1, 3-diaryl-1, 2, 4-triazole-containing terminated compound as well as preparation method and application of 1, 3-diaryl-1, 2, 4-triazole-containing terminated compound

Abstract

The invention provides a 1, 3-diaryl-1, 2, 4-triazole terminated compound as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The compound provided by the invention is modified with a characteristic 1, 3-diaryl-1, 2, 4-triazole group, so that the compound has relatively good selective inhibition activity on HDAC6, and a new choice is provided for an HDAC6 inhibitor. Results of the embodiment show that the 1, 3-diaryl-1, 2, 4-triazole-containing end-capped compound provided by the invention can effectively inhibit the activity of HDAC6 protein, and can resist myocardial cell injury caused by H2O2 and myocardial ischemia / reperfusion injury of mice, so that the 1, 3-diaryl-1, 2, 4-triazole-containing end-capped compound can be used for preparing an HDAC6 inhibitor and also can be used for preparing a medicine for resisting heart diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a 1,3-diaryl-1,2,4-triazole-capped compound and a preparation method and application thereof. Background Art

[0002] Epigenetic regulation plays an important role in cellular and molecular regulatory processes. Among them, histone acetylation and deacetylation are ubiquitous post-translational modification processes. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze and maintain the balance of acetylation levels of lysine residues in nuclear and cytoplasmic proteins, which is essential for cell homeostasis. The dysregulation of HDAC destroys this balance and leads to the occurrence of various diseases. The HDAC family has 18 isoforms, which are divided into four major categories. Class I includes HDAC1, HDAC2, HDAC3 and HDAC8; Class IIa includes HDAC4, HDAC5, HDAC7 and HDAC9; Class IIb includes HDAC6 and HDAC10; Class IV is HDAC11. Among them, HDAC6 is a structurally and functionally unique deacetylase consisting of 1215 amino acids and containing two independent catalytic domains. It can deacetylate specific cytoplasmic non-histone protein substrates, including heat shock protein (Hsp90), cortactin, peroxiredoxin, α-tubulin and heat shock transcription factor-1 (HSF-1).

[0003] Studies have shown that HDAC6 plays an important role in the cardiac system. It regulates the proliferation, differentiation, apoptosis and other mechanisms of cardiac cells. At the same time, it regulates physiological mechanisms such as inflammatory response and angiogenesis. In dilated heart disease, reducing the expression of HDAC6 or inhibiting its activity can improve cardiac function, and HDAC6 has become a molecular target for the prevention and treatment of heart disease.

[0004] Therefore, the development and design of highly effective HDAC6 inhibitors has become an important strategy for the development of anti-heart disease drugs, which is of great significance for the research and treatment of anti-heart disease drugs. Summary of the invention

[0005] The object of the present invention is to provide a 1,3-diaryl-1,2,4-triazole-capped compound and a preparation method and application thereof. The compound provided by the present invention can be used as an HDAC6 inhibitor and / or an anti-cardiac disease drug.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a 1,3-diaryl-1,2,4-triazole-terminated compound, characterized in that it has a chemical structure as shown in Formula I:

[0008]

[0009] The present invention also provides a method for preparing the 1,3-diaryl-1,2,4-triazole-terminated compounds described in the above technical solution, comprising the following steps:

[0010] (1) mixing phenylhydrazine, aromatic aldehyde and alcohol solution, and performing condensation reaction to obtain intermediate A;

[0011] (2) mixing the intermediate A obtained in step (1) with an organic solvent, a first catalyst and aminoethanol, and performing cyclization to obtain the intermediate B;

[0012] (3) mixing the intermediate B obtained in step (2) with an organic solvent and periodinane, and performing an oxidation reaction to obtain an intermediate C;

[0013] (4) mixing the intermediate C obtained in step (3) with an alcohol solvent, a second catalyst and methyl 4-aminobenzoate to carry out an aldehyde-amine condensation reaction to obtain an intermediate D;

[0014] (5) The intermediate D obtained in step (4) is mixed with an organic solvent, an aqueous solution of hydroxylamine and a base to carry out an amidation reaction to obtain a 1,3-diaryl-1,2,4-triazole-terminated compound.

[0015] Preferably, in step (1), the molar ratio of phenylhydrazine to aromatic aldehyde is 1:1.

[0016] Preferably, the temperature of the condensation reaction in step (1) is 20 to 80° C., and the time of the condensation reaction is 4 to 8 hours.

[0017] Preferably, the first catalyst in step (2) is elemental iodine and tert-butyl hydroperoxide, and the molar ratio of the elemental iodine to tert-butyl hydroperoxide is (0.1-0.5): (1.5-9.5).

[0018] Preferably, the molar ratio of the intermediate A to aminoethanol in the step (2) is (1-2.5):(3-9.5).

[0019] Preferably, the temperatures of the oxidation reaction in step (3) and the aldehyde-amine condensation reaction in step (4) are independently room temperature, and the times of the oxidation reaction and the aldehyde-amine condensation reaction are independently 4 to 15 hours.

[0020] The present invention also provides the use of the 1,3-diaryl-1,2,4-triazole-capped compounds described in the above technical scheme or the 1,3-diaryl-1,2,4-triazole-capped compounds prepared by the preparation method described in the above technical scheme in the preparation of HDAC6 inhibitors and / or anti-heart disease drugs.

[0021] Preferably, the anti-heart disease drug is a drug for preventing ischemic heart disease.

[0022] Preferably, the anti-heart disease drug is a drug for resisting oxidative stress damage to myocardial cells caused by strong oxidants.

[0023] The present invention provides a 1,3-diaryl-1,2,4-triazole-capped compound, which is modified with a characteristic 1,3-diaryl-1,2,4-triazole group, so that it has a better selective inhibitory activity on HDAC6, providing a new choice for HDAC6 inhibitors. The results of the examples show that the 1,3-diaryl-1,2,4-triazole-capped compound provided by the present invention can effectively inhibit the activity of HDAC6 protein, and can resist myocardial cell damage caused by H2O2, and resist myocardial ischemia / reperfusion injury in mice, so it can be used to prepare HDAC6 inhibitors and can also be used to prepare anti-heart disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The cell viability test of the protective effect of the compound of Example 1 of the present invention on H9c2 cardiomyocyte injury caused by H2O2;

[0025] Figure 2 This is a cell morphology diagram showing the protective effect of the compound of Example 1 of the present invention on H9c2 cardiomyocyte damage caused by H2O2;

[0026] Figure 3 After the H9c2 cardiomyocytes were treated with the compound of Example 1 of the present invention, the expression level of HDAC6 in the cells was detected by WB;

[0027] Figure 4 This is a graph showing the inflammation-related changes after H9c2 cardiomyocytes were treated with the compound of Example 1 of the present invention;

[0028] Figure 5 This is an echocardiogram of mice after administration of the compound of Example 1 of the present invention;

[0029] Figure 6 The analysis results of the echocardiogram of mice after administration of the compound of Example 1 of the present invention;

[0030] Figure 7The figure is the measurement result of myocardial infarction area in mice after administration of the compound of Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The present invention provides a 1,3-diaryl-1,2,4-triazole-terminated compound, characterized in that it has a chemical structure as shown in Formula I:

[0032]

[0033] The 1,3-diaryl-1,2,4-triazole-capped compounds provided by the present invention have the above structure, are novel in structure, and can effectively inhibit the activity of HDAC6 protein.

[0034] The present invention also provides a method for preparing the 1,3-diaryl-1,2,4-triazole-terminated compounds described in the above technical solution, comprising the following steps:

[0035] (1) mixing phenylhydrazine, aromatic aldehyde and alcohol solution, and performing condensation reaction to obtain intermediate A;

[0036] (2) mixing the intermediate A obtained in step (1) with an organic solvent, a first catalyst and aminoethanol, and performing cyclization to obtain the intermediate B;

[0037] (3) mixing the intermediate B obtained in step (2) with an organic solvent and periodinane, and performing an oxidation reaction to obtain an intermediate C;

[0038] (4) mixing the intermediate C obtained in step (3) with an alcohol solvent, a second catalyst and methyl 4-aminobenzoate to carry out an aldehyde-amine condensation reaction to obtain an intermediate D;

[0039] (5) The intermediate D obtained in step (4) is mixed with an organic solvent, an aqueous solution of hydroxylamine and a base to carry out an amidation reaction to obtain a 1,3-diaryl-1,2,4-triazole-terminated compound.

[0040] In the present invention, unless otherwise specified, the reagents used in the present invention are all commercially available products well known in the art.

[0041] The present invention mixes phenylhydrazine, aromatic aldehyde and alcohol solution, and performs condensation reaction to obtain intermediate A.

[0042] In the present invention, the structural formula of the aromatic aldehyde is preferably

[0043] In the present invention, the molar ratio of the phenylhydrazine to the aromatic aldehyde is preferably 1: 1. In the present invention, the amounts of the two are controlled within the above range, which is more conducive to the full reaction of the two.

[0044] In the present invention, the alcohol solution is preferably an ethanol aqueous solution. In an embodiment of the present invention, the mass percentage of ethanol in the ethanol aqueous solution may be 20%. The present invention uses an alcohol solution as a reaction medium, which is more conducive to promoting the reaction of phenylhydrazine and aromatic aldehyde.

[0045] The volume of the alcohol solution is not particularly limited in the present invention, and can be adjusted according to the amount of phenylhydrazine and aromatic aldehyde, as long as the phenylhydrazine and aromatic aldehyde are fully dissolved. In an embodiment of the present invention, when the amount of the phenyl substance is 10mmol, the volume of the alcohol solution can be 30mL.

[0046] In the present invention, the method of mixing the phenylhydrazine, the aromatic aldehyde and the alcohol solution is preferably: stirring and mixing the phenylhydrazine and the alcohol solution, and then adding the aromatic aldehyde in batches. The present invention has no particular limitation on the speed of the stirring, as long as it can promote uniform mixing of the components. The present invention adopts the above mixing method, which is more conducive to the full dissolution of phenylhydrazine and the aromatic aldehyde in the alcohol solution.

[0047] In the present invention, the temperature of the condensation reaction is preferably 20 to 80°C, more preferably 80°C; the time of the condensation reaction is preferably 6 to 8 hours, more preferably 7 to 8 hours. In the present invention, the condensation reaction is preferably refluxed at the above temperature. The above conditions adopted by the present invention are more conducive to promoting the condensation reaction more fully.

[0048] In the present invention, after the condensation reaction, the system of the condensation reaction is preferably cooled to room temperature, filtered, washed and dried in sequence to obtain intermediate A. The present invention has no particular limitation on the operation methods of cooling to room temperature, filtering, washing and drying, and conventional cooling to room temperature, filtering, washing and drying can be used. In an embodiment of the present invention, the washing reagent can be an ethanol aqueous solution.

[0049] After obtaining intermediate A, the present invention mixes intermediate A with an organic solvent, a first catalyst and aminoethanol to obtain intermediate B through cyclization.

[0050] In the present invention, the organic solvent preferably includes one or more of acetonitrile, tetrahydrofuran, dioxane, ethanol and DMF. The present invention adopts the above organic solvents, which have good solubility for intermediate A. The present invention does not specifically limit the capacity of the organic solvent, as long as it can fully dissolve each component. In an embodiment of the present invention, when the intermediate A is 2.5mmol, the organic solvent can be 15mL.

[0051] In the present invention, the first catalyst is preferably elemental iodine and tert-butyl hydroperoxide, and the molar ratio of the elemental iodine and tert-butyl hydroperoxide is preferably (0.1-0.5): (1.5-9.5), more preferably 0.5: 7.5. The present invention uses the above catalyst to catalyze the cyclization of intermediate A and aminoethanol.

[0052] In the present invention, the molar ratio of the intermediate A to aminoethanol is preferably (1-2.5): (3-9.5), and more preferably 2.5: 7.5. The present invention controls the amount of the two within the above range, which is more conducive to a more complete reaction of the two.

[0053] The present invention has no particular limitation on the method for mixing the intermediate A with the organic solvent, the first catalyst and aminoethanol, as long as each component can be dissolved in the organic solvent.

[0054] In the present invention, the cyclization temperature is preferably 60-80°C, more preferably 90°C; the cyclization time is preferably 4-5h, more preferably 4.5h. In the present invention, the cyclization is preferably carried out under reflux. The present invention can promote more complete cyclization at the above temperature and time.

[0055] In the present invention, after the cyclization, the product obtained by the cyclization is preferably extracted, washed and purified in sequence to obtain an intermediate B. The present invention can remove impurities in the intermediate B by extraction, washing and purification. The present invention has no special restrictions on the extraction, washing and purification methods, as long as the impurities in the intermediate B can be fully removed. In an embodiment of the present invention, the extraction method solvent can be water, and the number of extractions can be 3 times; the washing reagent can be a saturated sodium chloride solution; and the purification method can be silica gel column chromatography purification.

[0056] After obtaining intermediate B, the present invention mixes intermediate B with an organic solvent and periodinane to carry out an oxidation reaction to obtain intermediate C.

[0057] In the present invention, the organic solvent preferably includes one or more of dichloromethane, dichloroethane, toluene, DMF and dioxane. The present invention adopts the above organic solvents, which have good solubility for intermediate B. The present invention does not specifically limit the capacity of the organic solvent, as long as it can fully dissolve intermediate B. In an embodiment of the present invention, when the intermediate B is 1mmol, the organic solvent can be 8mL.

[0058] In the present invention, the method of mixing the intermediate B with the organic solvent and the periodinane is preferably: mixing the intermediate B with the organic solvent, and adding the periodinane in batches under stirring in an ice bath. The present invention adopts the above mixing method to reduce the generation of side reactions.

[0059] In the present invention, the oxidation reaction is preferably carried out at room temperature, and the oxidation reaction time is preferably 4 to 12 hours, more preferably 12 hours. Under the above conditions, the present invention is more conducive to promoting a more complete oxidation reaction.

[0060] In the present invention, after the oxidation reaction, the system obtained by the oxidation reaction is preferably quenched, extracted, washed and dried in sequence without purification to obtain intermediate C. In an embodiment of the present invention, the quenching reagent can be a saturated sodium thiosulfate and sodium bicarbonate solution quenching system; the extraction reagent can be ethyl acetate, and the number of extractions can be 3 times; the washing reagent can be a saturated sodium chloride solution.

[0061] After obtaining the intermediate C, the present invention mixes the intermediate C with an alcohol solvent, a second catalyst and methyl 4-aminobenzoate to carry out an aldehyde-amine condensation reaction to obtain an intermediate D.

[0062] In the present invention, the alcohol solvent preferably includes one or more of methanol, ethanol and n-butanol. The alcohol solvent used in the present invention is more conducive to dissolving the components and promoting the aldehyde-amine condensation reaction. The present invention has no special restrictions on the amount of the alcohol solvent, which can be adjusted according to the amount of intermediate C, and can fully dissolve intermediate C. In an embodiment of the present invention, when the amount of the intermediate C is 0.5mmol, the volume of the alcohol solvent can be 5mL.

[0063] In the present invention, the second catalyst is preferably acetic acid and sodium cyanoborohydride. The present invention utilizes the second catalyst to catalyze the aldehyde-amine condensation reaction of the intermediate C and methyl 4-aminobenzoate.

[0064] In the present invention, the molar ratio of the intermediate C, acetic acid, sodium cyanoborohydride and methyl 4-aminobenzoate is preferably (0.3-1.2): (0.03-0.3): (0.3-2): (0.3-2), and more preferably 0.5: 0.05: 0.55: 0.45. The present invention controls the amount of each component within the above range, which can promote a more complete reaction.

[0065] The present invention has no particular limitation on the method for mixing the intermediate C with the alcohol solvent, the second catalyst and methyl 4-aminobenzoate, as long as each component can be completely dissolved in the alcohol solvent.

[0066] In the present invention, the aldehyde-amine condensation reaction is preferably carried out at room temperature, and the time of the aldehyde-amine condensation reaction is preferably 4 to 12 hours, more preferably 12 hours. In the present invention, the aldehyde-amine condensation reaction is preferably carried out under reflux. The present invention is more conducive to making the aldehyde-amine condensation reaction more complete under the above conditions.

[0067] In the present invention, after the aldehyde-amine condensation reaction, the system of the aldehyde-amine condensation reaction is preferably quenched, washed and purified in sequence to obtain an intermediate D. The present invention has no particular limitation on the methods of quenching, washing and purification, and conventional quenching, washing and purification methods can be used. In an embodiment of the present invention, the quenching reagent can be water; the washing reagent can be a saturated sodium chloride solution; and the purification method can be silica gel column purification.

[0068] After obtaining the intermediate D, the present invention mixes the intermediate D with an organic solvent, an aqueous hydroxylamine solution and a base to carry out an amidation reaction to obtain a 1,3-diaryl-1,2,4-triazole-terminated compound.

[0069] In the present invention, the organic solvent is preferably a mixed solution of dimethylacetamide and methanol, and the volume ratio of dimethylacetamide to methanol is preferably 1: 2. The present invention uses the above solvent, which is more conducive to promoting the amidation reaction more fully.

[0070] In the present invention, when the amount of the intermediate D is 0.25 mmol, the volume of the organic solvent can be 8 mL.

[0071] In the present invention, the volume concentration of the hydroxylamine aqueous solution is preferably 20-80%, more preferably 50%.

[0072] In the present invention, the base is preferably one or more of sodium hydroxide, potassium carbonate, DBU and triethylamine.

[0073] In the present invention, the molar ratio of the intermediate D, the aqueous hydroxylamine solution and the base is preferably (0.1-2): (0.3-8): (0.3-8), and more preferably 0.25: 5: 1. The present invention controls the amount of each component within the above range, which is more conducive to promoting a more complete amidation reaction.

[0074] In the present invention, the method of mixing the intermediate D with the organic solvent, the hydroxylamine aqueous solution and the base is preferably: stirring and mixing the intermediate D, the organic solvent and the hydroxylamine aqueous solution for 30 minutes, and then adding the base and mixing evenly.

[0075] In the present invention, the amidation reaction is preferably carried out at room temperature, and the duration of the amidation reaction is preferably 0.5 to 1 hour, more preferably 1 hour. In the present invention, the amidation reaction is preferably carried out under stirring. The present invention is more conducive to a more complete amidation reaction under the above conditions.

[0076] In the present invention, after the amidation reaction, the solvent is preferably removed from the amidation reaction, the obtained solid precipitate is dissolved in water to obtain a solution, formic acid is added to adjust the pH value of the solution to 5-6, and the solution is filtered and dried to obtain a 1,3-diaryl-1,2,4-triazole end-capped compound.

[0077] In the present invention, the principle of the preparation method of the 1,3-diaryl-1,2,4-triazole end-capped compounds is preferably as shown in Formula II:

[0078]

[0079] The method provided by the invention is simple to operate, can introduce an aromatic group and a 4-amino-N-hydroxy-benzamide group into a 1,2,4-triazole base for modification, and synthesizes a series of 1,3-diaryl-1,2,4-triazole compounds with novel structures, and has the advantages of mild reaction conditions, simple operation, high yield, and the like.

[0080] The present invention also provides the use of the 1,3-diaryl-1,2,4-triazole-capped compounds described in the above technical solution in the preparation of HDAC6 inhibitors and / or anti-heart disease drugs.

[0081] The present invention has no special requirements on the content of the active component in the HDAC6 inhibitor, and those skilled in the art can select it based on experience.

[0082] The present invention has no special requirements for the preparation method of the HDAC6 inhibitor, and any drug preparation method known in the art can be used.

[0083] The present invention has no special requirements on the dosage form of the HDAC6 inhibitor, and those skilled in the art can select the dosage form of the HDAC6 inhibitor according to actual needs.

[0084] In the present invention, the anti-heart disease drug is preferably a drug for preventing ischemic heart disease.

[0085] In the present invention, the anti-cardiac disease drug is a drug for resisting oxidative stress damage of myocardial cells caused by strong oxidants. In the present invention, the strong oxidant is preferably H2O2; and the myocardial cells are H9c2 myocardial cells.

[0086] In the present invention, when preparing the anti-cardiac system disease drug, the anti-cardiac system disease drug may include a 1,3-diaryl-1,2,4-triazole end-capping compound and a pharmaceutically acceptable excipient. The present invention has no special requirements on the specific type of the pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0087] Since the 1,3-diaryl-1,2,4-triazole-terminated compounds provided by the present invention can significantly inhibit HDAC6, they can be used in the preparation of HDAC6 inhibitors and / or anti-heart disease drugs.

[0088] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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.

[0089] Example 1

[0090] A 1,3-diaryl-1,2,4-triazole-terminated compound having the structural formula:

[0091]

[0092] The preparation method of the 1,3-diaryl-1,2,4-triazole-terminated compound comprises the following steps:

[0093] (1) Add 10 mmol of phenylhydrazine and 30 mL of ethanol (20% aqueous solution) to a 100 mL round-bottom flask, then stir at room temperature and add aromatic aldehyde in batches; then, reflux the system at 80° C. for 8 h, monitor by TLC until complete conversion, cool to room temperature, filter, wash the solid with cold ethanol (20% aqueous solution), and dry in an oven to obtain intermediate A;

[0094] (2) 2.5 mmol of the intermediate A obtained in step (1) was dissolved in 15 mL of acetonitrile, and then 7.5 mmol of ethanolamine, 7.5 mmol of TBHP and 0.5 mmol of I2 were added to the obtained solution in sequence, and the system was refluxed at 90° C. for 4.5 h. After TLC monitoring until complete conversion, the mixture was cooled to room temperature, 10 mL of water was added, and the mixture was extracted with 15 mL of ethyl acetate, which was repeated three times; then, the organic layers were combined and washed with 25 mL of saturated sodium chloride solution; finally, the solvent was concentrated, and the precipitate was purified by silica gel column chromatography to obtain intermediate B;

[0095] (3) Dissolve 1 mmol of the intermediate B obtained in step (2) in 8 mL of dichloromethane, then continue stirring in an ice bath, and then add 1.5 mmol of DessMartin periodinane in batches; after stirring at room temperature for 12 h, quench the system with saturated sodium thiosulfate and sodium bicarbonate solutions, extract with 15 mL of ethyl acetate, and repeat three times; then, combine the organic layers and wash with 25 mL of saturated sodium chloride solution; remove the organic solvent in vacuo to obtain the intermediate C, which is directly used in the next step without purification;

[0096] (4) Dissolve 0.5 mmol of the intermediate C obtained in step (3) in 5 mL of methanol, stir at room temperature, add 0.45 mmol of methyl 4-aminobenzoate, and then add 0.05 mmol of acetic acid in a catalytic amount; after stirring for 0.5 h, add 0.55 mmol of sodium cyanoborohydride in batches, stir for another 12 h, then add 10 mL of water to quench the system, and extract three times with 15 mL of ethyl acetate; then, combine the organic layers and wash with 25 mL of saturated sodium chloride solution; finally, remove the solvent and purify by silica gel column chromatography to obtain intermediate D;

[0097] (5) Add 5 mmol of hydroxylamine (50% aqueous solution) to 0.25 mmol of the intermediate D obtained in step (4) in a mixture of 8 mL of DCM:MeOH = 1:2, and then stir at room temperature for 0.5 h, followed by adding 1 mmol of sodium hydroxide; stir the system for another 1 h, and monitor by TLC until complete conversion; then remove the solvent in vacuo, dissolve the precipitate in 5 mL of water, add formic acid to adjust the pH to 5, filter the solid, wash with water, and dry in an oven to obtain the compound of formula I; the Chinese name is: 4-(((3-(3-bromothiophene-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)methyl)amino)-N-hydroxybenzamide;

[0098] The H-NMR and C-NMR data of the 1,3-diaryl-1,2,4-triazole-terminated compounds prepared in this example are as follows:

[0099] Dark brown solid,yield:83%.Mp:231e232 C. 1 H NMR(400MHz,DMSO-d6,ppm)d10.79(s,1H,OH,D2O exchangeable),7.78(d,J1 / 41.6Hz,1H,NH,D2O exchangeable),7.69e7.64(m,2H,AreH),7.60(d,J1 / 42.0Hz,2H,AreH),7.58(t,J1 / 43.0Hz,2H,AreH),7.51(s,1H,AreH),7.49(s,1H,AreH),6.74(s,1H,NH,D2O exchangeable),6.53(d,J1 / 48.5Hz,2H,AreH),4.50(s,2H,eCH 2e ). 13C NMR(101MHz,DMSO-d6,ppm)d 164.73,155.60,154.71,150.17,136.51,134.71,129.57,129.33,128.18,1 27.89,125.09,124.77,120.34,111.23,109.54,38.72.HR-MS(ESI),calcd.C 20 H 16 BrN5O2S, 492.0106,found:4692.0099.

[0100] Test Example 1

[0101] The MTT method was used to detect the anti-H2O2 damage to H9c2 cardiomyocytes by the 1,3-diaryl-1,2,4-triazole-capped compounds (hereinafter referred to as compounds) provided in Example 1. The test process was as follows:

[0102] (1) Plating: H9c2 cells were digested and resuspended into a single cell suspension and plated in a 96-well plate with 5 × 10 cells per well. 3 The cells were cultured in a 37°C incubator overnight.

[0103] (2) Preparation: The compound provided in Example 1 and Tubastatin A (a selective HDAC6 inhibitor) were prepared into a 10 mM stock solution with DMSO. The compound obtained in Example 1 was diluted to 2 μM, 1 μM, and 0.5 μM, respectively; Tubastatin A was diluted to 2 μM, 100 μL per well, and 6 replicate wells per gradient;

[0104] (3) Adding drugs: aspirate and discard the cell supernatant of the 96-well plate, add 100 μL of different concentrations of the compound of Example 1 and Tubastatin A to each well, and culture in a 37° C. incubator for 24 h;

[0105] (4) Modeling: Aspirate the cell supernatant of the 96-well plate and add 100 μL of culture medium containing H2O2 (300 μM) to each well for 1.5 h;

[0106] (5) Cell viability test: Dilute the MTT stock solution (5 mg / mL) with serum-free cell culture medium at a ratio of 1:100, discard the cell supernatant, add MTT detection solution, 100 μL per well, and incubate at 37°C for 4 h; discard the supernatant, add 150 μL DMSO to each well, shake and dissolve for 10 min, and after the crystals are completely dissolved, detect the absorbance at 490 nm with an enzyme reader. The results are as follows Figure 1 shown.

[0107] from Figure 1It can be seen that as the concentration of the compound in Example 1 increases, the cell survival rate increases significantly. This indicates that the drug has a significant protective effect on H9c2 and has a significant concentration dependence. Compared with the CON group, the cell survival rate in the H2O2 group dropped to 51.0%, and the cell survival rate in the compound pre-protection group increased to 67.5%, 85.5%, and 93.3% with the increase in concentration.

[0108] Test Example 2

[0109] The cell morphology of the 1,3-diaryl-1,2,4-triazole-terminated compounds prepared in Example 1 against the damage of H2O2 to H9c2 cardiomyocytes was photographed. The test process is as follows:

[0110] (1) Plating: H9c2 cells were digested and resuspended into a single cell suspension and plated in a six-well plate with 3 × 10 cells per well. 4 The cells were cultured in a 37°C incubator overnight.

[0111] (2) Preparation: The compound of Example 1 was prepared into a 10 mM stock solution with DMSO, diluted to 2 μM, 1 μM, and 0.5 μM, and 2 mL was added to each well;

[0112] (3) Adding drugs: aspirate and discard the cell supernatant of the six-well plate, add 2 mL of the compound of Example 1 at different concentrations to each well, and culture in a 37° C. incubator for 24 h;

[0113] (4) Modeling: Aspirate the cell supernatant of the 96-well plate and add 100 μL of culture medium containing H2O2 (300 μM) to each well for 1.5 h;

[0114] (5) Photography: Photographed under a fluorescence inverted microscope, bar = 500 nm, 200×.

[0115] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the number of H9c2 in the CON group is relatively large, and the morphology is spindle-shaped, uniform in size and full; compared with the CON group, the number of cells in the H2O2 group is significantly reduced, the morphology is obviously wrinkled, the size is uneven, and there is an aggregated distribution; the drug-treated group has an obvious protective effect, which significantly reduces the damage caused by H2O2, and the protective effect is proportional to the concentration of the compound in Example 1.

[0116] Test Example 3

[0117] In vitro assay of HDAC6 enzyme activity: HDAC6 in vitro assay OptiPlate-96 black microplate (PerkinElmer) was used for in vitro assay of HDAC6 enzyme activity. A series of compounds as well as SAHA (HDAC pan-inhibitor) and TubastatinA (selective HDAC6 inhibitor) were diluted with HDAC assay buffer containing 1% DMSO. Human recombinant HDAC6 (BPS Bioscience, USA, catalog number 50006) was then incubated in a 96-well black plate at 37°C for 40 min in incubation buffer, BSA, fluorescent substrate (Z-(Ac)-Lys-AMC) and test compounds. After incubation, trypsin was added for 15 min to release the fluorescent fragment of AMC. Fluorescence intensity was measured using an excitation wavelength of 390 nm and an emission wavelength of 460 nm. IC of the compound 50 The results were calculated and fitted using GraphPadprism 6.0 and are shown in Table 1.

[0118] Table 1 In vitro assay results of HDAC6 enzyme activity

[0119] Compound Compound formula 1 SAHA Tubastatin A HDAC6 / IC50(nM) 54.94 59.3 64.04

[0120] As can be seen from Table 1, the compound of Example 1 has an IC of HDAC6 50 The concentration of HDAC6 in the sample was 54.94 nM, which was lower than that in SAHA (59.3 nM) and Tubastatin A (64.04 nM). This indicated that Example Compound 1 had a good inhibitory effect on HDAC6.

[0121] Test Example 4

[0122] WB was used to detect the regulation of intracellular HDAC6 and inflammatory proteins by the compound of Example 1. The test process was as follows:

[0123] (1) Plating: H9c2 cells were digested and resuspended into a single cell suspension and plated in a 6-well plate with 2-4×10 cells per well. 5 The cells were cultured in a 37°C incubator overnight.

[0124] (2) Adding drugs: aspirate and discard the cell supernatant of the 6-well plate, add 2 mL of the compound of Example 1 at different concentrations to each well, and culture in a 37° C. incubator for 24 h;

[0125] (3) Protein collection: Wash cells twice with pre-cooled PBS, add RIPA containing PMFS and phosphatase inhibitors, lyse on ice for 30 min, scrape cells to collect proteins, centrifuge at 12000 rpm for 10 min, aspirate supernatant, add loading buffer, and boil for 10 min;

[0126] (4) Electrophoresis: Separation by 10% SDS page gel electrophoresis;

[0127] (5) Transferring the membrane; after transferring to the PVDF membrane, the membrane was blocked with 5% BSA for 2 h, and antibodies HDAC6, IL-18, ASC, Caspase-1, NLRP1, AIM2, NLRP3, NLRC4, GPX4, and GAPDH were added and incubated at 4°C overnight;

[0128] (6) Wash three times with PBST, add secondary antibody diluted 1:10000; wash three times with PBST, add ECL luminescent solution for development and exposure; the results are as follows Figure 3 and 4 shown.

[0129] Depend on Figure 3 and 4 It can be seen that the addition of the compound of Example 1 of the present invention can significantly inhibit the expression of HDAC6 protein, and at the same time can reduce the expression of NLRP3, ASC, Caspase-1, IL-18 proteins of the inflammasome family, but has no significant effect on the expression of NLRP1, NLRC4 and AIM2. At the same time, it has a significant activation effect on the protein of GPX4, a key protein of ferroptosis. It shows that the compound of Example 1 of the present invention can effectively reduce the expression level and activity of inflammatory proteins in H9c2 cardiomyocytes and increase the expression of GPX4, a key protein of ferroptosis, which may be achieved by inhibiting the expression of HDAC6 in H9c2 cardiomyocytes.

[0130] Test Example 5

[0131] Example 1 Protective effect of the compound on myocardial ischemia-reperfusion

[0132] (1) Echocardiographic analysis

[0133] In vivo myocardial ischemia-reperfusion injury model was established; mice were anesthetized by inhalation of 2% isoflurane using an anesthesia device. Myocardial ischemia-reperfusion injury was induced by temporarily "popping" the heart through a small hole in the fourth intercostal space, while the left coronary artery (3 mm from the origin of LCA) was sutured with a 6-0 silk suture. Electrocardiogram monitoring showed significant ST segment elevation, indicating that MIR modeling was successful. After ligation for 30 minutes, the silk slip knot was removed, and myocardial reperfusion was induced within the specified time. Electrocardiogram showed ST segment recovery, indicating that MIR injury modeling was successful. One day before surgery, oral administration was performed by gavage. The mice were randomly divided into 5 groups on the second day, namely, sham operation group (Sham group), myocardial ischemia-reperfusion group (MIR group), MIR+Example 1 compound (5 mg / kg) group, MIR+Example 1 compound (10 mg / kg) group, and MIR+Example 1 compound (20 mg / kg) group.

[0134] The Vevo 2100 echocardiogram, which contains an in vivo imaging system (Fusion Film Visual Ultrasonics, Toronto, Canada) was used for echocardiography. Under anesthesia, the mouse chest hair was shaved, and two-dimensional long-axis images were acquired for end-diastolic and end-systolic volume measurements. The echocardiography system automatically calculated and recorded the left ventricular ejection fraction (EF%) and fractional shortening (FS%). Results are shown in Table 1. Figure 5 and 6 shown.

[0135] Depend on Figure 5 and 6 It can be seen that the compound of Example 1 has a protective effect on myocardial ischemia-perfusion injury in vivo. Through the summary of echocardiographic data, compared with the Sham group, the EF and FS of the MIR group were reduced to 54.18% and 27.57%, respectively. After the administration of the compound of Example 1, as the dose increased, the EF and FS increased, respectively. As the dose increased, the EF increased to 55.32%, 73.14%, and 81.19%, and as the dose increased, the EF increased to 27.98%, 41.87%, and 48.28%, indicating that the compound of Example 1 has a significant myocardial protective effect and improves the contractile and diastolic functions of the myocardium.

[0136] (2) Determination of myocardial infarction area

[0137] Myocardial infarct size determination included measurement of the area at risk and infarct size. At dissection, the heart was perfused with 1% Evans blue dye (Sigma-Aldrich, St. Louis, MO) to delineate the ischemic area at risk (AAR) and then snap-frozen at -20°C for 30 min. All sections were incubated in 1% 2,3,5-triphenylammonium chloride (TTC, Sigma Co., St Louis, Mo) buffer (pH 7.4) in the dark to identify unstained necrotic areas within the ischemic area at risk. These areas were segmented and digitally evaluated by using Image-Pro Plus 6.0. The myocardial infarct area appeared white or pale and was expressed as the ratio of infarcted myocardium to left ventricular area × 100%. The results are shown in Figure 7 shown.

[0138] Depend on Figure 7 It can be seen from the EB-TTC staining experiment that while the safety areas of each group remained basically the same, the proportion of the infarct area in the MIR group significantly increased to 58.96%. As the dosage increased, the infarct area gradually decreased by 51.97%, 21.47%, and 8.91%, respectively. This indicates that the compound in Example 1 can significantly reduce the area of ​​myocardial infarction in perfused mice caused by myocardial ischemia and has a significant myocardial protective effect.

[0139] From the above results, it can be seen that the 1,3-diaryl-1,2,4-triazole-terminated compounds provided by the present invention can effectively inhibit the activity of HDAC6 protein, and can resist myocardial cell damage caused by H2O2, and resist myocardial ischemia / reperfusion injury in mice, so they can be used to prepare HDAC6 inhibitors, and can also be used as new compounds for the preparation of anti-heart disease drugs. Therefore, the present invention can provide a new research and development direction for the development of HDAC6 inhibitors, and can provide a new structural selection for the development of new anti-heart disease drugs.

[0140] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A 1,3-diaryl-1,2,4-triazole-terminated compound, characterized in that: It has a chemical structure as shown in Formula I:

2. The method for preparing the 1,3-diaryl-1,2,4-triazole-terminated compound according to claim 1, comprising the following steps: (1) mixing phenylhydrazine, aromatic aldehyde and alcohol solution, and performing condensation reaction to obtain intermediate A; (2) mixing the intermediate A obtained in step (1) with an organic solvent, a first catalyst and aminoethanol, and performing cyclization to obtain the intermediate B; (3) mixing the intermediate B obtained in step (2) with an organic solvent and periodinane, and performing an oxidation reaction to obtain an intermediate C; (4) mixing the intermediate C obtained in step (3) with an alcohol solvent, a second catalyst and methyl 4-aminobenzoate to carry out an aldehyde-amine condensation reaction to obtain an intermediate D; (5) The intermediate D obtained in step (4) is mixed with an organic solvent, an aqueous hydroxylamine solution and a base to carry out an amidation reaction to obtain a 1,3-diaryl-1,2,4-triazole-terminated compound.

3. The preparation method according to claim 2, characterized in that: In the step (1), the molar ratio of phenylhydrazine to aromatic aldehyde is 1:

1.

4. The preparation method according to claim 2, characterized in that: The temperature of the condensation reaction in step (1) is 20 to 80° C.; the time of the condensation reaction is 4 to 8 hours.

5. The preparation method according to claim 2, characterized in that: The first catalyst in step (2) is elemental iodine and tert-butyl hydroperoxide, and the molar ratio of the elemental iodine to tert-butyl hydroperoxide is (0.1-0.5): (1.5-9.5).

6. The preparation method according to claim 2, characterized in that: The molar ratio of the intermediate A to aminoethanol in the step (2) is (1-2.5):(3-9.5).

7. The preparation method according to claim 2, characterized in that: The temperatures of the oxidation reaction in step (3) and the aldehyde-amine condensation reaction in step (4) are independently room temperature, and the times of the oxidation reaction and the aldehyde-amine condensation reaction are independently 4 to 15 hours.

8. Use of the 1,3-diaryl-1,2,4-triazole-terminated compound according to claim 1 or the 1,3-diaryl-1,2,4-triazole-terminated compound prepared by the preparation method according to any one of claims 2 to 7 in the preparation of HDAC6 inhibitors and / or anti-heart disease drugs.

9. The use according to claim 8, characterized in that: The anti-heart disease drug is a drug for resisting ischemic heart disease.

10. The use according to claim 8, characterized in that: The anti-cardiac disease drug is a drug for resisting oxidative stress damage of myocardial cells caused by strong oxidants.

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