A 1h-pyrazolo[3,4-b]pyridine derivative, and a preparation method and application thereof

Abstract

The present application relates to the technical field of compound synthesis, in particular to a 1H-pyrazolo[3,4-b]pyridine derivative and a preparation method and application thereof.The 1H-pyrazolo[3,4-b]pyridine derivative provided by the present application is a compound shown in formula I: The 1H-pyrazolo[3,4-b]pyridine derivative with the above structure designed by the present application has a novel structure, good selective inhibitory effect on histone deacetylase 6 (HDAC6), and good inhibitory activity on human gastric cancer cells.Therefore, the present application can provide a molecular basis and data support for the research and development of an HDAC6-targeted inhibitor or an anti-gastric cancer drug, and can also provide a potential lead compound for the development of a novel HDAC6-targeted inhibitor and an anti-gastric cancer drug.

Description

Technical Field

[0001] The present invention relates to the technical field of compound synthesis, and in particular to a 1H-pyrazolo[3,4-b]pyridine derivative, a preparation method and an application thereof. Background Art

[0002] With the accelerating pace of life, increasing environmental pollution, and a deepening aging population, a growing number of diseases are becoming serious challenges to global health, with cancer being a particular concern. While commonly used treatments such as radiotherapy and chemotherapy can control the disease to a certain extent, they can also cause significant side effects, such as nausea, vomiting, hair loss, and cardiotoxicity, which pose a serious threat to people's health and life. Therefore, in addition to improving early cancer diagnosis and prevention technologies, researching and developing highly effective, low-toxic anticancer drugs is also a key strategy in tumor treatment.

[0003] Histone lysine deacetylase 6 (HDAC6), a member of the class IIb HDAC family, not only participates in histone deacetylation but also targets a variety of non-histone substrates, regulating tumor cell growth and migration, thereby influencing tumor development and progression. Studies have found that HDAC6 plays an important role in the development and progression of gastrointestinal tumors such as gastric cancer. The positive expression rate of HDAC6 protein in gastric cancer tissue is significantly higher than that in normal or chronic mucosal inflammatory tissue, and is closely correlated with the patient's clinical pathological characteristics. Inhibiting HDAC6 activity can inhibit the growth and proliferation of gastric cancer cells, hindering their development and progression.

[0004] Therefore, the design and development of new HDAC6 inhibitors with high anti-tumor activity has very broad application prospects in the future treatment of gastric cancer and other digestive tract tumors. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a 1H-pyrazolo[3,4-b]pyridine derivative, which has good inhibitory activity against HDAC6 and can effectively inhibit the proliferation of gastric cancer cells.

[0006] The present invention also aims to provide a method for preparing the above-mentioned 1H-pyrazolo[3,4-b]pyridine derivatives, which has simple operation and mild conditions and can prepare 1H-pyrazolo[3,4-b]pyridine derivatives with strong inhibitory activity against HDAC6.

[0007] The present invention also aims to provide applications of the above-mentioned 1H-pyrazolo[3,4-b]pyridine derivatives.

[0008] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0009] A 1H-pyrazolo[3,4-b]pyridine derivative, a compound represented by formula I:

[0010]

[0011] In formula I, R1 is selected from one of hydrogen, methyl, ethyl, tert-butyl, and phenyl;

[0012] R2 is selected from one of the following groups:

[0013]

[0014] Based on the consideration of ensuring the inhibitory activity of the compound on HDAC6 and the anti-proliferative effect on gastric cancer tumor cells, preferably, the 1H-pyrazolo[3,4-b]pyridine derivative is selected from the following compounds:

[0015]

[0016]

[0017]

[0018] The preparation method of the above-mentioned 1H-pyrazolo[3,4-b]pyridine derivatives comprises the following steps:

[0019] Step (1): Mix the first raw material, diethyl oxalate, the first catalyst and the first good solvent, and perform a substitution reaction to obtain compound A, the structural formula of which is Alternatively, compound A is obtained directly without preparation, and the structural formula is

[0020] Step (2): Compound A, substituted pyrazole and a second good solvent are mixed and subjected to a cyclization reaction to obtain compound B; the structural formula of compound A is The structural formula of compound B is

[0021] Step (3): Compound B, the second catalyst and the third good solvent are mixed and subjected to a first hydrolysis reaction to obtain compound C having the structural formula

[0022] Step (4): Compound C, methyl 4-aminomethylbenzoate hydrochloride, a third catalyst, a fourth catalyst and a fourth good solvent are mixed to carry out a first condensation reaction to obtain compound D having the structural formula:

[0023] Step (5): Compound D, the second catalyst and the third good solvent are mixed and subjected to a second hydrolysis reaction to obtain compound E, the structural formula of which is

[0024] Step (6): Compound E, O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine, a third catalyst, a fourth catalyst, and a fifth good solvent are mixed to carry out a second condensation reaction to obtain a 1H-pyrazolo[3,4-b]pyridine derivative having a structure shown in Formula I;

[0025] The first raw material is one of 4-methylacetophenone, 3-methylacetophenone, 2-methylacetophenone, 4-chloroacetophenone, acetophenone, 3-chloroacetophenone, 2-chloroacetophenone, 4-bromoacetophenone, 4-methoxyacetophenone, 4-ethylacetophenone, 4-isopropylacetophenone, 4-dimethylaminoacetophenone, 4-hydroxyacetophenone, 4-trifluoromethylacetophenone, 2-acetylthiophene, 3-acetylthiophene, 2-acetylfuran, 1-acetonaphthone, 2-acetonaphthone, acetylcyclohexane, acetylcyclopentane, acetylcyclopropane, 4-fluoroacetophenone, 2-fluoroacetophenone, 3-fluoroacetophenone, 2-methoxyacetophenone, and 3-methoxyacetophenone.

[0026] Preferably, the substituted pyrazole is one of 1-methyl-5-aminopyrazole, 1-ethyl-5-aminopyrazole, 1-tert-butyl-5-aminopyrazole, 1-phenyl-5-aminopyrazole, and 2H-3-aminopyrazole.

[0027] Preferably, the temperature of the substitution reaction is 25-55°C, and the reaction time is 7-12h; the temperature of the cyclization reaction is 90-120°C, and the reaction time is 2-6h; the temperature of the first hydrolysis reaction and the second hydrolysis reaction is 50-75°C, and the reaction time is 0.5-4h; the temperature of the first condensation reaction and the second condensation reaction is 15-30°C, and the reaction time is 2-10h.

[0028] Preferably, the first good solvent, the second good solvent, the third good solvent, the fourth good solvent and the fifth good solvent are each independently selected from one or more of tetrahydrofuran, toluene, ethanol, acetic acid, methanol, dichloromethane, acetonitrile, N,N-dimethylformamide and water.

[0029] Preferably, the first catalyst is selected from one or more of potassium tert-butoxide, sodium hydride, sodium methoxide, and sodium ethoxide; the second catalyst is selected from one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide; the third catalyst is selected from one or more of HATU, HBTU, and EDCI; and the fourth catalyst is selected from one or more of TEA, DIPEA, HOBT, and DMAP.

[0030] The application of the above 1H-pyrazolo[3,4-b]pyridine derivatives in the preparation of HDAC6 inhibitors.

[0031] The application of the above 1H-pyrazolo[3,4-b]pyridine derivatives in the preparation of anti-gastric cancer drugs.

[0032] Preferably, the anti-gastric cancer drug works by inhibiting the activity of gastric cancer cells. More preferably, the gastric cancer cells are MGC-803 and / or MKN-45.

[0033] The beneficial effects of the technical solution of the present invention are:

[0034] 1. The 1H-pyrazolo[3,4-b]pyridine derivatives provided by the present invention are compounds with novel structures synthesized by the present invention. Experiments have confirmed that these compounds have good selective inhibition of histone deacetylase 6 (HDAC6) and have good inhibitory activity against human gastric cancer cells. Therefore, the present invention can provide a molecular basis and data support for the research and development of inhibitors or anti-gastric cancer drugs targeting HDAC6, and can also provide potential lead compounds for the development of new HDAC6-targeted inhibitors and anti-gastric cancer drugs.

[0035] 2. The preparation method of 1H-pyrazolo[3,4-b]pyridine derivatives provided by the present invention has simple steps, convenient operation, mild conditions, and can effectively prepare compounds with good inhibitory activity against HDAC6. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the embodiments. In the following examples, unless otherwise specified, the methods used are conventional methods in the art. The reagents used in the following examples, unless otherwise specified, are conventional reagents in the art and can be obtained from commercial sources.

[0037] Example 1

[0038] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is as follows:

[0039] (1) Weigh 4-methylacetophenone (10 mmol) and diethyl oxalate (12 mmol) separately into a dry three-necked flask containing toluene (30 mL); add potassium tert-butoxide (15 mmol) in batches under stirring, replace the air with nitrogen 5 times, react at room temperature overnight, and follow up with TLC detection; after the reaction, filter and rinse the solid with a small amount of toluene to obtain compound A-1, structural formula

[0040] (2) Compound A-1 (2 mmol) and 1-methyl-5-aminopyrazole (2 mmol) were placed in an eggplant-shaped flask containing glacial acetic acid (6 mL); refluxed at 100°C for 4 h under nitrogen protection, and followed by TLC detection; after the reaction was completed, the glacial acetic acid was removed, and the residue was dissolved in ethyl acetate. The ethyl acetate phase was concentrated to obtain compound B-1, structural formula:

[0041] (3) Compound B-1 (2 mmol) was weighed into a methanol / water (15 mL / 3 mL) solution; 2-4 mL of 33% sodium hydroxide / water solution was added, and the mixture was refluxed at 70°C for 1 h, followed by TLC detection; after the reaction, the methanol was removed, a small amount of water was added, and the solution was acidified with formic acid and cooled to room temperature, and filtered to obtain compound C-1, structural formula:

[0042] (4) Compound C-1 (1.5 mmol), methyl 4-aminomethylbenzoate hydrochloride (1.8 mmol), and HATU (1.8 mmol) were weighed separately into an eggplant-shaped flask containing DCM (15 mL); DIPEA (3 mmol) was added under stirring, and the mixture was reacted at room temperature for 6 h, followed by TLC detection; after the reaction, the DCM was removed by rotary evaporation, a small amount of water was added, and the DCM was completely removed by rotary evaporation until a solid gradually precipitated. The mixture was cooled to room temperature and filtered to obtain compound D-1, structural formula:

[0043] (5) Compound D-1 (1.5 mmol) was weighed and added to a methanol / water (15 mL / 3 mL) solution; 2-4 mL of 33% sodium hydroxide / water solution was added, and the mixture was refluxed at 70°C for 1.5 h and detected by TLC tracking; after the reaction was completed, the methanol was removed, a small amount of water was added, and the solution was acidified with formic acid and cooled to room temperature. The solid was filtered to obtain a solid, and the solid was washed with a small amount of petroleum ether / ethyl acetate (2:1) solution to obtain compound E-1, structural formula

[0044] (6) Compound E-1 (1 mmol), O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (1 mmol), and HATU (1.2 mmol) were weighed separately and placed in an eggplant-shaped flask containing DMF (5 mL); DIPEA (1.5 mmol) was added under stirring, and the mixture was reacted at room temperature for 2 h, followed by TLC detection; after the reaction, the reaction system was added to water, and solids were slowly precipitated. The solids were filtered and transferred to an eggplant-shaped flask containing dichloromethane (10 mL), and concentrated hydrochloric acid was slowly added until the protection was completely removed; the dichloromethane was removed, and sodium bicarbonate / water solution was added under stirring at room temperature, and the pH was adjusted to weak acidity. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 1 was obtained by filtration as an off-white solid with a melting point of 169-170°C and a structural formula of The structural analysis results are as follows:1 H NMR(400MHz,DMSO-d6)δ11.21(s,1H),9.61(s,1H),9.01(s,1H),8.43-8.14(m,4H),7.77(s ,2H),7.57-7.28(m,4H),4.68-4.63(m,2H),4.16(d,J=13.4Hz,3H),2.42(d,J=14.4Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ165.15,164.57,156.22,151.58,142.82,140.11,136.64,135.81 ,132.59,131.99,129.95,127.75,127.53,112.04,111.81,43.00,34.20,21.38.calcd.C 23 H 21 N5O3,[MH] - m / z:414.1571,found:414.1574.

[0045] Example 2

[0046] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 3-methylacetophenone, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 2 is prepared as a brown solid with a melting point of 172-174°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ9.61(s,1H),8.38(s,1H),8.26(s,1H),8.15-8.06(m,2H),7.75(d,J=8.2H z,2H),7.49-7.41(m,3H),7.34(d,J=7.5Hz,1H),4.64(d,J=5.3Hz,2H),4.15(s,3H),2.45(s,3H). 13 CNMR(101MHz,DMSO-d6)δ165.13,156.39,151.57,138.56,136.70,132.61,130.99,12 9.25,128.30,127.67,127.39,125.14,112.39,111.96,43.01,34.25,21.67.calcd.C 23 H 21 N5O3,[MH]- m / z:414.1571,found:414.1578.

[0047] Example 3

[0048] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-methylacetophenone, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 3 is prepared as a brown solid with a melting point of 177-179°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.51 (s, 1H), 8.41 (s, 1H), 7.85 (s, 1H), 7.74 (d, J = 7.9Hz, 2H), 7. 57(d,J=6.5Hz,1H),7.47-7.30(m,5H),4.60(d,J=6.0Hz,2H),4.11(s,3H),2.42(s,3H). 13 C NMR (101MHz, DMSO-d6) δ165.06,159.27,151.10,139.89,136.38,136.21,132.51,131.31,130. 48,129.23,127.70,127.39,126.42,115.84,111.38,43.01,34.25,20.81.HR-MS(ESI),calcd.C 23 H 21 N5O3,[MH] - m / z:414.1571,found:414.1576.

[0049] Example 4

[0050] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 4 is prepared as a white solid with a melting point of 200-201°C and a structural formula of The structural analysis results are as follows: 1H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.60(s,1H),9.01(s,1H),8.38(s,1H),8.30-8.08(m,3H),7.76(d, J=7.5Hz,2H),7.48(d,J=7.3Hz,2H),7.39(d,J=7.0Hz,2H),4.76-4.42(m,4H),2.40(s,3H),1.49(s,3H). 13 CNMR(101MHz,DMSO-d6)δ165.19,156.13,151.04,142.81,140.09,136.71,135.88,132.65,131 .98,129.96,127.91,127.63,112.15,111.94,43.00,42.02,21.39,15.30.HR-MS(ESI),calcd.C 24 H 23 N5O3,[M+H] + m / z:430.1873,found:430.1879

[0051] Example 5

[0052] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 3-methylacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 5 is prepared as a brown solid with a melting point of 174-175°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),9.63(s,1H),8.40(s,1H),8.26(s,1H),8.09(d,J=11.9Hz,2H),7.75(d,J =8.2Hz,2H),7.50-7.37(m,3H),7.34(d,J=7.5Hz,1H),4.66-4.55(m,4H),2.45(s,3H),1.49(t,J=7.2Hz,3H). 13C NMR (101MHz, DMSO-d6) δ165.15,164.25,156.30,151.03,141.96,138.66,138.55,136.78,133.03,132.67,130. 96,129.24,128.30,127.55,127.25,125.16,112.51,112.10,43.03,42.02,21.67,15.30.HR-MS(ESI),calcd.C 24 H 23 N5O3,[MH] - m / z:428.1728,found:428.1730.

[0053] Example 6

[0054] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-methylacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 6 is prepared as a light reddish brown solid with a melting point of 183-184°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.22(s,1H),9.52(s,1H),9.05(s,1H),8.42(s,1H),7.85(s,1H),7.74(d,J=8.3Hz, 2H),7.58(s,1H),7.44(d,J=7.9Hz,2H),7.38(s,3H),4.77-4.24(m,4H),2.43(s,3H),1.45(t,J=6.9Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ165.11,159.11,150.48,142.75,139.86,136.41,136.30,132.54,131.36,130. 53,129.23,127.71,127.47,126.44,115.93,111.53,42.99,42.20,20.80,15.39.HR-MS(ESI),calcd.C 24 H 23 N5O3,[MH] - m / z:428.1728,found:428.1733.

[0055] Example 7

[0056] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-chloroacetophenone, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 7 is prepared as a white solid with a melting point of 266-269°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ11.18(s,1H),9.62(s,1H),9.03(s,1H),8.39(s,1H),8.37-8.27(m,3H),7.7 6(d,J=8.2Hz,2H),7.65(d,J=8.4Hz,2H),7.48(d,J=7.8Hz,2H),4.65(d,J=5.4Hz,2H),4.15(s,3H). 13 CNMR(101MHz,DMSO-d6)δ164.99,154.87,151.48,142.72,137.38,136.85,135.27 ,132.67,132.01,129.54,129.40,127.76,127.52,112.19,43.02,34.25.calcd.C 22 H 18 ClN5O3,[MH] - m / z:434.1025,found:434.1029.

[0057] Example 8

[0058] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that 1-methyl-5-aminopyrazole in step (2) is replaced by 1-tert-butyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 8 is prepared as a white solid with a melting point of 197-199°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ11.19(s,1H),9.60(s,1H),9.04(s,1H),8.34(s,1H),8.24(s,1H),8.18(d,J=7.9Hz,2H),7 .76(d,J=8.0Hz,2H),7.47(d,J=8.0Hz,2H),7.40(d,J=7.9Hz,2H),4.65(d,J=5.4Hz,2H),2.40(s,3H),1.85(s,9H). 13 C NMR(101MHz,DMSO-d6)δ165.31,155.03,151.30,142.81,139.98,136.55,136.16,131.99, 131.25,130.03,127.53,113.25,111.61,60.19,42.97,29.46,21.38.HR-MS(ESI),calcd.C 26 H 27 N5O3,[M+H] + m / z:458.2185,found:458.2192.

[0059] Example 9

[0060] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by acetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-phenyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 9 is prepared as a white solid with a melting point of 220-221°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.22(s,1H),9.78(s,1H),9.02(s,1H),8.70(s,1H),8.45(s,1H),8.35(dd,J=1 3.2,7.8Hz,4H),7.78(d,J=7.9Hz,2H),7.67-7.47(m,8H),7.40(t,J=7.3Hz,2H),4.68(d,J=4.3Hz,2H). 13C NMR(101MHz,DMSO-d6)δ164.83,157.16,151.29,142.72,139.53,138.42,137.27,135.31,132.03,130.62,129.79,129.50,12 8.00,127.79,127.56,126.65,121.29,114.00,113.51,100.00,43.07,34.85,32.01,31.62,30.31,29.91.HRMS(ESI),calcd.C 27 H 21 N5O3,[M+H] + m / z:464.1716,found:464.1723.

[0061] Example 10

[0062] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that 1-methyl-5-aminopyrazole in step (2) is replaced by 1-phenyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 10 is prepared as a white solid with a melting point of 220-221°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.21(s,1H),9.71(s,1H),9.02(d,J=9.5Hz,1H),8.67(d,J=2.4Hz,1H),8.44-8.29(m,3H),8.22(d,J=7 .1Hz,2H),7.77(d,J=7.6Hz,2H),7.63(t,J=6.9Hz,2H),7.50(d,J=7.3Hz,2H),7.39(t,J=8.1Hz,3H),4.67(s,2H),2.41(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.87,157.16,151.30,142.71,140.45,139.57,137.17,135.66,135.29,132.04,130.10,129.77,127.88,127.67 ,126.59,123.19,121.24,116.06,113.79,113.16,43.07,34.95,32.01,31.62,30.31,29.91,21.41.HR-MS(ESI),calcd.C28H23N5O3,[M+H]+ m / z:478.1873,found:478.1879.

[0063] Example 11

[0064] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that: in step (1), instead of preparing compound A-1, compound A-11 (ethyl 2,4-dioxohexanoate) is directly purchased and subjected to subsequent reactions, and 1-methyl-5-aminopyrazole in step (2) is replaced with 1-phenyl-5-aminopyrazole, and the rest is the same as in Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 11 is prepared as a pink-white solid with a melting point of 175-177°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.53(s,1H),8.58(s,1H),8.31(d,J=8.0Hz,2H),7.75(t,J=9.5Hz,3H),7.59(t,J=7.9H z,2H),7.47(d,J=7.5Hz,2H),7.36(t,J=7.3Hz,1H),4.62(s,2H),3.02(q,J=7.4Hz,2H),1.38(t,J=7.5Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ165.07,164.68,151.01,142.72,139.62,136.74,134.95,132.01 ,129.67,127.77,127.51,126.49,121.11,115.68,113.08,43.03,31.62,13.92.calcd.C 23 H 21 N5O3,[MH] - m / z:414.1571,found:414.1576.

[0065] Example 12

[0066] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that: in step (1), instead of preparing compound A-1, compound A-12 (ethyl acetylacetonate) is directly purchased and used for subsequent reactions, and 1-methyl-5-aminopyrazole in step (2) is replaced with 1-phenyl-5-aminopyrazole, and the rest is the same as in Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 12 is prepared as a white solid with a melting point of 174-176°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),9.51(t,J=5.8Hz,1H),9.05(s,1H),8.57(s,1H),8.27(d,J=7.5Hz,2H),7.76(d,J=8. 3Hz,2H),7.70(s,1H),7.63-7.54(m,2H),7.47(d,J=8.4Hz,2H),7.37(t,J=7.4Hz,1H),4.61(d,J=6.0Hz,2H),2.73(s,3H). 13 C NMR(151MHz,DMSO-d6)δ165.03,159.86,151.04,142.69,139.56,136.64,134.91,132.01,1 29.66,127.75,127.48,126.57,121.30,116.57,112.85,43.02,25.29.HR-MS(ESI),calcd.C 22 H 19 N5O3,[MH] - m / z:400.1415,found:400.1422.

[0067] Example 13

[0068] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-chloroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 13 is prepared as a white solid with a melting point of 204-205°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ9.62(s,1H),8.41(s,1H),8.36-8.26(m,3H),7.76(d,J=8.2Hz,2H ),7.65(d,J=8.3Hz,2H),7.46(d,J=8.2Hz,2H),4.68-4.52(m,4H),1.49(t,J=7.2Hz,3H). 13C NMR (101MHz, DMSO-d6) δ165.03,164.62,154.73,150.93,148.21,137.48,136.94,135.19,132.72,129. 55,129.37,129.00,127.72,126.28,114.26,112.41,112.18,43.04,42.07,15.15.HR-MS(ESI),calcd.C 23 H 20 ClN5O3,[MH] - m / z:448.1182,found:448.1188.

[0069] Example 14

[0070] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 3-chloroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 14 is prepared as a brown solid with a melting point of 241-251°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.03(s,1H),9.62(s,1H),8.42(s,1H),8.36-8.23(m,3H),7.75(d,J=8. 2Hz,2H),7.61(d,J=7.0Hz,2H),7.43(d,J=8.2Hz,2H),4.74-4.51(m,4H),1.49(t,J=7.2Hz,3H). 13 CNMR(101MHz,DMSO-d6)δ164.55,154.37,150.90,148.21,140.80,137.33,134.31,132.85,131.25,130. 03,128.96,127.38,126.51,126.27,114.26,112.67,112.36,43.02,42.09,15.27.HR-MS(ESI),calcd.C 23 H 20 ClN5O3,[MH] - m / z:448.1182,found:448.1187.

[0071] Example 15

[0072] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-chloroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 15 is prepared as a gray solid with a melting point of 177-178°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.46 (s, 1H), 8.44 (s, 1H), 7.91 (s, 1H), 7.73 (d, J = 8.1Hz, 3H), 7.64 (s, 1H) ),7.56-7.50(m,2H),7.39(d,J=7.8Hz,2H),4.55(dd,J=14.0,6.8Hz,4H),1.46(t,J=7.2Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ164.89,156.27,150.49,139.00,136.24,132.33,131.09,130.50,1 30.12,127.93,127.57,116.23,111.97,108.88,43.05,42.32,15.31.HR-MS(ESI),calcd.C 23 H 20 ClN5O3,[MH] - m / z:448.1182,found:448.1188.

[0073] Example 16

[0074] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-fluoroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 16 is prepared as a white solid with a melting point of 218-220°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ9.68 (s, 1H), 8.47-8.14 (m, 4H), 7.76 (d, J = 7.5Hz, 2H), 7.51-7.28 (m, 4H), 4.74-4.49 (m, 4H), 1.49 (t, J = 7.1Hz, 3H). 13 C NMR(101MHz,DMSO-d6)δ165.08,155.09,150.97,142.63,136.89,135.18,132.70,132.17,130.14,127.72,12 7.47,116.49,116.28,112.33,112.08,43.00,42.07,32.01,31.62,30.31,29.91,15.28.HR-MS(ESI),calcd.C 23 H 20 FN5O3,[M+Na] + m / z:456.1441,found:456.1448.

[0075] Example 17

[0076] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-fluoroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 17 is prepared as a white solid with a melting point of 219-220°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),9.52(s,1H),9.01(s,1H),8.43(s,1H),8.02(dd,J=15.1,6.8Hz,2H ),7.75(d,J=8.0Hz,2H),7.61-7.53(m,1H),7.51-7.35(m,4H),4.66-4.52(m,4H),1.48(t,J=7.1Hz,3H). 13C NMR(101MHz,DMSO-d6)δ165.06,164.54,161.55,159.07,152.87,150.76,142.74,136.69,132.55,132.02 ,127.73(s),127.49,125.43,117.00,116.78,115.82,111.97,43.07,42.24,15.24.HR-MS(ESI),calcd.C 23 H 20 FN5O3,[M+Na] + m / z:456.1441,found:456.1448.

[0077] Example 18

[0078] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-bromoacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 18 is prepared as a white solid with a melting point of 204-205°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.88(s,1H),9.63(s,1H),8.41(s,1H),8.32-8.22(m,3H),7 .77(t,J=8.9Hz,4H),7.48(d,J=8.3Hz,2H),4.99-4.25(m,4H),1.49(t,J=7.2Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ165.02,164.41,154.85,150.94,142.67,137.80,136.94,132.74,132.33,1 32.07,129.81,127.73,127.49,124.09,112.36,112.26,43.01,42.10,15.27.HR-MS(ESI),calcd.C 23 H 20 BrN5O3,[MH] - m / z:492.0677,found:492.0682.

[0079] Example 19

[0080] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-methoxyacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 19 is prepared as a white solid with a melting point of 206-207°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ9.93(s,1H),9.60(s,1H),8.36(s,1H),8.31-8.18(m,3H),7.76(d,J=8.0Hz,2H ),7.43(d,J=7.9Hz,2H),7.12(d,J=8.4Hz,2H),4.72-4.50(m,4H),3.86(s,3H),1.49(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ164.85,161.25,155.90,151.03,148.17,137.08,132.69,131.13,129.31,128. 97,127.70,126.41,114.71,114.26,111.78,111.68,55.81,42.99,41.95,15.28.HR-MS(ESI),calcd.C 24 H 23 N5O4,[MH] - m / z:444.1677,found:444.1680.

[0081] Example 20

[0082] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-ethylacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 20 is prepared as an off-white solid with a melting point of 199-200°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ11.06(s,1H),9.60(s,1H),8.39(s,1H),8.30-8.17(m,3H),7.76(d,J=8.2Hz,2H),7.47(d,J=8. 2Hz, 2H), 7.41 (d, J = 8.3Hz, 2H), 4.70-4.54 (m, 4H), 2.70 (q, J = 7.6Hz, 2H), 1.49 (t, J = 7.2Hz, 3H), 1.24 (t, J = 7.6Hz, 3H). 13 C NMR(101MHz,DMSO-d6)δ165.20,164.39,156.23,151.05,146.31,142.71,136.72,136.20,132.63,132.06, 128.76,127.88,127.71,127.48,112.27,111.94,43.00,42.03,28.47,15.95,15.28.HR-MS(ESI),calcd.C 25 H 25 N5O3,[MH] - m / z:442.1884,found:442.1889.

[0083] Example 21

[0084] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-isopropylacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 21 is prepared as a white solid with a melting point of 197-198°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),9.68(t,J=6.0Hz,1H),8.38(s,1H),8.28(s,1H),8.22(d,J=8.4Hz,2H),7.76(d,J=8.3 Hz,2H),7.46(dd,J=15.4,8.4Hz,4H),4.68-4.55(m,4H),2.98(p,J=6.8Hz,1H),1.49(t,J=7.3Hz,3H),1.26(d,J=6.9Hz,6H). 13C NMR(101MHz,DMSO-d6)δ165.19,164.48,156.30,151.05,150.85,142.84,136.70,136.40,132.63,131.96,129 .97,127.96,127.73,127.51,127.28,112.41,111.93,42.98,42.03,33.76,24.20,15.28.HR-MS(ESI),calcd.C 26 H 27 N5O3,[MH] - m / z:456.2041,found:442.2049.

[0085] Example 22

[0086] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-dimethylaminoacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 22 is prepared as a white solid with a melting point of 219-220°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),9.55(t,J=6.1Hz,1H),9.01(s,1H),8.30(s,1H),8.20-8.13(m,3H),7.76(d,J=8.3Hz,2H) ,7.47(d,J=8.3Hz,2H),6.85(d,J=9.0Hz,2H),4.64(d,J=5.9Hz,2H),4.56(q,J=7.1Hz,2H),3.01(s,6H),1.48(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ165.44,164.57,156.61,151.90,151.18,142.91,136.35,132.54,131.96,128. 81,127.71,127.52,125.78,112.32,111.36,110.95,42.96,41.85,40.28,15.30.HR-MS(ESI),calcd.C 25 H 26 N6O3,[MH] -m / z:457.1993,found:457.2000.

[0087] Example 23

[0088] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-hydroxyacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 23 is prepared as a brownish yellow solid with a melting point of 227-228°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ9.73-9.50(m,1H),8.44-8.30(m,2H),8.18(q,J=8.7Hz,3H),7.81 -7.42(m,4H),6.96(dd,J=8.5,5.0Hz,2H),4.95-4.36(m,4H),1.48(td,J=6.9,2.8Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ165.30,159.90,156.34,151.06,136.53,132.58,131.96,130.61,129.40,129.16,1 28.64,128.22,128.00,127.72,127.50,116.15,111.69,111.34,42.97,41.92,15.31.HR-MS(ESI),calcd.C 23 H 21 N5O4,[MH] - m / z:430.1521,found:430.1527.

[0089] Example 24

[0090] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-trifluoromethylacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 24 is prepared as a light brown solid with a melting point of 199-200°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ9.66(s,1H),8.50(d,J=6.8Hz,2H),8.44(s,1H),8.36(s,1H),7.95(d,J=8 .3Hz,2H),7.75(d,J=8.0Hz,2H),7.44(d,J=7.5Hz,2H),4.67-4.47(m,4H),1.50(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ164.94,154.38,150.93,142.47,137.09,132.78,128.58,1 27.67,127.34,126.25,112.84,112.72,43.04,42.16,15.26.HR-MS(ESI),calcd.C 24 H 20 F3N5O3,[MH] - m / z:482.1445,found:482.1453.

[0091] Example 25

[0092] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-acetylthiophene, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 25 is prepared as a white solid with a melting point of 218-219°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.53(s,1H),9.01(s,1H),8.35(s,1H),8.22(s,1H),8.01(s,1H),7 .82-7.68(m,3H),7.48(d,J=7.8Hz,2H),7.25(d,J=3.7Hz,1H),4.79-4.43(m,4H),1.48(t,J=7.1Hz,3H). 13C NMR (101MHz, DMSO-d6) δ164.98,151.76,150.57,144.37,142.72,136.84,132.82,132.01,130. 25,129.06,127.99,127.82,127.62,111.91,111.43,43.02,42.08,15.16.HR-MS(ESI),calcd.C 21 H 19 N5O3S,[M+H] + m / z:422.1280,found:422.1287.

[0093] Example 26

[0094] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 3-acetylthiophene, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 26 is prepared as a white solid with a melting point of 247-248°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),9.52(s,1H),8.37(d,J=12.7Hz,2H),8.19(s,1H),7.94(d,J= 5.1Hz,1H),7.75(t,J=9.2Hz,3H),7.44(d,J=8.0Hz,2H),4.96-4.27(m,4H),1.48(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ165.15,152.54,150.83,141.95,136.78,132.65,128.99,127.91,127. 64,127.32,127.21,126.41,114.24,112.84,111.70,43.00,41.94,15.28.HR-MS(ESI),calcd.C 21 H 19 N5O3S,[M+H] + m / z:422.1280,found:422.1286.

[0095] Example 27

[0096] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-acetylfuran, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 27 is prepared as a white solid with a melting point of 200-201°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.16(s,1H),9.63(s,1H),8.36(s,1H),8.12(s,1H),7.97(s,1H),7.76(d,J=8.2Hz,2H),7.45(d,J=8.3 Hz,2H),7.38(d,J=3.5Hz,1H),6.76(dd,J=3.4,1.8Hz,1H),4.62(d,J=5.8Hz,2H),4.54(q,J=7.3Hz,2H),1.47(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ164.97,164.39,153.09,150.72,148.04,145.66,142.51,136.98,132.79,132. 31,127.70,127.41,114.23,113.17,111.82,111.63,111.16,43.04,41.98,15.25.HR-MS(ESI),calcd.C 21 H 19 N5O4,[MH] - m / z:404.1364,found:404.1372.

[0097] Example 28

[0098] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 1-naphthylacetonone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 28 is prepared as a white solid with a melting point of 197-198°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ11.18(s,1H),9.54(t,J=5.7Hz,1H),9.00(s,1H),8.48(s,1H),8.16(d,J=8.0Hz,1H),8.08(dd,J=13.3,8.3Hz,2H),7.99 (s,1H),7.81(d,J=5.8Hz,1H),7.75-7.64(m,3H),7.62-7.53(m,2H),7. 44(d,J=8.3Hz,2H), 4.57(dt,J=14.4,6.7Hz,4H), 1.47(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ165.10,164.55,158.27,150.73,142.76,137.93,136.52,133.94,132.65,131.97,131.06,129. 77,128.88,128.74,127.75,127.50,127.35,126.64,125.84,116.81,111.89,43.03,42.30,15.36.HR-MS(ESI),calcd.C 27 H 23 N5O3,[MH] - m / z:464.1728,found:464.1733.

[0099] Example 29

[0100] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-naphthylacetonone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 29 is prepared as a white solid with a melting point of 194-195°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ9.66 (s, 1H), 8.85 (s, 1H), 8.52-8.39 (m, 3H), 8.12 (d, J = 8.4Hz, 2H), 8.01 (t, J = 4.7Hz, 1H), 7.77(d,J=7.9Hz,2H),7.61(dd,J=6.3,3.2Hz,2H),7.50(d,J=7.9Hz,2H),4.71-4.60(m,4H),1.53(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ165.23,164.40,156.00,151.10,142.72,136.91,136.09,134.04,133.45,132.71,132.09,129. 17,128.93,128.15,127.74,127.63,127.49,127.21,125.24,112.75,112.15,43.06,42.08,15.34.HR-MS(ESI),calcd.C 27 H 23 N5O3,[MH] - m / z:464.1728,found:464.1734.

[0101] Example 30

[0102] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by acetylcyclohexane, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 30 is prepared as a white solid with a melting point of 190-191°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ11.13(s,1H),9.40(s,1H),8.28(s,1H),7.74(d,J=8.2Hz,2 H),7.59(s,1H),7.42(d,J=8.3Hz,2H),4.58(d,J=5.7Hz,2H),4.49(q,J=7.2Hz,2H) ,2.87(t,J=11.9Hz,1H),1.95(d,J=12.7Hz,2H),1.84(d,J=12.7Hz,2H),1.74(d,J= 12.7Hz, 1H), 1.63 (q, J = 12.0Hz, 2H), 1.43 (t, J = 7.2Hz, 5H), 1.30 (t, J = 12.7Hz, 1H). 13 C NMR(101MHz,DMSO-d6)δ166.50,165.36,150.61,136.25,132.26,127.70,127.41,11 3.58,111.37,46.65,42.94,41.89,32.82,26.38,26.07,15.22.HR-MS(ESI),calcd.C 23 H 27 N5O3,[MH] - m / z:420.2041,found:420.2045.

[0103] Example 31

[0104] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by acetylcyclopentane, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 31 is prepared as a white solid with a melting point of 186-188°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ9.40(s,1H),9.01(s,1H),8.27(s,1H),7.74(d,J=8.2Hz,2H),7.58(s,1H),7.43(d,J=8.2Hz,2H),4.59(d,J=6.0 Hz,2H),4.49(q,J=6.8Hz,2H),3.37(d,J=7.5Hz,1H),2.09(d,J=9.9Hz,2H),1.87(d,J=16.3Hz,4H),1.70(s,2H),1.43(t,J=7.1Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ165.92,165.41,150.58,142.83,136.21,132.23,131.98,127.70 ,127.47,114.13,111.28,48.07,42.94,41.89,33.64,25.89,15.20.HR-MS(ESI),calcd.C 22 H 25 N5O3,[MH] - m / z:406.1884,found:406.1890.

[0105] Example 32

[0106] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by acetylcyclopropane, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 32 is prepared as a white solid with a melting point of 180-182°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),9.39(s,1H),9.01(s,1H),8.24(s,1H),7.75(d,J=8.0Hz,2H),7.57-7.41(m, 3H), 4.59 (d, J = 5.3Hz, 2H), 4.51-4.38 (m, 2H), 2.37-2.21 (m, 1H), 1.41 (t, J = 7.2Hz, 3H), 1.11 (s, 2H), 1.10 (s, 2H). 13C NMR(101MHz,DMSO-d6)δ165.39,164.57,163.50,150.80,142.86,135.98,132.28,131 .95,127.59,113.51,111.13,42.93,41.82,17.98,15.17,11.35.HR-MS(ESI),calcd.C 20 H 21 N5O3,[M+H] + m / z:380.1786,found:380.1795.

[0107] Example 33

[0108] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that: in step (1), compound A-1 is not prepared, and compound A-33 (sodium salt of diethyl oxaloacetate) is directly purchased for subsequent steps, 1-methyl-5-aminopyrazole in step (2) is replaced with 1-ethyl-5-aminopyrazole, and the compound obtained in step (2) is B-33; step (3) first uses compound B-33 and iodomethane to carry out a substitution reaction in the presence of cesium carbonate as a catalyst and acetonitrile as a solvent, and then hydrolyzes to obtain C-33. The rest is the same as in Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 33 is prepared as a white solid with a melting point of 189-190°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.39(s,1H),8.17(s,1H),7.74(d,J=8.0Hz,2H),7.41(d,J=8.2Hz,2H) ,7.13(s,1H),4.56(d,J=6.0Hz,2H),4.41(q,J=7.7Hz,2H),4.01(s,3H),1.43(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ164.81,164.45,163.99,149.11,142.68,138.93,132.74,132. 00,127.61,127.44,108.30,105.20,54.33,42.90,41.91,15.09.HR-MS(ESI),calcd.C 18 H 19 N5O4,[MH] - m / z:368.1364,found:368.1372.

[0109] Example 34

[0110] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, the preparation method of which is basically the same as that of Example 1, except that: in step (1), instead of preparing compound A-1, compound A-34 (ethyl 2,4-dioxohexanoate) is directly purchased and then reacted, and 1-methyl-5-aminopyrazole in step (2) is replaced with 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 34 is prepared as a pink-yellow solid with a melting point of 169-170°C and a structural formula of The structural analysis results are as follows: 1 HNMR (400MHz, DMSO-d6) δ11.19(s,1H),9.40(t,J=4.7Hz,1H),9.02(s,1H),8.28(s,1H),7.74(d,J=8.3Hz,2H),7.57(s,1H),7.44( d,J=8.2Hz,2H),4.59(d,J=5.9Hz,2H),4.50(q,J=7.2Hz,2H),2.95(q,J=7.6Hz,2H),1.43(t,J=7.2Hz,3H),1.34(t,J=7.6Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ165.38,164.51,163.62,150.70,142.84,136.31,132.27,131.94, 127.71,127.48,114.56,111.18,42.94,41.88,31.55,15.25,14.13.HR-MS(ESI),calcd.C 19 H 21 N5O3,[MH] - m / z:366.1571,found:366.1577.

[0111] Example 35

[0112] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that: in step (1), instead of preparing compound A-1, compound A-35 (ethyl acetylacetonate) is directly purchased and then reacted. In addition, 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 35 is prepared as a white solid with a melting point of 162-163°C and a structural formula of The structural analysis results are as follows:1 H NMR (400MHz, DMSO-d6) δ9.39(t,J=6.1Hz,1H),8.27(s,1H),7.73(d,J=8.0Hz,2H),7.54(s,1H),7.4 0(d,J=7.9Hz,2H),4.57(d,J=5.6Hz,2H),4.48(q,J=7.2Hz,2H),2.67(s,3H),1.42(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ165.33,164.19,158.71,150.71,142.36,136.22,132.50,132. 27,127.61,127.31,115.55,110.96,42.96,41.85,25.04,15.29.HR-MS(ESI),calcd.C 18 H 19 N5O3,[MH] - m / z:352.1515,found:352.1522.

[0113] Example 36

[0114] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that: in step (1), instead of preparing compound A-1, compound A-36 (ethyl trimethylacetyl acetonate) is directly purchased for subsequent reaction, and 1-methyl-5-aminopyrazole in step (2) is replaced by 1-ethyl-5-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 36 is prepared as a white solid with a melting point of 162-163°C and a structural formula of The structural analysis results are as follows: 1 HNMR(400MHz,DMSO-d6)δ11.21(s,1H),9.49(s,1H),9.01(s,1H),8.30(s,1H),7.77(s,3H),7.45(s,2H),4.61(s,2H),4.51(s,2H),1.44(s,12H). 13 C NMR(101MHz,DMSO-d6)δ169.33,165.47,164.55,150.15,142.94,136.16,132.19,131.95, 127.72,127.52,111.75,110.98,42.94,41.92,38.53,30.55,15.16.HR-MS(ESI),calcd.C21 H 25 N5O3,[MH] - m / z:394.1884,found:394.1890.

[0115] Example 37

[0116] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 37 is prepared as a yellow solid with a melting point of 220-221°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.18(s,1H),9.64(t,J=6.3Hz,1H),9.00(s,1H),8.42(d,J=2.4Hz,1H),8.10(d,J=8.0Hz,2H),7 .72(d,J=8.1Hz,2H),7.68(s,1H),7.43(dd,J=8.3,3.1Hz,4H),6.99(d,J=2.4Hz,1H),4.57(d,J=6.3Hz,2H),2.44(s,3H). 13 C NMR (101MHz, DMSO-d6) δ164.60,163.55,149.47,148.49,147.17,146.30,142.97,141.97,131. 87,129.88,129.62,128.06,127.75,127.40,104.96,98.32,42.89,21.58.HR-MS(ESI),calcd.C 22 H 19 N5O3,[MH] - m / z:400.1415,found:400.1419.

[0117] Example 38

[0118] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-methylacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 38 is prepared as a yellow solid with a melting point of 229-230°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.21(s,1H),9.66(t,J=6.4Hz,1H),9.00(s,1H),8.35(d,J=2.4Hz,1H),7.74(d,J= 8.2Hz,2H),7.55-7.48(m,3H),7.46-7.36(m,4H),7.00(d,J=2.4Hz,1H),4.58(d,J=6.4Hz,2H),2.07(s,3H). 13 C NMR (101MHz, DMSO-d6) δ164.59,163.44,149.25,148.34,147.65,146.49,142.94,137.37,131.88,131. 67,130.86,130.64,129.98,127.78,127.42,126.46,106.78,98.42,42.91,19.59.HR-MS(ESI),calcd.C 22 H 19 N5O3,[MH] - m / z:400.1415,found:400.1424.

[0119] Example 39

[0120] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by acetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 39 is prepared as a yellow-brown solid with a melting point of 214-215°C and a structural formula of The structural analysis results are as follows: 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.75-9.49(m,1H),9.02(s,1H),8.71(s,1H),8.14(dd,J=16.2,7.9Hz,2H),7 .73(t,J=7.1Hz,2H),7.65-7.59(m,4H),7.43(dd,J=8.1,5.1Hz,2H),7.01(d,J=2.4Hz,1H),4.58(d,J=6.4Hz,2H). 13C NMR(101MHz,DMSO-d6)δ164.59,163.51,149.55,148.42,147.18,146.94,146.36,145.11,131.80,1 30.97,129.93,129.85,129.05,127.75,127.37,115.99,105.41,98.45,42.90.HR-MS(ESI),calcd.C 21 H 17 N5O3,[MH] - m / z:386.1258,found:386.1261.

[0121] Example 40

[0122] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-fluoroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 40 is prepared as a light yellow solid with a melting point of 209-210°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.15 (s, 1H), 9.73-9.60 (m, 1H), 9.02 (s, 1H), 8.43 (d, J = 2.4Hz, 1H), 8.27 (dd, J=8.9,5.4Hz,2H),7.75-7.68(m,3H),7.53-7.39(m,4H),7.01(d,J=2.5Hz,1H),4.57(d,J=6.4Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ165.33,163.48,162.85,149.54,148.41,146.36,146.15,142.91,1 32.64,131.90,127.74,127.37,116.26,116.04,105.40,98.50,42.90.HR-MS(ESI),calcd.C 21 H 16 FN5O3,[MH] - m / z:404.1164,found:404.1166.

[0123] Example 41

[0124] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-fluoroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 41 is prepared as a light yellow solid with a melting point of 215-216°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.67(t,J=6.3Hz,1H),8.38(d,J=2.5Hz,1H),7.87(t,J=7.4Hz,1H),7.71(d,J=7.9H z,3H),7.65(s,1H),7.51-7.45(m,2H),7.40(d,J=8.0Hz,2H),7.03(d,J=2.4Hz,1H),4.56(d,J=6.3Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.43,163.30,161.19,158.70,149.22,147.68,146.39,142.99,142.57,133.81,1 31.97,129.08,127.67,127.29,125.30,119.40,116.64,114.16,107.25,98.73,42.92.HR-MS(ESI),calcd.C 21 H 16 FN5O3,[MH] - m / z:404.1164,found:404.1169.

[0125] Example 42

[0126] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 3-fluoroacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 42 is prepared as a light yellow solid with a melting point of 205-206°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ11.14(s,1H),9.74-9.62(m,1H),9.02(s,1H),8.44(d,J=2.5Hz,1H),8.08(dt,J=10.1,2.2Hz,1H),7.99(dt ,J=7.9,1.2Hz,1H),7.76-7.63(m,4H),7.51(td,J=8.5,2.7Hz,1H),7.46-7.40(m,2H),7.03(d,J=2.4Hz,1H),4.57(d,J=6.3Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.49,163.35,160.93,149.55,148.38,146.42,145.64,142.88,133.01,131.92,131.2 8,131.19,127.74,127.37,126.20,118.78,118.57,117.08,116.84,105.79,98.65,42.91.HR-MS(ESI),calcd.C 21 H 16 FN5O3,[MH] - m / z:404.1164,found:404.1172.

[0127] Example 43

[0128] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 4-methoxyacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 43 is prepared as a light yellow-brown solid with a melting point of 220-223°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.59(t,J=6.4Hz,1H),8.41(d,J=2.4Hz,1H),8.23(dd,J=8.5,5.8Hz,2H),7.79-7.66( m,3H),7.42(d,J=7.9Hz,2H),7.17(d,J=8.5Hz,2H),6.96(d,J=2.4Hz,1H),4.57(d,J=6.3Hz,2H),3.88(s,3H). 13C NMR(101MHz,DMSO-d6)δ164.50,163.61,162.13,149.41,148.60,146.85,146.23,131.83,1 29.40,127.74,127.36,122.90,114.51,104.42,98.17,55.97,42.89.HR-MS(ESI),calcd.C 22 H 19 N5O4,[MH] - m / z:416.1364,found:416.1361.

[0129] Example 44

[0130] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 2-methoxyacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 44 is prepared as a yellow solid with a melting point of 235-236°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.64(t,J=6.3Hz,1H),8.31(d,J=2.4Hz,1H),7.72(d,J=8.3Hz,2H),7.63-7.56(m,2H),7.53(s,1H),7 .41(d,J=8.3Hz,2H),7.26(d,J=8.2Hz,1H),7.15(t,J=7.0Hz,1H),6.97(d,J=2.4Hz,1H),4.56(d,J=6.3Hz,2H),3.74(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.47,163.50,157.53,149.01,147.67,146.05,142.81,132.76,132.05,131.06,1 29.07,127.70,127.35,120.89,120.39,114.17,112.41,107.02,98.21,56.23,42.90.HR-MS(ESI),calcd.C 22 H 19 N5O4,[MH] - m / z:416.1364,found:416.1369.

[0131] Example 45

[0132] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that 4-methylacetophenone in step (1) is replaced by 3-methoxyacetophenone, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole. The rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 45 is prepared as a brownish yellow solid with a melting point of 230-231°C and a structural formula of The structural analysis results are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.65 (s, 1H), 8.42 (s, 1H), 7.78-7.65 (m, 6H), 7.53 (q, J = 8.0Hz, 2H), 7 .42(d,J=8.0Hz,2H),7.22(d,J=5.5Hz,1H),7.00(s,1H),4.56(d,J=5.9Hz,2H),3.85(s,3H). 13 C NMR (101MHz, DMSO-d6) δ164.42,163.50,159.55,149.52,148.43,146.97,146.37,132.18,130.25,129. 10,127.75,127.36,122.16,117.43,115.42,114.16,105.53,98.46,55.89,42.89.HR-MS(ESI),calcd.C 22 H 19 N5O4,[MH] - m / z:416.1364,found:416.1370.

[0133] Example 46

[0134] This embodiment provides a 1H-pyrazolo[3,4-b]pyridine derivative, and its preparation method is basically the same as that of Example 1, except that: in step (1), instead of preparing compound A-1, compound A-46 (ethyl acetylacetonate) is directly purchased and then reacted, and 1-methyl-5-aminopyrazole in step (2) is replaced by 2H-3-aminopyrazole, and the rest is the same as that of Example 1. The 1H-pyrazolo[3,4-b]pyridine derivative of Example 46 is prepared as a white solid with a melting point of 195-197°C and a structural formula of The structural analysis results are as follows: 1H NMR (400MHz, DMSO-d6) δ11.17(s,1H),9.63-9.46(m,1H),9.01(s,1H),8.39(d,J=2.4Hz,1H),7.71(d,J=8 .4Hz,2H),7.59(s,1H),7.40(d,J=8.3Hz,2H),6.91(d,J=2.5Hz,1H),4.54(d,J=6.4Hz,2H),2.83(s,3H). 13 C NMR (101MHz, DMSO-d6) δ164.60,163.65,149.01,148.04,147.13,146.01,142. 98,131.84,127.69,127.38,105.64,98.14,42.82,17.45.HR-MS(ESI),calcd.C 16 H 15 N5O3,[MH] - m / z:324.1102,found:324.1109.

[0135] Test Example 1

[0136] The inhibitory activity of the 1H-pyrazolo[3,4-b]pyridine derivatives obtained in Examples 1 to 46 on HDAC6 was determined at a concentration of 100 nM. Specifically: first, the compound was diluted in a gradient with the prepared HDAC6 buffer; then, bovine serum albumin, a fluorescent group with an HDAC6 substrate (Ac-Gly-Ala-Lys-AMC), and test compounds 1 to 46 were added to a 96-well plate, and incubated at 37°C. During the incubation process, the activity of HDAC6 will cause the fluorescent-labeled peptide to be deacetylated, releasing an AMC fluorescent fragment that can be hydrolyzed by Trypsin. Afterwards, an enzyme reader was used to measure the intensity of fluorescence in each well, the excitation wavelength was set to 355 nm, and the emission wavelength was set to 460 nm. By setting a series of different concentration gradients, the fluorescence intensity of the compound was measured, and the inhibition rate was calculated accordingly. GraphPad Prism 9.0 software was used for data analysis and curve fitting to determine the IC value of the compound. 50 The results are shown in Table 1. Inhibition rate (%) = (positive control test value - compound test value) / (positive control test value - negative control test value) × 100%.

[0137] Table 1 Inhibition rate of HDAC6 by compounds 1 to 46 prepared in Examples 1 to 46 at a concentration of 100 nM

[0138]

[0139] As shown in Table 1, the 1H-pyrazolo[3,4-b]pyridine derivatives provided by the present invention all have a certain HDAC6 inhibitory effect. In order to more accurately screen out compounds with high selectivity for HDAC6, the present invention further tested the HDAC6 inhibitory activity of some compounds with an inhibition rate greater than 80%, and calculated their IC 50 In addition, the inhibitory activity of these compounds against HDAC1 was measured. Most of the compounds had a certain degree of HDAC6 selectivity, and some of them had a higher HDAC6 selectivity. The results are shown in Table 2.

[0140] Table 2 Inhibitory effects of some compounds of the present invention on HDAC6 and HDAC1

[0141]

[0142] As shown in the test results in Table 2, compared with the positive control SAHA, the 1H-pyrazolo[3,4-b]pyridine derivatives provided by the present invention have poor inhibitory activity against HDAC1, but have good selective inhibitory effect on HDAC6.

[0143] Test Example 2

[0144] The compounds of the present invention that do not have obvious selectivity and those that have obvious selectivity (compounds corresponding to Examples 3, 15, 18, 24, 26, 28, 33, 34, and 37) were selected and tested for their proliferation inhibitory activity against two gastric cancer cell lines (MGC-803 and MKN-45) that highly express the HDAC6 protein using the MTT method. The assay method is as follows: Gastric cancer cells were cultured in DMEM medium containing 10% fetal bovine serum. The specific culture conditions were a constant temperature environment of 37°C, 5% CO2, and 90% relative humidity to allow cells to grow under optimal conditions. The cells were seeded on a 96-well plate at a density of 2500 cells per well and incubated in a cell culture incubator for 12 hours to allow the cells to attach. Subsequently, a pre-prepared stock solution of the test compound with different gradient concentrations was added to the 96-well plate at a volume of 200 μL per well and incubated in the incubator for 48 hours. Then, 20 μL of the prepared MTT solution was added. After 5 hours of incubation, the supernatant was removed and 200 μL of DMSO solution was added to each well. The wells were placed on a shaker and shaken at low speed for 15 minutes to ensure that the formed crystals were completely dissolved. Finally, the absorbance of each well was measured at a wavelength of 490 nm using a microplate reader. GraphPad Prism 9.0 software was used for data processing and curve fitting to calculate the IC 50 The results are shown in Table 3.

[0145] Table 3 IC values ​​of some compounds of the present invention against gastric cancer cells MGC-803 and MKN-45 50 value

[0146]

[0147] The test results in Table 3 show that, relative to MKN-45 gastric cancer cells, the 1H-pyrazolo[3,4-b]pyridine derivatives provided by the present invention have better inhibitory activity against gastric cancer cells MGC-803, and their inhibitory activity is better than that of the positive control SAHA.

[0148] In summary, the 1H-pyrazolo[3,4-b]pyridine derivatives provided by the present invention have good selective inhibitory effects on histone deacetylase 6 (HDAC6) and have good inhibitory activity against gastric cancer cells. They can be used to prepare anti-gastric cancer drugs, and are particularly suitable for HDAC6 small molecule inhibitors.

[0149] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A 1H-pyrazolo[3,4-b]pyridine derivative, characterized in that: It is a compound shown in formula I: In formula I, R1 is selected from one of hydrogen, methyl, ethyl, tert-butyl, and phenyl; R2 is selected from one of the following groups:

2. The 1H-pyrazolo[3,4-b]pyridine derivative according to claim 1, characterized in that: Selected from the following compounds:

3. A method for preparing a 1H-pyrazolo[3,4-b]pyridine derivative according to claim 1 or 2, characterized in that: The following steps are involved: Step (1): Mix the first raw material, diethyl oxalate, the first catalyst and the first good solvent, and perform a substitution reaction to obtain compound A, the structural formula of which is Alternatively, compound A is obtained directly without preparation, and the structural formula is Step (2): Compound A, substituted pyrazole and a second good solvent are mixed and subjected to a cyclization reaction to obtain compound B; the structural formula of compound A is The structural formula of compound B is Step (3): Compound B, the second catalyst and the third good solvent are mixed and subjected to a first hydrolysis reaction to obtain compound C having the structural formula Step (4): Compound C, methyl 4-aminomethylbenzoate hydrochloride, a third catalyst, a fourth catalyst and a fourth good solvent are mixed to carry out a first condensation reaction to obtain compound D having the structural formula: Step (5): Compound D, the second catalyst and the third good solvent are mixed and subjected to a second hydrolysis reaction to obtain compound E, the structural formula of which is Step (6): Compound E, O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine, a third catalyst, a fourth catalyst, and a fifth good solvent are mixed to carry out a second condensation reaction to obtain a 1H-pyrazolo[3,4-b]pyridine derivative having a structure shown in Formula I; The first raw material is one of 4-methylacetophenone, 3-methylacetophenone, 2-methylacetophenone, 4-chloroacetophenone, acetophenone, 3-chloroacetophenone, 2-chloroacetophenone, 4-bromoacetophenone, 4-methoxyacetophenone, 4-ethylacetophenone, 4-isopropylacetophenone, 4-dimethylaminoacetophenone, 4-hydroxyacetophenone, 4-trifluoromethylacetophenone, 3-acetylthiophene, 2-acetylfuran, 1-acetonaphthone, 2-acetonaphthone, acetylcyclohexane, acetylcyclopentane, acetylcyclopropane, 4-fluoroacetophenone, 2-fluoroacetophenone, 3-fluoroacetophenone, 2-methoxyacetophenone, and 3-methoxyacetophenone.

4. The method for preparing 1H-pyrazolo[3,4-b]pyridine derivatives according to claim 3, characterized in that: The substituted pyrazole is one of 1-methyl-5-aminopyrazole, 1-ethyl-5-aminopyrazole, 1-tert-butyl-5-aminopyrazole, 1-phenyl-5-aminopyrazole, and 2H-3-aminopyrazole.

5. The method for preparing 1H-pyrazolo[3,4-b]pyridine derivatives according to claim 3, characterized in that: The temperature of the substitution reaction is 25-55°C, and the reaction time is 7-12 hours; the temperature of the cyclization reaction is 90-120°C, and the reaction time is 2-6 hours; the temperature of the first hydrolysis reaction and the second hydrolysis reaction is 50-75°C, and the reaction time is 0.5-4 hours; the temperature of the first condensation reaction and the second condensation reaction is 15-30°C, and the reaction time is 2-10 hours.

6. The method for preparing 1H-pyrazolo[3,4-b]pyridine derivatives according to claim 3, characterized in that: The first good solvent, the second good solvent, the third good solvent, the fourth good solvent and the fifth good solvent are each independently selected from one or more of tetrahydrofuran, toluene, ethanol, acetic acid, methanol, dichloromethane, acetonitrile, N,N-dimethylformamide and water.

7. The method for preparing 1H-pyrazolo[3,4-b]pyridine derivatives according to claim 3, characterized in that: The first catalyst is selected from one or more of potassium tert-butoxide, sodium hydride, sodium methoxide, and sodium ethoxide; the second catalyst is selected from one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide; the third catalyst is selected from one or more of HATU, HBTU, and EDCI; and the fourth catalyst is selected from one or more of TEA, DIPEA, HOBT, and DMAP.

8. Use of the 1H-pyrazolo[3,4-b]pyridine derivative according to claim 1 or 2, characterized in that: Application in the preparation of anti-gastric cancer drugs.

9. The use of 1H-pyrazolo[3,4-b]pyridine derivatives according to claim 8, characterized in that: The anti-gastric cancer drug exerts its effect by inhibiting the activity of gastric cancer cells; the gastric cancer cells are MGC-803 and / or MKN-45.

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