PKMYT1 inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a PKMYT1 inhibitor and a preparation method and application thereof.The structural general formula of the compound is shown in the formula (I), the compound can effectively inhibit the activity of PKMYT1, it is indicated that the compound has the value of being further developed for medical application, and a new choice is provided for clinical development and preparation of drugs for treating PKMYT1 protein related diseases. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a PKMYT1 inhibitor and a preparation method and application thereof, belonging to the technical field of chemical synthesis. Background Art

[0002] PKMYT1 is a member of the WEE family and is mainly located in the endoplasmic reticulum and Golgi complex. The WEE protein kinase family includes: PKMYT1, WEE1 G2 checkpoint kinase (WEE1) and WEE2 oocyte meiosis inhibitory kinase (WEE1B). Functionally, WEE1 and PKMYT1 phosphorylate cyclin-dependent kinase 1 (CDK1) to inhibit its activity and prevent the formation of a complex with cyclin B1, thereby preventing the cell from undergoing mitosis to repair DNA damage. Mechanistically, WEE1 inhibits the binding of CDK to cyclin B1 by phosphorylating Tyr15 of CDK1, thereby inhibiting cell mitosis; while PKMYT1 has dual phosphorylation activity of Tyr15 and Thr14 on CDK1, which also achieves the effect of inhibiting cell mitosis. Premature entry into mitosis leads to increased unrepaired DNA damage and accumulation of genetic damage, ultimately leading to cell apoptosis or mitotic catastrophe. Although both WEE1 and PKMYT1 can phosphorylate CDK1, compared with WEE1, the function of PKMYT1 is more important for the normal mitosis of cancer cells than that of normal cells. Therefore, PKMYT1 is a promising target for cancer treatment.

[0003] PKMYT1 inhibitors inhibit the activation of CDK1, thereby releasing the regulatory process of the G2 / M phase of the cell cycle, causing cells to enter mitosis prematurely, and increasing the accumulation of unrepaired DNA damage, thereby inhibiting the division and growth of tumor cells. The CCNE1 gene is located on chromosome 19q12 and encodes G1-S phase-specific cyclin E1. During mitosis, cyclin E1 forms a complex with cyclin-dependent kinase 2 (CDK2) and drives cells from G1 phase to S phase. CCNE1 amplification forces cancer cells to enter mitosis prematurely, making CCNE1-amplified tumors more vulnerable. Based on the unique pharmacological effects of PKMYT1 in the cell cycle, the development of PKMYT1 inhibitors has a good inhibitory effect on CCNE1-amplified tumors. In summary, it is of great significance to continue to develop PKMYT1 inhibitors, enrich the types of compounds, and meet clinical needs.

[0004] Currently, the only PKMYT1 inhibitor, RP-6306, has entered Repare Therapeutics' Phase II clinical study, and is used in combination with gemcitabine, FOLFIRI or trastuzumab to treat patients with advanced, metastatic, recurrent or unresectable solid tumors. In addition, Matthias Schmidt's team screened out 10 PKMYT1 inhibitors containing 3 new skeletons in 2018. In 2019, the team screened out PKMYT1 inhibitors with a 2-aminopyrimidine skeleton through computational methods. PKMYT1 inhibitors also include research on other compounds with weaker activity and low selectivity. Therefore, PKMYT1 inhibitors are an unmet clinical need. Summary of the invention

[0005] Objectives of the invention: The first objective of the present invention is to provide a compound having the general formula (I) or a pharmaceutically acceptable salt thereof. The second objective of the present invention is to provide a method for preparing the compound having the general formula (I) or a pharmaceutically acceptable salt thereof. The third objective of the present invention is to provide the use of the compound having the general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug or inhibitor for treating, regulating and / or preventing tumor diseases mediated by PKMYT1.

[0006] Technical solution: The present invention provides a compound having a general formula (I) or a pharmaceutically acceptable salt thereof, wherein the general structural formula of the compound is as shown in formula (I):

[0007]

[0008] Wherein, R1 is -NH2, -OCH3, -OCH2CH3 or -NHCH3; R2 is selected from -Cl or one of the following structures:

[0009]

[0010] Furthermore, the compound is selected from any of the following structures:

[0011]

[0012] The preparation method of the compound of the general formula (I) or a pharmaceutically acceptable salt thereof of the present invention is synthesized according to the following route:

[0013] Route 1:

[0014]

[0015] Wherein, R3 is -OCH3, -OCH2CH3 or -NHCH3;

[0016] Route 2:

[0017]

[0018] Wherein, R4 is selected from any of the following structures:

[0019]

[0020] or route 3:

[0021] Wherein, R5 is selected from any of the following structures:

[0022]

[0023] R6 is selected from any of the following structures:

[0024]

[0025] Furthermore, the reaction conditions of step a in route 1 are: (a) 3-methoxy-2,6-dimethylaniline, Li HMDS, THF, 0-25°C.

[0026] Furthermore, the reaction conditions of step b in route 1 are: (b) PdCl2(dppf), NaH, DME, 0-90°C; or PdCl2(dppf), sodium tert-butoxide, DME, 0-90°C.

[0027] Furthermore, the reaction conditions of step c in route 1 are: (c) BBr3, CH2Cl2, -78-25°C, 4h.

[0028] Furthermore, the reaction conditions of step a in route 2 are: (a) 3-methoxy-2,6-dimethylaniline, Li HMDS, THF, 0°C-25°C.

[0029] Furthermore, the reaction conditions of step b in route 2 are: (b) PdCl2(dppf), NaH, DME, 0°C-90°C.

[0030] Furthermore, the reaction conditions of step c in route 2 are: (c) PdCl2(dppf), CS2CO3, 1,4-dioxane, H2O, 90°C, overnight or DIPEA, n-butanol, 130°C, 5h.

[0031] Further, the reaction conditions of step d in route 2 are: (d) H2SO4, 25°C; or H2O2, DMSO, 0°C-25°C;

[0032] Furthermore, the reaction conditions of step e in route 2 are: (e) BBr3, CH2Cl2, -78-25°C, 4h.

[0033] Furthermore, the reaction conditions of step a in route 3 are: (a) 2,4-dichloro-5-bromopyrimidine, LiHMDS, THF, 0-25°C.

[0034] Furthermore, the reaction conditions of step b in route 3 are: (b) malononitrile, PdCl2(dppf), NaH, DME, 0°C-90°C.

[0035] Furthermore, the reaction conditions of step c in route 3 are: (c) piperazine, DIPEA, n-butanol, 130° C., 5 h.

[0036] Furthermore, the reaction conditions of step d in route 3 are: (d) HATU, DIPEA, DMF, 60° C., 12 h.

[0037] Furthermore, the reaction conditions of step e in route 3 are: (e) H2O2, DMSO, 0-25°C.

[0038] Furthermore, the reaction conditions of step f in route 3 are: (f) BBr3, CH2Cl2, -78-25°C, 4h.

[0039] The invention relates to the use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug or inhibitor for treating, regulating and / or preventing tumor diseases mediated by PKMYT1.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0041] The present invention modifies the structure of the lead compound RP-6306, replaces pyridine with pyrimidine through the principle of skeleton migration, and introduces groups on the pyrimidine ring to increase the interaction site with the target, thereby obtaining a series of new compounds. The new compounds have strong inhibitory activity and are expected to become candidate compounds for more in-depth mechanism exploration and in vivo experiments, providing a basis for subsequent research on PKMYT1 inhibitors.

[0042] The compound of the present invention can effectively inhibit the activity of PKMYT1, has the value of further development for medical use, and provides a new option for clinical development and preparation of drugs for PKMYT1 protein-related diseases. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below.

[0044] Example 1: Synthesis of 6-amino-2-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (A1)

[0045] Synthesized according to the following route:

[0046]

[0047] Step 1: 2,4-Dichloro-5-bromopyrimidine (9 g, 39.5 mmol) and 3-methoxy-2,6-dimethylaniline (5 g, 33.1 mmol) were placed in a two-necked bottle, 10 mL of anhydrous tetrahydrofuran (THF) was added, and N2 was replaced. 1 M LiHMDS / THF solution (50 mL, 50 mmol) was slowly added dropwise under an ice bath. The reaction was allowed to proceed for 3 h. The disappearance of the starting material was monitored by TLC. N2 was removed, and 50 mL of NH4Cl aqueous solution was added to quench the reaction. THF was removed by distillation under reduced pressure, and then extracted with ethyl acetate (50 mL) 3 times. The organic phases were combined, washed with saturated NaCl aqueous solution, and finally dried over anhydrous Na2SO4, distilled under reduced pressure, and purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 10:1) to obtain white solid compound 2 (8 g).

[0048] Compound 2 was analyzed by hydrogen nuclear magnetic resonance spectrum, and the results were as follows:

[0049] 1 H NMR (300MHz, Chloroform-d) δ8.25 (s, 1H), 7.07 (d, J = 8.4Hz, 1H), 6.84–6.75 (m, 2H), 3.83 (s, 3H), 2.14 (s, 3H), 2.07 (s, 3H).

[0050] From the above results, it can be seen that the structure of compound 2 is as follows:

[0051]

[0052] Step 2: 2-Cyano-N-methylacetamide (431.6 mg, 4.40 mmol) was dissolved in ethylene glycol dimethyl ether (DME), and sodium tert-butoxide (422.7 g, 4.40 mmol) was slowly added under ice bath, replaced by N2, and stirred at room temperature for 30 min. Compound 2 (500 mg, 1.47 mmol) and PdCl2(dppf) (107.3 mg, 0.147 mmol) were added, and the reaction was carried out at 90°C overnight. The disappearance of the starting material was monitored by TLC, and the reaction was quenched by adding saturated NH4Cl aqueous solution. The mixture was extracted with ethyl acetate (50 mL) 3 times, and the organic phases were combined. The organic phases were washed 3 times with water (50 mL), and then washed 3 times with NaCl (50 mL), and finally dried over anhydrous Na2SO4. After concentration under reduced pressure, column chromatography was performed to obtain compound 3a (158.4 mg) as a white solid.

[0053] Step 3: Compound 3a (100 mg, 0.28 mmol) was dissolved in 5 mL of anhydrous dichloromethane (DCM), replaced with N2, stirred at -78°C for 10 min, and boron tribromide (1.4 mL, 1.4 mmol, 1.0 mol / L in DCM) was slowly added dropwise. After stirring at -78°C for 10 min, the temperature was raised to room temperature. The disappearance of the starting material was monitored by TLC. The reaction was stirred at -78°C, and 10 mL of methanol was slowly added dropwise to quench the reaction. The reaction solution was concentrated under reduced pressure and purified by column chromatography (SiO2, dichloromethane: methanol = 20: 1) to give compound A1 (48 mg).

[0054] Compound A1 was analyzed by H NMR and mass spectrometry, and the results were as follows:

[0055] 1 H NMR(300MHz,DMSO-d6)δ9.69(s,1H),8.91(s,1H),7.27(s,2H),7.19–7.07( m, 2H), 6.97 (d, J = 8.3Hz, 1H), 2.82 (d, J = 4.4Hz, 3H), 1.72 (d, J = 25.1Hz, 6H).

[0056] HRMS(ESI)m / z calcd for C 16 H 16 ClN5O2[M+H] + 346.10653, found 346.10562.

[0057] From the above results, it can be seen that the structure of compound A1 is as follows:

[0058]

[0059] Example 2: Synthesis of 6-amino-2-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylate (A2)

[0060] Follow these routes:

[0061]

[0062] Step 1: Same as step 1 in Example 1.

[0063] Step 2: Methyl cyanoacetate (436 mg, 4.40 mmol) was dissolved in ethylene glycol dimethyl ether (DME), and NaH (168.7 mg, 4.40 mmol, 60% dispersion in mineral oil) was slowly added under ice bath, replaced by N2, and stirred at room temperature for 30 min. Compound 2 (500 mg, 1.47 mmol) and PdCl2(dppf) (107.3 mg, 0.147 mmol) were added, and the reaction was allowed to proceed overnight at 90°C. The disappearance of the starting material was monitored by TLC, and the reaction was quenched by adding saturated NH4Cl aqueous solution. The mixture was extracted with ethyl acetate (50 mL) 3 times, and the organic phases were combined. The organic phases were washed 3 times with water (50 mL), and then washed 3 times with NaCl (50 mL), and finally dried over anhydrous Na2SO4, concentrated under reduced pressure, and then column chromatography was performed to obtain compound 3b (210 mg) as a white solid.

[0064] Step 3: Same as step 3 of Example 1 to obtain compound A2 (40 mg).

[0065] Compound A2 was analyzed by H NMR and mass spectrometry, and the results were as follows: 1 H NMR (300MHz, DMSO-d6) δ9.71(s,1H),8.62(s,1H),7.25(s,2H),7.11(d,J=8.3Hz,1H),6.98(d,J=8.3Hz,1H),3.83(s,3H),1.74(d,J=24.2Hz,6H).

[0066] HRMS(ESI)m / z calcd for C 16 H 15 ClN4O3[M+H] + 347.09054, found 347.09017.

[0067] From the above results, it can be seen that the structure of compound A2 is as follows:

[0068]

[0069] Example 3: Synthesis of 6-amino-2-chloro-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylate (A3)

[0070] Referring to the synthesis method of Example 2, the methyl cyanoacetate in step 2 was replaced by ethyl cyanoacetate, and other conditions remained unchanged to obtain compound A3 (45.2 mg).

[0071] Compound A2 was analyzed by H NMR and mass spectrometry, and the results were as follows: 1H NMR(300MHz,DMSO-d6)δ9.68(s,1H),8.61(s,1H),7.21(s,2H),7.14–6.91( m, 2H), 4.30 (q, J = 7.2Hz, 2H), 1.73 (d, J = 24.2Hz, 6H), 1.35 (t, J = 7.0Hz, 3H).

[0072] HRMS(ESI)m / z calcd for C 20 H 20 N6O3[M+H] + 361.10619, found 361.10503.

[0073] From the above results, it can be seen that the structure of compound A3 is as follows:

[0074]

[0075] Example 4: Synthesis of methyl 4-(6-amino-5-amino-carbamoyl-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)benzoate (B1)

[0076] Synthesized according to the following route:

[0077]

[0078] Step 1 is the same as step 1 of Example 1.

[0079] Step 2: Malononitrile (4.72 g, 71.46 mmol) was dissolved in DME, and NaH (2.86 g, 71.46 mmol, 60% dispersion in mineral oil) was slowly added under an ice bath, replaced by N2, and stirred at room temperature for 30 min. Compound 2 (8 g, 23.82 mmol) and PdCl2 (dppf) (1.74 g, 2.38 mmol) were then added. The reaction was allowed to proceed overnight at 90°C. The disappearance of the starting material was monitored by TLC. A saturated aqueous NH4Cl solution was added to quench the reaction. The mixture was extracted with ethyl acetate (100 mL) three times, and the organic phases were combined. The organic phases were washed three times with water and then with NaCl (100 mL) three times. Finally, the mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and then column chromatography (petroleum ether: ethyl acetate = 5:1) was performed to obtain compound 4 (2.3 g) as a white solid.

[0080] Compound 4 was analyzed by hydrogen nuclear magnetic resonance spectrum, and the results were as follows:

[0081] 1H NMR (300MHz, DMSO-d6) δ8.53(s,1H),7.65(s,2H),7.36–7.08(m,2H),3.85(s,3H),1.76(d,J=26.1Hz,6H).

[0082] From the above results, it can be seen that the structure of compound 4 is as follows:

[0083]

[0084] Step 3: Add p-methoxycarbonylphenylboronic acid (132 mg, 0.73 mmol), cesium carbonate (398.4 mg, 1.22 mmol) and PdCl2(dppf) (22.4 mg, 0.03 mmol) to a solution of compound 4 (200 mg, 0.61 mmol) in 1,4-dioxane (5 mL) and water (0.5 mL). Heat to 90°C under nitrogen protection and react overnight. After the reaction is completed, water and ethyl acetate are added for extraction, the organic layer is concentrated, and purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 2:1) to obtain compound 5a (195.9 mg).

[0085] Step 4: To compound 5a (150 mg, 0.35 mmol) were added 15 mL of dimethyl sulfoxide (DMSO), 0.4 mL of 1 mol / mL aqueous sodium hydroxide solution, and 4 mL of 30% H2O2 under ice bath. After the reaction was completed as monitored by TLC, 50 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL) three times. The organic layer was washed with water (50 mL) three times, washed with saturated aqueous sodium chloride solution (50 mL) five times, dried over anhydrous sodium sulfate, and concentrated to obtain 6a (123.9 mg).

[0086] Step 5: Same as step 3 of Example 1, except that 3a was replaced by 6a to obtain compound B1 (19.8 mg)

[0087] Compound B1 was analyzed by H NMR and mass spectrometry, and the results were as follows:

[0088] 1 H NMR (300MHz, DMSO-d6) δ9.65(s,1H),9.13(s,1H),8.25(d,J=8.3Hz,2H),7.99(d,J=8.3Hz,2H ),7.30(s,2H),7.14(d,J=8.3Hz,1H),7.07–6.92(m,3H),3.85(s,3H),1.77(d,J=25.0Hz,6H).

[0089] HRMS(ESI)m / z calcd for C 23 H 21N5O4[M+H] + 432.16663, found 432.16491.

[0090] From the above results, it can be seen that the structure of compound B1 is as follows:

[0091]

[0092] Example 5: Synthesis of 6-amino-2-(4-carbamoylphenyl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B2)

[0093] Referring to the synthesis method of Example 4, the p-methoxycarbonylphenylboronic acid in step 3 of Example 4 was replaced by 4-carbamoylphenylboronic acid, and other conditions remained unchanged to obtain compound B2 (21 mg).

[0094] Compound B2 was analyzed by H NMR and mass spectrometry, and the results were as follows:

[0095] 1 H NMR (300MHz, DMSO-d6) δ9.85(s,1H),9.12(s,1H),8.16(d,J=8.3Hz,2H),8.04(s,1H),7.90(d,J= 8.3Hz,2H),7.39(s,1H),7.28(s,2H),7.17–7.03(m,3H),7.01(s,2H),1.81(s,3H),1.72(s,3H).

[0096] HRMS(ESI)m / z calcd for C 22 H 20 N6O3[M+Na] + 439.14891, found 439.14999.

[0097] From the above results, it can be seen that the structure of compound B2 is as follows:

[0098]

[0099] Example 6: Synthesis of 6-amino-2-(azepine-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B3)

[0100] Synthesized according to the following route:

[0101]

[0102] The synthesis method of step 1 and step 2 is the same as step 1 and step 2 in embodiment 4.

[0103] Step 3: Add 17.9 mL of n-butanol, cyclohexylimide (2.73 g, 27.51 mmol), and N,N-diisopropylethylamine (DIPEA, 1.78 g, 13.76 mmol) to compound 5 (900 mg, 2.75 mmol), heat to 145°C for 3 hours, and concentrate to dryness after the reaction. Add 100 mL of ethyl acetate and 100 mL of water, wash the organic layer salt solution with water, dry over anhydrous sodium sulfate, and concentrate to obtain compound 5c (795 mg).

[0104] Step 4: Same as step 4 in Example 4, except that compound 5a was replaced by compound 5c, and the rest was the same, to obtain compound 6c (140.5 mg).

[0105] Step 5: Same as Step 5 in Example 4, except that compound 6a was replaced by compound 6c, and the rest was the same, to obtain compound B3 (45 mg).

[0106] Compound B3 was analyzed by H NMR and MS, the results are as follows:

[0107] 1 H NMR (300MHz, DMSO-d6) δ9.49 (s, 1H), 8.57 (s, 1H), 6.96 (dd, J=48.3, 8.2Hz, 2H), 6.64 (d, J= 15.9Hz,4H),3.52(t,J=5.5Hz,4H),1.75(d,J=25.0Hz,6H),1.58(s,4H),1.45–1.32(m,4H).

[0108] HRMS(ESI)m / z calcd for C 21 H 26 N6O2[M+H] + 395.219, found 395.22128.

[0109] From the above results, it can be seen that the structure of compound B3 is as follows:

[0110]

[0111] Example 7: Synthesis of 6-amino-2-(1,4-diazepine-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B4)

[0112] Referring to the synthesis method of Example 6, the cycloheximide in step 3 of Example 6 was replaced by homopiperazine, and other conditions remained unchanged to obtain compound B4 (18.9 mg).

[0113] Compound B4 was analyzed by H NMR and mass spectrometry, and the results were:

[0114] 1 H NMR(300MHz,DMSO-d6)δ9.68(s,1H),9.24(s,2H),8.65(s,1H),7.09–6.90(m,2H),6.71(d,J=15 .2Hz,4H),3.94–3.57(m,5H),3.19–2.97(m,5H),1.95(d,J=7.7Hz,2H),1.75(d,J=26.3Hz,6H).

[0115] HRMS(ESI)m / z calcd for C 20 H 25 N7O2[M+H] + 396.21425, found 396.21493.

[0116] From the above results, it can be seen that the structure of compound B4 is as follows:

[0117]

[0118] Example 8: Synthesis of 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-(piperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B5)

[0119] Referring to the synthesis method of Example 6, the cycloheximide in step 3 of Example 6 was replaced by piperazine, and other conditions remained unchanged to obtain compound B5 (23.5 mg).

[0120] Compound B5 was analyzed by H NMR and MS, the results are as follows:

[0121] 1 H NMR (300MHz, DMSO-d6) δ9.63(s,1H),8.89(s,1H),7.32(s,2H),7.10(d,J=8.3Hz,1H),6.96(d,J=8.9Hz,3 H), 6.73 (d, J = 26.1Hz, 1H), 3.22 (t, J = 5.0Hz, 5H), 2.61 (dd, J = 6.0, 3.9Hz, 4H), 1.76 (s, 3H), 1.68 (s, 3H).

[0122] HRMS(ESI)m / z calcd for C 19 H 23 N7O2[M+H] + 382.1986, found 382.19767.

[0123] From the above results, it can be seen that the structure of compound B5 is as follows:

[0124]

[0125] Example 9: Synthesis of 2-(4-(2-adamantan-1-yl)acetyl)piperazin-1-yl)-6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B6)

[0126] Synthesized according to the following route:

[0127]

[0128] Step 1 and step 2 are the same as step 1 and step 2 in Example 6.

[0129] Step 3: Referring to the synthesis method of Example 6, replace cycloheximide in Step 3 of Example 6 with piperazine and keep other conditions unchanged to obtain compound 7 (785 mg).

[0130] Compound 7 was analyzed by hydrogen nuclear magnetic resonance spectrum, and the results were as follows:

[0131] 1 H NMR (300MHz, DMSO-d6) δ8.23(s,1H),7.23(d,J=8.5Hz,1H),7.07(d,J=8.5Hz,1H),6.92 (s,2H),3.84(s,3H),3.45–3.39(m,4H),2.66(t,J=5.0Hz,4H),1.79(d,J=27.9Hz,6H).

[0132] From the above results, it can be seen that the structure of compound 7 is as follows:

[0133]

[0134] Step 4: Add HATU (362.9 mg, 0.954 mmol) and DIPEA (308.4 mg, 2.39 mmol) to adamantaneacetic acid (154.5 mg, 0.795 mmol) in DMF (5 mL), react at room temperature for 30 minutes under nitrogen protection, add intermediate 7 (300 mg, 0.795 mmol), and leave overnight. After the reaction, add water to quench the reaction, extract with ethyl acetate, wash the organic layer with water, dry over anhydrous sodium sulfate, and concentrate to obtain compound 8a (310 mg).

[0135] Step 5: Same as step 4 of Example 6, except that compound 5c in step 4 of Example 6 was replaced by compound 8a to obtain compound 9a (298 mg).

[0136] Step 6: Same as step 5 of Example 6, except that compound 6c in step 5 of Example 6 is replaced by compound 9a to obtain compound B6

[0137] Compound B6 was analyzed by H NMR and mass spectrometry, and the results were as follows:

[0138] 1 H NMR (300MHz, DMSO-d6) δ9.56(s,1H),8.66(s,1H),7.06(d,J=8.3Hz,1H),6.91(d,J=8.2Hz,1H),6.75(d ,J=25.7Hz,4H),3.53(s,8H),2.09(s,2H),1.88(s,3H),1.76(d,J=25.0Hz,6H),1.58(t,J=4.5Hz,12H).

[0139] HRMS(ESI)m / z calcd for C 31 H 39 N7O3[M+H] + 558.31871, found 558.31840.

[0140] From the above results, it can be seen that the structure of compound B6 is as follows:

[0141]

[0142] Example 10: Synthesis of 6-amino-2-(4-(cyclopentanecarbonyl)piperazin-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B7)

[0143] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced with cyclopentanecarboxylic acid, and other conditions remained unchanged to obtain compound B7 (32.5 mg).

[0144] Compound B7 was analyzed by H NMR and mass spectrometry, and the results were as follows: 1 H NMR (300MHz, DMSO-d6) δ9.55(s,1H),8.66(s,1H),7.06(d,J=8.3Hz,1H),6.91(d,J=8.2Hz,1 H),6.74(d,J=25.4Hz,4H),2.96(p,J=7.7Hz,1H),1.76(d,J=25.4Hz,8H),1.67–1.41(m,6H).

[0145] HRMS(ESI)m / z calcd for C 25 H 31 N7O3[M+H] + 478.25611, found 478.25752.

[0146] From the above results, it can be seen that the structure of compound B7 is as follows:

[0147]

[0148] Example 11: Synthesis of 6-amino-7-(3-hydroxy-2,6-dimethylphenyl)-2-(4-pentanoylpiperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B8)

[0149] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced by trimethylacetic acid, and other conditions remained unchanged to obtain compound B8 (40.5 mg).

[0150] Compound B8 was analyzed by H NMR and mass spectrometry, and the results were as follows:

[0151] 1 H NMR(300MHz,DMSO-d6)δ9.57(s,1H),8.64(s,1H),7.06(d,J=8.3Hz,1H),6.96–6.62( m,5H),3.55(dd,J=7.3,3.5Hz,8H),3.16(s,2H),1.75(d,J=25.1Hz,6H),1.16(s,9H).

[0152] HRMS(ESI)m / z calcd for C 24 H 31 N7O3[M+H] + 466.25611, found 466.25615.

[0153] From the above results, it can be seen that the structure of compound B8 is as follows:

[0154]

[0155] Example 12: Synthesis of 6-amino-2-(4-((1S,3S)-3-aminocyclopentane-1-carbonyl)piperazin-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B9)

[0156] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced with (1R,3S)-3-((tert-butoxycarbonyl)amino)cyclopentanecarboxylic acid, and other conditions remained unchanged to obtain compound B9 (19.4 mg).

[0157] Compound B9 was analyzed by H NMR and mass spectrometry, and the results were:

[0158] 1 H NMR (300MHz, DMSO-d6) δ9.65 (s, 1H), 8.66 (s, 1H), 7.11–6.94 (m, 2H), 6.75 (d, J = 24.9Hz, 4H), 3.16 (s, 2H), 2.05–1.62 (m, 12H).

[0159] HRMS(ESI)m / z calcd for C 25 H 32 N8O3[M+H] + 493.26701, found 493.26698.

[0160] From the above results, it can be seen that the structure of compound B9 is as follows:

[0161]

[0162] Example 13: Synthesis of 6-amino-2-(4-((1S,3R)-3-aminocyclopentane-1-carbonyl)piperazin-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B10)

[0163] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced with cis-3-(Boc-amino)cyclopentanecarboxylic acid, and other conditions remained unchanged to obtain compound B10 (20.5 mg).

[0164] Compound B10 was analyzed by H NMR and MS, and the results were as follows:

[0165] 1H NMR (300MHz, DMSO-d6) δ9.73 (s, 1H), 8.66 (s, 1H), 8.18 (s, 3H), 7.09–6.95 (m, 2H), 6.75 (d, J = 25.3Hz, 4H), 3.15 (t, J = 7.9Hz, 2H), 2.15–1.55 (m, 14H).

[0166] HRMS(ESI)m / z calcd for C 25 H 32 N8O3[M+H] + 493.26701, found 493.26801.

[0167] From the above results, it can be seen that the structure of compound B10 is as follows:

[0168]

[0169] Example 14: Synthesis of 6-amino-2-(4-((1S,3S)-3-aminocyclobutane-1-carbonyl)piperazin-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B11)

[0170] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced with trans-3-(tert-butoxycarbonylamino)cyclobutanecarboxylic acid, and other conditions remained unchanged to obtain compound B11 (17.5 mg).

[0171] Compound B11 was analyzed by H NMR and MS, and the results were as follows:

[0172] 1 H NMR (300MHz, DMSO-d6) δ9.61(s,1H),8.66(s,1H),7.06(d,J=8.3Hz,1H),6.93(d,J=8.3Hz,1H),6.75(d,J=25.5H z,4H),3.62–3.50(m,2H),3.42(d,J=13.9Hz,10H),2.46–2.33(m,2H),2.32–2.14(m,2H),1.75(d,J=26.7Hz,6H).

[0173] HRMS(ESI)m / z calcd for C 24 H 30 N8O3[M+H] + 479.25136, found 479.25296.

[0174] From the above results, it can be seen that the structure of compound B11 is as follows:

[0175]

[0176] Example 15: Synthesis of (S)-6-amino-2-(4-amino-2-cyclopropylacetyl)piperazin-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B12)

[0177] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced with Boc-L-cyclopropylglycine, and other conditions remained unchanged to obtain compound B12 (23.8 mg).

[0178] Compound B12 was analyzed by nuclear magnetic hydrogen spectrum and mass spectrometry, and the results were:

[0179] 1 H NMR (300MHz, DMSO-d6) δ9.68(s,1H),8.67(s,1H),7.06(d,J=8.3Hz,1H),6.96(d,J=8.2Hz,1H),6.75(d ,J=26.4Hz,4H),5.25(s,2H),3.65(s,1H),3.51(d,J=9.2Hz,8H),1.82–1.68(m,6H),1.37–1.21(m,5H).

[0180] HRMS(ESI)m / z calcd for C 24 H 30 N8O3[M+H] + 479.25136, found 479.25325.

[0181] From the above results, it can be seen that the structure of compound B12 is as follows:

[0182]

[0183] Example 16: Synthesis of 6-amino-2-(4-(6-aminohexanoyl)piperazin-1-yl)-7-(3-hydroxy-2,6-dimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (B13)

[0184] Referring to the synthesis method of Example 9, the adamantane acetic acid in step 4 of Example 9 was replaced with tert-butyloxycarbonyl 6-aminohexanoic acid, and other conditions remained unchanged to obtain compound B13 (24.4 mg).

[0185] Compound B13 was analyzed by H NMR and mass spectrometry, and the results were as follows:

[0186] 1 H NMR(300MHz,DMSO-d6)δ9.71(s,1H),8.66(s,1H),8.02(s,4H),7.09–6.94(m,2H),6.74(d,J=24.5Hz,4H),2.72( t,J=7.5Hz,2H),2.30(t,J=7.3Hz,2H),1.74(d,J=27.2Hz,6H),1.52(dt,J=21.1,7.3Hz,4H),1.31–1.20(m,2H).

[0187] HRMS(ESI)m / z calcd for C 25 H 34 N8O3[M+H] + 495.28266, found 495.28341.

[0188] From the above results, it can be seen that the structure of compound B13 is as follows:

[0189]

[0190] Example 17: Activity Test

[0191] 1. Reagent Preparation

[0192] 1ml Assay buffer: 200μL 5×Enzymatic Kinase Buffer (Cat#62EZBFDD, Cisbio), 50μL 1M MgCl2 (Cat#M8266, Sigma), 1μL 1M DTT (Cat#D5545, Sigma), 749μL H2O;

[0193] 2.5× ATP working solution: dilute 10 mM ATP (Cat#PV3227, Invitrogen) to 50 μM with Assay buffer.

[0194] 5×PKMYT1 enzyme working solution: dilute PKMYT1 enzyme (Cat#BP11517-12A, Biortus) to 150 nM with Assay buffer.

[0195] 2.5× compound working solution: dilute 10 mM compound to 10 μM and 1 μM with DMSO, then dilute 40-fold with Assaybuffer to obtain 2.5× working solution.

[0196] 2. Reaction steps

[0197] After the above reagents were prepared and equilibrated to room temperature, 4 μL of 2.5× compound working solution (final concentrations were 100 nM and 10 nM, respectively) and 2 μL of 5× PKMYT1 enzyme working solution were added to the reaction wells in sequence. After centrifugation, 4 μL of 2.5× ATP working solution was added to start the kinase reaction. After 1 hour of reaction at 37°C, 10 μL of ADP-GLO Reagent (Cat# V9102, Promega) was added, mixed and reacted at room temperature for 40 minutes, and then 20 μL of kinase detection reagent was added. After 30 minutes of reaction at room temperature, the luminescence signal was detected using a SpectraMax L instrument. The inhibition rate of each well was calculated based on the total activity wells and background signal wells, and the average value of the duplicate wells was taken. GraphPad Prism 6 (version 6.01) was used for graphical analysis to determine the inhibition rate of the compound at a given concentration.

[0198] The luminescence of the compound hole is recorded as Lum compound The luminescence of the control well is recorded as Luminescence vehicle The luminescence of the blank control well is recorded as Luminescence blank .

[0199] The inhibition rate was calculated using the following formula:

[0200] Inhibition rate = (Lum vehicle -Lum compound ) / (Lum vehicle -Lum blank )×100%

[0201] The test results are shown in Table 2, where the IC 50 The descriptions are classified as follows:

[0202] "A" stands for IC 50 The measured value is less than or equal to 10nM and greater than 0nM;

[0203] "B" stands for IC 50 The measured value is less than or equal to 100nM and greater than 10nM;

[0204] "C" stands for IC 50 The measured value is greater than 100nM

[0205] Table 1 Compounds obtained from Examples 1 to 16

[0206]

[0207]

[0208]

[0209]

[0210] Table 2 Inhibitory activity (IC) of the compounds in Table 1 against PKMYT1 50 )

[0211] Compound <![CDATA[IC 50 ]]> Compound <![CDATA[IC 50 ]]> Compound <![CDATA[IC 50 ]]> Compound <![CDATA[IC 50 ]]> Example 1 C Example 2 C Example 3 B Example 4 A Example 5 A Example 6 A Example 7 A Example 8 A Example 9 C Example 10 A Embodiment 11 A Example 12 B Example 13 B Embodiment 14 A Embodiment 15 B Example 16 A

[0212] As can be seen from Table 2, the compounds prepared in Examples 1 to 16 can all inhibit PKMYT1 enzyme activity, among which the compounds prepared in Examples 4 to 8, 10, 11, 14 and 16 have the best inhibitory effect, reaching Grade A, followed by Examples 3, 12 and 13, which are Grade B, and Examples 1, 2 and 9 are Grade C.

Claims

1. A compound of general formula (I) or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula of the compound is shown in formula (I): Wherein, R1 is -NH2, -OCH3, -OCH2CH3 or -NHCH3; R2 is selected from -Cl or one of the following structures:

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from any of the following structures:

3. A method for preparing a compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: Synthesized according to the following route: Route 1: Wherein, R3 is -OCH3, -OCH2CH3 or -NHCH3; Route 2: Wherein, R4 is selected from any of the following structures: or route 3: Wherein, R5 is selected from any of the following structures: R6 is selected from any of the following structures:

4. The preparation method according to claim 3, characterized in that: The reaction conditions of step a in route 1 are: (a) 3-methoxy-2,6-dimethylaniline, LiHMDS, THF, 0-25°C.

5. The preparation method according to claim 3, characterized in that: The reaction conditions of step b and step c in route 1 are: (b) PdCl2(dppf), NaH, DME, 0-90°C, or PdCl2(dppf), sodium tert-butoxide, DME, 0-90°C; (c) BBr3, CH2Cl2, -78-25°C, 4h.

6. The preparation method according to claim 3, characterized in that: The reaction conditions of step a and step b in route 2 are: (a) 3-methoxy-2,6-dimethylaniline, LiHMDS, THF, 0-25°C; (b) PdCl2(dppf), NaH, DME, 0-90°C.

7. The preparation method according to claim 3, characterized in that: The reaction conditions of step c to step e in route 2 are: (c) PdCl2(dppf), CS2CO3, 1,4-dioxane, H2O, 90°C, overnight or DIPEA, n-butanol, 130°C, 5h; (d) H2SO4, 25°C, or H2O2, DMSO, 0-25°C; (e) BBr3, CH2Cl2, -78-25°C, 4h.

8. The preparation method according to claim 3, characterized in that: The reaction conditions of step a to step c in route 3 are: (a) 2,4-dichloro-5-bromopyrimidine, LiHMDS, THF, 0-25°C; (b) malononitrile, PdCl2(dppf), NaH, DME, 0-90°C; (c) piperazine, DIPEA, n-butanol, 130°C, 5h.

9. The preparation method according to claim 3, characterized in that: The reaction conditions of step d to step f in route 3 are: (d) HATU, DIPEA, DMF, 60°C, 12h; (e) H2O2, DMSO, 0-25°C; (f) BBr3, CH2Cl2, -78-25°C, 4h.

10. Use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of a drug or inhibitor for treating, regulating and / or preventing tumor diseases mediated by PKMYT1.

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