Pyrazole amide compounds and preparation methods and applications thereof

Abstract

The present invention discloses a pyrazole amide compound and a preparation method and application thereof, and relates to the technical field of pharmaceutical chemistry. The pyrazole amide compound includes a compound as shown in formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof. The pyrazole amide compound involved in the present invention can effectively remove free radicals to play an antioxidant role and has a neuron protective effect. The pyrazole amide compound provided by the present invention can be used in the preparation of medicines for preventing or treating stroke, cardiovascular and cerebrovascular diseases, neurodegenerative diseases and complications thereof caused by excessive free radicals.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a pyrazole amide compound and a preparation method and application thereof. Background Art

[0002] Normal cellular aerobic metabolism produces oxygen- and nitrogen-containing free radicals, including superoxide anion free radicals, nitric oxide free radicals, etc. Although oxygen is essential for the survival of aerobic organisms, it also leads to the production of free radicals, especially reactive oxygen species (ROS) in biochemical processes, which are lethal. These ROS are free radicals that trigger a series of biochemical reactions that damage cellular components, leading to cell necrosis, and ultimately damage cellular components, leading to cell necrosis, ultimately leading to various pathological conditions, including liver and vascular diseases, inflammatory diseases, rheumatoid arthritis, oxidative stress, cancer, and aging. The brain is one of the organs with the highest oxygen-glucose ratio and the most active metabolism in the body. Therefore, brain tissues such as neurons and glial cells are more susceptible to the production of free radicals, which in turn leads to neuronal damage, which may further induce neurodegenerative diseases.

[0003] Cerebral vascular embolism causes ischemia and hypoxia of brain tissue, which in turn triggers a cascade of ischemic damage to brain tissue. Reperfusion after ischemia will induce a series of neuroinflammatory cascade reactions, further aggravating brain tissue death and blood-brain barrier damage. Blood reperfusion causes a sharp increase in oxygen content, releasing a large number of cytotoxic reactive oxygen free radicals, inducing the death of some neurons. Under the multiple effects of inflammatory mediators and a large amount of ROS enrichment, glial cells transform into proinflammatory types, causing further deterioration of inflammation. Therefore, the occurrence of neuroinflammation is spatiotemporally specific to ischemic brain damage and is closely related to glial cell activation and polarization.

[0004] When pyrazolone compounds exist in the form of isoanions, they can donate an electron in the structure to free radicals, thereby achieving the purpose of free radical scavenging. Among them, the pyrazolone compound Edaravone is a free radical scavenger. It was first launched in Japan in April 2001 and is mainly used clinically to treat ischemic stroke. Because its molecular structure contains lipophilic groups, it is easy to pass through the blood-brain barrier to reach brain tissue, and has a good protective effect on nerve cell damage caused by cerebral ischemia. Edaravone inhibits lipid peroxidation by scavenging oxygen free radicals, regulates inflammatory factors and inhibits cell apoptosis to achieve protection of nerve cells. Studies have shown that in addition to treating acute cerebral ischemia, Edaravone has a therapeutic effect on other diseases of the nervous system that involve oxidative stress. However, the neuroprotective effect of Edaravone basically depends on the free radical scavenging effect, so it is necessary to develop a drug that exerts a neuroprotective effect through multiple pathways. Summary of the invention

[0005] In view of the problem that the neuroprotective effect of edaravone in the prior art is relatively single, the purpose of the present invention is to provide a pyrazole amide compound and a preparation method and application thereof, which can be a drug that can exert neuroprotective effects in multiple pathways.

[0006] One of the purposes of the present invention is to provide a pyrazole amide compound, which comprises a compound as shown in formula (I) or a pharmaceutically acceptable salt, isomer or prodrug thereof:

[0007]

[0008] Wherein, Ar1 is selected from alkyl, alkoxy, and halogen substituents whose aromatic rings are substituted by different numbers and positions, and preferably Ar1 is:

[0009] X is selected from NH(CH2) n A group, wherein n is selected from an integer of 0 to 3;

[0010] Ar2 is selected from

[0011] The pyrazole amide compound is selected from any one of the following compounds or pharmaceutically acceptable salts or solvates thereof:

[0012] 5-Hydroxy-N-(6-methylpyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0013] 5-Hydroxy-N-(4-(hydroxymethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0014] 5-Hydroxy-N-(3-(hydroxymethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0015] N-(1,3-dihydroisobenzofuran-5-yl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0016] 5-Hydroxy-N-((6-methylpyridin-3-yl)methyl)-1-phenyl-1H-pyrazole-3-carboxamide;

[0017] 5-Hydroxy-N-(4-(hydroxymethyl)phenyl)-1-phenyl-1H-pyrazole-3-carboxamide;

[0018] N-(2-(Benzo[d][1,3]dioxol-4-yl)ethyl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0019] N-(3,5-dichlorobenzyl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0020] 5-Hydroxy-1-(pyridin-2-yl)-N-(pyridin-4-ylmethyl)-1H-pyrazole-3-carboxamide;

[0021] 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0022] 5-Hydroxy-N-(6-(hydroxymethyl)pyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0023] 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(4-(hydroxymethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0024] 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(6-(hydroxymethyl)pyridin-3-yl)-1H-pyrazole-3-carboxamide;

[0025] 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0026] N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-1-(5-fluoropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxamide;

[0027] N-(Benzo[d][1,3]dioxol-5-yl)-1-(5-fluoropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxamide;

[0028] 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(2-methylpyridin-4-yl)-1H-pyrazole-3-carboxamide;

[0029] 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(3-(hydroxymethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0030] 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0031] 1-(5-chloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0032] 1-(5-chloropyridin-2-yl)-5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0033] 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(pyrimidin-2-yl)-1H-pyrazole-3-carboxamide;

[0034] 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(3,5,6-trimethylpyrazin-2-yl)-1H-pyrazole-3-carboxamide;

[0035] 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0036] 5-Hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0037] 5-Hydroxy-N-(2-hydroxy-1-phenylethyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0038] 1-(5-fluoropyrimidin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0039] 1-(3,5-dichloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0040] 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(3-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide;

[0041] 1-(3-chloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide;

[0042] 1-(4,6-dimethylpyrimidin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide.

[0043] The second object of the present invention is to provide a method for preparing pyrazole amide compounds, comprising the following steps:

[0044] (1) Compound 1 is dissolved in methanol to prepare a solution with a molar concentration of 0.85 to 1.5 mol / L, and a methanol solution containing 0.9 to 1.5 eq of dimethyl butynedioate is added dropwise under ice bath conditions, and then 1.0 to 2.0 eq of sodium methoxide is added to the reaction solution, and the mixture is heated under reflux to react to obtain Compound 2;

[0045] (2) Compound 2 is dissolved in a mixed solution of methanol and tetrahydrofuran in a volume ratio of 4:3 to prepare a solution with a molar concentration of 0.3 to 0.6 mol / L, and 1.5 to 2.5 eq of a 2 mol / L sodium hydroxide aqueous solution is added dropwise under an ice bath to react and obtain Compound 3;

[0046] (3) Compound 3 is dissolved in N,N-dimethylformamide to prepare a solution with a molar concentration of 0.05-0.1 mol / L, and then 2.0-3.5 eq of N,N-diisopropylethylamine and 1.0-1.5 eq of condensing agent O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate are added to the reaction solution, and finally 1.0-1.5 eq of the corresponding amine compound is added to the reaction solution to prepare pyrazole amide compound 4 (I);

[0047] Wherein, the amine compound includes 6-methylpyridin-3-amine, (4-aminophenyl)methanol, (3-aminophenyl)methanol, 1,3-dihydroisobenzofuran-5-amine, 3,4-methylenedioxyphenethylamine, 3,5-dichlorobenzylamine, 4-methylaminopyridine, 2-(4-aminophenyl)ethanol, 2-(hydroxy)-5-aminopyridine, p-aminobenzyl alcohol, 3,4-methylenedioxyphenethylamine, 3,4-methylenedioxyaniline, 2-methyl-4-aminopyridine, 3-aminobenzyl alcohol, 2-(3-aminophenyl)ethanol or DL-phenylglycinol;

[0048] The synthetic route of the pyrazole amide compound 4 (I) is as follows:

[0049]

[0050] The third object of the present invention is to provide a pharmaceutical composition comprising the pyrazole amide compound, wherein the pyrazole amide compound is added with one or more pharmaceutically acceptable salts, isomers, prodrugs, polymorphs or solvent compounds.

[0051] Furthermore, the pharmaceutical composition is in the form of capsules, pills, tablets, granules or injections.

[0052] The fourth object of the present invention is to propose the application of the pyrazole amide compounds, including application in free radical scavenging, application in neuronal cell protection, and application in the preparation of medicines for preventing or treating or assisting in the treatment of stroke, cardiovascular and cerebrovascular diseases, neurodegenerative diseases and their complications caused by excessive free radicals.

[0053] Beneficial effects:

[0054] Compared with the existing technology, the present invention has the following significant characteristics: the pyrazole amide compounds proposed in the present invention can effectively remove free radicals and improve neuronal cell damage caused by peroxides. Therefore, the compounds play an important role in the preparation of prevention or treatment of stroke, cardiovascular and cerebrovascular diseases, and neurodegenerative diseases caused by excessive free radicals.

[0055] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is the ORAC antioxidant capacity characterization diagram of the target compound;

[0057] Figure 2A and 2B It is a characterization diagram of the chelating ability of the target compound with different metals;

[0058] Figure 3 This is a characterization diagram of the protective effect of the target compound on human neuroblastoma cells (SH-SY5Y) damaged by tert-butyl hydroperoxide (tBHP);

[0059] Figure 4 The figure represents the protective effect against oxygen glucose deprivation / reoxygenation (OGD / R)-induced microglial (BV2) injury;

[0060] Figure 5 The results show the effects of the target compounds on the level of reactive oxygen species (ROS) induced by tBHP. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The solvents used in the examples of the present invention are all commercially available chemically pure or analytically pure. The structures of the compounds were determined by nuclear magnetic resonance (NMR). NMR was measured using a Bruker AVANCE-300 / 500 NMR spectrometer, with CDCl3 or DMSO-d6 as the solvent and TMS as the internal standard.

[0063] The present invention provides a method for preparing a pyrazole amide compound, comprising the following steps:

[0064] (1) Compound 1 is dissolved in methanol to prepare a solution with a molar concentration of 0.85 to 1.5 mol / L, and a methanol solution containing 0.9 to 1.5 eq of dimethyl butynedioate is added dropwise under ice bath conditions, and then 1.0 to 2.0 eq of sodium methoxide is added to the reaction solution, and the mixture is heated under reflux to react to obtain Compound 2;

[0065] (2) Compound 2 is dissolved in a mixed solution of methanol and tetrahydrofuran in a volume ratio of 4:3 to prepare a solution with a molar concentration of 0.3 to 0.6 mol / L, and 1.5 to 2.5 eq of a 2 mol / L sodium hydroxide aqueous solution is added dropwise under an ice bath to react and obtain Compound 3;

[0066] (3) Compound 3 is dissolved in N,N-dimethylformamide to prepare a solution with a molar concentration of 0.05-0.1 mol / L, and then 2.0-3.5 eq of N,N-diisopropylethylamine and 1.0-1.5 eq of condensation agent O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate are added to the reaction solution. Finally, 1.0-1.5 eq of the corresponding amine compound is added to the reaction solution to prepare pyrazole amide compound 4(I).

[0067] Wherein, the amine compound includes 6-methylpyridin-3-amine, (4-aminophenyl)methanol, (3-aminophenyl)methanol, 1,3-dihydroisobenzofuran-5-amine, 3,4-methylenedioxyphenethylamine, 3,5-dichlorobenzylamine, 4-methylaminopyridine, 2-(4-aminophenyl)ethanol, 2-(hydroxy)-5-aminopyridine, p-aminobenzyl alcohol, 3,4-methylenedioxyphenethylamine, 3,4-methylenedioxyaniline, 2-methyl-4-aminopyridine, 3-aminobenzyl alcohol, 2-(3-aminophenyl)ethanol or DL-phenylglycinol;

[0068] The synthetic route of the pyrazole amide compounds proposed by the present invention is as follows:

[0069]

[0070] Wherein, Ar1 is selected from alkyl, alkoxy, and halogen substituents whose aromatic rings are substituted by different numbers and positions, and preferably Ar1 is:

[0071] X is selected from NH(CH2) n A group, wherein n is selected from an integer of 0 to 3;

[0072] Ar2 is selected from Any one of .

[0073] The preparation method of the above pyrazole amide compounds is exemplified below in conjunction with specific examples.

[0074] Example 1: Preparation of compound 5-hydroxy-1-phenyl-1H-pyrazole-3-carboxylic acid methyl ester (2a)

[0075]

[0076] Phenylhydrazine (1a, 12 g, 110 mmol) was dissolved in methanol (120 mL), and a solution of dimethyl butynedioate (15.2 mL, 124 mmol) in methanol (80 mL) was added dropwise under ice bath conditions. After 3 hours of low temperature reaction, sodium methoxide (9.2 g, 172 mmol) was added to the reaction solution, and refluxed at 70°C for 1 hour. After the reaction was completed, the reaction solution was concentrated to remove methanol, and 80 mL of water was added. Under ice bath conditions, 2 mol / L HCl was added dropwise until the pH of the reaction solution was 4, and a yellow precipitate was precipitated. The filter cake was washed with water and dried to obtain a yellow powder 2a with a yield of 60%.

[0077] Example 2: Preparation of compound 5-hydroxy-1-phenylpyrazole-3-carboxylic acid (3a)

[0078]

[0079] Methyl 5-hydroxy-1-phenyl-1H-pyrazole-3-carboxylate (2a, 17.4 g, 80 mmol) was dissolved in a mixture of methanol (120 mL) and tetrahydrofuran (90 mL). 2 mol / L NaOH (90 mL, 180 mmol) was added dropwise under ice bath, and the reaction solution was refluxed at 40°C for 5 hours. After the reaction was completed, 2 equivalents of water were added, the reaction solution was concentrated to remove methanol, and 2 mol / L HCl was added dropwise under ice bath until the pH of the reaction solution was 4. A large amount of light yellow solid precipitated, which was filtered by suction, and the filter cake was washed with water and dried to obtain light yellow solid 3a with a yield of 65%.

[0080] Example 3: Preparation of compound 5-hydroxy-1-pyridin-2-ylpyrazole-3-carboxylic acid (3b)

[0081]

[0082] Intermediate 3b is prepared by replacing phenylhydrazine (1a) with 2-hydrazinepyridine (1b) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is a yellow powder 3b with a yield of 55%. 1H NMR (300MHz, DMSO-d6) δ8.53 (ddd, J=5.0, 1.9, 0.9Hz, 1H), 8.09 (ddd, J=8.3, 7.5, 1.9 Hz, 1H), 7.81 (dt, J = 8.3, 1.0 Hz, 1H), 7.46 (ddd, J = 7.5, 5.0, 1.1 Hz, 1H), 5.99 (s, 1H).

[0083] Example 4: Preparation of compound 1-(5-chloropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxylic acid (3c)

[0084]

[0085] Intermediate 3c is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-5-fluoropyridine (1c) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is a yellow solid 3c with a yield of 58%. Melting point: 229.3°C-232.7°C. 1 HNMR (600MHz, DMSO-d6) δ 8.56 (d, J = 3.0 Hz, 1H), 7.99 (td, J = 8.6, 3.1 Hz, 1H), 7.80 (dd, J = 9.0, 3.9 Hz, 1H), 5.95 (s, 1H).

[0086] Example 5: Preparation of compound 1-(5-chloropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxylic acid (3d)

[0087]

[0088] Intermediate 3d is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-5-chloropyridine (1d) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is a dark yellow solid 3d with a yield of 70%. Melting point: 216.2°C-220.5°C. 1 H NMR (600MHz, DMSO-d6) δ12.14(s,1H),8.60(d,J=2.5Hz,1H),8.16(dd,J=8.7,2.6Hz,1H),7.80(d,J=8.7Hz,1H),5.98(s,1H).

[0089] Example 6: Preparation of compound 5-hydroxy-1-(pyrimidin-2-yl)-1H-pyrazole-3-carboxylic acid (3e)

[0090]

[0091] Intermediate 3e is prepared by replacing phenylhydrazine (1a) with 2-hydrazinopyrimidine (1e) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is an orange flaky solid 3e with a yield of 63%. Melting point: 162.4°C-172.9°C. 1 HNMR (600MHz, DMSO-d6) δ12.94 (s, 1H), 12.02 (s, 1H), 8.95 (d, J = 4.9Hz, 2H), 7.60 (t, J = 4.9Hz, 1H), 5.98 (s, 1H).

[0092] Example 7: Preparation of compound 5-hydroxy-1-(3,5,6-trimethylpyrazine-2-yl)-1H-pyrazole-3-carboxylic acid (3f)

[0093]

[0094] Intermediate 3f is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-3,5,6-trimethylpyrazine (1f) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is a yellow powder 3f with a yield of 55%.

[0095] Example 8: Preparation of compound 5-hydroxy-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxylic acid (3 g)

[0096]

[0097] Intermediate 3g is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-5-trifluoromethylpyridine (1g) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is an orange-red powder of 3g with a yield of 65%. Melting point: 137.4°C-144.5°C. 1 H NMR (600MHz, DMSO-d6) δ 13.05 (s, 1H), 12.31 (s, 1H), 8.95 (dt, J = 2.8, 1.0Hz, 1H), 8.44 (dd, J = 8.8, 2.5Hz, 1H), 8.02 (d, J = 8.6Hz, 1H), 6.01 (s, 1H).

[0098] Example 9: 1-(3-chloropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxylic acid (3h)

[0099]

[0100] Intermediate 3h is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-3-chloropyridine (1h), reacting with dimethyl butynedioate, and then hydrolyzing. The reaction process is similar to 3a, and the product is an off-white powder 3h with a yield of 60%.

[0101] Example 10: 5-Hydroxy-1-(3-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxylic acid (3i)

[0102]

[0103] Intermediate 3i is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-3-trifluoromethylpyridine (1i), reacting with dimethyl butynedioate, and then hydrolyzing. The reaction process is similar to 3a, and the product is a purple powder 3i with a yield of 65%.

[0104] Example 11: Preparation of compound 1-(5-fluoropyrimidin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxylic acid (3j)

[0105]

[0106] Intermediate 3j is prepared by replacing phenylhydrazine (1a) with 5-fluoro-2-hydrazinopyrimidine 1j, reacting with dimethyl butynedioate, and then hydrolyzing. The reaction process is similar to 3a, and the product is a yellow solid 3j with a yield of 70%.

[0107] Example 12: Preparation of compound 1-(3,5-dichloropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxylic acid (3k)

[0108]

[0109] Intermediate 3k is prepared by replacing phenylhydrazine (1a) with 3,5-dichloro-2-hydrazinopyridine (1k) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is a yellowish brown solid 3k with a yield of 73%.

[0110] Example 13: Preparation of compound 1-(4,6-dimethylpyrimidin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxylic acid (31)

[0111]

[0112] Intermediate 3l is prepared by replacing phenylhydrazine (1a) with 2-hydrazine-4,6-dimethylpyrimidine (1l) and reacting with dimethyl butynedioate, followed by hydrolysis. The reaction process is similar to 3a, and the product is a white solid 3l with a yield of 80%.

[0113] Example 14: Preparation of compound 5-hydroxy-N-(6-methylpyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-1)

[0114]

[0115] Compound 3b (205 mg, 1 mmol) was dissolved in DMF (15 mL), and DIPEA (704 μL, 4 mmol) was added. HATU (448 mg, 1.08 mmol) was added under ice bath, and compound 6-methylpyridin-3-amine (108 mg, 1 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain a yellowish brown powder 4-1 (190 mg), with a yield of 64%. Melting point: 169.3°C-172.4°C. 1 H NMR (300MHz, DMSO-d6) δ10.51(s,1H),9.00(d,J=2.5Hz,1H),8.58(dd,J=5.0,1.7Hz,1H),8.35(dd,J=8.6,2.5Hz,1H ),8.15(td,J=7.9,1.9Hz,1H),7.95(d,J=8.2Hz,1H),7.53(s,1H),7.50(d,J=3.6Hz,1H),6.11(s,1H),2.55(s,3H); 13 C NMR(75MHz,DMSO-d6)δ160.85,155.82,151.99,151.26,147.99,146.51,140.41, 137.92,134.48,131.85,125.22,123.21,116.11,88.47,21.97; HRMS(m / z):calcd for C 15 H 14 N5O2[M+H] + ,295.1190;found,295.1187.

[0116] Example 15: Preparation of compound 5-hydroxy-N-(4-(hydroxymethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-2)

[0117]

[0118] Compound 3b (205 mg, 1 mmol) was dissolved in DMF (15 mL), and DIPEA (528 μL, 3 mmol) was added. HATU (414 mg, 1 mmol) was added under ice bath, and compound (4-aminophenyl)methanol (123 mg, 1 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain a light yellow powder 4-2 (200 mg), with a yield of 64%. Melting point: 149.4°C-154.7°C. 1H NMR (300MHz, DMSO-d6) δ10.02(s,1H),8.56-8.52(m,1H),8.17-8.09(m,1H),7.99(d,J=8.2Hz,1H),7. 76(d,J=8.5Hz,2H),7.51-7.44(m,1H),7.29(d,J=8.5Hz,2H),6.08(s,1H),5.15(s,1H),4.47(s,2H). 13 C NMR (75MHz, DMSO-d6) δ160.19,155.94,152.39,147.68,147.38,140.53,138.32,137.56,127.31,122.85,120.48,115.51,88.34,63.11.

[0119] Example 16: Preparation of compound 5-hydroxy-N-(3-(hydroxymethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-3)

[0120]

[0121] Compound 3b (205 mg, 1 mmol) was dissolved in DMF (15 mL), and DIPEA (528 μL, 3 mmol) was added. HATU (1.45 g, 3.5 mmol) was added under ice bath, and compound (3-aminophenyl)methanol (123 mg, 1 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain gray-brown solid 4-3 (180 mg), with a yield of 58%. Melting point: 160.8°C-165.8°C. 1 H NMR (300MHz, DMSO-d6) δ10.02(s,1H),8.58-8.52(m,1H),8.17-8.09(m,1H),8.01(d,J=8.2Hz,1H),7.79(s,1H),7.69(d ,J=8.0Hz,1H),7.52-7.44(m,1H),7.30(t,J=7.8Hz,1H),7.06(d,J=7.6Hz,1H),6.09(s,1H),5.24(s,1H),4.50(s,2H). 13 C NMR(75MHz,DMSO-d6)δ160.21,155.93,152.46,147.62,147.34,143.59,140 .56,138.82,128.78,122.83,122.27,119.08,118.83,115.40,88.37,63.39.

[0122] Example 17: Preparation of Compound N-(1,3-dihydroisobenzofuran-5-yl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-4)

[0123]

[0124] Compound 3b (205 mg, 1 mmol) was dissolved in DMF (15 mL), and DIPEA (440 μL, 2.5 mmol) was added. HATU (448 mg, 1.08 mmol) was added under ice bath, and compound 1,3-dihydroisobenzofuran-5-amine (135 mg, 1 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain white powder 4-4 (150 mg), with a yield of 46%. Melting point: 188.8°C-190.2°C. 1 H NMR (300MHz, DMSO-d6) δ10.10(s,1H),8.55(ddd,J=4.9,1.8,0.8Hz,1H),8.13(ddd,J=8.2,7.5,1.8Hz,1H),7.98(d,J=8.3Hz,1H),7.8 2(s,1H),7.66(dd,J=8.2,1.7Hz,1H),7.48(ddd,J=7.4,5.0,1.0Hz,1H),7.28(d,J=8.2Hz,1H),6.08(s,1H),5.01(s,2H),4.99(s,2H). 13 CNMR(75MHz,DMSO-d6)δ160.27,155.89,152.37,147.69,147.30,140.52,139.98 ,138.23,134.55,122.88,121.60,119.99,115.54,113.40,88.37,73.07,72.86.

[0125] Example 18: Preparation of compound 5-hydroxy-N-((6-methylpyridin-3-yl)methyl)-1-phenyl-1H-pyrazole-3-carboxamide (4-5)

[0126]

[0127] Compound 3a (204 mg, 1 mmol) was dissolved in DMF (15 mL), and DIPEA (528 μL, 3 mmol) was added. HATU (448 mg, 1.08 mmol) was added under ice bath, and compound 6-methylpyridin-3-amine (108 mg, 1 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain light yellow powder 4-5 (180 mg), with a yield of 61%. Melting point: 171.1°C-195.6°C. 1 H NMR (300MHz, DMSO-d6) δ10.26(s,1H),8.90(d,J=2.3Hz,1H),8.19(dd,J=8.5,2.5Hz,1H),7.85(m,J=8.5,1 .0Hz,2H),7.57-7.50(m,2H),7.40(m,J=8.0,1.4Hz,1H),7.35(d,J=8.6Hz,1H),6.04(s,1H),2.48(s,3H). 13 C NMR (75MHz, DMSO-d6) δ161.04,154.38,151.89,145.69,139.52,138.58,134.07,130.29,129.46,127.43,124.27,122.75,88.22,22.76.

[0128] Example 19: Preparation of compound 5-hydroxy-N-(4-(hydroxymethyl)phenyl)-1-phenyl-1H-pyrazole-3-carboxamide (4-6)

[0129]

[0130] Compound 3a (204 mg, 1 mmol) was dissolved in DMF (15 mL), and DIPEA (528 μL, 3 mmol) was added. HATU (448 mg, 1.08 mmol) was added under ice bath, and compound (4-aminophenyl)methanol (123 mg, 1 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain yellow powder 4-6 (195 mg), with a yield of 63%. Melting point: 174.5°C-182.6°C. 1H NMR (300MHz, DMSO-d6) δ9.93(s,1H),7.88(d,J=7.9Hz,2H),7.77(d,J=8.1Hz,2H),7.54(t,J=7.8 Hz,2H),7.40(d,J=7.2Hz,1H),7.31(s,1H),7.28(s,1H),6.04(s,1H),5.16(s,1H),4.48(s,2H). 13 C NMR(75MHz,DMSO-d6)δ160.56,154.29,146.32,138.71,138.13,137.70,129.44,127.29,127.24,122.57,120.43,88.11,63.12; HRMS(m / z):calcd for C 17 H 16 N3O3[M+H] + ,310.1186;found,310.1185.

[0131] Example 20: Preparation of Compound N-(2-(Benzo[d][1,3]dioxol-4-yl)ethyl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-7)

[0132]

[0133] Compound 3b (410 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (696 μL, 4 mmol) was added. HATU (912 mg, 2.4 mmol) was added under ice bath, and compound 3,4-methylenedioxyphenylethylamine (375 μL, 2.6 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain a light yellow solid 4-7 (150 mg), with a yield of 21%. Melting point: 79.5-87.8°C. 1 H NMR (600MHz, CDCl3) δ8.32(d,J=5.1Hz,1H),7.93(d,J=7.2Hz,1H),7.90(d,J=8.0Hz,1H),7.27-7.25(m,1H),7.03(d,J=6.5Hz,1H),6.77-6 .74(m,2H),6.71-6.68(m,2H),6.07(d,J=4.7Hz,1H),5.94(s,1H),5.92(d,J=7.0Hz,1H),3.62(p,J=6.0,5.2Hz,2H),2.85(t,J=7.0Hz,2H). 13C NMR (151MHz, CDCl3) δ161.63,157.20,154.08,147.79,147.57,146.17,145.38,140 .20,132.72,121.76,121.04,112.40,109.23,108.35,100.89,88.33,40.61,35.67.

[0134] Example 21: Preparation of Compound N-(3,5-dichlorobenzyl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-8)

[0135]

[0136] Compound 3b (410 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (696 μL, 4 mmol) was added. HATU (912 mg, 2.4 mmol) was added under ice bath, and compound 3,5-dichlorobenzylamine (321 μL, 2.6 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain white solid 4-8 (210 mg), with a yield of 30%. Melting point: 122.3°C-132.5°C. 1 H NMR (600MHz, CDCl3) δ12.73(s,1H),8.33(d,J=5.1Hz,1H),7.93(d,J=8.0Hz,2H),7.32(t ,J=6.3Hz,1H),7.28-7.26(m,1H),7.25-7.23(m,2H),6.16(s,1H),4.58(d,J=6.3Hz,2H). 13 C NMR (151MHz, CDCl3) δ161.68,157.36,154.01,147.03,145.44,141.82,140.25,135.18,127.64,126.14,121.19,112.44,88.66,42.11.

[0137] Example 22: Preparation of compound 5-hydroxy-1-(pyridin-2-yl)-N-(pyridin-4-ylmethyl)-1H-pyrazole-3-carboxamide (4-9)

[0138]

[0139] Compound 3b (410 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (696 μL, 4 mmol) was added. HATU (912 mg, 2.4 mmol) was added under ice bath, and compound 4-methylaminopyridine (263 μL, 2.6 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (30 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain white solid 4-9 (218 mg), with a yield of 37%. Melting point: 99.5°C-101.7°C. 1 H NMR (600MHz, CDCl3) δ8.57(d,J=6.1Hz,2H),8.34(d,J=5.1Hz,1H),7.95-7.90(m,2H),7.42(t ,J=6.3Hz,1H),7.30(d,J=6.0Hz,2H),7.28-7.26(m,1H),6.16(s,1H),4.66(d,J=6.3Hz,2H); 13 C NMR(151MHz, CDCl3)δ161.93,157.37,153.99,149.75(×2),147.81,146.99,1 45.49,140.26,122.42(×2),121.24,112.44,88.65,41.94; HRMS(m / z):calcd for C 15 H 12 N5O2[MH] - ,294.0996;found,294.1026.

[0140] Example 23: Preparation of compound 5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-10)

[0141]

[0142] Compound 3b (410 mg, 2 mmol) was dissolved in DMF (5 mL), and DIPEA (528 μL, 3 mmol) was added. HATU (912 mg, 2 mmol) was added under ice bath, and compound (2-(4-aminophenyl)ethanol (328.96 mg, 2.4 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain yellow solid 4-10 (170 mg) with a yield of 27.5%. Melting point: 125.5°C-132.7°C. 1H NMR (600MHz, DMSO-d6) δ9.96(s,1H),8.54(dd,J=5.1,1.6Hz,1H),8.12(td,J=7.9,1.8Hz,1H),7.98(d,J=8.2Hz,1H),7.70(d,J=8.2 Hz,2H),7.47(dd,J=7.4,5.0Hz,1H),7.19(d,J=8.2Hz,2H),6.08(s,1H),4.65(s,1H),3.60(t,J=7.1Hz,2H),2.70(t,J=7.1Hz,2H). 13 CNMR(151MHz,DMSO-d6)δ160.12,155.93,152.36,147.71,147.40,140.48,136.86 ,135.35,129.43,122.85,120.68,115.58,88.33,62.70,38.97; HRMS(m / z):calcd for C 17 H 17 N4O3[M+H] + ,325.1295;found,325.1291.

[0143] Example 24: Preparation of compound 5-hydroxy-N-(6-(hydroxymethyl)pyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-11)

[0144]

[0145] Compound 3b (410 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (528 μL, 3 mmol) was added. HATU (836 mg, 2.2 mmol) was added under ice bath, and compound 2-(hydroxy)-5-aminopyridine (272.88 mg, 2.2 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain yellow solid 4-11 (185 mg), with a yield of 29%. Melting point: 169.3°C-174.7°C. 1H NMR (600MHz, DMSO-d6) δ10.29(s,1H),8.89(d,J=2.4Hz,1H),8.56(d,J=5.7Hz,1H),8.21(dd,J=8.5,2.5Hz,1H),8.13 (td,J=7.8,1.8Hz,1H),7.97(d,J=8.2Hz,1H),7.49(t,J=5.7Hz,1H),7.46(d,J=8.5Hz,1H),6.10(s,1H),4.55(s,2H). 13 C NMR(151MHz,DMSO-d6)δ160.67,157.22,155.85,152.22,147.82,146.91,141.35,1 40.46,134.04,128.56,123.01,120.51,115.79,88.41,64.45; HRMS(m / z):calcdfor C 15 H 14 N5O3[M+H] + ,312.1091;found,312.1086.

[0146] Example 25: Preparation of compound 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(4-(hydroxymethyl)phenyl)-1H-pyrazole-3-carboxamide (4-12)

[0147]

[0148] Compound 3c (223 mg, 1 mmol) was dissolved in DMF (5 mL), and DIPEA (216 μL, 1.2 mmol) was added. HATU (760 mg, 3 mmol) was added under ice bath, and compound p-aminobenzyl alcohol (184.60 mg, 1.2 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain off-white solid 4-12 (200 mg), with a yield of 60%. Melting point: 167.5℃-170.4℃. 1 H NMR (600MHz, DMSO-d6) δ9.95 (s, 1H), 8.57 (d, J = 3.0Hz, 1H), 8.02 (td, J = 8.5, 3.0Hz, 1H), 7.97 (dd, J = 9.0,4.1Hz,1H),7.75(d,J=8.2Hz,2H),7.27(d,J=8.2Hz,2H),5.99(s,1H),5.13(s,1H),4.46(s,2H); 13C NMR (151MHz, DMSO) δ160.39, 158.23 (d, J = 252.5Hz), 155.70, 147.88, 147.17, 138.20, 137.63, 136.17 (d, J=26.0Hz),127.27,126.90(d,J=20.5Hz),120.44,119.06(d,J=5.4Hz),87.98,63.11; HRMS(m / z):calcd for C 16 H 14 FN4O3[M+H] + ,329.1044;found,329.1044.

[0149] Example 26: Preparation of Compound 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(6-(hydroxymethyl)pyridin-3-yl)-1H-pyrazole-3-carboxamide (4-13)

[0150]

[0151] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (418 μL, 2.4 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(hydroxymethyl)-5-aminopyridine (297.73 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered, and the filter cake was subjected to silica gel column chromatography to obtain yellow solid 4-13 (210 mg), with a yield of 32%. Melting point: 190.1°C-200.3°C. 1 H NMR (600MHz, DMSO-d6) δ12.12(s,1H),10.29(s,1H),8.88(d,J=2.5Hz,1H),8.59(d,J=3.0Hz,1H),8.20(dd,J=8.5,2.5Hz,1 H),8.05(td,J=8.6,3.0Hz,1H),7.93(dd,J=9.0,4.0Hz,1H),7.45(d,J=8.4Hz,1H),6.05(s,1H),5.38(s,1H),4.54(s,2H). 13C NMR (151MHz, DMSO-d6) δ160.76,158.40(d,J=252.9Hz),157.13,155.08,147.64,146.71,141.35,136.34(d,J= 26.1Hz), 134.09, 128.54, 126.97 (d, J = 20.2Hz), 120.46, 119.40 (d, J = 5.4Hz), 88.26, 64.43; HRMS (m / z): calcd for C 15 H 13 FN5O3[M+H] + ,330.0997;found,330.0995.

[0152] Example 27: Preparation of compound 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-14)

[0153]

[0154] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (418 μL, 2.4 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (329 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was purified by silica gel chromatography to obtain yellow solid compound 4-14 (310 mg), with a yield of 45%. Melting point: 124.5°C-129.5°C. 1 H NMR(600MHz,DMSO-d6)δ9.93(s,1H),8.57(d,J=3.0Hz,1H),8.03(td,J=8.6,3.0Hz,1H),7.95(dd,J=9.0,4.0Hz,1H), 7.69(d,J=8.2Hz,2H),7.18(d,J=8.2Hz,2H),6.01(s,1H),4.64(s,1H),3.59(t,J=7.1Hz,2H),2.69(t,J=7.1Hz,2H). 13C NMR (151MHz, DMSO) δ160.24, 158.28 (d, J = 252.7Hz), 155.31, 147.83 (d, J = 2.5Hz), 147.19, 136.91, 136.19 (d, J =26.2Hz), 135.24, 129.40 (×2), 126.97 (d, J = 20.2Hz), 120.67 (×2), 119.12 (d, J = 5.1Hz), 88.14, 62.71, 38.98.

[0155] Example 28: Preparation of Compound N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-1-(5-fluoropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxamide (4-15)

[0156]

[0157] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (418 μL, 2.4 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 3,4-methylenedioxyphenylethylamine (396.17 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain off-white solid 4-15 (410 mg), with a yield of 55%. Melting point: 129.3°C-136.3°C. 1 H NMR (600MHz, DMSO-d6) δ11.91(s,1H),8.55(d,J=3.0Hz,1H),8.21(t,J=5.9Hz,1H),8.01(td,J=8.6,3.0Hz,1H),7.83(dd,J=9.3,3.9Hz, 1H),6.82(d,J=8.1Hz,1H),6.81(s,1H),6.68(dd,J=7.9,1.6Hz,1H),5.96(s,2H),5.90(s,1H),3.45-3.39(m,2H),2.75(t,J=7.4Hz,2H). 13C NMR(151MHz,DMSO-d6)δ161.57,158.18(d,J=252.3Hz),154.76,147.85,147.64,147.21,145.94,136.14(d,J=26.2Hz),13 3.69, 126.99 (d, J = 20.2Hz), 121.93, 118.72 (d, J = 4.5Hz), 109.44, 108.58, 101.10, 87.82, 40.66, 35.30; HRMS (m / z): calcd for C 18 H 16 FN4O4[M+H] + ,371.1150;found,371.1146.

[0158] Example 29: Preparation of Compound N-(Benzo[d][1,3]dioxol-5-yl)-1-(5-fluoropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxamide (4-16)

[0159]

[0160] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (418 μL, 2.4 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 3,4-methylenedioxyaniline (328.89 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain yellow powder 4-16 (500 mg), with a yield of 70%. Melting point: 165.6°C-169.4°C. 1 H NMR (600MHz, DMSO-d6) δ12.01(s,1H),9.95(s,1H),8.58(d,J=3.0Hz,1H),8.04(td,J=8.6,3.0Hz,1H),7.93(dd,J=9 .1,3.9Hz,1H),7.47(d,J=2.0Hz,1H),7.26(dd,J=8.4,2.0Hz,1H),6.88(d,J=8.4Hz,1H),6.02(s,1H),6.00(s,2H). 13C NMR(151MHz,DMSO-d6)δ160.12,158.32(d,J=252.8Hz),154.98,147.80,147.39,147.15,143.65,136.20(d,J=26.2 Hz), 133.34, 127.02 (d, J = 20.3Hz), 119.18 (d, J = 4.1Hz), 113.73, 108.35, 102.85, 101.43, 88.18; HRMS (m / z): calcd for C 16 H 12 FN4O4[M+H] + ,343.0837;found,343.0833.

[0161] Example 30: Preparation of Compound 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(2-methylpyridin-4-yl)-1H-pyrazole-3-carboxamide (4-17)

[0162]

[0163] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-methyl-4-aminopyridine (259.36 mg, 2.4 mmol) was added. The mixture was reacted at room temperature for 24 h under N2 protection. Water (35 mL) was added and filtered, and the filter cake was subjected to silica gel chromatography to obtain yellow powder compound 4-17 (200 mg) with a yield of 31%. 1 H NMR (600MHz, DMSO-d6) δ10.29(s,1H),8.57(d,J=2.9Hz,1H),8.32(d,J=5.6Hz,1H),8.02(td,J=8.6,2.9Hz ,1H),7.96(dd,J=9.5,3.9Hz,1H),7.73(d,J=1.9Hz,1H),7.65(d,J=5.7Hz,1H),5.99(s,1H),2.43(s,3H).

[0164] Example 31: Preparation of Compound 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(3-(hydroxymethyl)phenyl)-1H-pyrazole-3-carboxamide (4-18)

[0165]

[0166] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 3-aminobenzyl alcohol (295.36 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain a brown-red solid 4-18 (200 mg), with a yield of 30%. Melting point: 193.4°C-196.7°C. 1 H NMR (600MHz, DMSO-d6) δ9.98(s,1H),8.58(d,J=2.9Hz,1H),8.04(td,J=8.6,3.0Hz,1H),7.97(dd,J=9.0,4.0Hz,1H),7.79(t,J =1.8Hz,1H),7.67(dd,J=8.0,2.1Hz,1H),7.29(t,J=7.8Hz,1H),7.05(d,J=7.5Hz,1H),6.04(s,1H),5.22(s,1H),4.50(s,2H). 13 C NMR (151MHz, DMSO-d6) δ160.30, 158.29 (d, J = 252.6Hz), 155.08, 147.89, 147.11, 143.54, 138.84, 136.14 (d, J = 26. 2Hz), 128.74, 127.06 (d, J = 20.2Hz), 122.20, 119.07, 119.02 (d, J = 4.7Hz), 118.83, 88.22, 63.38; HRMS (m / z): calcd for C 16 H 14 FN4O3[M+H] + ,329.1044;found,329.1040.

[0167] Example 32: Preparation of compound 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-19)

[0168]

[0169] Compound 3c (446 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(3-aminophenyl)ethanol (329 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain yellow powder 4-19 (150 mg), with a yield of 22%. Melting point: 177.7°C-185.4°C. 1 H NMR (600MHz, DMSO-d6) δ12.04(s,1H),9.93(s,1H),8.58(d,J=3.0Hz,1H),8.04(td,J=8.6,3.0Hz,1H),7.96(dd,J=9.0,4.0Hz,1H), 7.70-7.61(m,2H),7.31-7.15(m,1H),6.95(d,J=7.5Hz,1H),6.04(s,1H),4.68(s,1H),3.62(t,J=7.1Hz,2H),2.72(t,J=7.1Hz,2H). 13 C NMR(151MHz,DMSO)δ160.27,158.30(d,J=252.5Hz),155.06,147.89,147.13,140.38,138.85,136.15(d,J=26.4Hz), 128.80,127.06(d,J=20.3Hz),124.73,121.18,119.06(d,J=5.2Hz),118.34,88.20,62.60,40.51; HRMS(m / z):calcd for C 17 H 16 FN4O3[M+H] + ,343.1201;found,343.1197.

[0170] Example 33: Preparation of Compound 1-(5-chloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-20)

[0171]

[0172] Compound 3d (478 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (329.24 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain a light yellow powder 4-20 (235 mg), with a yield of 32.8%. Melting point: 168.8°C-171.6°C. 1 H NMR (600MHz, DMSO-d6) δ9.97(s,1H),8.61(d,J=2.5Hz,1H),8.20(dd,J=8.7,2.6Hz,1H),7.97(d,J=8.8Hz,1H),7. 69(d,J=8.5Hz,2H),7.18(d,J=8.5Hz,2H),6.05(s,1H),4.64(s,1H),3.60(t,J=7.1Hz,2H),2.70(t,J=7.1Hz,2H). 13 CNMR(151MHz,DMSO-d6)δ160.14,155.74,150.19,147.58,146.79,139.51(×2),136. 87,135.31,129.41(×2),120.69(×2),118.05,88.41,62.70,38.97; HRMS(m / z):calcd forC 17 H 16 ClN4O3[M+H] + ,359.0905;found,359.0901.

[0173] Example 34: Preparation of compound 1-(5-chloropyridin-2-yl)-5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-21)

[0174]

[0175] Compound 3d (478 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(3-aminophenyl)ethanol (329.24 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain yellow powder 4-21 (239 mg), with a yield of 33.4%. Melting point: 203.3°C-206.9°C. 1H NMR (600MHz, DMSO-d6) δ12.12(s,1H),9.96(s,1H),8.62(d,J=2.6Hz,1H),8.21(dd,J=8.8,2.6Hz,1H),7.98(d,J=8.7Hz,1H),7.66(d,J =7.2Hz,1H),7.65(s,1H),7.30-7.19(m,1H),7.03-6.93(m,1H),6.06(s,1H),4.68(s,1H),3.62(t,J=7.1Hz,2H),2.72(t,J=7.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ160.17,155.35,150.22,147.49,146.78,140.40,139.60 ,138.80,129.52,128.82,124.79,121.20,118.36,118.09,88.46,62.60,40.49.

[0176] Example 35: Preparation of compound 5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(pyrimidin-2-yl)-1H-pyrazole-3-carboxamide (4-22)

[0177]

[0178] Compound 3e (412.32 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (329.24 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel chromatography to obtain a light yellow powder 4-22 (280 mg), with a yield of 43%. Melting point: 241.5°C-252.1°C. 1 H NMR (600MHz, DMSO-d6) δ10.00(s,1H),8.97(d,J=4.9Hz,2H),7.70(d,J=8.5Hz,2H),7.61(t,J=4.9Hz ,1H),7.18(d,J=8.5Hz,2H),6.07(s,1H),4.65(s,1H),3.59(t,J=7.2Hz,2H),2.69(t,J=7.1Hz,2H). 13C NMR (151MHz, DMSO-d6) δ160.21,159.57(×2),156.46,156.11,148.24,136.85,135.33,129.40(×2),120.78(×2),120.67,88.39,62.72,38.98.

[0179] Example 36: Preparation of compound 5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(3,5,6-trimethylpyrazin-2-yl)-1H-pyrazole-3-carboxamide (4-23)

[0180]

[0181] Compound 3f (372 mg, 1.5 mmol) was dissolved in DMF (7 mL), and DIPEA (392 μL, 2.25 mmol) was added. HATU (855 mg, 2.25 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (246.69 mg, 1.8 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered, and the filter cake was subjected to silica gel column chromatography to obtain yellow solid 4-23 (170 mg), with a yield of 31%. Melting point: 192.5°C-199.3°C. 1 H NMR (600MHz, DMSO-d6) δ11.96(s,1H),9.89(s,1H),7.68(d,J=8.5Hz,2H),7.15(d,J=8.3Hz,2H),5.99(s ,1H),4.63(s,1H),3.58(t,J=7.1Hz,2H),2.68(t,J=7.2Hz,2H),2.56(s,3H),2.50(s,3H),2.30(s,3H). 13 C NMR(151MHz,DMSO-d6)δ160.35,154.89,153.61,149.58,147.43,147.29,142.09,1 36.98,135.08,129.32(×2),120.71(×2),86.86,62.72,38.97,21.81,21.31,19.54.

[0182] Example 37: Preparation of compound 5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-24)

[0183]

[0184] Compound 3g (546 mg, 2 mmol) was dissolved in DMF (8 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (329.24 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel chromatography to obtain yellow solid 4-24 (180 mg), with a yield of 22%. Melting point: 172.5°C-179.4°C. 1 H NMR (600MHz, DMSO-d6) δ12.29(s,1H),10.05(s,1H),8.96(d,J=2.5Hz,1H),8.50(dd,J=8.8,2.3Hz,1H),8.19(d,J=8.6Hz, 1H),7.70(d,J=8.2Hz,2H),7.20(d,J=8.2Hz,2H),6.09(s,1H),4.66(s,1H),3.60(t,J=7.1Hz,2H),2.70(t,J=7.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ159.92,156.10,154.53,148.34,145.66(q,J=4.6Hz),137.48(q,J=4.0Hz),136.78,1 35.45, 129.44 (×2), 123.98 (q, J=272.0Hz), 123.59 (q, J=32.7Hz), 120.76 (×2), 116.12, 88.85, 62.69, 38.97.

[0185] Example 38: Preparation of compound 5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-25)

[0186]

[0187] Compound 3g (546 mg, 2 mmol) was dissolved in DMF (8 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(3-aminophenyl)ethanol (329.24 mg, 2.4 mmol) was added. Under N2 protection, the reaction was carried out at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain white powder 4-25 (220 mg), with a yield of 28%. Melting point: 158.4°C-163.0°C. 1H NMR (600MHz, DMSO-d6) δ12.32(s,1H),10.04(s,1H),9.05-8.86(m,1H),8.50(dd,J=8.8,2.4Hz,1H),8.21(d,J=8.6Hz,1H),7.75- 7.58(m,2H),7.25(t,J=7.7Hz,1H),6.97(d,J=7.5Hz,1H),6.09(s,1H),4.69(s,1H),3.62(t,J=7.1Hz,2H),2.72(t,J=7.1Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ159.99,156.11,154.58,148.27,145.65(q,J=4.3Hz),140.45,138.73,137.48(q,J=3.9H z), 128.84, 124.89, 123.99 (q, J = 272.0Hz), 123.58 (q, J = 33.2Hz), 121.27, 118.43, 116.09, 88.83, 62.59, 39.66.

[0188] Example 39: Preparation of compound 5-hydroxy-N-(2-hydroxy-1-phenylethyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-26)

[0189]

[0190] Dissolve compound 3g (546mg, 2mmol) in DMF (8mL), add DIPEA (522μL, 3mmol), add HATU (1.14g, 3mmol) under ice bath, add compound DL-phenylglycinol (329.24mg, 2.4mmol), react at room temperature for 24h under N2 protection. Add water (35mL) and filter, filter cake by silica gel column chromatography to obtain white solid 4-26 (300mg), yield 38.26%. Melting point: 126.4℃-131.5℃. 1H NMR (600MHz, DMSO-d6) δ12.28(s,1H),8.95(d,J=2.3Hz,1H),8.55-8.40(m,2H),8.13(d,J=8.8Hz,1H),7.39(d,J=7.6Hz,2H),7.33(t ,J=7.5Hz,2H),7.24(t,J=7.3Hz,1H),5.97(s,1H),5.04(q,J=7.4Hz,2H),3.75(dd,J=11.1,7.3Hz,1H),3.71(dd,J=11.3,5.3Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ161.14,155.86,154.40,148.22,145.73(q,J=4.5Hz),141.55,137.43(q,J=3.9Hz),1 28.57(×2),127.43(×2),127.32,123.99(q,J=271.9Hz),123.51(q,J=32.8Hz),116.04,88.56,64.69,55.46.

[0191] Example 40: Preparation of Compound 1-(5-fluoropyrimidin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-27)

[0192]

[0193] Compound 3j (360 mg, 1.5 mmol) was dissolved in DMF (7 mL), and DIPEA (392 μL, 2.25 mmol) was added. HATU (855 mg, 2.25 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (247 mg, 1.8 mmol) was added. The reaction was carried out at room temperature for 24 h under N2 protection. No solid was precipitated after adding water (20 mL), and the DMF was removed by vacuum concentration at 85°C. After adding water (15 mL), the mixture was extracted with EA three times. After concentrating the organic phase, the light yellow solid 4-27 (200 mg) was obtained by silica gel column chromatography, with a yield of 37%. Melting point: 70.5°C-82.5°C. 1 H NMR (600MHz, DMSO-d6) δ11.97(br s,1H),9.97(s,1H),9.07(s,2H),7.69(d,J=8.0Hz,2H),7.17(d,J=8.0Hz,2H),6.03(s,1H),4.63(br s,1H),3.59(t,J=7.3Hz,2H),2.70(d,J=7.4Hz,2H). 13C NMR(151MHz,DMSO-d6)δ160.21,156.74(d,J=261.1Hz),155.64,152.30(d,J=3.5Hz),148.0 2,147.54(d,J=22.7Hz,2H),136.88,135.29,129.37(×2),120.78(×2),88.23,62.71,38.98.

[0194] Example 41: Preparation of Compound 1-(3,5-dichloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-28)

[0195]

[0196] Compound 3k (409 mg, 1.5 mmol) was dissolved in DMF (5 mL), and DIPEA (392 μL, 2.25 mmol) was added. HATU (855 mg, 2.25 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (247 mg, 1.8 mmol) was added. The mixture was reacted at room temperature for 24 h under N2 protection. No solid was precipitated after adding water (25 mL), and the mixture was concentrated under reduced pressure at 85°C to remove DMF. After adding water (20 mL), the mixture was extracted with EA for 3 times. After concentrating the organic phase, the white solid 4-28 (200 mg) was obtained by silica gel column chromatography, with a yield of 34%. Melting point: 155.8°C-166.4°C. 1 H NMR(600MHz,DMSO-d6)δ11.98(br s,1H),10.00(s,1H),8.78(d,J=2.3Hz,1H),8.65(d,J=2.3Hz,1H),7.73(d,J=8.2Hz,2H),7.20(d,J=8.2Hz,2H),6.03(s,1H),4.67(br s,1H),3.63(t,J=7.1Hz,2H),2.73(t,J=7.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ160.20,155.29,148.04,146.92,145.79,139.58,136 .95,135.14,133.13,129.97,129.32(×2),120.75(×2),86.77,62.72,38.98.

[0197] Example 42: Preparation of compound 5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(3-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide (4-29)

[0198]

[0199] Compound 3i (448 mg, 2 mmol) was dissolved in DMF (7 mL), and DIPEA (522 μL, 3 mmol) was added. HATU (1.14 g, 3 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (329 mg, 2.4 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (25 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain white powder 4-26 (150 mg), with a yield of 22%. Melting point: 213.4°C-220.8°C. 1 H NMR(600MHz,DMSO-d6)δ9.78(s,1H),9.05-8.86(m,1H),8.49(d,J=8.0Hz,1H),7.93-7.77(m,1H),7.67(d,J=8 .1Hz,2H),7.14(d,J=8.1Hz,2H),5.90(s,1H),4.75-4.44(m,1H),3.57(t,J=7.2Hz,2H),2.67(t,J=7.2Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ160.42,156.57,153.43,147.42,147.14,138.01(q,J=4.6Hz),136.96,135.08 ,129.33(×2),126.18,123.55(q,J=32.8Hz),122.89(q,J=273.5Hz),120.64(×2),86.22,62.72,38.97.

[0200] Example 43: Preparation of Compound 1-(3,5-dichloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-30)

[0201]

[0202] Compound 3h (411 mg, 1.5 mmol) was dissolved in DMF (7 mL), and DIPEA (392 μL, 2.25 mmol) was added. HATU (855 mg, 2.25 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (247 mg, 1.8 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (35 mL) was added and filtered. The filter cake was subjected to silica gel column chromatography to obtain an orange-yellow solid 4-30 (120 mg), with a yield of 20%. Melting point: 145.6°C-158.4°C. 1H NMR (600MHz, DMSO-d6) δ12.13(s,1H),9.96(s,1H),8.67(dd,J=4.5,1.5Hz,1H),8.31(dd,J=8.1,1.6Hz,1H),7. 79-7.62(m,3H),7.20(d,J=8.2Hz,2H),6.02(s,1H),4.68(s,1H),3.63(t,J=7.2Hz,2H),2.73(t,J=7.1Hz,2H). 13 CNMR(151MHz,DMSO-d6)δ160.35,148.16,147.59,147.19,140.13,136.99,13 5.08,130.47,129.47,129.32(×2),127.27,120.72(×2),86.60,62.72,38.97.

[0203] Example 44: Preparation of Compound 1-(4,6-dimethylpyrimidin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide (4-31)

[0204]

[0205] Compound 31 (351 mg, 1.5 mmol) was dissolved in DMF (6 mL), and DIPEA (392 μL, 2.25 mmol) was added. HATU (855 mg, 2.25 mmol) was added under ice bath, and compound 2-(4-aminophenyl)ethanol (247 mg, 1.8 mmol) was added. Under N2 protection, the mixture was reacted at room temperature for 24 h. Water (20 mL) was added and filtered, and the filter cake was subjected to silica gel column chromatography to obtain white solid 4-31 (220 mg), with a yield of 41.5%. Melting point: 238.4°C-245.8°C. 1 H NMR (600MHz, DMSO-d6) δ12.27(s,1H),10.01(s,1H),7.69(d,J=8.4Hz,2H),7.36(s,1H),7.17(d ,J=8.2Hz,2H),6.07(s,1H),4.64(s,1H),3.67-3.49(m,2H),2.69(t,J=7.1Hz,2H),2.53(s,6H). 13C NMR(151MHz,DMSO-d6)δ169.25,160.25,156.31,156.21,148.22,136.86,135.35,129.38(×2),12 0.81(×2),119.26,88.23,62.70,40.38,40.25,40.11,39.97,39.83,39.69,39.55,38.98,23.78.

[0206] Example 45: DPPH free radical scavenging experiment

[0207] Preparation of DPPH test solution: 1.35 mg of DPPH solid was dissolved in 34.3 mL of methanol to prepare a 100 μM DPPH test solution. Preparation of sample solution: The sample was dissolved in a suitable solvent to prepare a 30 mM solution. The specific addition is shown in Table 1. Before the test, the 30 mM solution of the test compound was diluted with methanol to 300 μM, and Edaravone (Eda) was used as a positive control compound. It was then diluted with methanol to 5 concentrations (10, 20, 40, 60, 80 μM) for testing. A value measurement (test group): In a 96-well plate, 40 μL of the methanol solution of the test compound was taken with a pipette, and then 160 μL of DPPH solution was added. All the above tests were performed twice in parallel. At room temperature for 10 hours, the A519nm value was measured. A0 value measurement (blank group): 40 μL of methanol solution was taken with a pipette, and 160 μL of DPPH solution was added. The measured A value is A0. The experimental results (Table 2) show that all target compounds have scavenging ability against DPPH at concentrations of 4 to 16 μM. Among them, most target compounds show better DPPH scavenging ability than edaravone. The scavenging ability of some compounds against DPPH at a concentration of 16 μM reaches more than 70% (the scavenging rate of edaravone at the same concentration is 37.2%).

[0208] Table 1. DPPH test sample addition table

[0209] Test compound solution Methanol DPPH test solution Total volume 30μL 10μL 160μL 200μL 25μL 15μL 160μL 200μL 20μL 20μL 160μL 200μL 10μL 30μL 160μL 200μL 5μL 35μL 160μL 200μL

[0210] Table 2. Scavenging ability of compounds on DPPH (%)

[0211]

[0212]

[0213] Example 46: ORAC fluorescence test antioxidant capacity

[0214] The antioxidant activity of the compounds was determined by oxygen radical absorbance capacity-fluorescein (ORAC-FL). Trolox (Tro) was used as a positive control compound. The test compound (20 μL, 20 μM) and fluorescein solution (120 μL, 125 nM) were added to a 96-well black board in sequence. The test plate was incubated at 37 ° C for 15 minutes. Subsequently, azobisisobutylamidine hydrochloride (AAPH, 60 μL, 40 mM) was added to the system with a final volume of 200 μL to start the reaction. Immediately after the addition of AAPH, fluorescence was recorded once per minute in a Tecan 96 microplate reader (lex 485nm, lem 525nm, 37 ° C) for 120 minutes. The reaction mixture was prepared in triplicate. The blank control group was measured without the use of antioxidants or in the absence of AAPH and antioxidants. The ORAC value TE was expressed as Trolox equivalent (mean ± SD) using a standard curve calculated for each assay. Experimental results of the antioxidant test (Table 3 and Figure 1 All compounds are not shown in the table, indicating that all compounds can scavenge peroxyl radicals generated by the azo compound AAPH, and the compounds in the examples of the present invention all exhibit excellent antioxidant capacity compared to Trolox. Among them, compound 4-23 exhibits the best antioxidant capacity, and its ORAC value reaches 2.43.

[0215] Table 3. Antioxidant ability test data of compounds (TE)

[0216]

[0217]

[0218] Example 47: Metal Chelation Test

[0219] Taking compound 4-24 as an example, this example uses ultraviolet spectroscopy to explore the metal chelating ability of the target compound. The metal salts used in the test are aluminum chloride, cuprous chloride, cupric chloride, ferrous sulfate, ferric chloride and zinc chloride. A methanol solution of the test compound (1.0 mL, 300 μM) was added to a methanol solution containing metal ions (1.0 mL, 300 μM) and allowed to stand at room temperature for 2 hours. The solution was then transferred to a quartz cell to measure the absorbance of the solution at 200-550 nm. Experimental results ( Figure 2A and Figure 2B ) show that the representative compounds are respectively 2+ and Fe 2+ After incubation in the solution, the absorption spectrum line red-shifted, Cu 2+ The performance is more obvious.

[0220] Example 48: Evaluation of the protective effect of tert-butyl hydroperoxide-induced neuronal cell damage

[0221] The selection of neural cells includes but is not limited to SH-SY5Y and PC-12 cells. The experimental method is: take a bottle of cells in the logarithmic growth phase, add trypsin cell digestion solution to digest, so that the attached cells fall off, and prepare a concentration of 1×10 5 100 μL of cell suspension (1×10 cells / mL) was added to each well of a 96-well cell culture plate. 4 cells), and the plate was placed in a 37°C, 5% CO2 incubator for 24 hours. Cell damaging agents, such as tert-butyl hydroperoxide (tBHP), were prepared with DMEM culture medium. The compound to be tested was then prepared with DMEM culture medium containing the cell damaging agent, and the culture medium containing the compound to be tested was incubated with the corresponding cells for 36 hours. The culture medium was then discarded, washed once with PBS buffered saline solution, and 100 μL of the corresponding DMEM (containing damaging agent) culture medium containing different concentrations of the compound to be tested was added to each well; at the same time, a blank control group (only high-glucose DMEM culture medium) was set up. The 96-well plate was placed in a 37°C, 5% CO2 incubator for 48 hours, 15 μL of MTT solution (5 mg / mL) was added to each well, and the culture was continued in the incubator for 4 hours. The culture medium was discarded, 150 μL of DMSO was added to each well to dissolve, and the crystals were completely dissolved by shaking on a shaker for 5 minutes. Finally, the OD value was read at a wavelength of 490 nm with a multifunctional microplate reader to calculate the cell growth inhibition rate. The results showed that all compounds had protective effects on neuronal cell damage induced by tert-butyl hydroperoxide. The experimental results of some compounds were as follows: Figure 3 As shown, some of these compounds have better neuroprotective effects than edaravone.

[0222] Example 49: Evaluation of the protective effect on glial cell injury induced by oxygen glucose deprivation / reoxygenation (OGD / R)

[0223] The glial cells include but are not limited to BV2 cells. The experimental method is: take a bottle of cells in the logarithmic growth phase, add trypsin cell digestion solution to digest, so that the adherent cells fall off, and prepare a concentration of 4×10 4 100 μL of cell suspension (1×10 cells / mL) was added to each well of a 96-well cell culture plate. 4cells), and the plate was placed in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, the culture medium was changed to DMEM medium without glucose and serum, and placed in a three-gas incubator containing 5% CO2 and 95% N2 for 2 hours. The cells were then cultured with complete culture medium containing the compound to be tested and 10% FBS, and the cells were cultured in a humidified atmosphere containing 5% CO2 and 20% O2 for 48 hours. Finally, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation, the supernatant of each well was discarded, and 150 μL of DMSO was added to each well, and oscillated on a cell oscillator for 10 minutes. Finally, the OD value was read at a wavelength of 490 nm using a multifunctional microplate reader to calculate the cell growth inhibition rate. The experimental results of some compounds are shown in Figure 2. Figure 4 As shown: Representative compounds have a protective effect on BV2 cell damage induced by OGD / R and can significantly improve cell damage and shrinkage caused by oxygen and glucose deprivation ( Figure 4 ).

[0224] Example 50: Detection of intracellular reactive oxygen species (ROS) levels

[0225] Select SH-SY5Y cells in the logarithmic growth phase and adjust the cell concentration to 5.0×10 4 / mL, seeded in a 24-well plate at 0.4mL / well, and cultured in a constant temperature CO2 incubator for 24h. First, use the test compound for 1h, and then continue to use tBHP (175μM) containing the corresponding test compound for 24h. Remove the original culture medium, add 0.1mL of DCFH-DA with a concentration of 10μM prepared with serum-free culture medium (1:1000 dilution) to each well, and incubate in a 37℃ cell culture incubator for 20 minutes. Mix every 4 minutes to allow full contact between the probe and the cells. Remove the cell culture medium and wash the cells three times with serum-free culture medium to fully remove the DCFH-DA that has not entered the cells. Observe and record the green fluorescence under an inverted fluorescence microscope. The entire operation must be completed within 10 minutes to prevent fluorescence quenching. The intensity of green fluorescence is proportional to the content of ROS in the cell ( Figure 5 ). The experimental results show that the compound can effectively inhibit the generation of ROS in cells.

[0226] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pyrazole amide compound, characterized in that: It is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof: Wherein Ar1 is selected from X is selected from -NH(CH2) n A group, wherein n is selected from an integer of 0 to 3; Ar2 is selected from Any one of .

2. The pyrazole amide compound according to claim 1, characterized in that Selected from any one of the following compounds or pharmaceutically acceptable salts thereof: 5-Hydroxy-N-(6-methylpyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(hydroxymethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(3-(hydroxymethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; N-(1,3-dihydroisobenzofuran-5-yl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-((6-methylpyridin-3-yl)methyl)-1-phenyl-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(hydroxymethyl)phenyl)-1-phenyl-1H-pyrazole-3-carboxamide; N-(2-(Benzo[d][1,3]dioxol-4-yl)ethyl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; N-(3,5-dichlorobenzyl)-5-hydroxy-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-1-(pyridin-2-yl)-N-(pyridin-4-ylmethyl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(6-(hydroxymethyl)pyridin-3-yl)-1-(pyridin-2-yl)-1H-pyrazole-3-carboxamide; 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(4-(hydroxymethyl)phenyl)-1H-pyrazole-3-carboxamide; 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(6-(hydroxymethyl)pyridin-3-yl)-1H-pyrazole-3-carboxamide; 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-1-(5-fluoropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxamide; N-(Benzo[d][1,3]dioxol-5-yl)-1-(5-fluoropyridin-2-yl)-5-hydroxy-1H-pyrazole-3-carboxamide; 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(2-methylpyridin-4-yl)-1H-pyrazole-3-carboxamide; 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(3-(hydroxymethyl)phenyl)-1H-pyrazole-3-carboxamide; 1-(5-fluoropyridin-2-yl)-5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; 1-(5-chloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; 1-(5-chloropyridin-2-yl)-5-hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(pyrimidin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(3,5,6-trimethylpyrazin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(3-(2-hydroxyethyl)phenyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(2-hydroxy-1-phenylethyl)-1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide; 1-(5-fluoropyrimidin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; 1-(3,5-dichloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; 5-Hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1-(3-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide; 1-(3-chloropyridin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide; 1-(4,6-dimethylpyrimidin-2-yl)-5-hydroxy-N-(4-(2-hydroxyethyl)phenyl)-1H-pyrazole-3-carboxamide.

3. A method for preparing a pyrazole amide compound as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Compound 1 is dissolved in methanol to prepare a solution with a molar concentration of 0.85 to 1.5 mol / L, and a methanol solution containing 0.9 to 1.5 eq of dimethyl butynedioate is added dropwise under ice bath conditions, and then 1.0 to 2.0 eq of sodium methoxide is added to the reaction solution, and the mixture is heated under reflux to react to obtain Compound 2; (2) Compound 2 is dissolved in a mixed solution of methanol and tetrahydrofuran in a volume ratio of 4:3 to prepare a solution with a molar concentration of 0.3 to 0.6 mol / L, and 1.5 to 2.5 eq of a 2 mol / L sodium hydroxide aqueous solution is added dropwise under an ice bath to react and obtain Compound 3; (3) Compound 3 is dissolved in N,N-dimethylformamide to prepare a solution with a molar concentration of 0.05-0.1 mol / L, and then 2.0-3.5 eq of N,N-diisopropylethylamine and 1.0-1.5 eq of condensing agent O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate are added to the reaction solution, and finally 1.0-1.5 eq of the corresponding amine compound is added to the reaction solution to prepare pyrazole amide compound (I); Wherein, the amine compound is 6-methylpyridin-3-amine, (4-aminophenyl)methanol, (3-aminophenyl)methanol, 1,3-dihydroisobenzofuran-5-amine, 3,4-methylenedioxyphenethylamine, 3,5-dichlorobenzylamine, 4-methylaminopyridine, 2-(4-aminophenyl)ethanol, 2-(hydroxy)-5-aminopyridine, 3,4-methylenedioxyphenethylamine, 3,4-methylenedioxyaniline, 2-methyl-4-aminopyridine, 2-(3-aminophenyl)ethanol or DL-phenylglycinol; The synthetic route of the pyrazole amide compound (I) is as follows:

4. A pharmaceutical composition, characterized in that Contains the pyrazole amide compound according to claim 1 or 2.

5. A pharmaceutical preparation, characterized in that The preparation is prepared by adding one or more pharmaceutically acceptable excipients to the pyrazole amide compound according to claim 1 or 2.

6. The pharmaceutical preparation according to claim 5, characterized in that The preparation is in the form of capsules, pills, tablets, granules or injections.

7. Use of the pyrazole amide compound according to claim 1 or 2 or the pyrazole amide compound prepared by the preparation method according to claim 3 in the preparation of free radical scavenging drugs.

8. Use of the pyrazole amide compound according to claim 1 or 2 or the pyrazole amide compound prepared by the preparation method according to claim 3 in the preparation of a drug for protecting neuronal cells.

9. Use of the pyrazoleamide compound according to claim 1 or 2 or the pyrazoleamide compound prepared by the preparation method according to claim 3 in the preparation of a medicament for preventing or treating or assisting in treating cardiovascular and cerebrovascular diseases, neurodegenerative diseases and their complications caused by excessive free radicals.

Images