Hydroxamic acid small-molecule compound taking pyrazole as mother nucleus, application and preparation method of hydroxamic acid small-molecule compound

Abstract

The invention discloses a hydroxamic acid small-molecule compound taking pyrazole as a mother nucleus structure, a hydrate or pharmaceutically acceptable salt thereof, and application and a preparation method thereof, the structure of the compound is shown as a formula (I) or a formula (II), and the definition of each group and substituent group is shown in the specification. The compound can be used as a broad-spectrum HDAC inhibitor, not only shows high inhibitory activity at the enzyme level, but also can inhibit proliferation of various tumor cells, and the compound, the hydrate or the pharmaceutically acceptable salt thereof and the pharmaceutical composition containing the compound can be used for preventing and / or treating various tumor diseases and the like. The compound can also be used as a histone deacetylase inhibitor for preventing and treating thioredoxin-mediated diseases and the like.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis and drug treatment, and in particular to a hydroxamic acid small molecule compound with pyrazole as a parent core, a use thereof and a preparation method thereof. Background Art

[0002] Epigenetic modification is of great significance for the occurrence, diagnosis and treatment of tumors, mainly including DNA methylation, histone modification, chromatin remodeling and non-coding RNA regulation. Abnormal epigenetic modification can cause incorrect gene expression, leading to metabolic disorders and diseases and even tumors. With the rapid development of molecular oncology and molecular pharmacology, drug treatment of malignant tumors is no longer limited to ordinary chemotherapeutic drugs. The current research focus is shifting from traditional cytotoxic drugs to new anti-tumor drugs with multi-link effects targeting the mechanism.

[0003] Histone modifications mainly include histone methylation, acetylation, phosphorylation and histone ubiquitination. Among them, the most in-depth research is histone acetylation. Under normal circumstances, histone acetylation and deacetylation are in a dynamic equilibrium, which is jointly regulated by histone acetylase (HAT) and histone deacetylase (HDAC). HAT can transfer the acetyl group of acetyl-CoA to a specific lysine residue at the N-terminus of histone, neutralize its positive charge, make the chromatin present a loose structure, and promote transcription. HDAC can hydrolyze acetylated lysine, deacetylate it, and inhibit gene transcription.

[0004] Studies have shown that in tumor cells, histones are mostly hypoacetylated, and chromatin is densely curled, resulting in the inability of some tumor suppressor genes to express normally. HDAC inhibitors can inhibit the activity of highly expressed HDACs, restore the dynamic equilibrium state of HATs and HDACs, and inhibit the growth of tumor cells. With the in-depth study of HDACs in tumors, more and more HDAC inhibitors are being used in tumor research and have shown multiple anti-tumor mechanisms, such as: inhibiting the proliferation of tumor cells, promoting the differentiation and apoptosis of tumor cells, blocking the cell cycle of tumor cells, inhibiting the angiogenesis of tumor cells, and reducing the motility of tumor cells. There are currently four HDAC inhibitors approved by the FDA: SAHA (Merck) and FK228 (Celgene) for cutaneous T-cell lymphoma, PXD101 (Spectrum) for the treatment of peripheral T-cell lymphoma, and LBH589 (Novartis) through the FDA accelerated approval process. This drug can be used in combination with dexamethasone and bortezomib to treat multiple myeloma. In addition, there are more than 20 compounds in different clinical stages. Therefore, HDAC inhibitors are a very popular anti-tumor target with broad market prospects.

[0005] In addition, HDACs inhibitors are widely used in the prevention and treatment of autoimmune diseases, allergies, and inflammation. Studies on multiple animal models of inflammatory diseases have shown that HDAC inhibitors have anti-inflammatory effects. In addition to regulating the acetylation state of histones to cause the expression of inflammatory cytokines, HDAC inhibitors can also inhibit or activate the expression of inflammatory factors by regulating the acetylation state of non-histone proteins including transcription factors, the most important of which is achieved by inhibiting the nuclear transcription factor KB. However, different HDACs have different acetylation patterns and regulate different genes. For example, in the MRL-Ipr / 1pr mouse model of systemic lupus erythematosus, TSA and SAHA can downregulate the levels of inflammatory cytokines IL-12 and IL-6 mRNA and protein; in the mouse asthma model, TSA can reduce the concentrations of IL-4, IL-5, and IgE in bronchoalveolar lavage fluid, thereby slowing the development of asthma. However, there are also reports that HDAC inhibitors can promote the expression of inflammatory cytokines. Suuronen et al. reported that TSA can enhance the production of NO and IL-6 induced by lipopolysaccharide in microglial N9 cells. Therefore, HDAC inhibitors can regulate inflammatory responses as either gene transcription activators or inhibitors. Systemic sclerosis is an autoimmune disease characterized by extensive vascular lesions and progressive fibrosis of the skin and internal organs. In the study of the pathogenesis of systemic sclerosis, a variety of proinflammatory factors are involved, including TGF-β1, IL-4, PDGF, etc., and HDACs activity is an essential factor for the onset and progression of systemic sclerosis. Huber et al.'s study showed that in an animal model of bleomycin-induced skin fibrosis, TSA could attenuate the expression of type I collagen and fibronectin in skin fibroblasts by inhibiting the activity of HDACs, and reduce the accumulation of total collagen and extracellular matrix in skin fibroblasts.

[0006] However, further research and development of new HDAC inhibitors are still needed to obtain anti-tumor drugs with higher activity and stronger specificity. Summary of the invention

[0007] The present invention is based on the SAHA structure and uses pyrazole as the parent core structure to design and synthesize a series of anti-tumor drugs with novel structures, higher activities and stronger specificity. Specifically, a class of small molecule isohydroxamic acid compounds or their hydrates or pharmaceutically acceptable salts with pyrazole as the parent core structure is provided. The above compounds are a class of broad-spectrum HDAC inhibitors, which not only show strong inhibitory activity at the enzyme level, but also can inhibit the proliferation of various tumor cells.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention is to provide a class of hydroxamic acid small molecule compounds with pyrazole as the parent core structure, hydrates thereof or pharmaceutically acceptable salts thereof, wherein the structural formula of the compound is:

[0010]

[0011] Wherein, n is 0 to 7 CH2;

[0012] R1 is selected from the group consisting of methyl, isopropyl, tert-butyl, halogen, hydroxy, cyano, amino, nitro, aldehyde, acetyl, vinyl, ethynyl, propynyl, difluoromethyl, trifluoromethyl, carboxyl, phenyl, heterocyclic aromatic, substituted phenyl or heterocyclic aromatic, C 3-8 Cycloalkyl;

[0013] R2 and R3 are each independently selected from the following group: hydrogen, C 1-8 Alkyl and its isomers, C 3-8 R2 and R3 are connected to form a cycloalkyl group, alicyclic hydrocarbon, heterocyclic group, phenyl group, heterocyclic aromatic group, substituted phenyl group and substituted heterocyclic aromatic group with a chain length of 3 to 7;

[0014] R4 is selected from the group consisting of hydrogen, C 1-8 Alkyl and its isomers, C 3-8 Cycloalkyl, halogen, nitro, cyano, NR5R6; wherein R5 and R6 are independently selected from hydrogen, methyl, ethyl, propyl, isopropyl.

[0015] In a preferred embodiment, the compound is selected from the following group:

[0016] N-(1-adamantyl)-3-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-5-carboxamide (BE01);

[0017] N-(1-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE02);

[0018] N-(tert-butyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE03);

[0019] N-(2-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE04);

[0020] N-(2-Fluoro-3-trifluoromethylphenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE05);

[0021] N-(Morpholinyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE06);

[0022] N-(4H-1,2,4-triazolyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE07);

[0023] N-(3,5-dipyrimidinyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE08);

[0024] N-(4-chlorophenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE09);

[0025] N-(4-Fluorophenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE10);

[0026] N-(4-Fluorobenzyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE11);

[0027] (S)-N-(1-phenylethyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE12);

[0028] N-(3-(5-tert-butyl)isoxazolyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE13);

[0029] N-(1-adamantyl)-5-tert-butyl-1-(5-(hydroxyamino)-5-oxobutyl)-1H-pyrazole-3-carboxamide (BE14);

[0030] N-(1-adamantyl)-5-tert-butyl-1-(4-(hydroxyamino)-4-oxopropyl)-1H-pyrazole-3-carboxamide (BE15);

[0031] N-(1-adamantyl)-5-tert-butyl-1-(7-(hydroxyamino)-4-oxohexyl)-1H-pyrazole-3-carboxamide (BE16).

[0032] The structural formula of the above compounds is detailed in each embodiment. Preferably, the above compounds are BE01, BE02, BE03, BE04, BE05, BE16. More preferably, the compound is BE16.

[0033] In a preferred embodiment, the above-mentioned "pharmaceutically acceptable salt" is an acid addition salt formed by the compound or its hydrate with an acid, and the acid includes but is not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, salicylic acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid, and succinic acid.

[0034] In a preferred embodiment, the above compound or its hydrate can be combined with radioactivity, fluorescent group or biotin.

[0035] The second aspect of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of any one of the compounds described in the first aspect, its hydrate or its pharmaceutically acceptable salt.

[0036] In a preferred embodiment, the pharmaceutical composition is formulated into an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, a transdermal patch or an excipient, etc.

[0037] In a preferred embodiment, the pharmaceutical composition can be used alone or in combination with other drugs.

[0038] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers, wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0039] "Safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.

[0040] The third aspect of the present invention is to provide a prodrug of any of the compounds described in the first aspect, a hydrate thereof or a pharmaceutically acceptable salt thereof, and a drug metabolite. The above-mentioned prodrug refers to a compound obtained by chemically modifying the drug structure, which is inactive or less active in vitro and releases active drugs through enzymatic or non-enzymatic conversion in vivo to exert its drug effect; the above-mentioned drug metabolite refers to a substance with physiological or pharmacological activity.

[0041] The fourth aspect of the present invention is to provide an application of any one of the compounds described in the first aspect, its hydrate or pharmaceutically acceptable salt, or the pharmaceutical composition described in the second aspect, which is selected from at least one of the following applications: application in the preparation of histone deacetylase (HDACs) inhibitors, application in the preparation of preparations that promote the deacetylation of cellular histones H3 and H4 and α-Tubulin, and application as a histone deacetylase inhibitor in the preparation of drugs for preventing and / or treating thioredoxin-mediated diseases.

[0042] Furthermore, the histone deacetylase inhibitors include HDAC1 inhibitors, HDAC6 inhibitors and the like.

[0043] Furthermore, the thioredoxin-mediated diseases include, but are not limited to, autoimmune diseases, allergies or inflammation, neurodegenerative diseases, Alzheimer's disease, Charcot-Marie-Tooth disease, traumatic brain injury, hypertension and stroke.

[0044] The fifth aspect of the present invention is to provide the use of any one of the compounds described in the first aspect, its hydrate or its pharmaceutically acceptable salt, or the pharmaceutical composition described in the second aspect, which is selected from at least one of the following applications: use in the preparation of drugs for preventing and / or treating tumors, use in the preparation of drugs for inhibiting tumor cell proliferation, and use in the preparation of drugs for promoting tumor cell apoptosis.

[0045] In a preferred embodiment, the drugs for preventing and / or treating tumors include anti-tumor drugs used to induce acquired drug resistance after chemotherapy failure and drugs used to treat malignant tumors.

[0046] In a preferred embodiment, the compound, its hydrate or pharmaceutically acceptable salt, or the pharmaceutical composition is used to inhibit the proliferation, growth, infiltration and migration, and clone formation of tumor cells, promote the apoptosis of tumor cells, and inhibit the proliferation of tumor cells by blocking the cell cycle.

[0047] In a preferred example, the tumor includes but is not limited to lung cancer, breast cancer, skin cancer, rectal / colon cancer, liver cancer, stomach cancer, prostate cancer, pancreatic cancer, leukemia, ovarian cancer, bladder cancer, kidney cancer, oral cancer, melanoma, head and neck cancer, uterine cancer, brain tumor, esophageal cancer, lymphoma, etc.

[0048] In a preferred example, the tumor cells include but are not limited to lung cancer cells, breast cancer cells, epidermal cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, prostate cancer cells, pancreatic cancer cells, leukemia cells, ovarian cancer cells, bladder cancer cells, renal cancer cells, oral cancer cells, etc.; preferably, the tumor cells are non-small cell lung cancer, breast cancer, gastric cancer, colon cancer and promyelocytic leukemia; more preferably, the tumor cells are A549 cells (non-small cell lung cancer), MDA-MB-231 cells (breast cancer), SGC7901 (gastric cancer), HCT116 (colon cancer) and HL60 cells (promyelocytic leukemia).

[0049] The sixth aspect of the present invention is to provide a method for preparing any of the compounds described in the first aspect. The suitable reaction conditions of the reaction routes in the above-mentioned preparation methods are all known in the art, and appropriate changes in the reaction conditions, including replacement of solvents and reagents, are within the capabilities of those skilled in the art, and the preparation methods exemplified below have significant guiding value for small-scale tests, pilot tests and scale-up of the products.

[0050] In a preferred embodiment, the preparation method is method A or method B, wherein the method A comprises the steps of:

[0051] S1, reacting different α-ketones with diethyl oxalate to generate intermediates (preferably reacting at room temperature for 10 min), and forming corresponding substituted pyrazole rings under the action of hydrazine hydrate and glacial acetic acid (preferably reflux at 70°C for 3 h);

[0052] S2, hydrolyzing the corresponding substituted pyrazole ring into a carboxylic acid (preferably reacting at 40°C for 3h), and reacting with different amines to form a corresponding amide compound (preferably reacting at room temperature overnight);

[0053] S3, in anhydrous tetrahydrofuran, with potassium tert-butoxide as a base, the corresponding amide compound reacts with different bromine compounds to form two intermediates of different configurations (preferably refluxed at 70° C. under ice bath conditions overnight);

[0054] S4. The intermediate is coupled with hydroxylamine hydrochloride in a suitable solvent to generate the target compound (preferably at room temperature for 2 h).

[0055] Preferably, the reaction formula of method A is as follows:

[0056]

[0057] The method B comprises the steps of:

[0058] a), hydrolyzing different substituted pyrazole carboxylic acid ethyl esters into carboxylic acids, and condensing the carboxylic acids with different amines to form corresponding amide compounds;

[0059] b) In anhydrous tetrahydrofuran, with potassium tert-butoxide as a base, the corresponding amide compound reacts with different bromine compounds to form two intermediates with different configurations;

[0060] c), the intermediate is coupled with hydroxylamine hydrochloride in a suitable solvent to generate the target compound;

[0061] Preferably, the reaction formula of a method B is as follows:

[0062]

[0063] In a preferred embodiment, in the above preparation method, after the reaction is completed, it is generally quenched with ice water, quenched with a solvent such as ethyl acetate or dichloromethane, washed with water and saturated brine respectively, dried with anhydrous sodium sulfate, and then distilled off the solvent under reduced pressure. The final product is obtained by column chromatography, and the obtained product is verified by nuclear magnetic resonance, mass spectrometry, high performance liquid chromatography and other methods to prove the compound structure and purity.

[0064] In a preferred embodiment, the α-ketone in step S1 is pinacolone, and the corresponding substituted pyrazole ring or substituted pyrazole carboxylic acid ethyl ester in step S1 and step a) is 5-tert-butyl-1H-pyrazole-3-carboxylic acid ethyl ester; in step S2 and step a), the carboxylic acid obtained by hydrolysis is 5-tert-butyl-1H-pyrazole-3-carboxylic acid, and the amine is adamantane hydrochloride; in step S3 and step b), the corresponding amide compound is 3-tert-butyl-N-(1-adamantyl)-1H-pyrazole-5-carboxamide, the bromine compound is 6-bromohexanoic acid methyl ester, and the two intermediates of different configurations are 6-(5 -(1-adamantyl)formamido)-3-(tert-butyl)-1H-pyrazolyl)-hexanoic acid methyl ester, 6-(3-(1-adamantyl)formamido)-5-(tert-butyl)-1H-pyrazolyl)-hexanoic acid methyl ester; in step S4 and step c), the target compound is N-(1-adamantyl)-3-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-5-carboxamide, N-(1-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide.

[0065] In certain preferred embodiments, in the above steps, 1-adamantanamine hydrochloride can be replaced by tert-butylamine, 2-adamantanamine, 2-fluoro-3-trifluoromethylaniline, morpholine, 4-amino-1,2,4-triazole, 5-aminopyrimidine, 4-chloroaniline, 4-fluoroaniline, 4-fluorobenzylamine, (S)-α-phenylethylamine, 3-amino-5-tert-butylisoxazole; 6-bromohexanoic acid methyl ester is replaced by 5-bromovalerate methyl ester, 4-bromobutyrate methyl ester, 7-bromoheptanoic acid methyl ester. After the reactants are replaced, the structures of the intermediates and target products subsequently generated are also changed accordingly.

[0066] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0067] (1) The present invention provides a class of novel hydroxamic acid small molecule compounds with pyrazole as the parent core structure, which are HDAC inhibitors, including pharmaceutically acceptable salts, solvent compounds (such as hydrates), metabolites, isomers and prodrugs thereof, which can be used as anti-tumor drug lead compounds and clinical drug candidate compounds;

[0068] (2) The synthetic route of the compounds of the present invention is simple and the raw materials are cheap;

[0069] (3) The compounds of the present invention are a class of broad-spectrum HDAC inhibitors, which have a stronger activity improvement at the enzyme level compared with SAHA. In addition, in multiple in vitro anti-tumor proliferation experiments, the compounds of the present invention have shown good activity;

[0070] (4) Taking compound BE16 as an example, it has a promoting effect on apoptosis of non-small cell lung cancer A549;

[0071] (5) Taking compound BE16 as an example, it can block the cell cycle of non-small cell lung cancer A549, thereby inhibiting tumor cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0073] Figure 1 This is a diagram showing the effect of some compounds in one embodiment of the present invention on inhibiting the deacetylation of histones H3 and H4 and α-Tubulin;

[0074] Figure 2 This is a graph showing the effect of some compounds in one embodiment of the present invention on apoptosis of non-small cell lung cancer A549;

[0075] Figure 3 This is a diagram showing the effects of some compounds in an embodiment of the present invention on the cell cycle of non-small cell lung cancer A549 cells; wherein, part A is a schematic diagram of the cell cycle, and part B is a statistical calculation diagram. DETAILED DESCRIPTION

[0076] After extensive and in-depth research, the present inventors have discovered that the compound of formula (I) or formula (II) is a class of broad-spectrum HDAC inhibitors that not only exhibit strong inhibitory activity at the enzyme level, but also can inhibit the proliferation of various tumor cells.

[0077] Terms (In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.)

[0078] In the present invention, the halogen is F, Cl, Br or I.

[0079] In the present invention, the term "C 1-8 ” means having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, and so on.

[0080] In the present invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon moiety, for example, the term "C 1-8 The term "alkyl" refers to a straight or branched chain alkyl group having 1 to 8 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, etc.

[0081] In the present invention, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety, for example, the term "C 3-8 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

[0082] In the present invention, the term "alicyclic hydrocarbon" refers to a cyclic hydrocarbon having an aliphatic nature and containing a closed carbon ring in the molecule.

[0083] In the present invention, the term "heterocyclic aromatic group" refers to an aromatic cyclic group containing at least one (such as 1, 2, 3 or 4) ring heteroatom (such as N, O or S), such as pyridine, quinoline, pyrazine, furan, thiophene and the like.

[0084] In the present invention, the term "substituted" refers to the replacement of one or more hydrogen atoms on a specific group with a specific substituent. It should be understood by those skilled in the art that the combination of substituents contemplated by the present invention is a combination that is stable or chemically feasible. The substituents are, for example, halogen, hydroxyl, carboxyl, amino, C 1-8 Alkyl, C 1--8 Alkoxy, C 6-14 Aryl, C 3-8 Cycloalkyl, etc.

[0085] The following examples more specifically describe the preparation methods of the compounds of formula (I) or (II) of the present invention, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily carried out by those skilled in the art to which the present invention belongs.

[0086] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0087] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0088] In the following embodiments, 1 H-NMR was measured with a Bruker 500MHz instrument; MS was measured with a Bruker MicroTOF-Q LCMS instrument, and all were performed using the ESI method unless otherwise specified; all solvents were redistilled before use, and the anhydrous solvents used were obtained by drying according to standard methods; unless otherwise specified, all reactions were carried out under argon protection and tracked by TLC, and post-treatment was performed by washing with saturated brine and drying with anhydrous magnesium sulfate; the products were purified using silica gel (200-300 mesh) column chromatography unless otherwise specified; the silica gel used included 200-300 mesh and GF 254 Produced by Qingdao Ocean Chemical Plant or Yantai Yuanbo Silica Gel Company.

[0089] 1. Preparation Example

[0090] Example 1-1: Preparation of Compound N-(1-adamantyl)-3-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-5-carboxamide (BE-01)

[0091]

[0092] Pinacolone (1002 mg, 11.0 mmol) was dissolved in tetrahydrofuran (100 ml), and the reaction system was cooled with an ice-water bath. Potassium tert-butoxide (1459 mg, 14.3 mmol) was added, and the reaction was allowed to proceed at room temperature for 10 min. Then, diethyl oxalate (1461 mg, 12.1 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. Hydrazine hydrate and glacial acetic acid were added to the reaction system, and the reaction was refluxed at 70 ° C for 3 h. After the reaction was cooled to room temperature, the solvent was evaporated, and the mixture was extracted with ethyl acetate and water respectively. The organic phase was evaporated and then subjected to column chromatography to obtain the intermediate compound 5-tert-butyl-1H-pyrazole-3-carboxylic acid ethyl ester (1325 mg, 6.8 mmol).

[0093] 5-tert-Butyl-1H-pyrazole-3-carboxylic acid ethyl ester (1325 mg, 6.8 mmol) was dissolved in a mixed solvent of tetrahydrofuran, methanol and water, and LiOH·H2O was added, and the mixture was reacted at 40°C for 3 h. The solvent was evaporated to dryness, acidified with 3N hydrochloric acid solution, and extracted with ethyl acetate and water, and the organic phase was evaporated to dryness to obtain 5-tert-butyl-1H-pyrazole-3-carboxylic acid (1009 mg, 6.0 mmol).

[0094] 5-tert-butyl-1H-pyrazole-3-carboxylic acid (505 mg, 3.0 mmol) was dissolved in DMF, HATU (1341 mg, 3.6 mmol) and DIPEA (963 mg, 7.2 mmol) were added, and then adamantane hydrochloride (673 mg, 3.6 mmol) was added. After reacting at room temperature overnight, the mixture was extracted with ethyl acetate and water, and the organic phase was evaporated to dryness and then subjected to column chromatography to obtain the target compound 3-tert-butyl-N-(1-adamantyl)-1H-pyrazole-5-carboxamide (482 mg, 1.6 mmol).

[0095] 3-tert-butyl-N-(1-adamantyl)-1H-pyrazole-5-carboxamide (482 mg, 1.6 mmol) was dissolved in anhydrous tetrahydrofuran (5 ml), potassium tert-butoxide (210 mg, 1.8 mmol) was slowly added under ice bath conditions, 6-bromohexanoic acid methyl ester (374 mg, 1.8 mmol) was slowly added after 10 min, and refluxed at 70°C overnight. The solvent was evaporated to dryness, extracted with ethyl acetate and water, and the organic phase was evaporated to dryness and column chromatography was performed to obtain two intermediate compounds of different configurations, 6-(5-(1-adamantyl)carboxamido)-3-(tert-butyl)-1H-pyrazolyl)-hexanoic acid methyl ester (129 mg, 0.3 mmol) and 6-(3-(1-adamantyl)carboxamido)-5-(tert-butyl)-1H-pyrazolyl)-hexanoic acid methyl ester (172 mg, 0.4 mmol).

[0096] At 40°C, KOH (2.06 g, 36.7 mmol) was added to a solution of hydroxylamine hydrochloride (2.55 g, 36.7 mmol) in methanol (10 ml). After reacting for 10 min, the reaction system was placed in an ice bath. After 30 min, the filtrate was filtered to obtain the filtrate. The above ester 6-(5-(1-adamantyl)formamido)-3-(tert-butyl)-1H-pyrazolyl)-hexanoic acid methyl ester (129 mg, 0.3 mmol) was added to the filtrate. KOH (206 mg, 3.67 mmol) was added to the system and reacted at room temperature for 2 hours. After the solvent was evaporated by reduced pressure distillation, it was extracted with ethyl acetate and saturated ammonium chloride and then passed through a column to obtain the product N-(1-adamantyl)-3-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-5-carboxamide (BE-01, 43 mg, 33%). 1 H NMR (500MHz, DMSO) δ10.32(s,1H),8.64(s,1H),8.11(d,J=8.0Hz,1H),6.22(s,1H),4.48–4.40(m,2H),2.37– 2.30(m,2H),2.14–2.08(m,4H),2.07–1.98(m,4H),1.82–1.74(m,6H),1.66–1.56(m,4H),1.37–1.23(m,12H).

[0097] Example 1-2: Preparation of N-(1-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-02)

[0098]

[0099] At 40°C, KOH (2.06 g, 36.7 mmol) was added to a solution of hydroxylamine hydrochloride (2.55 g, 36.7 mmol) in methanol (10 ml). After reacting for 10 min, the reaction system was placed in an ice bath. After 30 min, the filtrate was filtered to obtain the filtrate. The above ester 6-(3-(1-adamantyl)formamido)-5-(tert-butyl)-1H-pyrazolyl)-hexanoic acid methyl ester (172 mg, 0.4 mmol) was added to the filtrate. KOH (206 mg, 3.67 mmol) was added to the system and reacted at room temperature for 2 hours. After the solvent was evaporated by reduced pressure distillation, it was extracted with ethyl acetate and saturated ammonium chloride and then passed through a column to obtain the product N-(1-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-02, 43 mg, 33%). 1H NMR (500MHz, DMSO) δ10.32(s,1H),8.64(s,1H),8.11(d,J=8.0Hz,1H),6.22(s,1H),4.48–4.40(m,2H),2.37– 2.30(m,2H),2.14–2.08(m,4H),2.07–1.98(m,4H),1.82–1.74(m,6H),1.66–1.56(m,4H),1.37–1.23(m,12H).

[0100] Example 1-3: Preparation of Compound N-(tert-butyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-03)

[0101]

[0102] The product was prepared by replacing 1-adamantanamine hydrochloride with tert-butylamine according to the method for preparing compound BE02. 1 H NMR (500MHz, DMSO) δ10.33 (s, 1H), 8.60 (s, 1H), 8.15 (d, J = 8.0Hz, 1H), 6.25 (s, 1H),4.44(t,J=7.4Hz,2H),1.87–1.70(m,4H),1.44(s,9H),1.39–1.21(m,13H).

[0103] Example 1-4: Preparation of Compound N-(2-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-04)

[0104]

[0105] The product was prepared by replacing 1-adamantanamine hydrochloride with 2-adamantanamine according to the method for preparing compound BE02. 1 H NMR (500MHz, DMSO) δ10.30(s,1H),8.75(s,1H),8.20(d,J=8.0Hz,1H),6.43(s,1H),4.48(t,J=7.4Hz,2H),4.25–4.10(m, 1H),2.10–2.02(m,2H),1.94–1.80(m,14H),1.76–1.72(m,2H),1.65–1.58(m,2H),1.40–1.33(m,9H),1.32–1.26(m,2H).

[0106] Example 1-5: Preparation of Compound N-(2-fluoro-3-trifluoromethylphenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-05)

[0107]

[0108] Prepare the product according to the method for preparing compound BE02, replacing 1-adamantanamine hydrochloride with 2-fluoro-3-trifluoromethylaniline. 1 H NMR(500MHz,DMSO)δ10.35(s,1H),8.76(s,1H),7.51–7.33(m,2H),7.29–7.27(m,1H),6. 36–6.28(m,1H),6.23(s,1H),4.50–4.32(m,2H),1.79–1.71(m,4H),1.35–1.20(m,13H).

[0109] Example 1-6: Preparation of Compound N-(morpholinyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-06)

[0110]

[0111] Prepare the compound BE02 by replacing 1-adamantanamine hydrochloride with morpholine. 1 H NMR (500MHz, DMSO) δ10.40(s,1H),8.70(s,1H),6.10(s,1H),4.21(t,J=7.4Hz,2H),3.72(s,8H),1.81–1.75(m,4H),1.33–1.24(m,13H).

[0112] Example 1-7: Preparation of Compound N-(4H-1,2,4-triazolyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-07)

[0113]

[0114] The 1-adamantanamine hydrochloride was replaced by 4-amino-1,2,4-triazole and prepared according to the method for preparing compound BE02. 1HNMR(500MHz,DMSO)δ10.60(s,1H),8.90(s,1H),8.77(d,J=5.8Hz,1H),8.49(d,J=8.4Hz,1H),7.50(dd ,J=8.4,4.5Hz,1H),7.20(s,1H),4.53–4.45(m,2H),1.87–1.82(m,4H),1.39(s,9H),1.34–1.28(m,4H).

[0115] Example 1-8: Preparation of Compound N-(3,5-dipyrimidinyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-08)

[0116]

[0117] The product was prepared according to the method for preparing compound BE02, except that 1-adamantanamine hydrochloride was replaced by 5-aminopyrimidine. 1 H NMR (500MHz, DMSO) δ10.36(s,1H),8.77(d,J=5.8Hz,1H),8.49(d,J=8.4Hz,1H),8.20(s,1H),7.50(dd, J=8.4,4.5Hz,1H),7.28(s,2H),4.53–4.44(m,2H),1.88–1.83(m,4H),1.39(s,9H),1.30–1.28(m,4H).

[0118] Example 1-9: Preparation of Compound N-(4-chlorophenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-09)

[0119]

[0120] Prepare the product by replacing 1-adamantanamine hydrochloride with 4-chloroaniline according to the method for preparing compound BE02. 1 H NMR (500MHz, DMSO) δ10.36(s,1H),8.72(s,1H),7.51–7.33(m,5H),6.25(s,1H),4.52–4.34(m,2H),1.77–1.71(m,4H),1.34–1.20(m,13H).

[0121] Example 1-10: Preparation of Compound N-(4-fluorophenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-10)

[0122]

[0123] The product was prepared by replacing 1-adamantanamine hydrochloride with 4-fluoroaniline according to the method for preparing compound BE02. 1 H NMR(500MHz,DMSO)δ10.45(s,1H),8.70(s,1H),7.53–7.33(m,2H),7.29–7.28(m,2H),6. 36–6.28(m,1H),6.23(s,1H),4.50–4.32(m,2H),1.79–1.71(m,4H),1.35–1.20(m,13H).

[0124] Example 1-11: Preparation of Compound N-(4-fluorobenzyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-11)

[0125]

[0126] The product was prepared by replacing 1-adamantanamine hydrochloride with 4-fluorobenzylamine according to the method for preparing compound BE02. 1 H NMR(500MHz,DMSO)δ10.47(s,1H),8.72(s,1H),7.34–7.24(m,2H),7.17–6.95(m,2H) ,6.31(s,1H),6.25(s,1H),4.62–4.40(m,4H),1.90–1.78(m,4H),1.38–1.21(m,13H).

[0127] Example 1-12: Preparation of Compound (S)-N-(1-phenylethyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-12)

[0128]

[0129] Prepare the compound BE02 by replacing 1-adamantanamine hydrochloride with (S)-α-phenylethylamine. 1HNMR(500MHz,DMSO)δ10.49(s,1H),8.82(s,1H),7.42–7.36(m,4H),7.32(dd,J=8.5,4.4Hz,1H),6.30(s,1H),6.16(d,J=7.5 Hz,1H),5.28(p,J=7.0Hz,1H),4.52–4.43(m,2H),1.85–1.78(m,4H),1.61(d,J=6.9Hz,3H),1.31(s,9H),1.30–1.22(m,4H).

[0130] Example 1-13: Preparation of Compound N-(3-(5-tert-butyl)isoxazolyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide (BE-13)

[0131]

[0132] Prepare the product according to the method for preparing compound BE02, replacing 1-adamantanamine hydrochloride with 3-amino-5-tert-butylisoxazole. 1 H NMR(500MHz,DMSO)δ10.50(s,1H),8.84(s,1H),6.21(s,1H),5.78(s,1H), 4.46(t,J=7.4Hz,2H),1.85–1.76(m,4H),1.46(s,9H),1.37–1.26(m,13H).

[0133] Example 1-14: Preparation of Compound N-(1-adamantyl)-5-tert-butyl-1-(5-(hydroxyamino)-5-oxobutyl)-1H-pyrazole-3-carboxamide (BE-14)

[0134]

[0135] Replace 6-bromohexanoic acid methyl ester with 5-bromovalerate methyl ester and prepare according to the method for preparing compound BE02. 1 H NMR (500MHz, DMSO) δ10.33(s,1H),8.67(s,1H),8.18(d,J=8.0Hz,,1H),6.24(s,1H),4.48–4.40(m,2H),2.38– 2.30(m,2H),2.14–2.08(m,4H),2.07–1.98(m,,4H),1.82–1.74(m,4H),1.66–1.52(m,4H),1.38–1.20(m,12H).

[0136] Example 1-15: Preparation of Compound N-(1-adamantyl)-5-tert-butyl-1-(4-(hydroxyamino)-4-oxopropyl)-1H-pyrazole-3-carboxamide (BE-15)

[0137]

[0138] Replace 6-bromohexanoic acid methyl ester with 4-bromobutyric acid methyl ester and prepare according to the method for preparing compound BE02. 1 H NMR (500MHz, DMSO) δ10.30(s,1H),8.61(s,1H),8.10(d,J=8.0Hz,,1H),6.21(s,1H),4.47–4.40(m,2H),2.38– 2.32(m,2H),2.12–2.08(m,4H),2.07–1.98(m,,4H),1.82–1.74(m,2H),1.68–1.52(m,4H),1.30–1.20(m,12H).

[0139] Example 1-16: Preparation of Compound N-(1-adamantyl)-5-tert-butyl-1-(7-(hydroxyamino)-4-oxohexyl)-1H-pyrazole-3-carboxamide (BE-16)

[0140]

[0141] Replace 6-bromohexanoic acid methyl ester with 7-bromoheptanoic acid methyl ester and prepare according to the method for preparing compound BE02. 1 H NMR (500MHz, DMSO) δ10.34(s,1H),8.66(s,1H),8.12(d,J=8.0Hz,,1H),6.27(s,1H),4.48–4.42(m,2H),2.47– 2.38(m,2H),2.20–2.18(m,4H),2.06–1.96(m,,4H),1.82–1.76(m,6H),1.68–1.56(m,6H),1.41–1.23(m,12H).

[0142] 2. Performance and efficacy evaluation

[0143] Example 2: Inhibition of HDAC1 and HDAC6 Enzyme Activity by Compounds

[0144] This example verifies the inhibitory effect of the compounds prepared in Examples 1-1 to 1-16 on the enzyme activities of HDAC1 and HDAC6, which specifically includes the steps of:

[0145] MDA-MB231 cells (purchased from the cell bank of the Chinese Academy of Sciences) were inoculated in a 6-cm cell culture dish. When the cells grew to a density of 80%, the prepared compounds and SAHA were added as control groups. After the cells were treated for 24 hours, the culture medium was removed and pre-cooled PBS was added. Then, the PBS was aspirated and cell lysis buffer was added. The cells were scraped into a 1.5-ml centrifuge tube. The centrifuge tube was placed on ice and vortexed once every five minutes. After vortexing five times in total, the tube was centrifuged at 4°C at 12,000 rpm for 15 minutes. The supernatant was transferred to a new pre-cooled centrifuge tube, and the protein concentration was measured by the BCA method. The loading buffer containing 40 μg of protein was run on an 8%-12% SDS protein separation gel. The gel was stopped after bromophenol blue ran out of the gel plate. The membrane was transferred at 100 volts for 1 hour, and the primary antibodies corresponding to HDAC1 and HDAC6 were incubated overnight at 4°C in a shaker. The cells were washed three times with TBST for 10 minutes each, and then incubated with the corresponding secondary antibodies for 1 hour. The cells were washed three times with TBST for 10 minutes each, and the target bands were exposed using an ECL kit.

[0146] The results are shown in Table 1. At a certain concentration, most compounds have the effect of inhibiting the enzyme activity of HDAC1 and HDAC6, and the inhibitory effect is equivalent to or better than that of the positive control SAHA. Among them, compound BE16 has a certain selectivity for the HDAC6 subtype.

[0147] Table 1 - Effects of compounds on inhibiting HDAC1 and HDAC6 enzyme activities

[0148] Example 3: Initial screening of compound cell activity

[0149] In this example, some compounds with good initial screening activity (BE01, BE02, BE03, BE04, BE05, BE16) and positive compounds (SAHA) were tested for cytotoxic activity on different types of cells. All of them were carried out on corresponding cells. There were 3000-10000 cells in each 100 μL of culture medium, which were placed in a 96-well plate (Falcon, CA) and incubated overnight. The cells to be tested were divided into 10 parts and treated with gradient concentrations of drugs. At least 3 replicates of each concentration were cultured at 37°C for 72 h and detected by the MTS method, that is, 20 μL CellTiter 96 Aqueous One Sollution was added to each well and incubated for 0.5-3h. The OD value was measured at 490 nm by an enzyme reader. The drug concentration at which 50% of the growth of the cells to be tested is inhibited is the IC 50 GraphPad Prism 5.0 software was used to calculate the IC of drugs. 50 .

[0150] The results are shown in Table 2. Compound BE16 showed strong activity in A549 cells (non-small cell lung cancer), MDA-MB-231 cells (breast cancer), SGC7901 (gastric cancer), HCT116 (colon cancer) and HL60 cells (promyelocytic leukemia) (purchased from the cell bank of the Chinese Academy of Sciences). Compared with the positive drug SAHA, the activity of compound BE16 was significantly improved.

[0151] Table 2 - Cytotoxic activity test results of compounds

[0152]

[0153] a A549 cells (non-small cell lung cancer); b MDA-MB-231 cells (breast cancer); c SGC7901 (gastric cancer); d HCT116 (colon cancer); e HL60 cells (promyelocytic leukemia)

[0154] Example 4: Compounds Promote Deacetylation of Histones H3 and H4 and α-Tubulin

[0155] This example is used to verify the deacetylation effect of compound BE16 on histones H3 and H4 and α-Tubulin. The test method is the same as that of Example 2. A549 cells (non-small cell lung cancer) are inoculated in a 6 cm cell culture dish. When the cells grow to 80% density, compound BE16, SAHA and DMSO are added as control groups. After the cells are treated for 24 hours, the culture medium is removed and pre-cooled PBS is added. Then, PBS is aspirated and cell lysis solution is added. The cells are scraped into a 1.5 ml centrifuge tube. The centrifuge tube is placed on ice and vortexed and centrifuged every five minutes. Tube, vortex 5 times, centrifuge at 12000 rpm at 4℃ for 15 minutes, aspirate the supernatant into a new pre-cooled centrifuge tube, measure protein concentration by BCA method, add loading buffer containing 40 μg protein, run on 8%-12% SDS protein separation gel, stop when bromophenol blue runs out of the gel plate, transfer to membrane at 100 volts for 1 hour, incubate H3, H4 acetylated antibodies in a shaker at 4℃ overnight, wash three times with TBST for 10 minutes each time, then incubate with corresponding secondary antibody for 1 hour, wash three times with TBST for 10 minutes each time, use ECL kit, and expose to display each target band.

[0156] The results are as follows Figure 1As shown, the compound BE16 of the present invention can effectively promote the acetylation of histones in cancer cells, and the level of acetylation promotion is gradient-dependent. Because α-tubulin is a specific substrate of HDAC6, BE16 not only helps the acetylation of H3 and H4, but also acts on α-tubulin, indicating that compound BE16 has an inhibitory effect on both HDAC1 and HDAC6.

[0157] Example 5: Effect of Compounds on Apoptosis of Non-Small Cell Lung Cancer A549

[0158] This example is used to verify the effect of compound BE16 on apoptosis of non-small cell lung cancer A549, and specifically includes the steps of: adding DMSO, SAHA (3μM, 8μM) and the compound BE16 of the present invention (0.3μM, 1μM, 3μM, 10μM) to non-small cell lung cancer cells A549. The cells were collected after 48 hours and analyzed using Annexin V-FITC Apoptosis Detection Kit (eBioscience / Bender MedSystems GmbH, Vienna, Austria). Annexin V-FITC and propidium iodide staining were used, and cell apoptosis was detected by flow cytometry. PARP cleavage in A549 cells was detected by western blotting to determine cell apoptosis.

[0159] Results of apoptosis in non-small cell lung cancer Figure 2 As shown, with the increase of the concentration of compound BE16 in A549 cells, the pro-apoptotic effect gradually increased, and the effect was significantly better than that of SAHA.

[0160] Example 6: Effects of the Compounds on the Cell Cycle of Non-Small Cell Lung Cancer A549 Cells

[0161] This example is used to verify the effect of compound BE16 on the cell cycle of non-small cell lung cancer A549 cells, and specifically includes the following steps: A549 cells were treated with DMSO, SAHA (8 μM) or BE16 (0.3 μM, 1 μM, 3 μM) for 24 hours, and then stained with propidium iodide (PI) and subjected to cell cycle detection and analysis using an American BD flow cytometer.

[0162] The results are as follows Figure 3 As shown in the figure, it can be found that when SAHA (8 μM) acts on A549 cells, cell cycle arrest occurs, the G2 / M phase is prolonged, and the S phase is shortened. It can be clearly seen from the experimental results that compound BE16 is concentration gradient dependent on cell cycle arrest, and the effect is better than SAHA. Therefore, the above experiments prove that BE16 can inhibit tumor cell proliferation by blocking the cell cycle.

[0163] It can be seen from the above embodiments that the present invention takes histone deacetylase as the target, based on the structural characteristics and anti-tumor effects of SAHA, retains some key structural skeletons and functional groups, and obtains a new type of HDAC inhibitor (hydroxamic acid small molecule compounds with pyrazole as the parent core structure) for the first time; the above experimental results show that such compounds not only have strong activity at the enzyme level, but also show good inhibitory activity in a variety of tumor cells, and can be used for the development of anti-tumor drug lead compounds and clinical drug candidate compounds.

[0164] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A class of small molecule hydroxamic acid compounds with pyrazole as the parent core structure, its hydrate or its pharmaceutically acceptable salt, characterized in that: The structural formula of the compound is: Wherein, n is 0 to 7 CH2; R1 is selected from the group consisting of methyl, isopropyl, tert-butyl, halogen, hydroxy, cyano, amino, nitro, aldehyde, acetyl, vinyl, ethynyl, propynyl, difluoromethyl, trifluoromethyl, carboxyl, phenyl, heterocyclic aromatic, substituted phenyl or heterocyclic aromatic, C 3-8 Cycloalkyl; R2 and R3 are each independently selected from the following group: hydrogen, C 1-8 Alkyl and its isomers, C 3-8 R2 and R3 are connected to form a cycloalkyl group, alicyclic hydrocarbon, heterocyclic group, phenyl group, heterocyclic aromatic group, substituted phenyl group and substituted heterocyclic aromatic group with a chain length of 3 to 7; R4 is selected from the group consisting of hydrogen, C 1-8 Alkyl and its isomers, C 3-8 Cycloalkyl, halogen, nitro, cyano, NR5R6; wherein R5 and R6 are independently selected from hydrogen, methyl, ethyl, propyl, isopropyl.

2. The hydroxamic acid small molecule compound with pyrazole as the parent core structure according to claim 1, characterized in that: The compound is selected from the group consisting of: N-(1-adamantyl)-3-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-5-carboxamide; N-(1-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(tert-butyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(2-adamantyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(2-fluoro-3-trifluoromethylphenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(Morpholinyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(4H-1,2,4-triazolyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(3,5-dipyrimidinyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(4-chlorophenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(4-fluorophenyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(4-fluorobenzyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; (S)-N-(1-phenylethyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(3-(5-tert-butyl)isoxazolyl)-5-tert-butyl-1-(6-(hydroxyamino)-6-oxopentyl)-1H-pyrazole-3-carboxamide; N-(1-adamantyl)-5-tert-butyl-1-(5-(hydroxyamino)-5-oxobutyl)-1H-pyrazole-3-carboxamide; N-(1-adamantyl)-5-tert-butyl-1-(4-(hydroxyamino)-4-oxopropyl)-1H-pyrazole-3-carboxamide; N-(1-adamantyl)-5-tert-butyl-1-(7-(hydroxyamino)-4-oxohexyl)-1H-pyrazole-3-carboxamide.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more safe and effective amounts of a hydroxamic acid small molecule compound with pyrazole as a parent core structure as described in any one of claims 1 to 2, a hydrate thereof or a pharmaceutically acceptable salt thereof.

4. A use of a hydroxamic acid small molecule compound with pyrazole as a parent core structure as claimed in any one of claims 1 to 2, a hydrate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 3, characterized in that: The application is selected from at least one of the following applications: application in the preparation of histone deacetylase inhibitors, application in the preparation of preparations that promote the deacetylation of cellular histones H3 and H4 and α-Tubulin, and application as a histone deacetylase inhibitor in the preparation of drugs for preventing and / or treating thioredoxin-mediated diseases.

5. The use according to claim 4, characterized in that: The histone deacetylase inhibitors include HDAC1 inhibitors and HDAC6 inhibitors.

6. The use according to claim 4, characterized in that: The thioredoxin-mediated diseases include autoimmune diseases, allergies or inflammation, neurodegenerative diseases, Alzheimer's disease, Charcot-Marie-Tooth disease, traumatic brain injury, hypertension and stroke.

7. A use of a hydroxamic acid small molecule compound with pyrazole as a parent core structure as claimed in any one of claims 1 to 2, a hydrate thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 3, characterized in that: The application is selected from at least one of the following applications: application in preparing drugs for preventing and / or treating tumors, application in preparing drugs for inhibiting tumor cell proliferation, and application in preparing drugs for promoting tumor cell apoptosis.

8. The use according to claim 7, characterized in that: The drugs for preventing and / or treating tumors include anti-tumor drugs used to induce acquired drug resistance after chemotherapy failure and drugs used to treat malignant tumors.

9. The use according to claim 7, characterized in that: The tumors include: lung cancer, breast cancer, skin cancer, rectal / colon cancer, liver cancer, gastric cancer, prostate cancer, pancreatic cancer, leukemia, ovarian cancer, bladder cancer, kidney cancer, oral cancer, melanoma, head and neck cancer, uterine cancer, brain tumor, esophageal cancer, and lymphoma; the tumor cells include: lung cancer cells, breast cancer cells, epidermal cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, prostate cancer cells, pancreatic cancer cells, leukemia cells, ovarian cancer cells, bladder cancer cells, kidney cancer cells, and oral cancer cells.

10. A method for preparing a hydroxamic acid small molecule compound with pyrazole as a parent core structure as claimed in any one of claims 1 to 2, characterized in that: The preparation method is method A or method B: Wherein, the method A comprises the steps of: S1, different α-ketones react with diethyl oxalate to generate intermediates, which then form corresponding substituted pyrazole rings under the action of hydrazine hydrate and glacial acetic acid; S2, hydrolyzing the corresponding substituted pyrazole ring into carboxylic acid, and reacting the carboxylic acid with different amines to form the corresponding amide compound; S3. In anhydrous tetrahydrofuran, with potassium tert-butoxide as a base, the corresponding amide compound reacts with different bromine compounds to form two intermediates with different configurations; S4, the intermediate is coupled with hydroxylamine hydrochloride in a suitable solvent to generate the target compound; And, the method B comprises the steps of: a), hydrolyzing different substituted pyrazole carboxylic acid ethyl esters into carboxylic acids, and condensing the carboxylic acids with different amines to form corresponding amide compounds; b) In anhydrous tetrahydrofuran, with potassium tert-butoxide as a base, the corresponding amide compound reacts with different bromine compounds to form two intermediates with different configurations; c) The intermediate is coupled with hydroxylamine hydrochloride in a suitable solvent to generate the target compound.

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