ,2H),7.68-7.62(m,4H),7.29(d,J=8.6Hz,2H),6.61(d,J=5.8Hz,1H),4.59(s,2H),2.55(s,3H). 13 C NMR(75MHz,DMSO-d6)δ196.2,166.9,160.1,159.4,157.9,139.5,133.2,131.5, 128.2,124.7,123.0,121.6,120.3,115.9,98.4,47.2,26.5.HRMS(ESI)cald.for C 20 H 17 ClN5O2H + [M+H] + :394.10653,found:394.10636.

[0104] The experimental data of 7ba are as follows: yield: 31.3%. HPLC purity: 100%. Melting point: 273.1-273.5℃. 1 HNMR(300MHz,DMSO-d6)δ10.89(brs,1H),9.60(brs,1H),8.19(d,J=5.8Hz,1H),7.86 –7.52(m,6H),7.41(d,J=8.6Hz,2H),6.62(d,J=5.8Hz,1H),4.59(s,2H),2.56(s,3H). 13CNMR(75MHz,DMSO-d6)δ196.2,166.8,160.1,159.3,157.8,139.9,133.2,131.6,131 .1,123.0,121.6,120.7,115.9,112.6,98.4,54.9,47.2,26.5.HRMS(ESI)cald.forC 20 H 17 BrN5O2H + [M+H] + :438.05601,found:438.05934.

[0105] The experimental data of 7bb are as follows: yield: 39.3%. HPLC purity: 100%. Melting point: 269.7-269.9℃. 1 HNMR(300MHz,DMSO-d6)δ10.88(brs,1H),9.34(brs,1H),8.15(d,J=5.7Hz,1H),7.63(m, 6H),7.06(d,J=8.0Hz,2H),6.55(d,J=5.7Hz,1H),4.59(s,2H),2.55(s,3H),2.24(s,3H). 13 CNMR(75MHz,DMSO-d6)δ196.2,166.9,160.0,159.7,157.9,137.9,133.1,131.8,131.5, 130.1,128.8,123.0,121.5,119.1,115.9,97.6,47.1,26.5,20.3.HRMS(ESI)cald.forC 21 H 20 N5O2H + [M+H] + :374.16115,found:374.16009.

[0106] The experimental data of 7bc are as follows: Yield: 41.2%. HPLC purity: 100%. Melting point: 282.3-282.8℃. 1 HNMR(300MHz,DMSO-d6)δ10.89(brs,1H),9.27(brs,1H),8.13(d,J=5.8Hz,1H),7.71–7.55( m,6H),6.85(d,J=8.9Hz,2H),6.52(d,J=5.8Hz,1H),4.58(s,2H),3.72(s,3H),2.55(s,3H). 13C NMR(75MHz,DMSO-d6)δ196.2,166.9,160.0,159.8,157.9,154.2,133.6,133.0,131.8, 131.5,123.1,121.6,120.8,115.9,113.6,97.4,55.1,47.1,26.5.HRMS(ESI)cald.for C 21 H 20 N5O3H + [M+H] + :390.15607,found:390.15589.

[0107] Example 30

[0108] Preparation of 7-methyloximino-4-((2-phenylamino)pyrimidin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (8)

[0109]

[0110] Compound 7ag (0.1 g, 0.28 mmol, 1.0 equiv), hydroxylamine hydrochloride (0.03 g, 0.45 mmol, 1.6 equiv), and sodium acetate (0.05 g, 0.56 mmol, 2.0 equiv) were placed in a 25 mL round-bottom flask and dissolved in ethanol / water (2:1). The mixture was placed in an oil bath and reacted at 95°C for 8 h. The reaction was monitored by TLC. After completion, the mixture was evaporated under reduced pressure and purified by column chromatography (200-300 mesh silica gel, DCM:MeOH = 10:1) to give 0.07 g of a white solid in a yield of 73.6%. HPLC purity: 91.7%. Melting point: 261.2-261.5°C. 1 H NMR (300MHz, DMSO-d6) δ11.26(brs,1H),10.79(brs,1H),9.40(brs,1H),8.12(d,J=5.8Hz,1H),7.72(d,J=8.1Hz,2H),7.52(d,J=8.4Hz ,1H),7.41(s,1H),7.34(d,J=8.5Hz,1H),7.25(t,J=7.7Hz,2H),6.93(t,J=7.3Hz,1H),6.52(d,J=5.8Hz,1H),4.61(s,2H),2.15(s,3H). 13C NMR(75MHz,DMSO-d6)δ167.3,160.3,159.6,157.6,152.0,140.6,133.9,131.8,128 .3,127.6,122.3,121.2,119.7,118.9,113.4,97.1,54.9,11.3.HRMS(ESI)cald.for C 20 H 19 N6O2H + [M+H] + :375.15460,found:375.15985.

[0111] Example 31

[0112] Preparation of 4-(2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-pyrimidin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (9a)

[0113]

[0114] Compound 7as (0.13 g, 0.33 mmol, 1.0 equiv), N-methylpiperazine (72.0 μL, 0.66 mmol, 2.0 equiv), lithium bis(trimethylsilyl)amide (1.6 mL, 1.64 mmol, 5.0 equiv), x-phos (0.03 g, 0.066 mmol, 0.2 equiv), and Pd2(dba)3 (0.0015 g, 0.002 mmol, 0.05 equiv) were placed in a 25 mL round-bottom flask and dissolved in 3 mL of anhydrous tetrahydrofuran. The nitrogen atmosphere was replaced three times, and the mixture was allowed to react in an oil bath at 70°C for 8 h. The reaction was monitored by TLC. After completion, the mixture was evaporated under reduced pressure and purified by column chromatography (200-300 mesh silica gel, DCM:MeOH = 5:1) to give 0.037 g of a white solid in a 27.8% yield. HPLC purity: 100% with a melting point of 261.2-261.5°C. 1 H NMR (300MHz, DMSO-d6) δ10.72(brs,1H),9.14(brs,1H),8.04(d,J=5.9Hz,1H),7.52(dd,J=16.6,8.1Hz,3H),7.17(d,J=7.9Hz ,1H),7.08(t,J=8.4Hz,2H),6.88(d,J=8.1Hz,2H),6.40(d,J=5.9Hz,1H),4.60(s,2H),3.08(s,4H),2.28(s,4H),2.11(s,3H) 13C NMR(75MHz,DMSO-d6)δ167.6,160.4,159.8,157.5,145.8,132.8,132.1,127.3,125.4 ,122.8,122.2,120.3,116.8,115.9,96.1,54.5,48.6,46.8,45.4.HRMS(ESI)cald.for C 23 H 25 N7OH + [M+H] + :416.21933,found:416.21906.

[0115] Examples 32-33

[0116] Except for using N-ethylpiperazine instead of N-methylpiperazine or using compound 7av instead of 7as, the operation and purification methods were the same as Example 31 to prepare 9b and 9c, respectively.

[0117]

[0118] The experimental data of 9b are as follows: Yield: 23.7%. HPLC purity: 100%. Melting point: 261.3-261.9 ℃. 1 H NMR(300MHz,DMSO-d6)δ10.72(brs,1H),9.14(brs,1H),8.04(d,J=5.8Hz,1H ),7.56(d,J=8.7Hz,2H),7.49(d,J=7.8Hz,1H),7.19(d,J=6.8Hz,1H),7.08( t,J=6.5Hz,2H),6.89(d,J=8.8Hz,2H),6.40(d,J=5.8Hz,1H),4.60(s,2H),3 .36(s,4H),2.70(s,4H),2.25(dd,J=2.9,7.5Hz,2H),1.09(t,J=7.0Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ167.6,160.4,159.7,157.5,133.0,132.1,127.3,125.4,12 2.8,122.2,120.3,116.7,115.9,96.1,51.9,51.4,48.4,46.8.HRMS(ESI)cald.for C 24 H 27 N7OH + [M+H] + :430.23498,found:430.23310.

[0119] The experimental data of 9c are as follows: Yield: 25.7%. HPLC purity: 100%. Melting point: 274.1-274.8℃. 1 HNMR(300MHz,DMSO-d6)δ10.72(brs,1H),9.14(brs,1H),8.04(d,J=5.9Hz,1H),7.56(s,1H),7.55–7.47(m,2H),7.24–7.14(m ,1H),7.09(d,J=6.3Hz,2H),6.87(d,J=8.9Hz,1H),6.40(d,J=5.9Hz,1H),4.60(s,2H),3.05(s,4H),2.25(s,4H),1.23(s,3H). 13 C NMR(75MHz,DMSO-d6)δ168.0,160.9,160.2,158.0,146.5,133.6,132.6,131.4,127.9, 125.9,123.3,122.7,120.8,117.2,116.3,96.5,55.1,49.3,46.1.HRMS(ESI)cald.for C 22 H 24 N8OH + [M+H] + :417.21458,found:417.21441.

[0120] Example 34

[0121] Preparation of 4-(3,4-dihydroquinoxalin-1(2H)yl)-N-phenylpyrimidin-2-amine (10a)

[0122]

[0123] Compound 7aa (0.1 g, 0.32 mmol, 1.0 equiv) was placed in a 25 mL two-necked flask and the atmosphere was purged with nitrogen three times. 3 mL of borane tetrahydrofuran solution (3.2 mmol, 10.0 equiv) was then injected into the flask using a 1 mL syringe. The mixture was allowed to react at 0°C for 10 h. The reaction was monitored by TLC. Upon completion, methanol was added to quench the reaction mixture. The mixture was then evaporated under reduced pressure and purified by column chromatography (200-300 mesh silica gel, PE:EA = 15:1) to afford 0.046 g of a white solid with a yield of 48.9%. HPLC analysis revealed a 90.1% yield. Melting point: 132.1-132.5°C. 1H NMR (300MHz, DMSO-d6) δ9.24(brs,1H),7.99(d,J=5.9Hz,1H),7.72(d,J=8.0Hz,2H),7.24(t,J=7.7Hz,2H),7.17(d,J=7.7H z,1H),6.90(t,J=7.1Hz,2H),6.66(d,J=8.0Hz,1H),6.54(d,J=4.1Hz,2H),6.45(d,J=5.9Hz,1H),3.97(s,2H),3.32(s,2H). 13 CNMR(75MHz,DMSO-d6)δ167.4,160.3,159.5,157.8,140.5,133.6,128.4,126.7,124.7,124.7,121.2,118.9,116.7,97.0,46.9,29.0.HRMS(ESI)cald.for C 18 H 17 N5H + [M+H] + :304.15567found:304.15870.

[0124] Examples 35 to 37

[0125] The operation and purification methods were the same as in Example 34, except that compounds 7ac, 7ad, and 7aj were used instead of 7aa, to prepare compounds 10b, 10c, and 10d, respectively.

[0126]

[0127] 10b experimental data are as follows: Yield 47.5%. HPLC: 93.6%. Melting point: 167.8-168.4 ℃. 1 H NMR (300MHz, DMSO-d6) δ8.36(brs,1H),8.00(d,J=7.1Hz,1H),7.57(d,J=7.8Hz,2H),7.37(t,J=7.7Hz,2H), 7.22(d,J=8.7Hz,1H),7.15(t,J=7.2Hz,1H),6.74–6.69(m,1H),6.65–6.46(m,3H),3.91(s,2H),2.13(s,2H) 13C NMR(75MHz,DMSO-d6)δ160.6,154.5,154.0,141.2,138.0,130.8,129.2,124.7,122.5,121.5,114.4,113.7,97.7,65.5,31.6.HRMS(ESI)cald.for C 18 H 16 ClN5H + [M+H] + :338.11670,found:338.11690.

[0128] 10c experimental data are as follows: Yield 41.5%. HPLC: 100%. Melting point: 163.1-164.0 ℃. 1 H NMR (300MHz, DMSO-d6) δ9.24(brs,1H),8.02(d,J=5.6Hz,1H),7.71(d,J=7.8Hz,2H),7.23(t,J=7.5Hz,2H),7.13(d,J=8.3Hz, 1H), 6.90 (t, J = 7.1Hz, 1H), 6.82 (s, 1H), 6.65 (d, J = 8.1Hz, 1H), 6.49 (s, 1H), 6.42 (d, J = 5.7Hz, 1H), 3.94 (s, 2H), 3.31 (s, 2H). 13 C NMR(75MHz,DMSO-d6)δ160.7,159.6,157.1,140.8,140.6,128.3,125.5,123.0,120.9,118.7,117.1,116.7,116.0,97.1,29.0.HRMS(ESI)cald.for C 18 H 16 BrN5H + [M+H] + :382.06621,found:382.06618.

[0129] 10d experimental data are as follows: Yield 44.2%. HPLC: 93.3%. Melting point: 167.1-167.9 ℃. 1H NMR (300MHz, DMSO-d6) δ8.35(brs,1H),7.98(d,J=6.9Hz,1H),7.59(d,J=8.0Hz,2H),7.38(t,J=7.7Hz,2H),7.28–7.10(m ,2H),6.94(dt,J=16.9,7.4Hz,2H),6.69(d,J=8.0Hz,1H),6.56(dd,J=16.6,7.1Hz,1H),3.93(s,2H),2.29–1.95(m,2H). 13 C NMR(75MHz,DMSO-d6)δ160.5,154.4,153.8,139.9,138.1,129.2,128.8,127.0, 124.6,122.6,122.3,115.0,114.9,97.8,55.4,49.1,41.4.HRMS(ESI)cald.for C 18 H 16 ClN5H + [M+H] + :338.11670,found:338.11690.

[0130] Example 38

[0131] Preparation of 4-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-N-phenylpyrimidin-2-amine (11a)

[0132]

[0133] The operation and purification methods were the same as those in step (5) of Example 1, except that benzomorpholine was used instead of compound 3. The yield was 46.8%. HPLC: 100%. Melting point: 141.2-141.7°C. 1 H NMR (300MHz, DMSO-d6) δ9.36(brs,1H),8.11(d,J=5.9Hz,1H),7.71(d,J=8.0Hz,2H),7.50(d,J=7.8Hz,1H),7.25(t, J=7.7Hz,2H),7.10–7.02(m,1H),6.92(t,J=8.1Hz,3H),6.60(d,J=5.9Hz,1H),4.31–4.24(m,2H),4.14–4.07(m,2H). 13C NMR(75MHz,DMSO-d6)δ160.5,158.7,155.9,146.9,140.2,128.4,126.4,125.1,123.3,121.5,119.8,119.2,117.2,97.6,65.7,41.8.HRMS(ESI)cald.for C 18 H 16 N6OH + [M+H] + :305.13969,found:305.13978.

[0134] Example 39

[0135] Preparation of 1-(2-(phenylamino)pyrimidin-4-yl)-2,3-dihydroquinolin-4(1H)-one (11b)

[0136]

[0137] The operation and purification methods were the same as those in step (5) of Example 1, except that 2,3-dihydroquinolin-4(1H)-one was used instead of compound 3. The yield was 48.4%. HPLC: 98.2%. Melting point: 155.6-155.5°C. 1 H NMR (300MHz, DMSO-d6) δ9.39(s,1H),8.17(d,J=5.8Hz,1H),7.90(d,J=7.7Hz,1H),7.72(d,J=8.2Hz,2H),7.58(q,J=8.5,7. 4Hz, 2H), 7.24 (t, J = 7.4Hz, 3H), 6.91 (t, J = 7.3Hz, 1H), 6.62 (d, J = 5.8Hz, 1H), 4.40 (t, J = 5.9Hz, 2H), 2.78 (t, J = 5.9Hz, 2H). 13 C NMR(75MHz,DMSO-d6)δ193.8,161.0,159.8,157.7,145.0,140.6,134.1,128.3, 127.1,124.9,123.6,122.3,121.1,118.9,98.7,54.9,44.6.HRMS(ESI)cald.for C 19 H 17 N4OH + [M+H] + :317.13969,found:317.14070.

[0138] Below are some pharmacological experiments and results described in the present invention:

[0139] 1. Tumor cell proliferation inhibition experiment (MTT experiment)

[0140] (1) Experimental methods

[0141] The compounds described herein were tested for their inhibitory activity against human hepatocellular carcinoma cell lines (HepG2) and human cervical carcinoma cell lines (HeLa). All cell lines were cryopreserved and passaged in this laboratory. Methods: Cells were seeded in 96-well flat-bottom plates (approximately 4,000-6,000 cells per well) and incubated at 37°C, 5% CO₂ for 24 hours. Test compounds or positive control drugs were added to each well at varying concentrations. After 72 hours of incubation, 50 μL of MTT-containing PBS buffer (1 mg / mL) was added to each well. The cells were incubated at 37°C for 4 hours. The MTT and culture medium were removed, and 100 μL of DMSO was added to each well. The concentrations were measured at 570 nm (Power Wave XS, Bio-Tek, USA). Palbociclib and colchicine were used as positive controls. Statistical analysis was performed on the data.

[0142] (2) Experimental results

[0143] The experimental results are shown in Table 1.

[0144] Table 1 MTT test results of compounds

[0145]

[0146]

[0147]

[0148] Note: The compounds Palbociclib and Cochicine in the table are positive control drugs and are not included in the scope of patent protection.

[0149] The results in Table 1 show that this class of compounds generally have strong cell growth inhibition effects. The half inhibitory concentration (IC 50 ) reached the micromolar level (μM). Among them, 9a was comparable to the positive control drug palbociclib.

[0150] 2. Tubulin polymerization inhibition experiment

[0151] (1) Experimental methods

[0152] The inhibitory effect of the compounds described herein on tubulin polymerization was tested using the Cytoskeleton BK011P kit. A 96-well plate was placed in a multifunctional microplate reader (CLARIOstar, BMG LABTECH, Germany) and preheated at 37°C for 10 minutes. According to the instructions, colchicine (10×), test compound solution (10×), buffer 1, GTP stock solution, tubulin glycerol buffer, and tubulin stock solution were prepared for later use. Buffer 1, GTP stock solution, tubulin glycerol buffer, and tubulin stock solution were mixed in appropriate proportions according to the experiment to prepare a microtubule reaction solution, which was then placed in an ice bath. 5 μL of control buffer, colchicine solution (5 μM), and test compound solution (9a, 5 μM, with 1 replicate) were added to the 96-well plate. The plate was preheated at 37°C for 1 minute. The prepared microtubule reaction solution was quickly added, and the fluorescence intensity change was recorded over 80 minutes.

[0153] Preparation of Buffer 1: ① Dissolve the contents of each bottle of Buffer 1 in 10 mL of Milli-Q ultrapure water (two bottles total); ② Combine the contents of the two bottles; ③ Aliquot the buffer into 13 cryovials, 1.5 mL per tube, and store at -70°C.

[0154] Tubulin glycerol buffer, stored at 4°C.

[0155] Preparation of GTP stock solution: ① Dissolve the substance in each tube of GTP stock solution with 100 μL of ice-cold sterile distilled water (a total of 3 tubes); ② Place it on ice; ③ Mix the substances in the three tubes; ④ Dispense the above solution into 13 cryovials, 20 μL per tube, and store at -70°C.

[0156] Preparation of tubulin solution: ① Place the vial containing lyophilized 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 Dissolve the lyophilized tubulin powder in 1.1mL of the above mixture; ⑥ Place on ice for 2 minutes until the powder is completely dissolved and suspended; ⑦ Aliquot the above tubulin solution into 12 cell cryovials, 88μL per tube, and quickly freeze in liquid nitrogen; ⑧ Store at -70℃.

[0157] (2) Experimental results

[0158] The results showed that compound 9a had a significant inhibitory effect on tubulin polymerization, such as Figure 1 shown.

[0159] 3. Cell cycle experiment

[0160] (1) Experimental methods

[0161] The effects of the compounds of the present invention on the cell cycle of cancer cells (human hepatocellular carcinoma cell line HepG2) were tested using propidium iodide (PI) staining. Methods: Cells were seeded in 6-well flat-bottom plates (approximately 100,000 cells per well) and incubated at 37°C, 5% CO₂ for 24 hours. Different concentrations of 9a (final concentrations of 0.5 μM, 1 μM, and 2 μM) were then added to each well. A blank control group (no compound 9a) was set up and incubated at 37°C, 5% CO₂ for 24 hours. Cells were harvested, washed with pre-chilled PBS (4°C, 0.01 mol / L), and fixed in pre-chilled 70% (v / v) ethanol (4°C). The fixed cells were then stored 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 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).

[0162] (2) Experimental results

[0163] The results are as follows Figure 2 As shown, Figure 2 It can be seen that compound 9a can significantly arrest cells in the G2 / M growth phase at 2 μM.

[0164] IV. CDK6 activity detection experiment

[0165] Using ADP-Glo TMtu The kit detects compound inhibition of CDK6 enzymatic activity. (a) Prepare 2× ATP / substrate solution and 2× kinase solution (containing kinase reaction buffer). (b) Using an Echo 655, transfer 100 nL of compound (positive drug palbociclib or compound 9a) dilution to a 384-well assay plate (maximum final concentration of 10 μM, followed by three-fold dilution to the next concentration, using a 10-step dilution process, down to a minimum concentration of 0.508 nM). After centrifugation, add 5 μL of 2× kinase solution, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 10 minutes. (c) Add 5 μL of 2× substrate and ATP solution to the 384-well assay plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 60 minutes. (d) Add 5 μL of ADP-Glo ​​reagent to the 384-well assay plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 40 minutes. (e) Transfer 10 μL of the assay solution to a 384-well plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 40 minutes. (f) Detect the luminescent signal using a multi-functional microplate reader. Finally, perform statistical analysis of the data.

[0166] (2) Experimental results

[0167] The results are as follows Figure 3 As shown, Figure 3 It can be seen that compound 9a has a significant inhibitory effect on CDK6 activity, with a half-maximal inhibitory concentration of 2.70 μmol·L -1 .

Claims

1. A diarylamine structure compound, characterized in that: The general structural formula is shown below: where R 1 represents hydrogen, halogen, methyl, cyano, methoxy, acetyl, methoxycarbonyl, or oxime; R 2 represents hydrogen, halogen, methyl, methoxy or cyano; R 3 represents hydrogen or halogen, R 4 represents hydrogen, halogen or methyl, R 5 represents hydrogen, halogen, methyl, methoxy, N-methylpiperazinyl, N-ethylpiperazinyl, W represents a carbon atom or a nitrogen atom, X represents an oxygen atom or NH, and Y represents a methylene group or a carbonyl group.

2. A biologically acceptable salt formed by the diarylamine structural compound according to claim 1 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

3. The method for preparing the diarylamine structure compound according to claim 1, characterized in that: The synthetic route of this type of compound is as follows: R 1 represents hydrogen, F, Cl, Br, methyl, cyano, methoxy, acetyl, methoxycarbonyl; R 2 represents hydrogen, F, Cl, Br, methyl or cyano; R 3 Represents hydrogen, F, Cl, Br, R 4 represents hydrogen, F, Cl, Br or methyl, R 5 represents hydrogen, F, Cl, Br, methyl, methoxy, N-methylpiperazinyl or N-ethylpiperazinyl, W represents a carbon atom or a nitrogen atom, Het is The specific steps are: (a) Using substituted o-fluoronitrobenzene as the starting material, a nucleophilic substitution reaction with glycine methyl ester hydrochloride under alkaline conditions was performed to obtain compound 2; (b) Compound 2 is reduced with hydrogen gas under the catalysis of palladium on carbon to obtain compound 3; (c) Compound 4 reacts with a substituted aromatic amine under acidic conditions to give compound 5 through nucleophilic substitution reaction; (d) Compound 5 is reacted with phosphorus oxychloride to obtain compound 6; (e) Compound 3 reacts with compound 6 under acidic conditions to obtain compound 7 through nucleophilic substitution reaction; (f)R 1 Compound 7 with an acetyl group is subjected to dehydration condensation with hydroxylamine hydrochloride under alkaline conditions to obtain compound 8; (g)R 4 or R 5 Compound 7 containing F, Cl, or B is reacted with N-methylpiperazine or N-ethylpiperazine via Bach-Wald coupling to obtain compound 9; (h) Compound 7 is reduced by borane to obtain compound 10; (i) Compound 6 reacts with benzomorpholine or 2,3-dihydro-1H-quinolin-4-one to obtain compound 11 through affinity substitution reaction.

4. A pharmaceutical composition, characterized in that Contains the compound according to claim 1 or the biologically acceptable salt according to claim 2.

5. Use of the compound according to claim 1 or the biologically acceptable salt according to claim 2 in the preparation of a tubulin polymerization inhibitor and / or a CDK4 / 6 inhibitor.

6. Use of the compound according to claim 1 or the biologically acceptable salt according to claim 2 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 disease is breast cancer, ovarian cancer, cervical cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, prostate cancer, melanoma or leukemia.

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