2,3-dihydropyrimido[4,5-d]pyrimidin-4(1h)-one derivatives, processes for their preparation and uses thereof

Abstract

The application belongs to the technical field of chemical medicine, and particularly relates to a kind of 2,3-dihydropyrimidino [4,5-d] pyrimidine-4 (1H) -ketone derivatives and preparation method and purposes thereof.The existing Wee1 small molecule inhibitors still have one or more defects in activity, safety or pharmacokinetic properties, and a new generation of Wee1 small molecule inhibitors with novel structure, low toxicity, high efficiency and excellent pharmacokinetic properties needs to be developed.The application provides a kind of 2,3-dihydropyrimidino [4,5-d] pyrimidine-4 (1H) -ketone derivatives, specifically including 95 new compounds, or pharmaceutically acceptable salt thereof, or stereoisomer thereof.Experiments show that the compound of formula (I) disclosed in the application has good inhibitory activity on Wee1, and therefore the compound of the application can be used for treating diseases caused by abnormal Wee1 activity.

Description

Technical Field

[0001] This invention belongs to the field of chemical and pharmaceutical technology, specifically relating to a class of 2,3-dihydropyrimidine[4,5-d]pyrimidine-4(1H)-one derivatives, their preparation methods, and uses. Background Technology

[0002] The cell cycle refers to the entire process of a cell from the completion of one division to the end of the next. The cell cycle includes four growth phases: the G1 phase, in which proteins and RNA are synthesized in large quantities after mitosis; the S phase, in which DNA is synthesized and replicated; the G2 phase, a preparatory phase before mitosis; and the M phase, in which the cell undergoes mitosis.

[0003] Before cells enter the M phase, DNA replication must be completed. Due to interference from various endogenous and exogenous factors, DNA often mutates or is damaged. These abnormal DNA molecules must be repaired before entering the next stage of the cell cycle; otherwise, mitotic catastrophe will occur, leading to cell death. Cell cycle checkpoints can pause the cell cycle, allowing cells to repair abnormal DNA before entering the M phase.

[0004] Cell cycle checkpoints are a complex regulatory system that ensures the quality of DNA replication and chromosome segregation during the cell cycle. Their core component is the CDKs / Cyclins complex, formed by the binding of cyclin-dependent kinases (CDKs) and cyclins. When abnormal events occur during the cell cycle, such as DNA damage or DNA replication inhibition, this regulatory system is activated, pausing the cell cycle. The cell cycle resumes once the cell repairs itself or the fault is resolved. There are multiple cell cycle checkpoints throughout the cell cycle. The G1 / S checkpoint at the end of G1 phase and the G2 / M checkpoint in G2 phase are two major checkpoints. The G1 / S checkpoint determines whether to enter the cell cycle by examining the state inside and outside the cell, thus determining whether the cell enters the S phase for DNA synthesis. The G2 / M checkpoint examines whether there is DNA damage or defects after DNA synthesis, thus determining whether the cell enters the M phase for mitosis. The core component of the G2 / M checkpoint is the CDK1 (human homolog also known as CDC2) / Cyclin B complex. CDC2 is maintained in an inactive state by phosphorylation before the cell enters mitosis, allowing the cell to detect and repair damaged DNA. Its phosphorylation state is regulated by protein kinases such as Wee1.

[0005] Wee1 protein kinase belongs to the nuclear serine and threonine protein kinase family and is a key inhibitory regulator of CDC2 activity. It can phosphorylate the Tyr15 site on CDC2, inhibit the activation of the CDC2 / Cyclin B complex, and arrest cells from entering the M phase until the damaged DNA is repaired. This ensures that DNA can enter mitosis without damage. However, the loss or inactivation of Wee1 can cause cells to enter mitosis prematurely, leading to mitotic failure and cell death.

[0006] Normal cells can complete DNA repair during the G1 phase using the G1 / S checkpoint. However, nearly 50% of cancer cells have mutations in the tumor suppressor gene p53, leading to G1 / S checkpoint dysfunction and a greater reliance on the G2 / M checkpoint for DNA repair. Cancer cells can evade DNA-damaging agents and radiation therapy through the G2 / M checkpoint. Weel kinase is highly expressed in many types of cancer cells. Inhibiting Weel activity can lead to G2 / M checkpoint dysfunction, causing tumor cells to skip the G2 phase of DNA repair, prematurely enter mitosis, and ultimately die, thus achieving the goal of cancer treatment.

[0007] In summary, inhibiting Wee1 activity can selectively induce the death of cancer cells with G1 / S checkpoint dysfunction, and targeted inhibition of Wee1 has great potential for cancer treatment.

[0008] AstraZeneca's Weel inhibitor AZD1775 is currently in clinical trials, with over 30 clinical trials under development showing promising therapeutic effects. Related patents include US20070254892, WO2007126122, EP2213673, WO2008133866, and WO2011034743. Abbott and Abbvie have also conducted research on Weel inhibitors, with related patents including US2012220572, WO2013126656, WO2013012681, WO2013059485, WO2013013031, and WO2013126656. Almac's patents related to Wee1 inhibitors include WO2014167347, WO2015019037, and WO2015092431.

[0009] WO2008133866 discloses compound AZD1775, with the following structure:

[0010] Summary of the Invention

[0011] The technical problem to be solved by this invention is that existing Wee1 small molecule inhibitors still have one or more defects in terms of activity, safety or pharmacokinetic properties, and there is a need to develop a new generation of Wee1 small molecule inhibitors with novel structure, low toxicity, high efficiency and excellent pharmacokinetic properties.

[0012] The technical solution of this invention to solve the above-mentioned technical problems is as follows: a class of 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivatives is provided. The general structural formula of this derivative is shown in Formula I below:

[0013] Where R1 is

[0014] At least one of them; R2 is At least one of them; R3 and R4 are independently selected from H, At least one of them; R5 and R6 are independently selected from H, At least one of the following; n = 0, 1, 2 or 3.

[0015] Preferably, when n=0, the general structural formula of the derivative is shown in Formula II:

[0016] The R7 is

[0017] Preferably, when n=1, the general structural formula of the derivative is shown in Formula III:

[0018] R8 is

[0019] More preferably, the specific structure of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative is any one of the following:

[0020]

[0021]

[0022] The present invention also provides a pharmaceutical composition comprising a 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, and / or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or polymorph of the derivative.

[0023] The aforementioned pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0024] The present invention also provides a method for preparing the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, comprising the following steps:

[0025] a. Preparation of intermediate 1-A

[0026] Sodium hydride was added to a solution of ethyl 2-methylmercapto-4-aminopyrimidine-5-carboxylate in N,N-dimethylformamide, followed by o-chlorophenyl isocyanate. The mixture was reacted at room temperature for 4 hours. Sodium chloride solution was added, and after the mixture stopped producing bubbles, water was added. The mixture was extracted with ethyl acetate, and the aqueous phase was extracted again with ethyl acetate. The aqueous phase was collected, the pH was adjusted to 4-5, and the solid precipitated was filtered and dried to obtain 1-A.

[0027] b. Preparation of intermediate 1-B

[0028] Phosphorus oxychloride and N,N-diisopropylethylamine were added sequentially to 1-A, and the reaction was carried out for 2 hours. The reaction solution was heated to 90°C, and most of the phosphorus oxychloride and N,N-diisopropylethylamine were removed by vacuum concentration. Ice water and sodium bicarbonate solution were added, followed by extraction with ethyl acetate to obtain a solid. The solid was washed sequentially with water and diethyl ether and dried to obtain 1-B.

[0029] c. Preparation of intermediate 1-C

[0030] Add 3-amino-1-propanol to the acetonitrile solution of 1-B, react at room temperature for 2 hours, concentrate under reduced pressure to remove acetonitrile, add diethyl ether and stir thoroughly, filter to collect the solid, and dry to obtain intermediate 1-C;

[0031] d. Preparation of intermediate 1-D

[0032] Phosphorus oxychloride was added to 1-C and reacted for 4 hours. Phosphorus oxychloride was removed by vacuum concentration. Ice water and sodium bicarbonate solution were added to adjust the pH value. Ethyl acetate was added, and the mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was used to obtain intermediate 1-D.

[0033] e. Preparation of intermediate 1-E

[0034] Add m-chloroperoxybenzoic acid in portions to a dichloromethane solution of 1-D, react for 3 hours, add sodium bicarbonate solution and sodium thiosulfate solution, extract and separate the liquid, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure to obtain a grayish-yellow solid, add diethyl ether to disperse the solid, stir vigorously, filter to obtain a solid, dry to obtain intermediate 1-E;

[0035] f. Preparation of 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivatives

[0036] 4-Cyclohexaniline was added to the isopropanol solution of 1-E and reacted for 5 hours. The isopropanol was removed by concentration under reduced pressure. Ammonium chloride solution was added, followed by ethyl acetate. The mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was used to obtain the final product, 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative.

[0037] In the preparation method of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, the o-chlorophenyl isocyanate in step a is replaced by 2-chloro-6-methylphenyl isocyanate, 2,6-dimethylisocyanate, 1-chloro-3-fluoro-2-phenyl isocyanate or 2,6-dichlorophenyl isocyanate.

[0038] In the preparation method of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, 3-amino-1-propanol in step c is replaced with cis-2-aminocyclohexanol, 4-amino-1-butanol, 3-amino-2,2-dimethyl-1-propanol, 3-amino-3-methylbut-1-ol, 3-amino-2,2-difluoroprop-1-ol, 5-amino-1-pentanol, 3-aminotetrahydro-2H-pyran-4-ol, (R)-3-aminobutanol, (S)-3-aminobutanol, (2S)-3-amino-2-fluoropropane-1-ol, (2R)-3-amino-2-fluoropropane-1-ol, 3-amino-2-methylpropanol, 2-aminocyclopentanol, 3-aminocyclobutanol, or 3-aminocyclohexanol.

[0039] In the preparation method of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, the 4-cyclohexaneaniline in step f is replaced by 4-piperidineaniline, 4-(1-methylpiperidin-4-yl)aniline, 4-(4-methylpiperazin)aniline, 4-piperazinylaniline, 4-(4-morpholinyl)aniline, 4-(4-aminophenyl)morpholino-3-one, 3-fluoro-4-(4-methylpiperazinyl)aniline, 2-fluoro-4-(4-methyl-1-piperazinyl)aniline, 1-methyl-4-(6-aminopyridin-3-yl)piperidine, etc. At least one of the following: azimine, 2-methoxy-4-(4-methylpiperazin-1-yl)aniline, 2-chloro-4-(4-methylpiperazin-1-yl)aniline, 3-(4-methylpiperazin-1-yl)aniline or 2-(4-methyl-1-piperazin)aniline, 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine, 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine, 4-((2S,6R)-2,6-dimethylmorpholino)aniline or 4-((3S,5R)-3,4,5-trimethylpiperazin-1-yl)aniline.

[0040] In the preparation method of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, 4-cyclohexaneaniline in step f is replaced by 15-B or 16-B. The preparation steps of 15-B or 16-B are as follows: sodium hydride is added to the N,N-dimethylformamide solution of 6-nitroindoline or 7-nitrotetrahydroquinoline, iodomethane is slowly added dropwise to the reaction solution, the reaction solution is restored to room temperature, and stirring is continued at room temperature for 1 hour. Sodium chloride solution is added, a solid is precipitated, filtered, and dried to obtain intermediate 15-A or 16-A; palladium on carbon and hydrazine hydrate solution are added to the methanol solution of intermediate 15-A or 16-A, and the reaction is carried out overnight in a sealed container at room temperature. After the reaction is completed, palladium on carbon is removed by diatomaceous earth filtration, and methanol and water are removed by vacuum concentration to obtain 15-B or 16-B.

[0041] In the preparation method of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, 4-cyclohexaneaniline in step f replaces 17-B. The preparation steps of 17-B include: adding formaldehyde solution and sodium triacetoxyborohydride to a dichloromethane solution of 7-nitro-1,2,3,4-tetrahydroisoquinoline, stirring overnight at room temperature, filtering with diatomaceous earth to obtain filtrate, concentrating under reduced pressure, diluting with ethyl acetate, adding sodium bicarbonate aqueous solution, stirring vigorously, extracting with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, and concentrating to obtain 17-A; adding palladium on carbon and hydrazine hydrate solution to a methanol solution of intermediate 17-A, reacting overnight at room temperature in a sealed environment, filtering with diatomaceous earth to remove palladium on carbon, and concentrating under reduced pressure to remove methanol and water to obtain 17-B.

[0042] In the above method for preparing the 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, 4-cyclohexaneaniline in step f replaces 18-D. The 18-D preparation step includes: adding N,N-dimethylformamide solution to isoquinoline-1,3(2H,4H)-dione, potassium carbonate, and tetrabutylammonium bisulfate, then adding iodomethane, reacting for 5 hours, cooling to room temperature, extracting with ammonium chloride aqueous solution and ethyl acetate, extracting the organic phase obtained by separation with ammonium chloride aqueous solution twice more, collecting the organic phase, and anhydrous... Sodium sulfate was dried, concentrated under reduced pressure, and column chromatography was used to obtain 18-A; 18-A was dissolved in concentrated sulfuric acid, concentrated nitric acid was added dropwise, the reaction was stirred, and a solid precipitated. The solid was filtered, washed with water, and dried under vacuum to obtain 18-B; Palladium on carbon and hydrazine hydrate solution were added to the methanol solution of intermediate 18-B, and the reaction was carried out overnight at room temperature in a sealed environment. After the reaction was completed, palladium on carbon was removed by diatomaceous earth filtration, and methanol and water were removed by concentration under reduced pressure to obtain 18-C; 18-C was dissolved in tetrahydrofuran, a tetrahydrofuran solution of borane dimethyl sulfide was added, the reaction was carried out, tetrahydrofuran was removed by concentration under reduced pressure, and column chromatography was used to obtain 18-D.

[0043] In the preparation method of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative, 4-cyclohexaneaniline in step f replaces 19-B. The preparation steps of 19-B include: adding acetonitrile to a mixture of cis-1,2,6-trimethylpiperazine or cis-2,6-dimethylmorpholine, potassium carbonate, and p-fluoronitrobenzene, reacting for 5 hours, cooling to room temperature, concentrating under reduced pressure to remove acetonitrile, adding ethyl acetate and water, extracting and separating the liquid, drying the organic phase with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 19-A; dissolving 19-A in tetrahydrofuran, adding a tetrahydrofuran solution of borane dimethyl sulfide, reacting and concentrating under reduced pressure to remove tetrahydrofuran, and obtaining 19-B by column chromatography.

[0044] Furthermore, the present invention also provides the use of the above-mentioned 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivatives, and / or pharmaceutically acceptable salts, solvates, hydrates, isomers, and polymorphs of the derivatives in the preparation of pharmaceuticals.

[0045] The aforementioned drugs are for the prevention or treatment of tumor diseases.

[0046] Furthermore, the drug in question is a Weel inhibitor.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0048] This invention provides a class of 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivatives, specifically 95 new compounds. These derivatives exhibit excellent Weel inhibitory activity and can be used as Weel inhibitors for the prevention or treatment of tumor-related diseases, providing a novel targeted therapeutic strategy for anti-tumor therapy and possessing significant pharmaceutical potential. Detailed Implementation

[0049] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0050] Example 1: Preparation of compound CY1

[0051]

[0052] Step a: Preparation of intermediate 1-A

[0053] Under ice bath conditions, sodium hydride (60% dispersed in mineral oil, 5.6 g, 1.5 eq) was added in portions to a 100 mL solution of ethyl 2-methylmercapto-4-aminopyrimidine-5-carboxylate (20.0 g, 1 eq) in N,N-dimethylformamide. The mixture was stirred in an ice bath for 10 minutes. Then, o-chlorophenyl isocyanate (13.6 mL, 1.2 eq) was slowly added dropwise to the reaction mixture under ice bath conditions. The reaction solution was gradually brought back to room temperature and stirred for 4 hours at room temperature. After the reaction was completed by TLC, 100 mL of saturated sodium chloride solution was slowly added to the stirred reaction solution under ice bath conditions. After the mixture stopped producing bubbles, 500 mL of water was added, followed by 400 mL of ethyl acetate for extraction. The aqueous phase obtained by separation was extracted again with 400 mL of ethyl acetate. The aqueous phase was collected and the pH was adjusted to 4-5 with an appropriate amount of 5% citric acid. A large amount of solid precipitated. After filtration, the obtained solid was dried under vacuum overnight to obtain 1-A, a white powder with a yield of 92%. 1 H NMR (400MHz, DMSO-d6) δ12.62(s,1H),8.99(s,1H),7.57–7.66(m,1H),7.43–7.50(m,3H),2.51(s,3H).ESI-MS m / z 321.0[M+H] + .

[0054] Step b: Preparation of intermediate 1-B

[0055] At room temperature, phosphorus oxychloride (160 mL, 20 eq) and N,N-diisopropylethylamine (160 mL, 10.7 eq) were added sequentially to 1-A (27.5 g, 1 eq). The reaction solution was heated to 90 °C and maintained at that temperature for about 2 hours. After the reaction was completed by TLC, most of the phosphorus oxychloride and N,N-diisopropylethylamine were removed by vacuum concentration. Ice water (200 mL) and saturated sodium bicarbonate solution (100 mL) were slowly added at room temperature, followed by extraction with 100 mL of ethyl acetate. A solid that was insoluble in both phases appeared. The solid was collected by filtration and washed sequentially with 100 mL of water and 50 mL of diethyl ether. The solid was dried under vacuum overnight to give intermediate 1-B, a grayish-white solid with a yield of 73%. 1 H NMR(400MHz, DMSO-d6)δ9.33(s,1H),7.74–7.85(m,2H),7.52–7.71(m,2H),2.67(s,3H).MS-MS m / z 339.0[M+H] + .

[0056] Step c: Preparation of intermediate 1-C

[0057] At room temperature, 3-amino-1-propanol (5 mL, 1.1 eq) was added to a 200 mL acetonitrile solution of 1-B (20 g, 1 eq). The reaction was carried out at room temperature for 2 hours. TLC showed that the reaction was complete. The acetonitrile was removed by concentration under reduced pressure. 50 mL of diethyl ether was added and stirred thoroughly. The solid was collected by filtration and dried to obtain intermediate 1-C, which can be used for the next step without further purification. It is a brownish-yellow solid with a yield of 85%. 1 HNMR(400MHz,Chloroform-d)δ9.84(s,1H),7.76(s,1H),7.54(ddd,J=7.1,4.8,2.0Hz,2H),7.30(td,J=7.5,2.1Hz,1H),7.25(dd,J=7 .4,2.1Hz,1H),3.64(t,J=7.7Hz,2H),3.41(td,J=7.6,4.9Hz,2H),2.44(s,3H),1.61(p,J=7.7Hz,2H),1.21(t,J=5.0Hz,1H).MS-MSm / z 378.1[M+H] + .

[0058] Step d: Preparation of intermediate 1-D

[0059] At room temperature, 80 mL of phosphorus oxychloride was added to 1-C (16.5 g, 1 eq). The reaction solution was gradually heated to 80 °C and stirred for 4 hours. TLC showed that the reaction was complete. Phosphorus oxychloride was removed by vacuum concentration. Ice water (100 mL) and saturated sodium bicarbonate solution (200 mL) were slowly added at room temperature. The mixture was stirred at room temperature for 30 minutes until no more bubbles were generated. Sodium bicarbonate solid was added slowly in batches to adjust the pH to about 7. Then 300 mL of ethyl acetate was added, and the mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and obtained by rapid column chromatography in a PE / EA system (20-100%). Intermediate 1-D was obtained as a light yellow solid with a yield of 71%. 1 H NMR (400MHz, Chloroform-d) δ8.91 (s, 1H), 7.55–7.52 (m, 1H), 7.39 (ddd, J = 7.4, 4.4, 2.0Hz, 2H), 7.31–7. 28(m,1H),4.28–4.21(m,1H),4.18–4.07(m,1H),3.55–3.46(m,2H),2.62(s,3H),2.02–1.95(m,2H).MS-MS m / z 360.1[M+H] + .

[0060] Step e: Preparation of intermediate 1-E

[0061] At room temperature, m-chloroperoxybenzoic acid (5.3 g, 1.1 eq) was added in portions to a 200 mL solution of 1-D (10 g, 1 eq) in dichloromethane (ensuring complete dissolution). The mixture was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. A saturated sodium bicarbonate solution (150 mL) and a sodium thiosulfate solution (50 mL) were added, and the mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a grayish-yellow solid. 50 mL of diethyl ether was added to disperse the solid, and the mixture was stirred vigorously for 30 minutes. The solid was filtered and dried. It was ready for use in the next step without purification. The yield was 87%. 1 H NMR(400MHz,Chloroform-d)δ9.65(s,1H),7.51–7.46(m,1H),7.26-7.22(m,2H),7. 18(m,1H),4.43-4.37(m,2H),3.53(t,J=5.4Hz,2H),3.01(s,3H),2.06(m,2H).MS-MS m / z 392.1[M+H] + .

[0062] Step f: Preparation of compound CY1

[0063] At room temperature, 132 μL of 4-cyclohexaneaniline (1.5 eq) was added to a 5 mL isopropanol solution of 1-E (0.2 g, 1 eq). The reaction solution was heated to 80 °C and stirred for 5 hours. TLC showed that the starting material was completely consumed. Isopropanol was removed by concentration under reduced pressure. 10 mL of saturated ammonium chloride solution was added, followed by 10 mL of ethyl acetate. The mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the final product CY1 was obtained by rapid column chromatography in a PE / EA system (0-100%). The product was a white solid with a yield of 27%. 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),7.94(s,1H),7.57–7.29(m,6H),7.26–7.17(m,2H),4.15(ddd,J=44.9,13.3,6.7Hz,2H),3.48(dd,J=12.7, 4.2Hz,2H),2.58–2.42(m,1H),1.99(p,J=6.8Hz,2H),1.87(dd,J=17.4,9.7 Hz,4H),1.76(d,J=13.1Hz,1H),1.46–1.33(m,4H),1.28–1.22(m,1H).HRMS m / z(ESI)calculated for C 27 H 27 ClN6O[M+H] +487.2013, found 487.2017.

[0064] Example 2: Preparation of compound CY2

[0065]

[0066] The preparation method of compound CY2 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 4-piperidineaniline; white solid, yield 23%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.92(s,1H),7.53–7.44(m,3H),7. 37(pd,J=7.5,1.9Hz,2H),7.30(dd,J=7.3,2.1Hz,1H),6.98(d,J=8.5Hz,2H), 4.11(ddt,J=45.2,12.8,6.0Hz,2H),3.48(q,J=5.8Hz,2H),3.16(t,J=5.4Hz, 4H),1.96(p,J=6.0Hz,2H),1.74(p,J=5.6Hz,4H),1.59(p,J=6.0Hz,2H).HRMS m / z(ESI)calculated for C 26 H 26 ClN7O[M+H] + 488.1966, found 488.1966.

[0067] Example 3: Preparation of compound CY3

[0068]

[0069] The preparation method of compound CY3 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 4-(1-methylpiperidin-4-yl)aniline; white solid, yield 16%; 1H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.79(s,1H),7.58–7.50(m,3H),7.37(td,J=7.3,2.0 Hz,2H),7.29(dd,J=7.3,2.2Hz,1H),7.24(d,J=2.1Hz,2H),4.20(dt,J=12.1,5.8Hz,1H),4.09( dt,J=12.8,6.2Hz,1H),3.49(q,J=5.8Hz,2H),3.09(d,J=11.2Hz,2H),2.53(dq,J=11.2,6.3,5. 7Hz,1H),2.40(s,3H),2.18(td,J=11.3,3.7Hz,2H),2.02–1.93(m,3H),1.92–1.87(m,3H).HRMS m / z(ESI)calculatedfor C 27 H 28 ClN7O[M+H] + 502.2122, found 502.2119.

[0070] Example 4: Preparation of compound CY4

[0071]

[0072] The preparation method of compound CY4 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 4-(4-methylpiperazine)aniline; white solid, yield 15%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),8.23(s,1H),7.54–7.45(m,3H),7.41–7.27(m,3H),6.98–6.91(m,2H),4.21–4 .02(m,2H),3.47(q,J=5.8Hz,2H),3.22(t,J=5.0Hz,4H),2.60(t,J=5.0Hz,4H),2.37(s,3H),1.95(p,J=6.0Hz,2H).HRMS m / z(ESI)calculated for C 26 H 27 ClN8O[M+H] + 503.2075, found 503.2076.

[0073] Example 5: Preparation of compound CY5

[0074]

[0075] The preparation method of compound CY5 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 4-piperazinylaniline; white solid, yield 20%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.85(s,1H),7.55–7.49(m,3H),7.36(tt,J=7.6,3.6Hz,2H),7.29(dd,J=7.3,2.2Hz,1H),6.96(d,J=8 .8Hz,2H),4.11(ddt,J=44.9,12.8,6.0Hz,2H),3.52–3.44(m,3H),3.27(q,J=7.5,6.0Hz,4H),3.18(t,J=4.9Hz,4H),1.96(p,J=6.1Hz,2H).HRMS m / z(ESI)calculated for C 25 H 25 ClN8O[M+H] + 489.1918, found 489.1917.

[0076] Example 6: Preparation of compound CY6

[0077]

[0078] The preparation method of compound CY6 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 4-(4-morpholino)aniline; white solid, yield 41%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.19(s,1H),7.56–7.47(m,3H),7.42–7.28(m,3H),6.94(d,J=8.6Hz,2H),4.12(d dt,J=45.2,12.8,6.0Hz,2H),3.88(t,J=4.8Hz,4H),3.49(q,J=5.6Hz,2H),3.16(t,J=4.8Hz,4H),1.96(p,J=6.1Hz,2H).HRMS m / z(ESI)calculated for C 25 H 24 ClN7O2[M+H] + 490.1758, found 490.1760.

[0079] Example 7: Preparation of compound CY7

[0080]

[0081] The preparation method of compound CY7 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 4-(4-aminophenyl)morpholin-3-one; pale yellow solid, yield 32%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.84(s,1H),7.71–7.66(m,2H),7.55–7.51(m,1H),7.43–7.29(m,5H),4.36(s,2H),4.21(dt, J=12.0,5.8Hz,1H),4.10(q,J=6.5Hz,1H),4.07–4.03(m,2H),3.82–3.73(m,2H),3.51(q,J=5.2Hz,2H),1.99(h,J=5.3,4.8Hz,2H).HRMS m / z(ESI)calculated for C 25 H 22 ClN7O3[M+H] + 504.1551, found 504.1550.

[0082] Example 8: Preparation of compound CY8

[0083]

[0084] The preparation method of compound CY8 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 3-fluoro-4-(4-methylpiperazinyl)aniline; pale yellow solid, yield 23%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.80(s,1H),7.62–7.51(m,2H),7.37(pd, J=7.5,1.9Hz,2H),7.29(dd,J=7.3,2.2Hz,1H),7.16(dd,J=8.7,2.4Hz,1H),6.96(t, J=9.1Hz,1H),4.14(ddt,J=45.3,12.8,5.9Hz,2H),3.49(td,J=7.0,6.3,4.4Hz,2H), 3.14(t,J=4.8Hz,4H),2.65(t,J=4.9Hz,4H),2.39(s,3H),1.98(p,J=6.1Hz,2H).HRMS m / z(ESI)calculated for C 26 H 26 ClFN8O[M+H] + 521.1980, found 521.1983.

[0085] Example 9: Preparation of compound CY9

[0086]

[0087] The preparation method of compound CY9 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 2-fluoro-4-(4-methyl-1-piperazinyl)aniline; pale yellow solid, yield 28%; 1 H NMR (400MHz, Chloroform-d) δ8.90 (s, 1H), 8.32 (dt, J=12.7, 6.5Hz, 2H), 7.53 (dd, J=7. 5,1.9Hz,1H),7.38(qt,J=7.6,3.6Hz,2H),7.29(dd,J=7.2,2.2Hz,1H),6.91(ddd,J=16 .4,8.8,2.8Hz,2H),4.22(dt,J=12.1,5.8Hz,1H),4.11(dt,J=12.7,6.2Hz,1H),3.54–3 .47(m,2H),2.98(t,J=4.8Hz,4H),2.73(s,4H),2.44(s,3H),2.01(q,J=5.7Hz,2H).HRMS m / z(ESI)calculated for C 26 H 26 ClFN8O[M+H] + 521.1980, found 521.1980.

[0088] Example 10: Preparation of compound CY10

[0089]

[0090] The preparation method of compound CY10 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 1-methyl-4-(6-aminopyridin-3-yl)piperazine; white solid, yield 35%; 1H NMR(400MHz,Chloroform-d)δ9.08(s,1H),8.13(d,J=3.0Hz,1H),7.53(td,J=7.5,3.1Hz,1H),7.43–7.25(m,5H),4.20(dq,J=10.2,5.3,4.9Hz,1H) ,4.15–4.07(m,1H),3.47(dq,J=5.8,3.8,3.1Hz,2H),3.32(t,J=5.1Hz,4 H),2.61(t,J=5.1Hz,4H),2.38(s,3H),1.90(dt,J=10.5,5.7Hz,2H).HRMS m / z(ESI)calculated for C 25 H 26 ClN9O[M+H] + 504.2027, found 504.2025.

[0091] Example 11: Preparation of compound CY11

[0092]

[0093] The preparation method of compound CY11 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 2-methoxy-4-(4-methylpiperazin-1-yl)aniline; pale yellow solid, yield 31%; 1 H NMR (400MHz, Chloroform-d) δ8.85 (s, 1H), 8.22 (d, J = 8.7Hz, 1H), 7.93 (s, 1H), 7. 52(dd,J=7.5,2.0Hz,1H),7.42–7.33(m,2H),7.29(dd,J=7.3,2.1Hz,1H),6.60–6 .54(m,2H),4.16(ddd,J=39.0,13.1,6.8Hz,2H),3.91(s,3H),3.54–3.43(m,2H), 3.25–3.20(m,4H),2.62(t,J=5.0Hz,4H),2.38(s,3H),1.99(q,J=5.9Hz,2H).HRMS m / z(ESI) calculated for C 27 H 29 ClN8O2[M+H] + 533.2180, found 533.2183.

[0094] Example 12: Preparation of compound CY12

[0095]

[0096] The preparation method of compound CY12 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 2-chloro-4-(4-methylpiperazin-1-yl)aniline; pale yellow solid, yield 45%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.54–7.47(m,3H),7.37(pd,J=7.5,1.9Hz,2H),7.29(dd,J=7.3,2.1Hz,1H),6.98–6.93(m HRMS m / z(ESI)calculated for C 26 H 26 Cl2N8O[M+H] + 537.1685, found 537.1684.

[0097] Example 13: Preparation of compound CY13

[0098]

[0099] The preparation method of compound CY13 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 3-(4-methylpiperazin-1-yl)aniline; white solid, yield 25%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),8.02(s,1H),7.52(d,J=7.6Hz,1H),7.40– 7.32(m,3H),7.31–7.28(m,1H),7.27–7.22(m,1H),7.07(d,J=8.0Hz,1H),6.75–6.67( m,1H),4.21(dt,J=12.3,5.9Hz,1H),4.11(dt,J=12.9,6.2Hz,1H),3.53–3.47(m,2H), 3.27(t,J=5.0Hz,4H),2.61(t,J=5.0Hz,4H),2.37(s,3H),1.96(h,J=5.9Hz,2H).HRMS m / z(ESI)calculated for C 26 H 27 ClN8O[M+H] + 503.2075, found 503.2079.

[0100] Example 14: Preparation of compound CY14

[0101]

[0102] The preparation method of compound CY14 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 2-(4-methyl-1-piperazine)aniline; white solid, yield 44%; 1 H NMR(400MHz,Chloroform-d)δ8.91(s,1H),8.65(s,1H),8.42(dd,J=8.1,1.5Hz,1H),7.53(dd,J =7.4,2.0Hz,1H),7.38(pd,J=7.5,6.6,2.5Hz,2H),7.30(dd,J=7.3,2.1Hz,1H),7.25–7.18(m,2 H),7.10(td,J=7.6,1.5Hz,1H),4.26(dt,J=12.1,5.8Hz,1H),4.15(dt,J=12.7,6.3Hz,1H),3.5 7–3.44(m,2H),3.00(t,J=4.9Hz,4H),2.73(s,4H),2.44(s,3H),2.01(h,J=5.2,4.8Hz,2H).HRMS m / z(ESI)calculated forC 26 H 27 ClN8O[M+H] + 503.2075, found 503.2078.

[0103] Example 15: Preparation of compound CY15

[0104]

[0105] Step a: Preparation of intermediate 15-A

[0106] Under ice bath conditions, sodium hydride (60% dispersed in mineral oil, 0.37 g, 1.5 eq) was added in portions to a 5 mL solution of 6-nitroindoline (1 g, 1 eq) in N,N-dimethylformamide, and the mixture was stirred for 10 minutes under ice bath conditions. Then, methyl iodoform (0.49 mL, 1.3 eq) was slowly added dropwise to the reaction mixture under ice bath conditions. The reaction mixture was gradually brought to room temperature and stirred for 1 hour at room temperature. After the reaction was complete, 30 mL of saturated sodium chloride solution was slowly added to the stirred reaction mixture under ice bath conditions, precipitating a large amount of solid. This solid was filtered, dried under vacuum overnight to give intermediate 15-A, a yellow solid with a yield of 95%, which could be used directly in the next step without purification. MS-MS m / z 165.1 [M+H] + .

[0107] Step b: Preparation of intermediate 15-B

[0108] At room temperature, 0.1 g of palladium on carbon and 1 mL of hydrazine hydrate solution were added to a methanol solution (10 mL) of intermediate 15-A (1 g, 1 eq). The reaction was carried out overnight in a sealed container at room temperature. After the reaction was completed by TLC, palladium on carbon was removed by diatomaceous earth filtration, and methanol and water were removed by vacuum concentration to obtain the target product, a light yellow oil with a yield of 97%. 1 H NMR(400MHz,Chloroform-d)δ6.88–6.84(m,1H),6.03(dd,J=7.7,2.1Hz,1H),5.90(d,J=2.0 Hz,1H),3.56(s,2H),3.28(t,J=8.0Hz,2H),2.85(td,J=8.0,1.1Hz,2H),2.73(s,3H).MS-MS m / z 149.1[M+H] + .

[0109] The preparation method of compound CY15 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 15-B, followed by a substitution reaction with 1-E. The resulting CY15 is a white solid with a yield of 37%. 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.62(s,1H),7.53(dd,J=7.3,2.2Hz,1H),7.38(p d,J=7.4,1.9Hz,2H),7.30(dd,J=7.4,2.0Hz,1H),7.07–7.03(m,1H),6.94(s,1H),6.78(dd, J=7.8,2.0Hz,1H),4.23(dt,J=12.1,5.8Hz,1H),4.12(dt,J=12.6,6.1Hz,1H),3.50(d,J=5. 8Hz,2H),3.37(t,J=8.1Hz,2H),3.00–2.91(m,2H),2.79(s,3H),1.98(q,J=5.9Hz,2H).HRMS m / z(ESI)calculatedfor C 24 H 22 ClN7O[M+H] + 460.1653, found 460.1651.

[0110] Example 16: Preparation of compound CY16

[0111] The preparation method of compound CY16 is the same as in Example 15, except that 6-nitroindoline is replaced with 7-nitrotetrahydroquinoline (16-B); pale yellow solid, yield 47%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.82(s,1H),7.52(dd,J=7.5,2.0Hz, 1H),7.42–7.33(m,2H),7.30(dd,J=7.4,2.2Hz,1H),7.02–6.89(m,2H),6.81(dd, J=7.9,2.1Hz,1H),4.31–4.08(m,2H),3.49(dd,J=10.8,5.3Hz,2H),3.26(q,J=6 .1Hz,2H),2.92(s,3H),2.76(t,J=6.4Hz,2H),1.98(dp,J=12.0,6.2Hz,4H).HRMS m / z(ESI)calculated for C 25 H 24 ClN7O[M+H] + 474.1809, found 474.1810.

[0112] Example 17: Preparation of compound CY17

[0113]

[0114] Step a: Preparation of intermediate 17-A

[0115] At room temperature, 37% formaldehyde solution (0.17 mL, 1.1 eq) and sodium triacetoxyborohydride (5.9 g, 5 eq) were added to a 20 mL solution of 7-nitro-1,2,3,4-tetrahydroisoquinoline (1 g, 1 eq) in dichloromethane. The mixture was stirred overnight at room temperature. After the reaction was completed by TLC, the filtrate was filtered through diatomaceous earth, concentrated under reduced pressure, diluted with 10 mL of ethyl acetate, and then slowly added with 20 mL of saturated sodium bicarbonate aqueous solution. The mixture was stirred vigorously for 10 minutes, and then extracted with 20 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the target product, a brown oily substance with a yield of 89%. 1 H NMR(400MHz,Chloroform-d)δ7.94(dd,J=8.4,2.4Hz,1H),7.87(d,J=2.4Hz,1H),7.22(d,J =8.4Hz,1H),3.61(s,2H),2.97(t,J=6.0Hz,2H),2.69(t,J=5.9Hz,2H),2.45(s,3H).MS-MS m / z 193.1[M+H] + .

[0116] Step b: Preparation of intermediate 17-B

[0117] The preparation method of intermediate 17-B is similar to that of intermediate 16-B, except that intermediate 16-A is replaced with 17-A. It is a yellow oily substance with a yield of 96%. 1 H NMR(400MHz,Chloroform-d)δ6.76(dd,J=7.8,1.3Hz,1H),6.05–5.98(m,2H),3.39(s,2H),3.23–3 .16(m,2H),2.87(d,J=1.2Hz,3H),2.69(t,J=6.5Hz,2H),1.97(qdd,J=6.1,4.4,2.1Hz,2H).MS-MS m / z 163.1[M+H] + .

[0118] The preparation method of compound CY17 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 17-B, followed by a substitution reaction with 1-E. The resulting CY17 is a white solid with a yield of 21%. 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.06(s,1H),7.51(dd,J=7.5,1.9H z,1H),7.37(qd,J=7.6,2.0Hz,3H),7.31–7.26(m,2H),7.11(d,J=8.3Hz,1H),4 .11(ddt,J=44.4,12.7,6.0Hz,2H),3.59(s,2H),3.48(q,J=5.9Hz,2H),2.91(t ,J=5.9Hz,2H),2.71(t,J=5.9Hz,2H),2.47(s,3H),1.96(p,J=6.0Hz,2H).HRMS m / z(ESI)calculated for C 25 H 24 ClN7O[M+H] + 474.1809, found 474.1809.

[0119] Example 18: Preparation of compound CY18

[0120]

[0121] Step a: Preparation of intermediate 18-A

[0122] At room temperature, 25 mL of N,N-dimethylformamide solution was added to isoquinoline-1,3(2H,4H)-dione (5.0 g, 1 eq), potassium carbonate (12.9 g, 3 eq), and tetrabutylammonium hydrogen sulfate (1.1 g, 0.1 eq). Then, 5.8 mL of iodomethane (3 eq) was added. The reaction mixture was gradually heated to 70 °C and reacted for 5 hours. TLC showed the reaction was complete. The reaction mixture was cooled to room temperature and extracted with 100 mL of saturated ammonium chloride aqueous solution and 30 mL of ethyl acetate. The resulting organic phase was extracted twice more with 100 mL of saturated ammonium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to rapid column chromatography (0-100%) in a PE / EA system to obtain the target product as a yellow solid, with a yield of 86%. 1 H NMR (400MHz, Chloroform-d) δ8.22(dd,J=7.9,1.5Hz,1H),7.62(ddd,J=8.1,7.2,1.5Hz,1H),7.48–7.38(m,2H),3.37(s,3H),1.62(s,6H).MS-MS m / z 204.1[M+H] + .

[0123] Step b: Preparation of intermediate 18-B

[0124] At room temperature, intermediate 18-A (5 g, 1 eq) was dissolved in concentrated sulfuric acid (35 mL). The solution was then cooled to -5 °C, and concentrated nitric acid (15 mL) was slowly added dropwise while maintaining this temperature. After the addition was complete, the mixture was stirred at this temperature for 10 minutes. The reaction solution was then gradually raised to room temperature, and the reaction solution was slowly poured into ice water, resulting in the precipitation of a large amount of solid. The solid was filtered, washed with water, and dried under vacuum overnight to obtain the target product, which could be used for the next step without further purification. The product was a yellow solid with a yield of 97%. 1 H NMR (400MHz, Chloroform-d) δ9.21(d,J=2.0Hz,1H),8.46(dd,J=7.5,2.0Hz,1H),7.87(d,J=7.4Hz,1H),3.01(s,3H),1.51(s,6H).MS-MS m / z 249.1[M+H] + .

[0125] Step c: Preparation of intermediate 18-C

[0126] The preparation method of intermediate 18-C is similar to that of intermediate 16-B, except that intermediate 16-A is replaced with 18-B. It is a yellow oily substance with a yield of 95%. 1H NMR(500MHz,Chloroform-d)δ7.33(d,J=7.4Hz,1H),7.21(d,J=2.0Hz,1H),6.80(dd,J=7.5,2.0Hz,1H),3.93(s,2H),3.05(s,3H),1.59(s,6H).MS-MS m / z219.1[M+H] + .

[0127] Step d: Preparation of intermediate 18-D

[0128] At room temperature, intermediate 18-C (4.5 g, 1 eq) was dissolved in tetrahydrofuran (50 mL), and a tetrahydrofuran solution of borane dimethyl sulfide (2 M, 41 mL, 4 eq) was added. The reaction solution was heated to 70 °C and refluxed at this temperature overnight. TLC showed that the starting material was completely consumed. The reaction solution was cooled to room temperature, and tetrahydrofuran was removed by concentration under reduced pressure. The target product was obtained by rapid column chromatography in a PE / EA system (0-100%), which was a yellow oil with a yield of 83%. 1 H NMR(400MHz,Chloroform-d)δ7.09(d,J=8.3Hz,1H),6.54(dd,J=8.3,2.5Hz,1H), 6.32(d,J=2.5Hz,1H),3.43(s,4H),2.39(s,3H),2.36(s,2H),1.26(s,6H).MS-MS m / z 191.1[M+H] + .

[0129] The preparation method of compound CY18 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 18-D, followed by a substitution reaction with 1-E. The resulting CY18 is a pale yellow solid with a yield of 30%. 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.72(s,1H),7.52(dd,J=7.5,2.0Hz,1H),7.44(d,J=8.7Hz,1H),7.37(td,J=7.5,1.9Hz,2H),7.30(dd,J=15 .7,7.9Hz,3H),4.14(ddd,J=37.8,12.9,6.7Hz,2H),3.56(s,2H),3.49(d,J =6.2Hz,2H),2.43(d,J=6.6Hz,5H),1.98(p,J=6.0Hz,2H),1.32(s,6H).HRMS m / z(ESI)calculated for C 27 H 28 ClN7O[M+H] +502.2122, found 502.2124.

[0130] Example 19: Preparation of compound CY19

[0131]

[0132] Step a: Preparation of intermediate 19-A

[0133] Acetonitrile was added to a mixture of cis-1,2,6-trimethylpiperazine (2 g, 1 eq), potassium carbonate (6.5 g, 3 eq), and p-fluoronitrobenzene (2.6 g, 1.2 eq) at room temperature. The reaction solution was then heated to 70 °C and maintained at this temperature for 5 hours. TLC showed that the starting material was completely consumed. The reaction solution was cooled to room temperature, concentrated under reduced pressure to remove acetonitrile, and ethyl acetate (20 mL) and water (20 mL) were added. The mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the target product, which was a brownish-yellow solid with a yield of 97% and could be used in the next step without further purification. 1 H NMR (400MHz, Chloroform-d) δ7.58–7.51(m,2H),7.07–6.98(m,2H),3.63-3.57(m,4H),2.61(p,J=6.8Hz,2H),2.32(s,3H),1.17(d,J=6.1Hz,6H).MS-MS m / z 250.2[M+H] + .

[0134] Step b: Preparation of intermediate 19-B

[0135] The preparation method of intermediate 19-B is similar to that of intermediate 16-B. Only intermediate 16-A needs to be replaced with 19-A. The resulting intermediate can be used in the next step without further purification. It is a yellow solid with a yield of 96%. 1 H NMR(400MHz,Chloroform-d)δ6.63–6.54(m,2H),6.33–6.22(m,2H),3.91(s,2H),3 .82-3.72(m,2H),2.53(p,J=6.8Hz,2H),2.46(s,3H),1.15(d,J=6.8Hz,6H).MS-MS m / z 220.2[M+H] + .

[0136] The preparation method of compound CY19 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 19-B, followed by a substitution reaction with 1-E. The resulting CY19 is a white solid with a yield of 44%. 1H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.58–7.45(m,3H),7.35(td,J=7.6, 2.0Hz,2H),7.29(dt,J=8.4,5.2Hz,1H),6.93(d,J=8.6Hz,2H),4.15(dt,J=12. 5,6.0Hz,1H),4.09–4.01(m,1H),3.51–3.43(m,4H),2.59(t,J=11.2Hz,2H),2. 44–2.37(m,2H),2.33(s,3H),1.94(p,J=5.9Hz,2H),1.18(d,J=6.1Hz,6H).HRMS m / z(ESI)calculated for C 28 H 31 ClN8O[M+H] + 531.2388, found 531.2390.

[0137] Example 20: Preparation of compound CY20

[0138]

[0139] The preparation method of compound CY20 is the same as in Example 19, except that cis-1,2,6-trimethylpiperazine is replaced with cis-2,6-dimethylmorpholine.

[0140] The preparation method of compound CY20 is the same as in Example 1, except that 4-cyclohexaneaniline is replaced with 20-B, followed by a substitution reaction with 1-E. The resulting CY20 is a white solid with a yield of 26%. 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.23(s,1H),7.51(t,J=8.2Hz,3H),7.37(tt ,J=7.7,3.7Hz,2H),7.31(dd,J=7.4,2.3Hz,1H),6.95(d,J=8.6Hz,2H),4.18(dt,J=12. 0,5.7Hz,1H),4.08(q,J=9.8,8.0Hz,1H),3.83(ddt,J=11.9,8.1,4.0Hz,2H),3.52–3.4 2(m,4H),2.43(t,J=11.0Hz,2H),1.97(p,J=7.0,6.2Hz,2H),1.27(d,J=6.3Hz,6H).HRMS m / z(ESI)calculated forC 27 H 28 ClN7O2[M+H] +518.2071, found 518.2075.

[0141] Example 21: Preparation of compound CY21

[0142]

[0143] The preparation method of compound CY21 is the same as in Example 4, except that 3-amino-1-propanol is replaced with cis-2-aminocyclohexanol; grayish-white solid, yield 28%; 1 H NMR(400MHz,Chloroform-d)δ8.78(s,1H),7.83(s,1H),7.58–7.54(m,1H),7.49(d,J=8.5Hz,2H),7.42–7. 37(m,3H),6.97–6.92(m,2H),4.65(q,J=7.1Hz,1H),4.17(dd,J=8.7,4.4Hz,1H),3.23(t,J=5.0Hz,4H),2.6 3(t,J=5.0Hz,4H),2.39(s,3H),2.14(td,J=8.3,7.9,3.9Hz,1H),1.92(ddd,J=18.4,8.6,4.2Hz,2H),1.74 (td,J=9.7,5.1Hz,1H),1.63(dd,J=12.4,5.6Hz,1H),1.53(dq,J=11.1,5.7Hz,2H),1.44–1.36(m,1H).HRMS m / z(ESI)calculated for C 29 H 31 ClN8O[M+H] + 543.2388, found 543.2392.

[0144] Example 22: Preparation of compound CY22

[0145]

[0146] The preparation method of compound CY22 is the same as in Example 4, except that o-chlorophenyl isocyanate is replaced with 2-chloro-6-methylphenyl isocyanate; white solid, yield 20%; 1H NMR(400MHz,Chloroform-d)δ8.86(d,J=1.5Hz,1H),7.52–7.47(m,2H),7.34(dt,J=7.4,1.9Hz,1H),7.24–7.19(m,2H),6.96(dd,J=8.9,1. HRMS m / z(ESI)calculated forC 27 H 29 ClN8O[M+H] + 517.2231, found 517.2233.

[0147] Example 23: Preparation of compound CY23

[0148]

[0149] The preparation method of compound CY23 is the same as in Example 22, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 47%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),7.45(dd,J=8.6,2.3Hz,1H),7.38–7.28(m,3H),7.28(d,J=2.2Hz,1H),7.26–7.18(m,2H),4.14 HRMS m / z(ESI)calculatedfor C 28 H 30 ClN7O[M+H] + 516.2279, found 516.2276.

[0150] Example 24: Preparation of compound CY24

[0151]

[0152] The preparation method of compound CY24 is the same as in Example 22, except that 3-amino-1-propanol is replaced with 4-amino-1-butanol; white solid, yield 21%; 1 H NMR(400MHz,Chloroform-d)δ9.03(s,1H),7.59–7.45(m,4H),7.42(d,J=7.7Hz,1H),7.37–7.34(m,1H),6.94(d,J=8.9Hz,2H),4.51(t,J=5 .7Hz,1H),3.53(t,J=6.0Hz,3H),3.29(t,J=5.0Hz,4H),2.75(t,J=5.0Hz,4H),2.47(s,3H),2.20(s,3H),1.74(dq,J=19.8,6.5Hz,4H).HRMS m / z(ESI)calculated for C 28 H 31 ClN8O[M+H] + 531.2388, found 531.2386.

[0153] Example 25: Preparation of compound CY25

[0154]

[0155] The preparation method of compound CY25 is the same as in Example 24, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 36%; 1 H NMR(400MHz,Chloroform-d)δ9.06(s,1H),7.51–7.40(m,4H),7.35(d,J=7.6Hz,1H),7.32–7.28(m,2H),4.52(t,J=5.7H z,1H),3.59(s,2H),3.54(t,J=5.9Hz,3H),2.43(s,3H),2.42(s,2H),2.21(s,3H),1.80–1.70(m,4H),1.31(s,6H).HRMS m / z(ESI)calculated for C 29 H 32 ClN7O[M+H] + 530.2435, found 530.2439.

[0156] Example 26: Preparation of compound CY26

[0157]

[0158] The preparation method of compound CY26 is the same as in Example 4, except that o-chlorophenyl isocyanate is replaced with 2,6-dimethylisocyanate; yellowish-white solid, yield 13%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.69(s,1H),7.50(d,J=8.6Hz,2H),7.19(dd,J=8.7,6.2Hz,1H),7.13(d,J=7.4Hz,2H),6.99–6.93(m, 2H),4.12(t,J=6.1Hz,2H),3.51–3.47(m,2H),3.26(t,J=5.0Hz,4H),2.6 8(t,J=4.9Hz,4H),2.42(s,3H),2.13(s,6H),1.95(p,J=5.7Hz,2H).HRMS m / z(ESI)calculatedfor C 28 H 32 N8O[M+H] + 497.2777, found 497.2773.

[0159] Example 27: Preparation of compound CY27

[0160]

[0161] The preparation method of compound CY27 is the same as that in Example 26, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 32%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.90(s,1H),7.48–7.38(m,1H),7.32(d,J=2.2Hz,1H),7.24–7.11(m,4H),4.14(q,J=7.5,6.0Hz, HRMS m / z(ESI)calculated forC 27 H 29 N7O[M+H] + 468.2512, found 468.2515.

[0162] Example 28: Preparation of compound CY28

[0163]

[0164] The preparation method of compound CY28 is the same as that in Example 26, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 39%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),7.78(s,1H),7.45(dd,J=8.5,2.3Hz,1H),7.34–7.27(m,2H),7.20(dd,J=8.6,6.2Hz,1H),7.14(d,J=6.7Hz, 2H),4.15(t,J=6.1Hz,2H),3.57(s,2H),3.49(t,J=5.6Hz,2H),2.44(d,J=5 .2Hz,5H),2.14(s,6H),1.97(qd,J=8.7,7.7,3.5Hz,2H),1.33(s,6H).HRMS m / z(ESI)calculated for C 29 H 33 N7O[M+H] + 496.2825, found 496.2826.

[0165] Example 29: Preparation of compound CY29

[0166]

[0167] The preparation method of compound CY29 is the same as that in Example 26, except that 4-(4-methylpiperazine)aniline is replaced with 4-((2S,6R)-2,6-dimethylmorpholino)aniline; grayish-white solid, yield 37%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.50(d,J=8.5Hz,2H),7.19(dd,J=8.6,6.2Hz,1H),7.13(d,J=7.5Hz,2H),6.93(d,J=8.9Hz,2H),4.11(t,J= 6.0Hz,2H),3.82(dqd,J=12.5,6.2,2.1Hz,2H),3.46–3.41(m,4H),2.42(t, J=11.0Hz,2H),2.13(s,6H),1.94(q,J=5.9Hz,2H),1.28–1.25(m,6H).HRMS m / z(ESI)calculated for C 29 H 33 N7O2[M+H]+ 512.2774, found 512.2772.

[0168] Example 30: Preparation of compound CY30

[0169]

[0170] The preparation method of compound CY30 is the same as in Example 26, except that 4-(4-methylpiperazine)aniline is replaced with 4-((3S,5R)-3,4,5-trimethylpiperazine-1-yl)aniline; white solid, yield 38%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.75(s,1H),7.50(d,J=8.5Hz,2H),7.20(dd,J=8.6,6.3Hz,1H),7.13(d,J=7.5Hz,2H),6.98–6.93(m, 2H),4.11(t,J=6.1Hz,2H),3.49–3.45(m,4H),2.69(s,2H),2.50(s,2H), 2.39(s,3H),2.13(s,6H),1.94(t,J=6.0Hz,2H),1.27–1.22(m,6H).HRMS m / z(ESI)calculated for C 30 H 36 N8O[M+H] + 525.3090, found 525.3091.

[0171] Example 31: Preparation of compound CY31

[0172]

[0173] The preparation method of compound CY31 is the same as in Example 4, except that o-chlorophenyl isocyanate is replaced with 1-chloro-3-fluoro-2-phenyl isocyanate; grayish-white solid, yield 26%; 1H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.71(s,1H),7.48(d,J=8.8Hz,2H),7.33(dd,J=6.6,4.0Hz,2H),7.13(ddd,J=9.4,6.7,3.0Hz,1H),7.0 2–6.91(m,2H),4.21–4.03(m,2H),3.48(td,J=5.6,2.0Hz,2H),3.24(t,J =5.0Hz,4H),2.63(t,J=5.0Hz,4H),2.39(s,3H),2.01–1.92(m,2H).HRMS m / z(ESI)calculatedfor C 26 H 26 ClFN8O[M+H] + 521.1980, found 521.1977.

[0174] Example 32: Preparation of compound CY32

[0175]

[0176] The preparation method of compound CY32 is the same as that in Example 31, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 43%; 1 H NMR (400MHz, Chloroform-d) δ8.87(s,1H),7.84(s,1H),7.41(d,J=8.4Hz,1H),7.37–7.29(m,3H),7.17–7.09(m,2H),4.11(ddt,J=25. HRMS m / z(ESI)calculated for C 25 H 23 ClFN7O[M+H] + 492.1715, found 492.1711.

[0177] Example 33: Preparation of compound CY33

[0178]

[0179] The preparation method of compound CY33 is the same as that in Example 31, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 18%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),8.17(s,1H),7.43(dd,J=8.4,2.3Hz,1H),7.31(dt,J=5.5,3.2Hz,3H),7.27(d,J=2.2Hz,1H),7.12(ddd,J=9.3,6 .7,3.2Hz,1H),4.13(qt,J=12.7,5.8Hz,2H),3.55(s,2H),3.49(td,J=5.8,2. 1Hz,2H),2.43(d,J=4.7Hz,5H),1.99(q,J=7.3,6.5Hz,2H),1.32(s,6H).HRMS m / z(ESI)calculated for C 27 H 27 ClFN7O[M+H] + 520.2028, found 520.2026.

[0180] Example 34: Preparation of compound CY34

[0181]

[0182] The preparation method of compound CY34 is the same as that in Example 31, except that 4-(4-methylpiperazine)aniline is replaced with 4-((3S,5R)-3,4,5-trimethylpiperazine-1-yl)aniline; white solid, yield 41%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.84(s,1H),7.48(d,J=8.6Hz,2H),7.36–7.29(m,2H),7.13(ddd,J=9.4,6.7,3.0Hz,1H),6.98–6.92(m,2H), 4.10(ddt,J=25.3,12.9,6.4Hz,2H),3.51–3.42(m,4H),2.61(s,2H),2.38( d,J=29.1Hz,5H),1.97(dtt,J=7.4,5.1,2.3Hz,2H),1.23–1.15(m,6H).HRMS m / z(ESI)calculated for C 28 H 30 ClFN8O[M+H] +549.2293, found549.2293.

[0183] Example 35: Preparation of compound CY35

[0184]

[0185] The preparation method of compound CY35 is the same as that in Example 31, except that 4-(4-methylpiperazine)aniline is replaced with 4-((2S,6R)-2,6-dimethylmorpholino)aniline; white solid, yield 23%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.15(s,1H),7.49(d,J=8.6Hz,2H),7 .37–7.30(m,2H),7.16–7.09(m,1H),6.99–6.92(m,2H),4.10(ddt,J=25.4,12.9 ,6.5Hz,2H),3.88–3.80(m,2H),3.52–3.42(m,4H),2.44(dd,J=11.8,10.3Hz,2H ),1.97(td,J=5.9,3.2Hz,2H),1.27(d,J=6.3Hz,6H).HRMSm / z(ESI)calculated for C 27 H 27 ClFN7O2[M+H] + 536.1977, found 536.1974.

[0186] Example 36: Preparation of compound CY36

[0187]

[0188] The preparation method of compound CY36 is the same as in Example 31, except that 3-amino-1-propanol is replaced with 4-amino-1-butanol; white solid, yield 28%; 1 H NMR(400MHz,Chloroform-d)δ9.02(s,1H),7.56–7.44(m,5H),7.28(dd,J=8.5,1.4Hz,1H),6.94(d,J=8.7Hz,2H),4.60(t HRMS m / z(ESI)calculated for C 27 H 28ClFN8O[M+H] + 535.2137, found 535.2139.

[0189] Example 37: Preparation of compound CY37

[0190]

[0191] The preparation method of compound CY37 is the same as that in Example 36, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 46%; 1 H NMR(400MHz,Chloroform-d)δ9.05(s,1H),7.57–7.49(m,2H),7.46(d,J=8.1Hz,2H),7.29(dd,J=8.5,4.1Hz,3H),4.63(t,J= 5.7Hz,1H),3.59(s,2H),3.55(t,J=6.0Hz,3H),2.43(d,J=6.5Hz,5H),1.77(ddd,J=16.8,7.7,4.6Hz,4H),1.31(s,6H).HRMS m / z(ESI)calculated for C 28 H 29 ClFN7O[M+H] + 534.2184, found 534.2185.

[0192] Example 38: Preparation of compound CY38

[0193]

[0194] The preparation method of compound CY38 is the same as in Example 31, except that 3-amino-1-propanol is replaced with 3-amino-2,2-dimethyl-1-propanol; white solid, yield 20%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.88(s,1H),7.48(d,J=8.5Hz,2H),7.32(d,J=3.4Hz,2H),7.16–7.11(m,1H),6.96(d,J=8.7Hz,2 H),3.87(d,J=12.6Hz,1H),3.67(d,J=12.7Hz,1H),3.23(t,J=5.0Hz,4H),3.17(s,2H),2.62(t,J=4.9Hz,4H),2.38(s,3H),1.06(s,6H).HRMS m / z(ESI)calculated forC28 H 30 ClFN8O[M+H] + 549.2293, found 549.2297.

[0195] Example 39: Preparation of compound CY39

[0196]

[0197] The preparation method of compound CY39 is the same as that in Example 38, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 30%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),7.38(d,J=11.5Hz,1H),7.38–7.27(m,5H),7.14(ddd,J=9.2,6.9,3.1Hz,1H),3.9 0(d,J=12.6Hz,1H),3.71(d,J=12.6Hz,1H),3.57(s,2H),3.19(s,2H),2.43(d,J=4.2Hz,6H)1.33(s,10H),1.08(s,7H).HRMS m / z(ESI)calculated for C 29 H 31 ClFN7O[M+H] + 548.2341, found 548.2342.

[0198] Example 40: Preparation of compound CY40

[0199]

[0200] The preparation method of compound CY40 is the same as in Example 31, except that 3-amino-1-propanol is replaced with 3-amino-3-methylbut-1-ol; white solid, yield 35%; 1H NMR(400MHz,Chloroform-d)δ8.84(s,1H),7.83(s,1H),7.49(d,J=8.6Hz,2H),7.34–7.27(m,2H),7.09(td,J=7.9,6.8,2.4Hz,1H),6.96(d,J= HRMS m / z(ESI)calculated forC 28 H 30 ClFN8O[M+H] + 549.2293, found 549.2294.

[0201] Example 41: Preparation of compound CY41

[0202]

[0203] The preparation method of compound CY41 is the same as that in Example 40, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; grayish-white solid, yield 32%; 1 H NMR (400MHz, Chloroform-d) δ8.86(d,J=1.4Hz,1H),7.58(s,1H),7.39(d,J=8.3Hz,1H),7.34–7.27(m,3H),7.14–7.07(m,2H),4.08(t,J=6. HRMS m / z(ESI)calculated for C 27 H 27 ClFN7O[M+H] + 520.2028, found 520.2029.

[0204] Example 42: Preparation of compound CY42

[0205]

[0206] The preparation method of compound CY42 is the same as that in Example 40, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 27%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.74(s,1H),7.46(d,J=8.5Hz,1H),7.34–7.26(m,4H),7.13–7.07(m,1H ),4.10(t,J=6.4Hz,2H),3.56(s,2H),2.44(d,J=6.0Hz,5H),1.81(t,J=6.3Hz,2H),1.32(s,6H),1.09(s,6H).HRMS m / z(ESI)calculated for C 29 H 31 ClFN7O[M+H] + 548.2341, found 548.2343.

[0207] Example 43: Preparation of compound CY43

[0208]

[0209] The preparation method of compound CY43 is the same as in Example 31, except that 3-amino-1-propanol is replaced with 3-amino-2,2-difluoroprop-1-ol; white solid, yield 42%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.80(s,1H),7.44(d,J=8.7Hz,2H),7.40–7.32(m,2H),7.14(td,J=8.2,7.7,2.0Hz,1H),6.97(d, J=8.5Hz,2H),4.31(ddd,J=30.0,19.3,11.8Hz,2H),3.75(t,J=12.1Hz,2H),3.25(t,J=4.9Hz,4H),2.63(t,J=4.9Hz,4H),2.39(s,3H).HRMS m / z(ESI)calculated forC 26 H 24 ClF3N8O[M+H] + 557.1792, found 557.1789.

[0210] Example 44: Preparation of compound CY44

[0211]

[0212] The preparation method of compound CY44 is the same as that in Example 43, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 18%; 1 H NMR(400MHz,Chloroform-d)δ8.92(s,1H),7.62(s,2H),7.45–7.29(m,4H),7.15(td,J=8.3,7.8,2.0 HRMS m / z(ESI)calculated for C 27 H 25 ClF3N7O[M+H] + 556.1839, found 556.1836.

[0213] Example 45: Preparation of compound CY45

[0214]

[0215] The preparation method of compound CY45 is the same as in Example 4, except that o-chlorophenyl isocyanate is replaced with 2,6-dichlorophenyl isocyanate; white solid, yield 39%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.63(s,1H),7.49(d,J=8.8Hz,2H),7.43(d,J=8.0Hz,2H),7.32–7.28(m,1H),6.99–6.9 3(m,2H),4.11(t,J=6.1Hz,2H),3.48(t,J=5.6Hz,2H),3.25(t,J=5.0Hz,4H),2.64(s,4H),2.40(s,3H),1.97(p,J=5.7Hz,2H).HRMS m / z(ESI)calculated for C 26 H 26 Cl2N8O[M+H] + 537.1685, found 537.1682.

[0216] Example 46: Preparation of compound CY46

[0217]

[0218] The preparation method of compound CY46 is the same as that in Example 45, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 43%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.93(s,1H),7.44(s,1H),7.43–7.40(m,2H),7.33–7.27(m,2H),7.13(d,J=8.3Hz,1H),4.14(dt,J=1 HRMS m / z(ESI)calculated for C 25 H 23 Cl2N7O[M+H] + 508.1419, found 508.1416.

[0219] Example 47: Preparation of compound CY47

[0220]

[0221] The preparation method of compound CY47 is the same as that in Example 45, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 45%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),8.22(s,1H),7.46–7.40(m,3H),7.33–7.26(m,3H),4.14(t,J=5 .9Hz,2H),3.56(s,2H),3.51–3.48(m,2H),2.43(d,J=4.2Hz,5H),1.98(p,J=5.9Hz,2H),1.32(s,6H).HRMS m / z(ESI)calculated for C 27 H 27 Cl2N7O[M+H] + 536.1732, found 536.1735.

[0222] Example 48: Preparation of compound CY48

[0223]

[0224] The preparation method of compound CY48 is the same as that in Example 45, except that 4-(4-methylpiperazine)aniline is replaced with 4-((3S,5R)-3,4,5-trimethylpiperazine-1-yl)aniline; grayish-white solid, yield 23%; 1 H NMR (400MHz, Chloroform-d) δ8.86(s,1H),7.77(s,1H),7.48(d,J=8.5Hz,2H),7.43(d,J=8.1Hz,2H),7.32–7.27(m,1H),6.95(d,J=9. 0Hz,2H),4.11(t,J=6.0Hz,2H),3.51–3.44(m,4H),2.61(s,2H),2.39(d,J=29.3Hz,5H),1.97(p,J=5.8Hz,2H),1.24–1.17(m,6H).HRMS m / z(ESI)calculatedfor C 28 H 30 Cl2N8O[M+H] + 565.1998, found 565.1999.

[0225] Example 49: Preparation of compound CY49

[0226]

[0227] The preparation method of compound CY49 is the same as that in Example 45, except that 4-(4-methylpiperazine)aniline is replaced with 4-((2S,6R)-2,6-dimethylmorpholino)aniline; white solid, yield 29%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.96(s,1H),7.50(d,J=8.6Hz,2H),7.46–7.41(m,2H),7.29(ddd,J=8.6,7.5,1.0Hz,1H),6.95(d,J=8.6Hz,2H ),4.12(t,J=6.1Hz,2H),3.83(ddd,J=14.9,7.1,3.8Hz,2H),3.53–3.41(m,4 H),2.44(t,J=11.0Hz,2H),1.97(p,J=5.7Hz,2H),1.27(d,J=6.6Hz,6H).HRMS m / z(ESI)calculated for C 27 H 27 Cl2N7O2[M+H] + 552.1682, found 552.1687.

[0228] Example 50: Preparation of compound CY50

[0229]

[0230] The preparation method of compound CY50 is the same as in Example 45, except that 3-amino-1-propanol is replaced with 4-amino-1-butanol; white solid, yield 28%; 1 H NMR(400MHz,Chloroform-d)δ7.57(d,J=8.0Hz,2H),7.54–7.43(m,3H),7.39(s,1H),6.95(d,J=8.8Hz,2H),4.46(t,J=5.7Hz,1 H),3.66–3.53(m,3H),3.22(t,J=5.0Hz,4H),2.64(t,J=4.9Hz,5H),2.39(s,3H),1.86–1.69(m,5H).HRMSm / z(ESI)calculated for C 27 H 28 Cl2N8O[M+H] + 551.1841, found 551.1841.

[0231] Example 51: Preparation of compound CY51

[0232]

[0233] The preparation method of compound CY51 is the same as that in Example 50, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 32%; 1 H NMR(400MHz,Chloroform-d)δ9.07(s,1H),7.70(s,1H),7.57(d,J=7.8Hz,2H),7.50–7.47(m,1H),7.44–7.37(m,2H),7.10(d,J=8.2 HRMS m / z(ESI)calculated for C 26 H 25 Cl2N7O[M+H] + 522.1576, found 522.1578.

[0234] Example 52: Preparation of compound CY52

[0235]

[0236] The preparation method of compound CY52 is the same as that in Example 50, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 44%; 1 H NMR(400MHz,Chloroform-d)δ9.05(s,1H),7.61–7.54(m,3H),7.49–7.43(m,2H),7.32–7.27(m,2H),4.55(t,J=5 .7Hz,1H),3.59(d,J=4.7Hz,3H),3.54(t,J=6.0Hz,2H),2.45–2.41(m,5H),1.82–1.71(m,4H),1.31(s,6H).HRMS m / z(ESI)calculated for C 28 H 29 Cl2N7O[M+H] + 550.1889, found 550.1887.

[0237] Example 53: Preparation of compound CY53

[0238]

[0239] The preparation method of compound CY53 is the same as in Example 45, except that 3-amino-1-propanol is replaced with 5-amino-1-pentanol; white solid, yield 47%; 1 H NMR(400MHz,Chloroform-d)δ9.02(s,1H),7.56(s,2H),7.53–7.41(m,4H),6.96–6.92(m,2H),3.57(q,J=6.6Hz,2H),3.51(t,J=6.4Hz,2H) HRMS m / z(ESI)calculated for C 28 H 30 Cl2N8O[M+H] + 565.1998, found 565.1995.

[0240] Example 54: Preparation of compound CY54

[0241]

[0242] The preparation method of compound CY54 is the same as that in Example 53, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 19%; 1 H NMR(400MHz,Chloroform-d)δ9.05(s,1H),7.62(s,1H),7.58–7.55(m,2H),7.46(ddd,J=8.8,7.2,1.0Hz,2H),7.32–7.27(m,2H),3.59(s,2H),3. 55(t,J=6.5Hz,2H),3.50(t,J=6.5Hz,2H),2.43(d,J=4.7Hz,5H),1.76(p,J=6.7Hz,2H),1.64–1.56(m,2H),1.47–1.42(m,2H),1.31(s,6H).HRMS m / z(ESI)calculated for C 29 H 31 Cl2N7O[M+H] + 564.2045, found 564.2049.

[0243] Example 55: Preparation of compound CY55

[0244]

[0245] The preparation method of compound CY55 is the same as in Example 45, except that 3-amino-1-propanol is replaced with 3-aminotetrahydro-2H-pyran-4-ol; grayish-white solid, yield 17%; 1 H NMR(400MHz,Chloroform-d)δ8.81(s,1H),8.61(s,1H),7.44(dd,J=8.4,2.8Hz,4H),7.32(t,J=8.1Hz,1H),6.93(d,J=8.7Hz,2H),4.76(q,J=6.6Hz,1H) ,4.09(dt,J=8.5,4.3Hz,1H),3.83(qt,J=7.6,4.7Hz,3H),3.63(dt,J=11.6 ,5.8Hz,1H),3.21(t,J=5.0Hz,4H),2.60(t,J=4.9Hz,4H),2.36(s,5H).HRMS m / z(ESI)calculated for C 28 H28 Cl2N8O2[M+H] + 579.1791, found 579.1792.

[0246] Example 56: Preparation of compound CY56

[0247]

[0248] The preparation method of compound CY56 is the same as that in Example 55, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 29%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.76(s,1H),7.50–7.45(m,2H),7.37–7.29(m,4H),4.80(dd,J=9.9,4.6Hz,1H),4.15(dt,J=8.7,4.4H z,1H),3.87(ddt,J=25.5,12.8,6.2Hz,3H),3.71–3.65(m,1H),3.54(s,2H),2.43(d,J=6.3Hz,5H),2.38(dd,J=8.3,3.5Hz,2H),1.32(s,6H).HRMS m / z(ESI)calculated for C 29 H 29 Cl2N7O2[M+H] + 578.1838, found 578.1834.

[0249] Example 57: Preparation of compound CY57

[0250]

[0251] The preparation method of compound CY57 is the same as in Example 45, except that 3-amino-1-propanol is replaced with 3-amino-2,2-dimethyl-1-propanol; white solid, yield 39%; 1H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.75(s,1H),7.49(d,J=8.5Hz,2H),7.43(dd,J=8.0,1.4Hz,2H),7.32–7.27(m,1H),6.98–6.9 2(m,2H),3.78(s,2H),3.23(t,J=4.9Hz,4H),3.19–3.16(m,2H),2.62(t,J=4.8Hz,4H),2.38(d,J=1.5Hz,3H),1.07(d,J=1.5Hz,6H).HRMS m / z(ESI)calculated for C 28 H 30 Cl2N8O[M+H] + 565.1998, found 565.1996.

[0252] Example 58: Preparation of compound CY58

[0253]

[0254] The preparation method of compound CY58 is the same as that in Example 57, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 34%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.83(s,1H),7.45–7.41(m,3H),7.33–7.27(m,2H),7.12(d,J=8.2Hz,1H) ,3.80(s,2H),3.61(s,2H),3.18(s,2H),2.93(t,J=6.0Hz,2H),2.73(t,J=5.9Hz,2H),2.48(s,3H),1.08(s,6H).HRMS m / z(ESI)calculated for C 27 H 27 Cl2N7O[M+H] + 536.1732, found 536.1737.

[0255] Example 59: Preparation of compound CY59

[0256]

[0257] The preparation method of compound CY59 is the same as that in Example 57, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 25%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.57(s,1H),7.43(td,J=8.0,3.0Hz,3H),7.37–7.29(m,3H),3.8 1(s,2H),3.56(s,2H),3.19(s,2H),2.43(d,J=4.3Hz,5H),1.33(d,J=1.5Hz,6H),1.09(d,J=1.4Hz,6H).HRMS m / z(ESI)calculated for C 29 H 31 Cl2N7O[M+H] + 564.2045, found564.2045.

[0258] Example 60: Preparation of compound CY60

[0259]

[0260] The preparation method of compound CY60 is the same as in Example 45, except that 3-amino-1-propanol is replaced with (R)-3-aminobutanol; white solid, yield 44%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.52(d,J=8.4Hz,2H),7.44–7.38(m,3H), 7.29(t,J=8.5Hz,1H),6.95(d,J=8.7Hz,2H),4.27(ddd,J=13.2,6.6,4.6Hz,1H),3.9 7–3.90(m,1H),3.62–3.58(m,1H),3.24(t,J=5.0Hz,4H),2.65(t,J=4.9Hz,4H),2.40 (s,3H),2.08–2.02(m,1H),1.64(dq,J=13.0,3.7Hz,1H),1.09(d,J=6.5Hz,3H).HRMS m / z(ESI)calculated for C 27 H 28 Cl2N8O[M+H] + 551.1841, found 551.1845.

[0261] Example 61: Preparation of compound CY61

[0262]

[0263] The preparation method of compound CY61 is the same as that in Example 60, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 14%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),8.03(s,1H),7.45–7.38(m,3H),7.31( d,J=8.5Hz,1H),7.29–7.25(m,2H),4.35–4.28(m,1H),3.94(ddd,J=13.4,9.2,4. 5Hz,1H),3.60(td,J=7.0,4.2Hz,1H),3.55(s,2H),2.42(d,J=6.2Hz,5H),2.13–2 .03(m,1H),1.65(tt,J=9.2,4.2Hz,1H),1.32(s,6H),1.10(d,J=6.7Hz,3H).HRMS m / z(ESI) calculated for C 28 H 29 Cl2N7O[M+H] + 550.1889, found 550.1888.

[0264] Example 62: Preparation of compound CY62

[0265]

[0266] The preparation method of compound CY62 is the same as in Example 45, except that 3-amino-1-propanol is replaced with (S)-3-aminobutanol; white solid, yield 34%; 1H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.13(s,1H),7.52(d,J=8.5Hz,2H),7.45–7.40(m, 2H),7.30–7.27(m,1H),6.97(d,J=8.7Hz,2H),4.32–4.25(m,1H),3.94(ddd,J=13.8,9.1,4.2H z,1H),3.60(td,J=7.0,4.4Hz,1H),3.24(t,J=5.0Hz,4H),2.64(t,J=5.0Hz,4H),2.40(s,3H) ,2.06(dd,J=13.2,5.9Hz,1H),1.65(dtd,J=13.0,8.1,4.6Hz,1H),1.11(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated forC 27 H 28 Cl2N8O[M+H] + 551.1841, found 551.1836.

[0267] Example 63: Preparation of compound CY63

[0268]

[0269] The preparation method of compound CY63 is the same as that in Example 62, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 34%; 1 H NMR (400MHz, Chloroform-d) δ8.88(s,1H),7.91(s,1H),7.45–7.39(m,3H),7.34–7.26(m,3H),4.31(dt,J=12.0,5.4Hz,1H),3.95(ddd,J=13.2,9.0,4. 4Hz,1H),3.64–3.54(m,3H),2.43(d,J=6.1Hz,5H),2.07(dq,J=10.6,4.7Hz ,1H),1.66(dp,J=13.0,4.2Hz,1H),1.32(s,6H),1.11(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated for C 28 H 29 Cl2N7O[M+H] + 550.1889, found 550.1887.

[0270] Example 64: Preparation of compound CY64

[0271]

[0272] The preparation method of compound CY64 is the same as in Example 45, except that 3-amino-1-propanol is replaced with 3-amino-2,2-difluoroprop-1-ol; white solid, yield 19%; 1 H NMR(400MHz,Chloroform-d)δ8.90(s,1H),7.63(s,1H),7.48–7.43(m,4H),7.34–7.29(m,1H),6.97(d,J=8.6Hz, 2H),4.33(t,J=11.7Hz,2H),3.75(t,J=12.0Hz,2H),3.30(d,J=8.6Hz,4H),2.76–2.61(m,4H),2.43(s,3H).HRMS m / z(ESI)calculated for C 26 H 24 Cl2F2N8O[M+H] + 573.1496, found 573.1491.

[0273] Example 65: Preparation of compound CY65

[0274]

[0275] The preparation method of compound CY65 is the same as that in Example 64, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 33%; 1 H NMR(400MHz,Chloroform-d)δ8.93(s,1H),7.73(s,1H),7.48–7.29(m,6H),4.36(t, J=11.7Hz,2H),3.77(t,J=12.1Hz,2H),3.64(s,2H),2.50(s,5H),1.36(s,6H).HRMS m / z(ESI)calculated for C 27 H 25 Cl2F2N7O[M+H] + 572.1544, found 572.1547.

[0276] Example 66: Preparation of compound CY66

[0277]

[0278] The preparation method of compound CY66 is the same as in Example 45, except that 3-amino-1-propanol is replaced with (2S)-3-amino-2-fluoropropane-1-ol; white solid, yield 20%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.89(s,1H),7.49–7.42(m,4H),7.32 –7.27(m,1H),6.96(d,J=8.8Hz,2H),5.19(t,J=2.8Hz,0.5H),5.07(t,J=2.7Hz,0 .5H),4.87(t,J=13.5Hz,1H),3.84(dd,J=17.0,13.9Hz,1H),3.77–3.63(m,1H),3 .62–3.49(m,1H),3.28(t,J=4.9Hz,4H),2.70(t,J=4.8Hz,4H),2.43(s,3H).HRMS m / z(ESI) calculated for C 26 H 25 Cl2FN8O[M+H] + 555.1591, found 555.1593.

[0279] Example 67: Preparation of compound CY67

[0280]

[0281] The preparation method of compound CY67 is the same as that in Example 66, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 14%; 1 H NMR(400MHz,Chloroform-d)δ8.90(s,1H),8.10(s,1H),7.46–7.41(m,2H),7.36 (d,J=8.3Hz,1H),7.30(d,J=8.2Hz,1H),7.24(s,1H),7.12(d,J=8.2Hz,1H),5.2 0(t,J=2.8Hz,0.5H),5.08(t,J=2.8Hz,0.5H),4.92–4.82(m,1H),3.89–3.64(m, 3H),3.61(s,2H),2.93(t,J=6.0Hz,2H),2.73(t,J=6.0Hz,2H),2.49(s,3H).HRMS m / z(ESI) calculated for C 25 H 22 Cl2FN7O[M+H] +526.1325, found 526.1321.

[0282] Example 68: Preparation of compound CY68

[0283]

[0284] The preparation method of compound CY68 is the same as that in Example 66, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 34%; 1 H NMR(400MHz,Chloroform-d)δ8.91(s,1H),7.65(s,1H),7.46–7.40(m,3H),7.35–7.27(m,2H),7.23(s,1H),5.22(d,J=3.5Hz,0.5H),5. HRMS m / z(ESI)calculatedfor C 27 H 26 Cl2FN7O[M+H] + 554.1638, found 554.1635.

[0285] Example 69: Preparation of compound CY69

[0286]

[0287] The preparation method of compound CY69 is the same as in Example 45, except that 3-amino-1-propanol is replaced with (2R)-3-amino-2-fluoropropane-1-ol; white solid, yield 24%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.98(s,1H),7.50–7.42(m,4H),7.31(d,J=8.1Hz,1H),6.96(d,J=8.5Hz,2H),5.19(d,J=2.8Hz, HRMS m / z(ESI)calculated forC 26H 25 Cl2FN8O[M+H] + 555.1591, found 555.1590.

[0288] Example 70: Preparation of compound CY70

[0289]

[0290] The preparation method of compound CY70 is the same as that in Example 69, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; beige solid, yield 39%; 1 H NMR(400MHz,Chloroform-d)δ8.91(s,1H),8.07(s,1H),7.43(dt,J=8.1,1.5Hz, 2H),7.35(d,J=8.3Hz,1H),7.32–7.27(m,1H),7.23(s,1H),7.12(d,J=8.2Hz,1H ),5.23–5.17(m,0.5H),5.12–5.05(m,0.5H),4.93–4.82(m,1H),3.90–3.64(m,3 H),3.60(s,2H),2.92(t,J=6.0Hz,2H),2.72(t,J=5.9Hz,2H),2.48(s,3H).HRMS m / z(ESI) calculated for C 25 H 22 Cl2FN7O[M+H] + 526.1325, found 526.1322.

[0291] Example 71: Preparation of compound CY71

[0292]

[0293] The preparation method of compound CY71 is the same as that in Example 69, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 43%; 1H NMR(400MHz,Chloroform-d)δ8.91(s,1H),7.74(s,1H),7.46–7.40(m,3H),7.34–7.28(m,2H),7.23(d,J=2.3Hz,1H),5.23–5.20(m, HRMS m / z(ESI)calculated forC 27 H 26 Cl2FN7O[M+H] + 554.1638, found 554.1633.

[0294] Example 72: Preparation of compound CY72

[0295]

[0296] The preparation method of compound CY72 is the same as in Example 45, except that 3-amino-1-propanol is replaced with 3-amino-2-methylpropanol; white solid, yield 25%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.75(s,1H),7.51–7.39(m,4H),7 .29(d,J=8.1Hz,1H),7.00–6.91(m,2H),4.44–4.35(m,1H),3.55–3.40(m,2H ),3.23(t,J=5.0Hz,4H),3.08(dd,J=15.5,8.8Hz,1H),2.61(t,J=5.1Hz,4H) ,2.38(s,3H),2.07(ddt,J=13.8,8.5,4.6Hz,1H),1.09(d,J=6.7Hz,3H).HRMS m / z(ESI)calculated for C 27 H 28 Cl2N8O[M+H] + 551.1841, found 551.1846. CY72-R and CY72-S were obtained through chiral separation, CY72-R; 1H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.76(s,1H),7.50–7.36(m,4H),7.28-7.30(m,1H),6.96(d,J=9.0Hz,2H),4.46–4.37(m,1H) ),3.59–3.44(m,2H),3.24(t,J=5.0Hz,4H),3.08(m,1H),2.63(t,J=5.0Hz,4H),2.41(s,3H),2.07(m,1H),1.12(d,J=6.7Hz,3H).HRMS m / z(ESI)calculated for C 27 H 28 Cl2N8O[M+H] + 551.1841,found551.1846.CY72-S; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.76(s,1H),7.49(d,J=8.5Hz,2H),7.45–7.4 0(m,2H),7.29(d,J=8.1Hz,1H),6.96(d,J=9.0Hz,2H),4.43–4.36(m,1H),3.51(ddd,J=15 .5,4.3,2.2Hz,1H),3.44(t,J=11.8Hz,1H),3.23(t,J=5.0Hz,4H),3.08(dd,J=15.5,8.8H z,1H),2.61(t,J=5.1Hz,4H),2.37(s,3H),2.12–2.02(m,1H),1.08(d,J=6.7Hz,3H).HRMS m / z(ESI)calculated for C 27 H 28 Cl2N8O[M+H] + 551.1841, found 551.1846.

[0297] Example 73: Preparation of compound CY73

[0298]

[0299] The preparation method of compound CY73 is the same as that in Example 72, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 14%; 1H NMR(400MHz,Chloroform-d)δ8.89(s,1H),8.13(s,1H),7.45–7.35(m,4H),7.33–7.26(m,2H),4.45(ddd,J=12.7,4.7,2.3Hz,1H),3.56(s, HRMS m / z(ESI)calculated for C 28 H 29 Cl2N7O[M+H] + 550.1889, found 550.1889. CY73-R and CY73-S were obtained through chiral separation, CY73-R; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),8.12(s,1H),7.44–7.41(m,2H),7.40–7.34(m,2H),7.31–7.26(m,2H),4.45(m,1H) ,3.53(s,2H),3.50–3.39(m,2H),3.05(m,1H),2.45(s,3H),2.42(s,2H),2.09(m,1H),1.31(s,6H),1.09(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated for C 28 H 29 Cl2N7O[M+H] + 550.1889, found 550.1889.CY73-S; 1HNMR(400MHz,Chloroform-d)δ8.90(s,1H),8.14(s,1H),7.43(dd,J=8.2,2.4Hz,2H),7.38(d ,J=7.7Hz,2H),7.31(d,J=8.7Hz,1H),7.27(d,J=7.0Hz,1H),4.45(ddd,J=12.7,4.7,2.3Hz,1 H),3.56(s,2H),3.53–3.49(m,1H),3.46(dt,J=13.0,6.2Hz,1H),3.09(dd,J=15.5,9.1Hz,1H ),2.43(d,J=3.7Hz,5H),2.10(p,J=8.8,7.0Hz,1H),1.33(s,6H),1.11(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated for C 28 H 29 Cl2N7O[M+H] + 550.1889, found550.1889.

[0300] Example 74: Preparation of compound CY74

[0301]

[0302] The preparation method of compound CY74 is the same as that in Example 72, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 29%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),7.89(s,1H),7.45–7.38(m,3H),7.34(dd,J=8.2, 2.2Hz,1H),7.29(d,J=8.1Hz,1H),7.12(d,J=8.3Hz,1H),4.43(ddd,J=12.7,4.7,2.3Hz,1H), 3.61(s,2H),3.55–3.48(m,1H),3.45–3.38(m,1H),3.08(dd,J=15.5,9.1Hz,1H),2.93(t,J=6 .0Hz,2H),2.73(t,J=5.9Hz,2H),2.48(s,3H),2.13–2.03(m,1H),1.09(d,J=6.7Hz,3H).HRMS m / z(ESI)calculated for C 26 H 25 Cl2N7O[M+H] +522.1576, found 522.1578. CY74-R and CY74-S were obtained through chiral separation, CY74-R; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),7.91(s,1H),7.47–7.39(m,3H),7.35(dd,J=8.2, 2.2Hz,1H),7.31(d,J=8.1Hz,1H),7.14(d,J=8.3Hz,1H),4.45(ddd,J=12.7,4.7,2.3Hz,1H), 3.63(s,2H),3.55–3.49(m,1H),3.47–3.39(m,1H),3.09(dd,J=15.5,9.1Hz,1H),2.95(t,J=6 .0Hz,2H),2.75(t,J=5.9Hz,2H),2.49(s,3H),2.14–2.05(m,1H),1.10(d,J=6.7Hz,3H).HRMS m / z(ESI)calculated for C 26 H 25 Cl2N7O[M+H] + 522.1576, found 522.1578.CY74-S; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.86(s,1H),7.42(dq,J=8.5,1.5Hz,2H),7.39–7. 32(m,2H),7.29(d,J=8.1Hz,1H),7.11(d,J=8.3Hz,1H),4.43(ddd,J=12.7,4.7,2.3Hz,1H),3. 61(s,2H),3.55–3.48(m,1H),3.41(d,J=10.2Hz,1H),3.07(dd,J=15.5,9.1Hz,1H),2.92(t,J= 6.0Hz,2H),2.73(t,J=5.9Hz,2H),2.48(s,3H),2.13–2.03(m,1H),1.09(d,J=6.7Hz,3H).HRMS m / z(ESI)calculatedfor C 26 H 25 Cl2N7O[M+H] + 522.1576, found 522.1578.

[0303] Example 75: Preparation of compound CY75

[0304]

[0305] The preparation method of compound CY75 is the same as in Example 4, except that o-chlorophenyl isocyanate is replaced with 2,6-difluorophenyl isocyanate; white solid, yield 38%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.90(s,1H),7.48(d,J=8.6Hz,2H),7.36(tt,J=8.5,6.1Hz,1H),7.05–6.93(m,4H),4. 10(t,J=6.0Hz,2H),3.49(t,J=5.6Hz,2H),3.25(t,J=5.0Hz,4H),2.66(t,J=4.9Hz,4H),2.41(s,3H),1.97(p,J=5.8Hz,2H).HRMS m / z(ESI)calculated for C 26 H 26 F2N8O[M+H] + 505.2276, found505.2277.

[0306] Example 76: Preparation of compound CY76

[0307]

[0308] The preparation method of compound CY76 is the same as that of Example 75, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 18%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.51(s,1H),7.37(tt,J=8.5,6.1Hz,2H),7.28(s,1H),7.12(d,J=8.2Hz,1H),7.06–6.99(m,2H),4.11 HRMS m / z(ESI)calculated for C 25 H 23 F2N7O[M+H] + 476.2010, found 476.2010.

[0309] Example 77: Preparation of compound CY77

[0310]

[0311] The preparation method of compound CY77 is the same as that in Example 75, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; grayish-white solid, yield 14%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.74(s,1H),7.44(dd,J=8.5,2.3Hz,1H),7.40–7.30(m,2H),7.27(d,J=2.7Hz,1H),7. 05–6.98(m,2H),4.13(t,J=6.1Hz,2H),3.61(s,2H),3.50(t,J=5.6Hz,2H),2.48(s,5H),1.99(p,J=5.8Hz,2H),1.34(s,6H).HRMS m / z(ESI)calculated forC 27 H 27 F2N7O[M+H] + 504.2323, found 504.2320.

[0312] Example 78: Preparation of compound CY78

[0313]

[0314] The preparation method of compound CY78 is the same as in Example 76, except that 3-amino-1-propanol is replaced with 4-amino-1-butanol; grayish-white solid, yield 20%; 1 H NMR(400MHz,Chloroform-d)δ9.06(d,J=1.5Hz,1H),7.70(s,1H),7.57–7.37(m,4H),7.29(dd,J=8.5,1.6Hz,1H),7.10(d,J=8.1Hz,1H),4.63(t,J=5.8 Hz,1H),3.64(s,2H),3.61–3.52(m,3H),2.91(t,J=6.0Hz,2H),2.72(t,J=5 .9Hz,2H),2.48(d,J=1.6Hz,3H),1.78(ddt,J=19.5,14.6,6.7Hz,4H).HRMS m / z(ESI)calculated for C 26 H 25 F2N7O[M+H] + 490.2167, found 490.2168.

[0315] Example 79: Preparation of compound CY79

[0316]

[0317] The preparation method of compound CY79 is the same as in Example 75, except that 3-amino-1-propanol is replaced with 2-aminocyclopentanol; white solid, yield 37%; 1 H NMR(400MHz,Chloroform-d)δ8.77(s,1H),7.88(s,1H),7.59–7.48(m,2H),7.40(tt,J=8.5,6.1Hz ,1H),7.05(td,J=8.7,2.9Hz,2H),6.97–6.91(m,2H),4.90(t,J=7.2Hz,1H),4.63(td,J=7.4,1.8Hz ,1H),3.22(t,J=5.0Hz,4H),2.62(t,J=5.0Hz,4H),2.40–2.36(m,3H),2.32(dd,J=13.7,5.9Hz,1H ),1.90(s,2H),1.80(dt,J=12.4,6.0Hz,1H),1.75–1.68(m,1H),1.60(tt,J=12.1,6.0Hz,1H).HRMS m / z(ESI)calculatedfor C 28 H 28 F2N8O[M+H] + 531.2432, found 531.2432.

[0318] Example 80: Preparation of compound CY80

[0319]

[0320] The preparation method of compound CY80 is the same as that in Example 79, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 37%; 1H NMR(400MHz,Chloroform-d)δ8.79(s,1H),7.97(s,1H),7.46–7.35(m,3H),7 .29(d,J=8.5Hz,1H),7.08–7.02(m,2H),4.93(t,J=7.2Hz,1H),4.66(t,J=6.9 Hz,1H),3.55(s,2H),2.43(d,J=7.1Hz,5H),2.38(d,J=10.4Hz,1H),1.96–1. 88(m,2H),1.84–1.72(m,2H),1.60(dq,J=12.1,5.9Hz,1H),1.32(s,6H).HRMS m / z(ESI)calculated for C 29 H 29 F2N7O[M+H] + 530.2480, found 530.2476.

[0321] Example 81: Preparation of compound CY81

[0322]

[0323] The preparation method of compound CY81 is the same as in Example 75, except that 3-amino-1-propanol is replaced with 3-aminocyclobutanol; white solid, yield 26%; 1 H NMR(400MHz,Chloroform-d)δ8.88(s,1H),7.92(s,1H),7.45(d,J=8.7Hz,2H ),7.42–7.35(m,1H),7.05(t,J=8.2Hz,2H),6.97(d,J=8.6Hz,2H),5.78(d,J= 6.0Hz,1H),4.27(dd,J=7.1,3.6Hz,1H),3.26(t,J=5.0Hz,4H),2.66(t,J=4.9 Hz,4H),2.41(s,3H),2.36(h,J=3.7Hz,2H),1.49(dd,J=7.1,2.5Hz,2H).HRMS m / z(ESI)calculated for C 27 H 26 F2N8O[M+H] + 517.2276, found 517.2279.

[0324] Example 82: Preparation of compound CY82

[0325]

[0326] The preparation method of compound CY82 is the same as in Example 75, except that 3-amino-1-propanol is replaced with 3-aminocyclohexanol; white solid, yield 43%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),8.17(s,1H),7.46(d,J=8.6Hz,2H) ,7.36(td,J=8.5,4.3Hz,1H),7.01(td,J=8.7,3.9Hz,2H),6.94(d,J=8.9Hz,2 H),5.14(s,1H),3.82(s,1H),3.22(t,J=5.0Hz,4H),2.61(t,J=4.9Hz,4H),2. 37(s,3H),2.18(d,J=13.4Hz,1H),1.92–1.80(m,3H),1.67–1.53(m,4H).HRMS m / z(ESI)calculated for C 29 H 30 F2N8O[M+H] + 545.2589, found 545.2585.

[0327] Example 83: Preparation of compound CY83

[0328]

[0329] The preparation method of compound CY83 is the same as that in Example 82, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 32%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),8.05(s,1H),7.39–7.32(m,3H),7.29(d,J=8.9Hz,1H),7.01(td,J=8.6,3.9Hz,2H),5.17(s HRMS m / z(ESI)calculatedfor C 30 H 31 F2N7O[M+H] + 544.2636, found 544.2631.

[0330] Example 84: Preparation of compound CY84

[0331]

[0332] The preparation method of compound CY84 is the same as in Example 72, except that 3-amino-1-propanol is replaced with 3-amino-2,2-dimethyl-1-propanol; white solid, yield 30%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.92(s,1H),7.48(d,J=8.5Hz,2H),7.36(tt,J=8.6,6.1Hz,1H),7.01(t,J=8 .2Hz,2H),6.95(d,J=8.7Hz,2H),3.76(s,2H),3.25–3.16(m,6H),2.61(t,J=4.9Hz,4H),2.37(s,3H),1.05(s,6H).HRMS m / z(ESI)calculated for C 28 H 30 F2N8O[M+H] + 533.2589, found 533.2584.

[0333] Example 85: Preparation of compound CY85

[0334]

[0335] The preparation method of compound CY85 is the same as that in Example 84, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 27%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.93(s,1H),7.40(d,J=2.9Hz,1H),7.33(ddd,J=15.4,7.3,2.3Hz,2H),7.11(d,J=8.3Hz,1H), HRMS m / z(ESI)calculatedfor C 27 H 27 F2N7O[M+H] + 504.2323, found 504.2327.

[0336] Example 86: Preparation of compound CY86

[0337]

[0338] The preparation method of compound CY86 is the same as that in Example 84, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 32%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),8.04(s,1H),7.42–7.35(m,2H),7.32(t,J=8.0Hz,2H),7.01(t, J=8.1Hz,2H),3.80(s,2H),3.56(s,2H),3.20(s,2H),2.43(d,J=3.9Hz,5H),1.33(s,6H),1.08(s,6H).HRMS m / z(ESI)calculated for C 29 H 31 F2N7O[M+H] + 532.2636, found 532.2634.

[0339] Example 87: Preparation of compound CY87

[0340]

[0341] The preparation method of compound CY87 is the same as in Example 75, except that 3-amino-1-propanol is replaced with (2S)-3-amino-2-fluoropropane-1-ol; white solid, yield 16%; 1 H NMR(400MHz,Chloroform-d)δ8.87(s,1H),8.02(s,1H),7.44(d,J=8.8Hz,2H),7.3 7(tt,J=8.5,6.1Hz,1H),7.02(t,J=8.6Hz,2H),6.96(d,J=8.7Hz,2H),5.18(t,J=2 .8Hz,0.5H),5.06(t,J=2.7Hz,0.5H),4.87(t,J=13.5Hz,1H),3.90–3.51(m,3H),3 .23(t,J=5.0Hz,4H),2.61(t,J=5.0Hz,4H),2.37(s,3H).HRMSm / z(ESI)calculated for C 26 H 25 F3N8O[M+H] + 523.2182, found 523.2181.

[0342] Example 88: Preparation of compound CY88

[0343]

[0344] The preparation method of compound CY88 is the same as that of Example 87, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 27%; 1 H NMR(400MHz,Chloroform-d)δ8.89(s,1H),8.15(s,1H),7.41–7.34(m,2H),7.24(s,1H),7.12(d,J=8.2Hz,1H),7.02(t,J=8.4Hz,2H),5.22–5.1 9(m,0H),5.12–5.05(m,1H),4.93–4.82(m,1H),3.90–3.64(m,3H),3.61 (s,2H),2.93(t,J=6.0Hz,2H),2.74(t,J=5.9Hz,2H),2.49(s,3H).HRMS m / z(ESI)calculated for C 25 H 22 F3N7O[M+H] + 494.1916, found 494.1912.

[0345] Example 89: Preparation of compound CY89

[0346]

[0347] The preparation method of compound CY89 is the same as that of Example 87, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; pale yellow solid, yield 38%; 1 H NMR(400MHz,Chloroform-d)δ8.90(s,1H),7.80(s,1H),7.44–7.35(m,2H),7.32(d,J=8.6Hz,1H),7.22(d,J=2.2Hz,1H),7.06–7.00(m,2H) HRMS m / z(ESI)calculated for C 27 H26 F3N7O[M+H] + 522.2229, found 522.2228.

[0348] Example 90: Preparation of compound CY90

[0349]

[0350] The preparation method of compound CY90 is the same as in Example 75, except that 3-amino-1-propanol is replaced with (S)-3-aminobutanol; white solid, yield 37%; 1 H NMR(400MHz,Chloroform-d)δ8.85(s,1H),7.69(s,1H),7.48(d,J=8.5Hz,2H),7.40–7 .31(m,1H),7.05–6.93(m,4H),4.31(dt,J=13.1,5.3Hz,1H),3.88(d,J=10.7Hz,1H),3 .58(td,J=7.2,4.5Hz,1H),3.24(t,J=4.9Hz,4H),2.64(t,J=4.9Hz,4H),2.40(s,3H), 2.06(dt,J=13.8,5.0Hz,1H),1.63(dp,J=13.2,4.3Hz,1H),1.11(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated for C 27 H 28 F2N8O[M+H] + 519.2432, found 519.2433.

[0351] Example 91: Preparation of compound CY91

[0352]

[0353] The preparation method of compound CY91 is the same as that of Example 90, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 39%; 1H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.72(s,1H),7.35(td,J=8.6,4.4Hz,2H),7.28(d, J=2.2Hz,1H),7.12(d,J=8.2Hz,1H),7.00(q,J=7.9Hz,2H),4.33(dt,J=12.7,5.2Hz,1H),3.8 6(ddd,J=13.4,9.7,4.2Hz,1H),3.60(s,3H),2.92(t,J=5.9Hz,2H),2.72(t,J=6.0Hz,2H),2. 48(s,3H),2.09–2.06(m,1H),1.64(ddd,J=13.3,8.5,4.6Hz,1H),1.11(d,J=6.7Hz,3H).HRMS m / z(ESI)calculated for C 26 H 25 F2N7O[M+H] + 490.2167, found 490.2169.

[0354] Example 92: Preparation of compound CY92

[0355]

[0356] The preparation method of compound CY92 is the same as that of Example 90, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 20%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.58–7.41(m,2H),7.39–7.26(m,3H),7.00(q,J=8.9Hz,2H),4.36(dd,J=13.0,5.4Hz,1H),3.94–3.83(m ,1H),3.58(d,J=15.5Hz,3H),2.43(d,J=7.1Hz,5H),2.08(ddt,J=12.1,6. 8,3.7Hz,1H),1.68–1.61(m,1H),1.32(s,6H),1.12(d,J=7.0Hz,3H).HRMS m / z(ESI)calculated for C 28 H 29 F2N7O[M+H] + 518.2480, found 518.2479.

[0357] Example 93: Preparation of compound CY93

[0358]

[0359] The preparation method of compound CY93 is the same as in Example 75, except that 3-amino-1-propanol is replaced with (R)-3-aminobutanol; white solid, yield 26%; 1 H NMR(400MHz,Chloroform-d)δ8.84(s,1H),7.79(s,1H),7.48(d,J=8.6Hz,2H),7.34(tt,J =8.4,6.0Hz,1H),7.04–6.93(m,4H),4.31(dt,J=13.0,5.3Hz,1H),3.86(t,J=9.7Hz,1H), 3.58(td,J=7.3,4.3Hz,1H),3.25(t,J=4.9Hz,4H),2.66(t,J=4.9Hz,4H),2.41(s,3H),2. 06(dt,J=13.8,5.1Hz,1H),1.62(dtd,J=13.3,8.4,4.6Hz,1H),1.11(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated for C 27 H 28 F2N8O[M+H] + 519.2432, found 519.2436.

[0360] Example 94: Preparation of compound CY94

[0361]

[0362] The preparation method of compound CY94 is the same as that of Example 93, except that 4-(4-methylpiperazine)aniline is replaced with 2-methyl-1,2,3,4-tetrahydroisoquinoline-7-amine; white solid, yield 43%; 1H NMR(400MHz,Chloroform-d)δ8.86(d,J=1.2Hz,1H),7.58(s,1H),7.41–7.33(m,2H),7.3 0(s,1H),7.12(d,J=8.2Hz,1H),7.00(q,J=7.9Hz,2H),4.33(dt,J=13.2,5.2Hz,1H),3.91 –3.83(m,1H),3.61(d,J=14.3Hz,3H),2.93(t,J=6.0Hz,2H),2.74(t,J=6.0Hz,2H),2.50 (d,J=1.2Hz,3H),2.10–2.06(m,1H),1.66–1.57(m,1H),1.11(dd,J=6.6,1.2Hz,3H).HRMS m / z(ESI)calculated forC 26 H 25 F2N7O[M+H] + 490.2167, found 490.2168.

[0363] Example 95: Preparation of compound CY95

[0364]

[0365] The preparation method of compound CY95 is the same as that of Example 93, except that 4-(4-methylpiperazine)aniline is replaced with 2,4,4-trimethyl-1,2,3,4-tetrahydroisoquinoline-7-amine; grayish-white solid, yield 45%; 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.80(s,1H),7.43(dd,J=8.5,2.3Hz,1H) ,7.38–7.26(m,3H),7.03–6.96(m,2H),4.35(dt,J=13.1,5.2Hz,1H),3.88(ddd,J=13 .6,9.8,4.3Hz,1H),3.63–3.53(m,3H),2.42(d,J=7.4Hz,5H),2.07(ddd,J=15.6,7. 9,3.0Hz,1H),1.64(dp,J=13.4,4.5Hz,1H),1.32(s,6H),1.12(d,J=6.6Hz,3H).HRMS m / z(ESI)calculated for C 28 H 29 F2N7O[M+H] + 518.2480, found 518.2479.

[0366] The bioactivity of the derivatives prepared in Examples 1-95 was determined, and the specific experiments are as follows:

[0367] Inhibitory activity of compound 1 against Wee1

[0368] The enzyme potency assay of the compounds was provided by Eurofins, France. In short, the assay system containing Wee1(h) included 20 mM Tris-HCl, pH 8.5, 0.2 mM EDTA, 500 μM peptide substrate, 10 mM magnesium acetate, and 10 μM [γ-]... 33 [P]-ATP (intensity approximately 500 cpm / pmol), and the compound of the examples at different concentrations (1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 nM). Mg was added. 2+ After mixing with ATP, incubate at room temperature for 40 minutes. Add 3% phosphate buffer to terminate the reaction. Filter 10 μL of the reaction solution through a continuous filter P30, wash four times with 0.425% phosphate buffer for 4 minutes each, and wash once with methanol for 1 minute each. After drying, read the count using time-resolved fluorescence analysis. Determine the homogeneous time-resolved fluorescence (HTRF) signal according to the formula HTRF = 10000 × (Em665nm / Em620nm). Fit the IC50 based on the inhibition rates of nine concentrations. 50 Values ​​were measured using replicate wells. Each group was repeated three times, and the average value was taken. The results are shown in Tables 1 and 2, where AZD1775 is a known highly active Wee1 inhibitor.

[0369] Table 1. Inhibitory activity of the compounds in the examples against Wee1 at different concentrations.

[0370]

[0371]

[0372] Table 2 shows the IC50 inhibition of Wee1 by the compounds in the examples. 50 value

[0373] cmpd <![CDATA[Wee1 IC 50 (nM)]]> cmpd <![CDATA[Wee1 IC 50 (nM)]]> AZD1775 47 CY47 83 CY2 268 CY48 51 CY3 202 CY50 151 CY4 449 CY53 275 CY5 248 CY57 589 CY6 364 CY60 143 CY12 379 CY62 545 CY17 110 CY64 83 CY18 99 CY66 64 CY19 380 CY67 51 CY20 396 CY68 35 CY22 228 CY69 314 CY24 1042 CY71 166 CY26 352 CY72 435 CY31 97 CY73 11 CY32 12 CY74 52 CY33 53 CY85 49 CY34 121 CY86 43 CY36 919 CY87 689 CY43 112 CY90 1354 CY44 164 CY93 10 CY45 44 CY94 75 CY46 58 CY95 60

[0374] As can be seen from the conclusions in Tables 1 and 2, some of the compounds in this application have a good inhibitory effect on Wee1 kinase, which is superior to the positive molecule AZD1775.

[0375] Inhibitory activity of compound in Experiment Example 2 against H1299 and H1975 cells

[0376] The inhibitory effect of the compounds in the examples on the growth of H1299 and H1975 cells was detected using the CCK-8 assay. Newly revived H1299 and H1975 cells were passaged to the third generation, and those with good growth and approximately 90% confluence were used for the experiment. H1299 and H1975 cells were digested with trypsin, centrifuged at 1000 pm for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh medium and counted. Cells were seeded at a density of 2000 cells per well in 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator. Stock solutions of the test substances (including the test compounds and AZD1775) were diluted with DMSO at ratios of 1:3 and 1:10 to eight concentrations (the last concentration being a DMSO negative control): 10 μM, 3.3 μM, 1 μM, 0.33 μM, 0.1 μM, 0.033 μM, 0.01 μM, and 0 μM (final DMSO concentration less than 1‰). Add 5 μL of each concentration to 120 μL of culture medium (25-fold dilution) and vortex to mix. Collect cells cultured overnight, remove the culture medium, add 195 μM fresh culture medium to each well, and then add 5 μL of the diluted culture medium containing the corresponding concentration of the test substance. Incubate the plate at 37°C in a 5% CO2 incubator for 3 days. Remove the stock solution and add 20 μL of CCK-8 to each well for continued incubation. After 2-4 hours, shake in the dark for 10 min and read the absorbance (OD) at 450 nm using a multi-function reader. Analyze the data using Graph Pad Prism 5.0 software; plot the inhibitory activity of the compound on cell proliferation against cell viability and compound concentration. IC50 50 The value was fitted with an S-type dose-response curve equation, which is: Y = 100 / (1 + 10^(LogC - LogIC)) 50 )), where C is the concentration of the compound.

[0377] The experimental results are shown in Tables 3 and 4 below.

[0378] Table 3. Inhibitory effect of the compounds in the examples on the growth of H1299 cells.

[0379]

[0380]

[0381] Table 4. Inhibitory effect of the compounds in the examples on the growth of H1975 cells.

[0382]

[0383]

[0384] Some compounds showed good inhibitory effects on the growth of both H1299 and H1975 cells, superior to the positive control molecule AZD1775. Among them, compounds numbered 23, 33, 34, 45, 46, 47, 48, 61, 66, 67, 68, 84, 93, 94, and 95 showed relatively good activity in H1299 cells, comparable to or slightly weaker than the positive control. Due to varying sensitivity in different cell types, some compounds performed better than the positive control in H1975 cells. Compounds superior to the positive control molecule were numbered 19, 30, 31, 32, 33, 34, 35, 43, 45, 48, 60, 61, 64, 72, 84, 93, and 94.

[0385] As can be seen from the above experimental results, this invention provides a class of 2,3-dihydropyrimidine[4,5-d]pyrimidine-4(1H)-one derivatives with good Weel inhibitory activity, which can be used as Weel inhibitors for the prevention or treatment of tumor-related diseases.

Claims

1,2,3-Dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivatives, characterized in that: The specific structural formula is any one of the following: 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

2. A pharmaceutical composition comprising the 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative of claim 1, and / or a pharmaceutically acceptable salt of said derivative.

3. The pharmaceutical composition according to claim 2, characterized in that: It also includes pharmaceutically acceptable excipients.

4. The use of the 2,3-dihydropyrimidino[4,5-d]pyrimidin-4(1H)-one derivative of claim 1, and / or a pharmaceutically acceptable salt thereof, in the preparation of a medicament.

5. The application according to claim 4, characterized in that: The aforementioned drug is a drug for the prevention or treatment of tumor diseases.

6. The application according to claim 4, characterized in that: The drug in question is a Weel inhibitor.

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