CN105669556B - It is a kind of with the amides compound of 3 substituted pyrazolecarboxylic 5 of antitumor activity and its application - Google Patents

Abstract

The invention belongs to pharmaceutical technology field; it is related to a kind of sulfenyl class compound with antitumor activity; more particularly to the amides compound of 3 substituted pyrazolecarboxylic 5 containing sulfenyl; and its pharmaceutically acceptable salt, hydrate; with the pharmaceutical composition using the compound as active component, and prepare histon deacetylase (HDAC) inhibitor and its for treat and/or pre- anti-cancer in purposes.Compound and its pharmaceutically acceptable salt, hydrate structure shown in involved formula I is as follows：Wherein, R₁、R₂As described in claim and specification.

Description

A kind of 3-substituted pyrazole-5-amide compound with antitumor activity and its application

Technical field:

The invention belongs to the technical field of medicine, and relates to a sulfur-based compound with anti-tumor activity, in particular to a thio-containing 3-substituted pyrazole-5-amide compound, and pharmaceutically acceptable salts and hydrates thereof. And a pharmaceutical composition with the compound as an active ingredient, as well as the preparation of histone deacetylase inhibitor and its use in treating and/or preventing cancer.

Background technique:

Epigenetic research is becoming the hope for human beings to conquer tumors. Epigenetic changes mostly occur in the early stages of tumorigenesis, when tumor cells have not yet caused substantial damage to the human body, and intervention at this time is likely to kill them in their infancy. In addition, whereas genetic modifications are almost impossible to reverse, abnormal epigenetic modifications can be reversed, returning tumor cells to a normal state. Therefore, epigenetic research has a broader application prospect.

Histone modification is an important way of epigenetic modification. Most human tumor cells have abnormal histone modification, which can cause tumor suppressor gene silencing and lead to tumor formation. Histone deacetylase (Histonedeacetylase, HDAC) is an enzyme family that includes multiple members. There are currently 18 known subtypes, which are divided into the following four categories according to their species and homology with yeast: and yeast Rpd3 , HoS1, class I homologous to HoS2, including HDAC1, 2, 3, 8; class IIa homologous to yeast Hda1, HoS3 including HDAC4, 5, 7, 9, class IIb including HDAC6, 10; homologous to yeast Sir2 Class III includes SIRT1～SIRT7; it has partial homology with class I and class II HDAC, but its germline is different from class IV, HDAC11. Among them, classes I, II, and IV are classic Zn ²⁺ dependent HDACs, while class III belongs to the Sirtuin family and is NAD ⁺ dependent HDACs. Studies have shown that class I and class II HDACs can inhibit tumor cell differentiation and apoptosis, and promote tumor cell proliferation, etc., which are closely related to the occurrence and development of tumors. The research on inhibitors targeting HDACs has become the focus of anti-tumor drug research. One of the hot spots.

The present invention designs and synthesizes a series of thiol-containing 3-substituted pyrazole-5-amide compounds on the basis of references, and the in vitro antitumor activity test results show that it has good antitumor activity and exhibits Excellent HDAC inhibition.

Invention content:

The object of the present invention is to provide a thio-based compound with anti-tumor activity, specifically a thio-containing 3-substituted pyrazole-5-amide compound and a preparation method thereof, and the compound as a histone deacetylase Application of enzyme inhibitors in preventing and/or treating tumors.

The present invention relates to compounds represented by general formula I, and pharmaceutically acceptable salts and hydrates thereof:

in,

R ₁ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, substituted or unsubstituted C ₆ -C ₁₀ aryl or heteroaryl, the substituents can be C ₁ -C ₄ alkoxy, C ₁ -C ₄ alkyl, C ₆ ~C ₁₀ aryl, the heteroaryl contains 1-3 N, O or S heteroatoms;

R ₂ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₁ -C ₄ acyl, C ₁ -C ₄ alkenyloxycarbonyl, benzyloxycarbonyl, C ₆ -C ₁₂ arylthio, Oleyl, 3-pyridyl-1H-pyrazole-5-amido-1,6-hexyl-thio; preferably H, methyl, acetyl, propionyl, n-butyryl, isobutyryl, acryloxy Carbonyl, benzyloxycarbonyl, pyridylthio, oleic acid, 3-pyridyl-1H-pyrazole-5-amido-1,6-hexyl-thio;

Preferably, R ₁ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, substituted or unsubstituted phenyl, naphthyl, pyridyl, pyrazinyl, ind Indolyl, said substituent is C ₁ -C ₄ alkoxy, phenyl; preferably H, cyclohexyl, methyl, methoxyphenyl, biphenyl, naphthyl, pyridyl, pyrazinyl, indyl Indolyl.

The present invention preferably relates to compounds defined as general formula I and pharmaceutically acceptable salts thereof, hydrates:

R1 is independently selected from the following substituents _: H, cyclohexyl, methyl, methoxyphenyl, biphenyl, 6-10 membered aryl;

R ₂ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₁ -C ₄ acyl, C ₁ -C ₄ alkenyloxycarbonyl, benzyloxycarbonyl, C ₆ -C ₁₂ arylthio, Oleic acid group, 3-pyridyl-1H-pyrazole-5-amido-1,6-hexyl-thiol;

The present invention preferably relates to compounds defined as general formula I and pharmaceutically acceptable salts thereof, hydrates:

in,

R is independently selected from the following substituents _: H, cyclohexyl, methyl, phenyl, methoxyphenyl, biphenyl, naphthyl, pyridyl, pyrazinyl, indolyl;

R ₂ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₁ -C ₄ acyl, C ₁ -C ₄ alkenyloxycarbonyl, benzyloxycarbonyl, C ₆ -C ₁₂ arylthio, Oleyl, 3-pyridyl-1H-pyrazole-5-amido-1,6-hexyl-thio.

Specifically, the following compounds are preferred in the present invention:

3-methyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

N-(6-Mercaptohexyl)-1H-pyrazole-5-amide;

3-cyclohexyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-Phenyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(4-methoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(3.4-Dimethoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(3,4,5-trimethoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(4-biphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(1-naphthyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(2-naphthyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(2-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(3-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(4-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(2-pyrazinyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

3-(3-indolyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide;

N-(6-methylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-(2-pyridyldithio)hexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-propionylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-n-butyrylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-isobutyrylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-propenyloxycarbonylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-Benzyloxycarbonylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-oleylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-acetylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide;

N-(6-methylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-(2-pyridyldithio)hexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-propionylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-n-butyrylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-isobutyrylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-propenyloxycarbonylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-Benzyloxycarbonylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-oleylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N-(6-acetylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide;

N,N'-(bis 1,6-hexyldithio)-(bis 3-(4-pyridyl)-1H-pyrazole-5-amide);

N,N'-(bis 1,6-hexyldithio)-(bis 3-(3-pyridyl)-1H-pyrazole-5-amide).

Furthermore, the present invention also includes prodrugs of the compounds of the present invention. According to the invention, prodrugs are compounds of the general formula I which themselves may have weak or even no activity, but which, after administration, are converted under physiological conditions (e.g. by metabolism, solvolysis or otherwise) into the corresponding biologically active form.

The present invention includes a pharmaceutical composition, which contains the sulfur-based compound of the above formula I containing a 3(5)-substituted phenyl-5(3)-amide moiety and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field. The compounds according to the invention can be used in combination with other active ingredients, provided they do not produce other adverse effects, such as allergic reactions.

The pharmaceutical composition of the present invention can be formulated into several dosage forms, which contain some excipients commonly used in the pharmaceutical field, for example, oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as Injectable solution or suspension, or injectable dry powder, add water for injection immediately before injection); topical preparations (eg ointment or solution).

The carrier used for the pharmaceutical composition of the present invention is the common type available in the pharmaceutical field, including: adhesives for oral preparations, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents, pigments, Flavoring agents, etc.; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for topical preparations. Pharmaceutical preparations can be administered orally or parenterally (eg intravenously, subcutaneously, intraperitoneally or topically), and if certain drugs are unstable under gastric conditions, they can be formulated as enteric-coated tablets.

Through in vitro enzyme inhibition test screening, we found that the compound of the present invention can inhibit the activity of histone deacetylase, therefore, the compound of the present invention can be used in the application of diseases related to the abnormal expression of histone deacetylase activity, such as various cancers .

Through in vitro activity screening and in vivo pharmacodynamic studies, we found that the compound of the present invention has anti-tumor activity, so the compound of the present invention can be used to prepare drugs for the treatment and/or prevention of various cancers, such as breast, lung, colon, rectum, stomach , prostate, bladder, uterus, pancreas and ovarian cancer.

The active compound of the present invention can be used as the sole anticancer drug, or used in combination with one or more other anticancer drugs. Combination therapy is achieved by simultaneous, sequential or spaced administration of the individual therapeutic components.

The Examples and Preparations provided hereinafter further illustrate and illustrate the compounds of the present invention and methods for their preparation. It should be understood that the scope of the following examples and preparations does not limit the scope of the invention in any way.

The following synthetic schemes describe the preparation of the compounds of formula I of the present invention, all starting materials are prepared by the methods described in these schemes, by methods well known to those of ordinary skill in the art of organic chemistry or are commercially available. All final compounds of the invention are prepared by the methods described in these schemes or by methods analogous thereto, which methods are well known to those of ordinary skill in the art of organic chemistry. All variables used in these schemes are as defined below or as defined in the claims.

According to the compound of formula I of the present invention, when R ₂ is H, the target compound can be obtained according to the method of Route 1. Each substituent is as defined in the Summary of the Invention.

According to the formula I compound of the present invention, when R ₂ is a methyl group, the target compound can be obtained according to the method of Route 2. Each substituent is as defined in the Summary of the Invention.

According to the compound of formula I of the present invention, when R ₂ is pyridylthio, the target compound can be obtained according to the method of Route 3. Each substituent is as defined in the Summary of the Invention.

According to the compound of formula I of the present invention, when R ₂ is 3-pyridyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide, the target compound can be obtained according to the method of route 4. Each substituent is as defined in the Summary of the Invention.

According to the formula I compound of the present invention, when R ₂ is other substituents, the target compound can be obtained according to the method of Route 4. Each substituent is as defined in the Summary of the Invention.

The preparation method of the invention has simple operation and mild conditions, and the obtained compounds all have histone deacetylase inhibitory activity, and have remarkable antitumor effect.

detailed description

The present invention will be described in detail below through specific examples, but the use and purpose of these exemplary embodiments are only used to exemplify the present invention, and do not constitute any form of any limitation to the actual protection scope of the present invention, nor will the present invention The scope of protection is limited to this.

Embodiment 1: the preparation of 3-methyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

Step A: Preparation of methyl 2,4-dioxopentanoate

Add 20ml of dry methanol dropwise to 1g of sodium at 0°C, stir until the sodium is completely dissolved, and add the solution dropwise to a solution of 2.1g (36mmol) of acetone and 4.3g (36mmol) of dimethyl oxalate in 150ml of ether at room temperature. React for 4h, filter with suction, and dry the filter cake. A white solid was obtained with a yield of 99.2%. .

Step B: Preparation of 3-methyl-1H-pyrazole-5-carboxylic acid methyl ester

Add 150ml of acetic acid to 5.1g (35.7mmol) of methyl 2,4-dioxopentanoate, stir to dissolve, add 2.14ml (43.2mmol) of 98% hydrazine hydrate, reflux for 2 hours, and pour the reaction solution into a large amount of cold water , precipitated solid, suction filtered, and the filter cake was dried. A white solid was obtained with a yield of 99.7%.

Step C: Preparation of 3-methyl-1H-pyrazole-5-carboxylic acid

Add 75ml of methanol to 10.1g (71.8mmol) of 3-methyl-1H-pyrazole-5-carboxylic acid methyl ester, stir to dissolve, add 4.4g (110mmol) of sodium hydroxide in 150ml of aqueous solution, and react at 50°C for 2h, then 2M hydrochloric acid was used to adjust the pH to 2, a solid was precipitated, filtered with suction, and the filter cake was dried. A white solid was obtained with a yield of 99.8%.

Step D: Preparation of N-(6-bromohexyl)-3-methyl-1H-pyrazole-5-amide

Dissolve 1.2g (9.7mmol) of 3-methyl-1H-pyrazole-5-carboxylic acid in 50ml of tetrahydrofuran, add 0.5ml of triethylamine and 1.6g (11.6mmol) of 1-hydroxybenzotriazepam at 0°C Azole (HOBt), stirred and dissolved, added 3.0g (11.6mmol) 6-bromohexylamine, stirred for 10min, added 2.3g (12.0mmol) 1-ethyl-3(3-dimethylpropylamine) carbodiimide ( EDCI), reacted at room temperature for 24 hours, added 50ml of water, extracted twice with ethyl acetate, combined the organic layers, washed the organic layer once with 0.5M hydrochloric acid, washed once with water, washed once with saturated aqueous sodium bicarbonate solution, and washed once with saturated aqueous sodium chloride solution After washing twice, the organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to obtain a brown solid with a yield of 79.4%.

Step E: Preparation of 3-methyl-N-(6-acetylmercaptohexyl)-1H-pyrazole-5-amide

Dissolve 2.9g (10mmol) of N-(6-bromohexyl)-3-methyl-1H-pyrazole-5-amide in 100ml of ethanol, add 3.42g (30mmol) of potassium thioacetate, and heat up to 60°C for reaction After 4 hours, the insoluble matter was removed by filtration, the filtrate was evaporated to dryness, dissolved in dichloromethane, the dichloromethane layer was washed twice with water, twice with saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. After concentration, the crude product was separated by column chromatography (silica gel, petroleum ether/ethyl acetate, 5/1) to obtain a brown solid with a yield of 71.5%.

Step F: Preparation of 3-methyl-N-(6-acetylmercaptohexyl)-1H-pyrazole-5-amide

Dissolve 2.8g (10mmol) of S-(6-(5-(4-fluorophenyl)-1H-pyrazole-3-amido)hexyl)acetylthioester in 100ml of ethanol, and add 25ml of 1.2mol /L LiOH aqueous solution, reacted at room temperature for 4 hours, distilled off ethanol, added 100ml of water, adjusted pH=2 with 2M hydrochloric acid, extracted twice with ethyl acetate, collected the organic layer, washed the organic layer twice with saturated aqueous sodium chloride solution, anhydrous Na2SO4 dried. After concentration, the crude product was separated by column chromatography (silica gel, petroleum ether/ethyl acetate, 5/1) to obtain a white solid with a yield of 65.2%.

According to the preparation method of Example 1, appropriate raw materials were selected to prepare the compounds of Example 2-Example 15.

Example 2: Preparation of N-(6-mercaptohexyl)-1H-pyrazole-5-amide

Example 3: Preparation of 3-cyclohexyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:335.1[M+H] ⁺ .

Example 4: Preparation of 3-phenyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:338.2[M+H] ⁺ .

Example 5: Preparation of 3-(4-methoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:308.4[M+H] ⁺ .

Example 6: Preparation of 3-(3.4-dimethoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:304.2[M+H] ⁺ .

Example 7: Preparation of 3-(3,4,5-trimethoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:320.3[M+H] ⁺ .

Example 8: Preparation of 3-(4-biphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:359.3[M+H] ⁺ .

Example 9: Preparation of 3-(1-naphthyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

Example 10: Preparation of 3-(2-naphthyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:359.5[M+H] ⁺

Example 11: Preparation of 3-(2-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:334.6[M+H] ⁺

Example 12: Preparation of 3-(3-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:358.3[M+H] ⁺

Example 13: Preparation of 3-(4-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:370.2[M+H] ⁺

Example 14: Preparation of 3-(2-pyrazinyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:370.6[M+H] ⁺

Example 15: Preparation of 3-(3-indolyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide

LC-MS m/z:370.1[M+H] ⁺

Example 16: Preparation of N-(6-methylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

Dissolve 3.5g (10mmol) N-(6-bromohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide in 100ml ethanol, add 1.4g (30mmol) mercaptomethane, and heat up to 60°C After reacting for 4 hours, the insoluble matter was removed by filtration, the filtrate was evaporated to dryness, dissolved in dichloromethane, the dichloromethane layer was washed twice with water, twice with saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. After concentration, the crude product was separated by column chromatography (silica gel, petroleum ether/ethyl acetate, 5/1) to obtain a brown solid with a yield of 64.1%

LC-MS m/z:366.2[M+H] ⁺

Example 17: Preparation of N-(6-(2-pyridyldithio)hexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

Dissolve 1.7g (5mmol) of 3-(4-pyridyl)-N-(6-acetylmercaptohexyl)-1H-pyrazole-5-amide in 100ml of DMF, add 1ml of ethylamine, dropwise add 3.3g (15mmol) 2,2'-dimercaptopyridine, react at room temperature for 12h. Stop the reaction, add 200ml of water, extract 3 times with ethyl acetate, concentrate, flash preparative liquid phase separation to obtain a white solid. Yield 37.8%.

LC-MS m/z:376.5[M+H] ⁺

Example 18: Preparation of N-(6-propionylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

Dissolve 1.5g (5mmol) 3-(4-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide in 100ml dichloromethane, add 0.46g (5mmol) propane dropwise at 0°C Acyl chloride, after the dropwise transfer to room temperature to continue the reaction for 1h. Stop the reaction, add 200ml of water, extract 3 times with dichloromethane, concentrate, flash preparative liquid phase separation to obtain a white solid. Yield 76.3%.

LC-MS m/z:322.7[M+H] ⁺

According to the preparation method of Example 18, appropriate raw materials were selected to prepare the compounds of Example 19-Example 24.

Example 19: Preparation of N-(6-n-butyrylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:336.2[M+H] ⁺

Example 20: Preparation of N-(6-isobutyrylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:348.4[M+H] ⁺

Example 21: Preparation of N-(6-propyleneoxycarbonylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:291.5[MH] ^-

Example 22: Preparation of N-(6-Benzyloxycarbonylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:294.2[MH] ^-

Example 23: Preparation of N-(6-oleylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:264.3[MH] ^-

Example 24: Preparation of N-(6-acetylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:308.8[M+H] ⁺

According to the preparation method of Example 16, the compound of Example 25 was prepared by selecting appropriate raw materials.

Example 25: Preparation of N-(6-methylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:366.5[M+H] ⁺

According to the preparation method of Example 17, appropriate raw materials were selected to prepare the compound of Example 26.

Example 26: Preparation of N-(6-(2-pyridyldithio)hexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:376.0[M+H] ⁺

According to the preparation method of Example 18, appropriate raw materials were selected to prepare the compound of Example 27.

Example 27: Preparation of N-(6-propionylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:322.9[M+H] ⁺

According to the preparation method of Example 27, appropriate raw materials were selected to prepare the compounds of Example 28-Example 33.

Example 28: Preparation of N-(6-n-butyrylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:336.0[M+H] ⁺

Example 29: Preparation of N-(6-isobutyrylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:348.1[M+H] ⁺

Example 30: Preparation of N-(6-propyleneoxycarbonylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:291.7[MH] ^-

Example 31: Preparation of N-(6-Benzyloxycarbonylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:294.3[MH] ^-

Example 32: Preparation of N-(6-oleylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:264.1[MH] ^-

Example 33: Preparation of N-(6-acetylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide

LC-MS m/z:308.5[M+H] ⁺

Example 34: Preparation of N,N'-(bis 1,6-hexyldithio)-(bis 3-(4-pyridyl)-1H-pyrazole-5-amide)

Dissolve 1.9g (5mmol) N-(6-(2-pyridyldithio)hexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide in 100mL dichloromethane, add 1.8g (5mmol) of 3-(4-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide was reacted at room temperature for 24h. Stop the reaction, add 200 mL of water, extract 3 times with 80 mL of dichloromethane, concentrate, flash preparative liquid phase separation, and obtain a white solid. Yield 61.2%.

LC-MS m/z:607.8[M+H] ⁺

According to the preparation method of Example 34, appropriate raw materials were selected to prepare the compound of Example 35.

Example 35: Preparation of N,N'-(bis 1,6-hexyldithio)-(bis 3-(3-pyridyl)-1H-pyrazole-5-amide)

LC-MS m/z:608.0[M+H] ⁺

Example 36. Research on the pharmacological action of the product of the present invention

The experiment set up a blank control group (no drug) and a positive control group (vorinostat). The compound to be tested and Hela nuclear extract or HDAC1 or HDAC6 were pre-incubated for 15 min at room temperature, and the fluorescent substrate Boc–Lys(Ac)–AMC was added. After incubating at 37° C. for 60 min, a terminator (containing Trypsin and SAHA) was added to terminate the reaction. After 15 minutes, use a full-wavelength multifunctional microplate reader to detect the fluorescence intensity when the excitation and emission wavelengths are 355 nm and 460 nm, respectively, and calculate the inhibition rate. See Tab.1 for the inhibitory activity of target compounds on HDACs enzymes.

Tab.1 embodiment 1-15 is to HDACs, HDAC1, HDAC6 inhibitory activity

In vitro inhibitory activity test against cell proliferation

1. Cell recovery

Carefully remove the cells (cryopreservation tube) from liquid nitrogen and thaw them all quickly in a 37-40°C water bath, so that the cells can quickly cross the extremely vulnerable temperature range of 0-5°C. Under sterile conditions, use a pipette gun to suck out the cells and put them into a centrifuge tube, centrifuge at 1300r/min for 3 minutes, gently discard the supernatant, add the culture medium, blow and mix the cells, transfer them into a culture bottle and put them in carbon dioxide culture Cultured in the box, the medium was changed once after 4 hours.

2. Cell passage

After recovery, the cells need to be cultured and passaged 2-3 times until they are stable before the experiment can be carried out. Each passage is based on the fact that the cells cover 90% of the bottom of the culture flask.

3. Cell embedding

Trypsin solution (0.25%) was used to digest the cells from the bottom of the culture flask when the cells grew to the bottom of the culture flask. Gently discard the upper trypsin solution, add 10mL of culture medium, pipette and mix the cells, draw 10uL of cell suspension and add it to a cell counting plate for counting, and adjust the cell concentration to 3.5×10 ⁴ cells/well. In the 96-well plate, 100 uL of cell suspension was added to well A1 except well A1, which was a blank well and no cells were added. The 96-well plate was placed in an incubator for 24 h.

4. Cell dosing

First dissolve the drug with 50 μL DMSO. Then add an appropriate amount of culture solution to dissolve the drug into a 2 mmol/mL drug solution. Drugs were then dissolved at 100, 50, 25, 12.5, 6.25 μmol/mL in a 96-well plate. Each concentration was added to 3 wells, and the growth of cells in the surrounding two rows and two columns was greatly affected by the environment, so they were only used as blank cell wells. The 96-well plate was placed in an incubator for 24 h.

MTT test method

Embed the cells in a 96-well plate at a cell density of 1.5-3×104, ^100uL per well, add different concentrations of drugs (100uL/well) for 24 hours after the cells adhere to the wall, add MTT after the drugs and cells are incubated for 96 hours, and put them in the incubator for 4 hours Afterwards, the MTT (tetrazolium) solution was discarded, and 200uL of DMSO was added. Vibrate on a magnetic oscillator to fully dissolve the formazan, the product of the reaction between the surviving cells and MTT, put it into a microplate reader and read the OD value at a wavelength of 570nm. Calculate the inhibition rate according to the following formula:

Using SAHA as a positive control drug, the in vitro inhibitory activity test of the target compound against cell proliferation. The test results are shown in Tab.2.

Tab.2 Examples 1-23 have growth inhibitory activity on breast cancer cell MCF-7 in vitro

The above test results show that the compound of the general formula to be protected in the present invention has good antitumor activity and HDAC inhibitory effect. The compound of the invention has good industrial application prospect.

Claims (8)

1. Compounds as shown in general formula I, and pharmaceutically acceptable salts thereof: in, R ₁ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, substituted or unsubstituted naphthyl, pyridyl, pyrazinyl, indolyl, the substituted The base is C ₁ -C ₄ alkoxy, phenyl; R ₂ is independently selected from the following substituents: H, C ₁ -C ₄ alkyl, C ₁ -C ₄ acyl, C ₁ -C ₄ alkenyloxycarbonyl, benzyloxycarbonyl, C ₆ -C ₁₂ arylthio, Oleyl, 3-pyridyl-1H-pyrazole-5-amido-1,6-hexyl-thio. 2. The compound of claim 1, and pharmaceutically acceptable salts thereof: in, R1 is independently selected from the following substituents _: H, cyclohexyl, methyl, naphthyl, pyridyl, pyrazinyl, indolyl. 3. The compound according to any one of claims 1-2, and pharmaceutically acceptable salts thereof: in, R2 is independently selected from the following substituents: H, methyl, acetyl, propionyl, n _- butyryl, isobutyryl, propenyloxycarbonyl, benzyloxycarbonyl, pyridylthio, oleyl, 3-pyridyl-1H -pyrazole-5-amido-1,6-hexyl-thio. 4. The following compounds, and pharmaceutically acceptable salts thereof, are selected from: 3-methyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; N-(6-Mercaptohexyl)-1H-pyrazole-5-amide; 3-cyclohexyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-Phenyl-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(4-methoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(3.4-Dimethoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(3,4,5-trimethoxyphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(4-biphenyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(1-naphthyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(2-naphthyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(2-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(3-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(4-pyridyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(2-pyrazinyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; 3-(3-indolyl)-N-(6-mercaptohexyl)-1H-pyrazole-5-amide; N-(6-(2-pyridyldithio)hexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-methylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-acetylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-propionylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-n-butyrylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-isobutyrylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-propenyloxycarbonylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-Benzyloxycarbonylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N-(6-oleylmercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-amide; N,N'-(bis 1,6-hexyldithio)-(bis 3-(4-pyridyl)-1H-pyrazole-5-amide); N-(6-(2-pyridyldithio)hexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-methylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-acetylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-propionylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-n-butyrylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-isobutyrylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-propenyloxycarbonylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-Benzyloxycarbonylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N-(6-oleylmercaptohexyl)-3-(3-pyridyl)-1H-pyrazole-5-amide; N,N'-(bis 1,6-hexyldithio)-(bis 3-(3-pyridyl)-1H-pyrazole-5-amide). 5. A pharmaceutical composition, characterized in that it comprises a compound according to any one of claims 1-4 and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 6. Use of the compound according to any one of claims 1-4 and the pharmaceutically acceptable salt thereof or the pharmaceutical composition described in claim 5 in the preparation of medicines for diseases related to the abnormal expression of histone deacetylase activity. 7. The compound of any one of claims 1-4 and the pharmaceutically acceptable salt thereof or the application of the pharmaceutical composition described in claim 5 in the preparation of antitumor drugs. 8. The use according to claim 7, wherein the tumor is prostate cancer, breast cancer, cervical cancer or leukemia.