A kind of thiophenol compound and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and in particular to a thiophenol compound and its preparation method and application. The present invention uses the prepared thiophenol compound for human kidney cells HK-2 and live mice, and finds that the above-mentioned compound can significantly inhibit the peroxidation of cell membrane lipid molecules induced by hypoxia-reoxygenation, and then induces cell ferroptosis, and has a long-term stable inhibitory effect. The levels of creatinine and urea nitrogen in serum after kidney damage can be significantly reduced by pre-treating the compound, indicating that it can alleviate acute kidney injury. In addition, the compound pre-treatment prepared by the present invention significantly reduces the damage to the outer medullary region of the kidney caused by ischemia-reperfusion, and effectively alleviates lesions such as renal tubular epithelial cell necrosis, and its application in the repair of renal ischemia-reperfusion injury provides a new treatment strategy for ferroptosis inhibitors.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a thiophenol compound and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Renal ischemia-reperfusion injury (IRI) refers to further damage to the kidney caused by oxidative stress, cell damage, and inflammatory response when blood is reperfused after ischemia. The occurrence of renal ischemia-reperfusion injury is related to multiple factors, including the generation of oxygen free radicals, mitochondrial dysfunction, cell apoptosis, and necrosis. Studies have found that IRI not only involves traditional cell death pathways, such as apoptosis and necrosis, but is also closely related to a new type of cell death, ferroptosis.

[0004] Ferroptosis is a non-apoptotic cell death caused by cell membrane lipid peroxidation mediated by iron ions. It plays a key role in the pathological process of renal ischemia-reperfusion injury and becomes a new therapeutic target. Studies have shown that ferroptosis is not only involved in the occurrence of acute kidney injury, but is also closely related to the recovery of renal function. Therefore, intervention against ferroptosis has become an emerging research direction for the prevention and treatment of renal ischemia-reperfusion injury. Inhibiting ferroptosis can not only reduce oxidative damage to cells, but also improve kidney function, reduce inflammatory response and cell necrosis, thus providing new ideas and strategies for the treatment of acute kidney injury.

[0005] Although a variety of methods and drugs have been proposed to reduce renal ischemia-reperfusion injury, especially to reduce cell damage through antioxidant and anti-inflammatory pathways, these methods still have certain limitations. For example, existing antioxidants usually reduce oxidative damage by improving the antioxidant capacity of cells, but their effects in inhibiting ferroptosis are relatively limited. In addition, many drugs may have adverse reactions or toxicity during clinical application, which limits their long-term use. Treatment strategies for ferroptosis have gradually attracted attention, but existing ferroptosis inhibitors are still in the preliminary research stage, and there is a lack of clinical drugs that can effectively and stably inhibit ferroptosis.

[0006] Thiophenol compounds play an important role in the field of drug development due to their unique chemical structure and wide range of pharmacological effects. As a biologically active molecule, thiophenol compounds have multiple pharmacological effects such as anti-inflammatory, anti-fibrotic, antibacterial, and anti-tumor, and can regulate cell growth, differentiation, and apoptosis. Studies have shown that thiophenol compounds can help improve organ function and reduce kidney damage by regulating multiple cell signaling pathways, reducing the accumulation of extracellular matrix, and inhibiting pathological fibrosis.

[0007] Therefore, the preparation of a class of thiophenol-based ferroptosis inhibitors with high activity, good pharmacological properties and safety is of great significance in regulating renal inflammatory response and reducing cell damage. It can also be used as an inhibitor of drugs for the treatment of diseases such as neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular, renal failure and diabetic complications, providing a new therapeutic strategy for the prevention and treatment of ferroptosis. Summary of the invention

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide a thiophenol compound and a preparation method and application thereof. The present invention utilizes the design of the thiophenol structure and the amino substituent to enhance the antioxidant activity of the thiophenol compound, which can play a strong protective effect in cells; it has a long-term and stable inhibitory effect, can continuously and effectively reduce the occurrence of ferroptosis, and reduce oxidative stress and inflammatory response; especially in renal ischemia-reperfusion injury, it can effectively inhibit ferroptosis through multiple mechanisms, significantly reduce the damage to the outer medullary region of the kidney, and alleviate the necrosis of renal tubular epithelial cells, and has broad clinical application prospects.

[0009] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0010] In a first aspect of the present invention, there is provided a thiophenol compound or a pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer thereof, having a structure shown in Formula I:

[0011] Formula I;

[0012] Wherein, R is independently hydrogen, C1~C2 alkyl, , , , , , , , , .

[0013] Preferably, the thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer is one of the following compounds:

[0014]

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer, comprising the following steps:

[0016] S1, mixing methyl 3-amino-4-mercaptobenzoate and hydrazine hydrate in an organic solvent, heating to react to obtain 3-amino-4-mercaptobenzoyl hydrazide, mixing the 3-amino-4-mercaptobenzoyl hydrazide with KOH and CS2 to obtain 2-(3-amino-4-mercaptobenzoyl)hydrazine-1-aminodithioic acid potassium, and mixing and reacting with hydrazine hydrate to obtain compound 1;

[0017] S2. Mix compound 1, KOH and compound R to react to obtain compounds 2~12.

[0018] Preferably, in step S1, the molar ratio of methyl 3-amino-4-mercaptobenzoate to hydrazine hydrate is 1-1.5:1; the organic solvent is selected from one or more of anhydrous ethanol and methanol; the temperature of the heating reaction is 80-90° C., and the time is 5-10 h.

[0019] Preferably, in step S1, the molar ratio of 3-amino-4-mercaptobenzohydrazide, KOH and CS2 is 1:1~2:1~2.

[0020] Preferably, in step S1, the molar ratio of the 2-(3-amino-4-mercaptobenzoyl)hydrazine-1-aminodithioic acid potassium and hydrazine hydrate is 1:1.5~3.5.

[0021] Preferably, in step S1, the temperature of the mixing reaction is 100-110° C., and the time is 20-40 min.

[0022] Preferably, in step S2, the compound R is selected from benzyl chloride, 4-methylbenzyl chloride, 4-methoxybenzyl chloride, 4-cyanobenzyl chloride, 4-fluorobenzyl chloride, 2-(chloromethyl)naphthalene, methyl chloride, ethyl chloride, 1-(chloromethyl)-4-nitrobenzene, 4-(chloromethyl)-N,N-diethylimine, and 4-(trifluoromethyl)benzyl chloride.

[0023] Preferably, in step S2, the molar ratio of compound 1, KOH and compound R is 1-1.5:1-1.5:1.

[0024] Preferably, in step S2, the reaction temperature is 0°C and the reaction time is 0.5 to 24 h;

[0025] The crude product obtained by the reaction needs to be further purified, specifically by adding 98% by mass concentrated sulfuric acid to the crude product for purification, and then filtering, washing, and recrystallizing in sequence.

[0026] The third aspect of the present invention provides a pharmaceutical composition, which comprises an active dose of the thiophenol compound described in the first aspect, or its pharmaceutically acceptable salts, pharmaceutically acceptable esters, solvates, hydrates and stereoisomers as the main ingredients, and is prepared by adding biopharmaceutically acceptable auxiliary ingredients.

[0027] Preferably, the dosage of the thiophenol compound is an effective dosage, and the pharmaceutical composition is in a dosage form that is easy for precise administration.

[0028] Preferably, when the pharmaceutical composition is used to prepare a pharmaceutical preparation for in vivo administration, the pharmaceutical preparation is sterile.

[0029] The fourth aspect of the present invention provides a use of the thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer described in the first aspect and / or the pharmaceutical composition described in the third aspect in the preparation of a ferroptosis inhibitor.

[0030] The fifth aspect of the present invention provides a use of the thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer described in the first aspect and / or the pharmaceutical composition described in the third aspect in preparing a ferroptosis pathway inhibition model.

[0031] Preferably, the ferroptosis pathway inhibition model is to use the thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer described in the first aspect and / or the pharmaceutical composition described in the third aspect as a model drug for the preparation of a basic research model.

[0032] The sixth aspect of the present invention provides a use of the thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer described in the first aspect and / or the pharmaceutical composition described in the third aspect in the preparation of a lipid peroxidation inhibitor.

[0033] The seventh aspect of the present invention provides a use of the thiophenol compound or its pharmaceutically acceptable salt, pharmaceutically acceptable ester, solvate, hydrate and stereoisomer described in the first aspect and / or the pharmaceutical composition described in the third aspect in the preparation of a drug for treating acute kidney injury.

[0034] The eighth aspect of the present invention provides a use of the thiophenol compound or its salt, or its stereoisomer described in the first aspect and / or the pharmaceutical composition described in the third aspect in the preparation of a drug for preventing, improving and / or treating diseases related to ferroptosis.

[0035] Preferably, the ferroptosis-related diseases are neurodegenerative diseases, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, diabetic complications, and pancreatic cancer.

[0036] Further preferably, the neurodegenerative disease is Parkinson's syndrome; and the stroke is ischemic stroke.

[0037] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0038] (1) The present invention proposes for the first time a novel strategy targeting the ferroptosis pathway and improves the repair effect of thiophenol compounds in renal ischemia-reperfusion injury by optimizing their structure. Compared with the prior art, the compounds provided by the present invention not only have significant advantages in inhibiting ferroptosis, but also show long-term and stable inhibitory effects in kidney cells and mouse models, and can continuously and effectively reduce the occurrence of ferroptosis, and alleviate oxidative stress and inflammatory response, and have broad application prospects in the prevention and treatment of acute kidney injury and ferroptosis-related diseases.

[0039] (2) The ferroptosis inhibitor obtained by the present invention can effectively target the key process of ferroptosis, especially play a role in the repair of renal ischemia-reperfusion injury.

[0040] Through molecular design, synthesis, activity screening and pharmacological action testing of the compound, the present invention verifies the unique advantages of the compound in preventing and treating ferroptosis and alleviating renal ischemia-reperfusion injury. The compound blocks the expansion of free radicals on the lipid membrane, reduces the peroxidation of membrane lipids, inhibits the occurrence of ferroptosis, and thus reduces the damage and death of kidney cells.

[0041] The experimental results show that the thiophenol compounds prepared by the present invention can effectively reduce the levels of serum creatinine (Cr) and urea nitrogen (BUN) after kidney injury, alleviate pathological damage, and significantly improve kidney function, providing an effective therapeutic strategy for the repair of acute kidney injury.

[0042] (3) The thiophenol compounds provided by the present invention can not only be used to treat acute kidney injury, but can also be applied to other diseases related to ferroptosis, such as neurodegenerative diseases, cardiovascular diseases, etc., providing a new therapeutic strategy for the prevention and treatment of ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The broad-spectrum anti-ferroptosis activity evaluation test of compound 3 in Experimental Example 2 of the present invention is performed;

[0044] Figure 2The protective effect of compound 3 in Experimental Example 3 of the present invention on hypoxia / reoxygenation injury; wherein (A) is the result of lipid peroxidation biochemical detection, and (B) is the result of cell survival rate detection;

[0045] Figure 3 The results of the treatment of kidney damage in mice administered with compound 3 in Experimental Example 4 of the present invention, wherein (A) is the result of the biochemical test of urea nitrogen (BUN), and (B) is the result of the biochemical test of creatinine (Cr);

[0046] Figure 4 The following are pathological HE staining images (20X) of kidney damage in mice treated with compound 3 in Experimental Example 4 of the present invention, wherein (A) is the control group, (B) is the I / R group, (C) is the I / R+compound 3 (2 mg / kg) group, and (D) is the I / R+compound 3 (10 mg / kg) group;

[0047] Figure 5 The immunohistochemical images of pathological HNE of kidney damage in mice treated with compound 3 in Experimental Example 4 of the present invention are shown, wherein (A) is the control group, (B) is the I / R group, (C) is I / R + compound 3 (2 mg / kg), and (D) is I / R + compound 3 (10 mg / kg). DETAILED DESCRIPTION

[0048] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0049] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0050] Ferrostatin-1, an inhibitor of ferroptosis, and erastin, RSL3, ML210, FIN56, DL-buthionine-(S,R) sulfoximine (BSO), and sulfasalazine (SFZ) were purchased from Cayman and dissolved in sterile dimethyl sulfoxide (DMSO) to prepare the required concentrations. The fluorescent probe C11-BODIPY 581 / 591 was purchased from Invitrogen. The CCK-8 kit was purchased from Shanghai Beibo Biotechnology Co., Ltd.

[0051] The culture conditions of human renal proximal tubule HK-2 cells were: RPMI-1640 medium (GIBCO) containing 10% FBS (GIBCO), 37°C, 5% CO2 saturated humidity incubator.

[0052] The statistical analysis was performed using R software, and the experimental data were expressed as Mean ± SEM. Tukey's test (ANOVA) was used for intergroup comparisons in cell and animal experiments, and Student's t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant, and different letters indicate P < 0.05.

[0053] The raw materials and reagents used in the present invention are as follows:

[0054] Table 1 Main raw materials and reagents

[0055]

[0056] The specific synthesis process of the compound of the present invention is as follows:

[0057]

[0058] Example 1: This example provides the preparation process and verification data of compound 1

[0059] Compound 1: 4-amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazole-3-thiol

[0060] 3-Amino-4-mercaptobenzoic acid methyl ester (18.30 g, 0.1 mol) was dissolved in anhydrous ethanol (30 mL), and 35% mass fraction of hydrazine hydrate (6.16 mL, 0.1 mol) was added dropwise to the mixture under stirring, and refluxed at 85°C for 6 hours. The reaction was monitored by thin layer chromatography using ethyl acetate and petroleum ether (1:1) as eluents and ultraviolet light on a silica gel plate. After the reaction was completed, the excess ethanol was distilled off and cooled and filtered, and the formed crystals were washed with water and dried to obtain 3-amino-4-mercaptobenzoic acid hydrazide with a yield of 13.73 g (0.075 mol, 75%).

[0061] Potassium hydroxide (8.42 g, 0.15 mol) was dissolved in anhydrous ethanol (200 mL), and 3-amino-4-mercaptophenylhydrazine (18.31 g, 0.1 mol) was added, and the mixture was cooled in ice, and then carbon disulfide (11.42 g, 0.15 mol) was added in 4 portions, while stirring continuously, and the reaction was carried out at room temperature for 15 hours. The reaction mixture was diluted with anhydrous ether, and the precipitate was collected by filtration, further washed with anhydrous ether (100 mL) and dried, and used directly in the next step without further purification to obtain 2-(3-amino-4-mercaptobenzoyl)hydrazine-1-aminodithioic acid potassium, with a yield of 24.35 g (0.082 mol, 82%).

[0062] Potassium 2-(3-amino-4-mercaptobenzoyl)hydrazine-1-aminodithioate (29.70 g, 0.1 mol) was suspended in water (5 mL), and hydrazine hydrate (18.48 mL, 0.3 mol) was added. The mixture was stirred and refluxed at 105°C for 30 minutes. The reaction was monitored on a silica gel plate by thin layer chromatography using ethyl acetate and petroleum ether (1:1) as eluents. After the reaction was completed, the mixture was cooled to room temperature and diluted with water (100 mL). It was acidified with concentrated hydrochloric acid and filtered, washed thoroughly with cold water, and recrystallized from ethanol to obtain 4-amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazole-3-thiol with a yield of 16.25 g (0.068 mol, 68%).

[0063] 1 H NMR (400 MHz, DMSO-d6) δ 7.33 (s, 1H), 6.93 (dd, J = 7.0, 2.2 Hz,1H), 6.74 (d, J = 2.1 Hz, 1H), 5.77 (s, 2H), 5.14 (s, 1H), 4.90 (s, 2H), 4.09(s, 1H). HRMS (ESI) m / z: calcd for C8H9N5S2:[M+H] 239.03, found 239.32. HPLCpurity = 95.2%.

[0064] Example 2: This example provides the preparation process and verification data of compound 2

[0065] Compound 2: 2-amino-4-(4-amino-5-(benzylthio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0066] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) prepared in Example 1 was suspended in water (10 mL), and a potassium hydroxide solution (5.5 mL, 0.1 mol) with a mass fraction of 85% was added while stirring at room temperature. After 10 minutes, the solution was cooled to 0°C and placed in an ice bath, and benzyl chloride (12.60 g, 0.1 mol) was added under vigorous stirring. The reaction progress was monitored using thin layer chromatography (TLC, ether as eluent). When the reaction was completed, concentrated sulfuric acid (10 mL, 0.184 mol) with a mass fraction of 98% was added, and a white precipitate was slowly precipitated from the crude product. The precipitate was filtered, washed with water, and crystallized from ethanol to give 2-amino-4-(4-amino-5-(benzylthio)-4H-1,2,4-triazol-3-yl)benzenethiol in a yield of 28.30 g (0.086 mol, 86%).

[0067] 1 H NMR (400 MHz, DMSO-d6) δ 7.42 – 7.36 (m, 3H), 7.38 – 7.28 (m, 3H),7.28 – 7.21 (m, 1H), 6.93 (dd, J = 7.1, 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01 (s, 2H), 4.90 (s, 2H), 4.40 (t, J = 0.9 Hz, 2H), 4.09 (s, 1H). HRMS (ESI)m / z: calcd for C 15 H 15 N5S2:[M+H] 329.08, found 329.44. HPLC purity = 94.6%.

[0068] Example 3: This example provides the preparation process and verification data of compound 3

[0069] Compound 3: 2-amino-4-(4-amino-5-((4-methylbenzyl)thio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0070] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 4-methylbenzyl chloride (14.00 g, 0.1 mol) was added dropwise under vigorous stirring. The progress of the reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 3 with a yield of 27.44 g (0.080 mol, 80%).

[0071] 1 H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 7.1 Hz, 1H), 7.18 (dt, J = 7.7,1.1 Hz, 2H), 7.13-7.07 (m, 2H), 6.99-6.90 (m, 2H), 6.74 (d, J = 1.9 Hz, 1H), 5.65 (s, 2H), 4.97 (s, 2H), 4.44 (t, J = 0.9 Hz, 2H). HRMS (ESI) m / z: calcd forC 16 H 17 N5S2[M+H] 343.09, found: 343.47. HPLC purity = 96.1%.

[0072] Example 4: This example provides the preparation process and verification data of compound 4

[0073] Compound 4: 2-amino-4-(4-amino-5-((4-methoxybenzyl)thio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0074] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 4-methoxybenzyl chloride (15.60 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 4 with a yield of 25.85 g (0.072 mol, 72%).

[0075] 1 H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 7.1 Hz, 1H), 7.26 (dt, J = 7.7,1.0 Hz, 2H), 6.99-6.90 (m, 2H), 6.88-6.82 (m, 2H), 6.74 (d, J = 1.9 Hz, 1H), 5.65 (s, 2H), 4.97 (s, 2H), 4.44 (t, J = 1.0 Hz, 2H), 3.78 (s, 2H). HRMS (ESI)m / z: calcd for C 16 H 17 N5OS2:[M+H] 359.09, found: 359.47. HPLC purity = 93.7%.

[0076] Example 5: This example provides the preparation process and verification data of compound 5

[0077] Compound 5: 4-(((4-amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazol-3-yl)thio)methyl)benzonitrile

[0078] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 4-cyanobenzyl chloride (15.10 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 5 with a yield of 29.39 g (0.083 mol, 83%).

[0079] 1 H NMR (400 MHz, DMSO-d6) δ 7.68 – 7.62 (m, 2H), 7.40 – 7.33 (m, 3H), 6.93 (dd, J = 7.1, 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01 (s, 2H), 4.90 (s,2H), 4.37 (d, J = 0.9 Hz, 2H), 4.09 (s, 1H). HRMS (ESI) m / z: calcd forC 16 H 14 N6S2:[M+H] 354.07, found: 354.45. HPLC purity = 95.7%.

[0080] Example 6: This example provides the preparation process and verification data of compound 6

[0081] Compound 6: Amino-5-((4-fluorobenzyl)thio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0082] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 4-fluorobenzyl chloride (14.40 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 6 with a yield of 26.37 g (0.076 mol, 76%).

[0083] 1 H NMR (400 MHz, DMSO-d6) δ 7.42 – 7.35 (m, 2H), 7.15 – 7.07 (m, 2H), 6.93 (dd, J = 7.1, 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01 (s, 2H), 4.90 (s,2H), 4.37 (d, J = 0.9 Hz, 2H), 4.09 (s, 1H). HRMS (ESI) m / z: calcd forC 15 H 14 FN5S2:[M+H] 347.07, found: 347.43. HPLC purity = 93.9%.

[0084] Example 7: This example provides the preparation process and verification data of compound 7

[0085] Compound 7: 2-amino-4-(4-amino-5-((naphthalen-2-ylmethyl)thio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0086] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 2-(chloromethyl)naphthalene (17.60 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 7 with a yield of 28.05 g (0.074 mol, 74%).

[0087] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 – 7.75 (m, 2H), 7.68 – 7.60 (m, 2H),7.51 (ddd, J = 8.5, 6.7, 1.5 Hz, 1H), 7.44 – 7.35 (m, 2H), 6.93 (dd, J = 7.1, 2.1Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01 (s, 1H), 4.90 (s, 1H), 4.44 (t, J = 0.9Hz, 1H), 4.09 (s, 1H). HRMS (ESI) m / z: calcd for C 19 H 17 N5S2: [M+H] 379.09, found: 379.50. HPLC purity = 96.7%.

[0088] Example 8: This example provides the preparation process and verification data of compound 8

[0089] Compound 8: 2-amino-4-(4-amino-5-(methylthio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0090] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and methyl chloride (5.00 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 8 with a yield of 22.52 g (0.089 mol, 89%).

[0091] 1 H NMR (400 MHz, DMSO-d6) δ 7.38 (s, 1H), 6.93 (dd, J = 7.0, 2.1 Hz,1H), 6.74 (d, J = 2.1 Hz, 1H), 4.90 (s, 2H), 4.83 (s, 2H), 4.09 (s, 1H), 2.68(s, 3H). HRMS (ESI) m / z: calcd for C9H 11 N5S2:[M+H] 253.05, found: 253.34. HPLC purity = 94.1%.

[0092] Example 9: This example provides the preparation process and verification data of compound 9

[0093] Compound 9: 2-amino-4-(4-amino-5-(ethylthio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0094] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and ethyl chloride (6.40 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 9 with a yield of 18.69 g (0.070 mol, 70%).

[0095] 1 H NMR (400 MHz, DMSO-d6) δ 7.38 (s, 1H), 6.93 (dd, J = 7.0, 2.1 Hz,1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01 (s, 2H), 4.90 (s, 2H), 4.09 (s, 1H), 3.08(q, J = 6.2 Hz, 3H), 1.37 (d, J = 12.4 Hz, 2H). HRMS (ESI) m / z: calcd forC 10 H 13 N5S2[M+H] 267.06, found: 267.37. HPLC purity = 96.2%.

[0096] Example 10: This example provides the preparation process and verification data of compound 10

[0097] Compound 10: N-(4-(((4-amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazol-3-yl)thio)methyl)phenyl)-N-oxyhydroxylammonium

[0098] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 1-(chloromethyl)-4-nitrobenzene (17.20 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 10 with a yield of 31.88 g (0.085 mol, 85%).

[0099] 1 H NMR (400 MHz, DMSO-d6) δ 8.17 – 8.11 (m, 2H), 7.60 (dt, J = 7.9, 0.9Hz, 2H), 7.37 (s, 1H), 6.93 (dd, J = 7.0, 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01 (s, 2H), 4.90 (s, 2H), 4.37 (d, J = 0.9 Hz, 2H), 4.09 (s, 1H). HRMS (ESI)m / z: calcd for C 15 H 15 N6O2S2:[M+H] 375.07, found: 375.44. HPLC purity = 98.4%.

[0100] Example 11: This example provides the preparation process and verification data of compound 11

[0101] Compound 11: 2-amino-4-(4-amino-5-((4-(diethylamino)benzyl)thio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0102] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0 °C in an ice bath and 4-(chloromethyl)-N,N-diethylimine (19.71 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to obtain Compound 11 with a yield of 29.21 g (0.073 mol, 73%).

[0103] 1 H NMR (400 MHz, DMSO-d6) δ 7.15 (dq, J = 7.7, 0.9 Hz, 1H), 6.93 (dd, J =7.0, 2.1 Hz, 0H), 6.74 (d, J = 2.1 Hz, 0H), 6.72 – 6.66 (m, 1H), 5.01 (s, 1H), 4.90 (s, 1H), 4.30 (d, J = 0.9 Hz, 1H), 3.27 (q, J = 7.0 Hz, 2H), 1.19 (t, J = 7.0Hz, 3H). HRMS(ESI) m / z: calcd for C 19 H 24 N6S2:[M+H] 400.15, found: 400.56. HPLCpurity = 92.4%.

[0104] Example 12: This example provides the preparation process and verification data of compound 12

[0105] Compound 12: 2-amino-4-(4-amino-5-((4-(trifluoromethyl)benzyl)thio)-4H-1,2,4-triazol-3-yl)benzenethiol

[0106] 4-Amino-5-(3-amino-4-mercaptophenyl)-4H1,2,4-triazole-3-thiol (23.90 g, 0.1 mol) (Compound 1) was suspended in water (10 mL) and 85% potassium hydroxide solution (5.5 mL, 0.1 mol) was added under stirring at room temperature. After 10 minutes, the solution was brought to 0°C in an ice bath and 4-(trifluoromethyl)benzyl chloride (19.40 g, 0.1 mol) was added dropwise under vigorous stirring. The reaction mixture was checked by TLC (ether as eluent). When the reaction was complete, 98% concentrated sulfuric acid (10 mL, 0.184 mol) was added and a white precipitate slowly formed from the crude mixture. It was filtered, washed with water, and crystallized from ethanol to give Compound 12 with a yield of 33.75 g (0.085 mol, 85%).

[0107] 1 H NMR (400 MHz, DMSO-d6) δ 7.58 (dq, J = 6.7, 1.3 Hz, 2H), 7.31 (dt, J =6.5, 0.9 Hz, 2H), 6.93 (dd, J = 7.1, 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 5.01(s, 2H), 4.90 (s, 2H), 4.37 (d, J = 0.9 Hz, 2H), 4.09 (s, 1H). HRMS (ESI) m / z:calcd for C 16 H 14 F3N5S2:[M+H] 397.06, found: 397.44. HPLC purity = 93.1%.

[0108] Test Example 1: This test example screens the activity of the synthetic compounds 1 to 12 prepared in Examples 1 to 12

[0109] After the cultured HK-2 cells adhered to the wall, DMSO, RSL-3 (3 μM), and RSL-3 (3 μM) + 2-fold gradient dilution of the synthetic compound were added to the culture medium. The cell viability was detected by CCK-8 colorimetry, and the average value data was obtained after each group of experiments was repeated 3 times. The activity test results of the specifically prepared (4-amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazole-3-thiol derivatives on the inhibition of cell ferroptosis are shown in Table 2:

[0110] Table 2

[0111]

[0112] As shown in Table 2, the 4-amino-5-(3-amino-4-mercaptophenyl)-4H-1,2,4-triazole-3-thiol compounds prepared by the present invention can effectively inhibit cell ferroptosis, and the best effect is achieved when the R group is 4-methylbenzyl.

[0113] Experimental Example 2: This experimental example evaluates the broad-spectrum anti-ferroptosis activity of compound 3, which is the best inhibitor of cell ferroptosis activity screened out in Experimental Example 1

[0114] After the cells adhered to the wall, DMSO, six known ferroptosis inducers, and their combined treatment groups with compound 3 (final concentration of 1 μM) were added to the culture medium. After 24 h of treatment, the cell viability was determined by CCK-8 method. The experiment was repeated three times to ensure the reliability and reproducibility of the results.

[0115] The six ferroptosis inducers used were: RSL3 (3 μM), Erastin (10 μM), Sulfasalazine (SFZ, 250 μM), ML210 (90 μM), FIN56 (8 μM) and BSO (800 μM). The concentrations of these inducers were determined in the laboratory.

[0116] Cell viability assay (CCK-8 method): The treated HK-2 cells were detected by colorimetry using the CCK-8 kit. The specific steps are as follows: CCK-8 reagent was added to each well, and after incubation for 1 to 4 hours, the absorbance value (OD value) was read at a wavelength of 450 nm using a microplate reader. The cell viability was calculated using the following formula:

[0117]

[0118] The results are as follows Figure 1As shown, the experimental results showed that compared with the control group (Ctrl, DMSO), the six ferroptosis inducers significantly reduced the survival rate of HK-2 cells, indicating that they effectively induced ferroptosis of cells. Specifically, these inducers promote the accumulation of intracellular iron ions and the increase of cell membrane lipid free radicals (ROS) through different mechanisms, and ultimately induce cell ferroptosis. In the experimental group treated with compound 3, the presence of compound 3 significantly inhibited the cell death caused by these ferroptosis inducers. The results of statistical analysis showed that compound 3 showed significant cytoprotective effects under the treatment of each inducer (p<0.01), showing its potential in anti-ferroptosis. Specifically, compound 3 had a significant inhibitory effect on cell death caused by RSL3, Erastin, SFZ, ML210, FIN56 and BSO, indicating that it has a broad spectrum of anti-ferroptosis activity.

[0119] Experimental Example 3: This experimental example explores the protective effect of compound 3, which is the best inhibitor of cell ferroptosis activity screened out in Experimental Example 1, on hypoxia / reoxygenation injury

[0120] In order to evaluate the protective effect of compound 3 in hypoxia / reoxygenation (H / R) injury, human renal tubular epithelial cells (HK-2) were used as a cell model to simulate hypoxia / reoxygenation-induced renal injury. First, HK-2 cells were exposed to hypoxia (volume fraction: 1% oxygen, 2 h), and then restored to conventional culture conditions (volume fraction: 21% oxygen) for reoxygenation for 24 h. The experimental groups were pretreated with different concentrations of compound 3 before hypoxia / reoxygenation. Cell viability was detected by CCK-8 method; in addition, lipid free radicals were labeled with C11-BODIPY 581 / 591 and analyzed by laser confocal imaging.

[0121] The experiment was divided into four groups: control group (no drug treatment), H / R group (hypoxia / reoxygenation model), compound 3 pretreatment group (H / R + compound 3), positive control group (H / R + Fer-1);

[0122] Lipid peroxidation results and cell survival Figure 2 As shown in (A) and (B), the experimental results showed that pretreatment with compound 3 significantly reduced the cell death rate induced by hypoxia / reoxygenation, and the cell survival rate of the group treated with compound 3 was significantly higher than that of the H / R group. Further experiments showed that compound 3 could inhibit hypoxia / reoxygenation-induced ferroptosis. Compared with the control group, the hypoxia / reoxygenation model significantly increased the level of lipid free radicals in the cell membrane. At the same time, compound 3 could significantly scavenge lipid free radicals induced by hypoxia / reoxygenation (p<0.01) and its activity was comparable to that of the positive control group (Fer-1 pretreated with compound 3).

[0123] Experimental Example 4: This experimental example investigates the alleviating effect of compound 3, which is the best inhibitor of ferroptosis activity selected in Experimental Example 1, on acute renal injury in mice induced by bilateral renal ischemia-reperfusion.

[0124] In order to evaluate the alleviating effect of compound 3 on renal ischemia-reperfusion injury, this experimental example used a mouse bilateral renal ischemia-reperfusion model.

[0125] The experiment was divided into four groups: control group (Control, no treatment), ischemia-reperfusion group (I / R, ischemia-reperfusion only), low-dose compound 3 pretreatment group (I / R+compound 3, compound 3 was given 24 h before ischemia, the dose was 2 mg / kg), and high-dose compound 3 pretreatment group (I / R+compound 3, compound 3 was given 24 h before ischemia, the dose was 10 mg / kg). All experimental mice were evaluated 24 h after ischemia-reperfusion treatment.

[0126] The results are as follows Figure 3 As shown, the experimental results showed that compared with the control group and the ischemia-reperfusion group, compound 3 pretreatment significantly improved the renal function of mice. The levels of serum creatinine (Cr) and urea nitrogen (BUN) were significantly decreased compared with the ischemia-reperfusion group (P<0.05) and were dose-dependent, indicating that compound 3 can effectively alleviate renal damage; and the levels of serum creatinine (Cr) and urea nitrogen (BUN) in the high-dose group pretreated with compound 3 were more significantly reduced than those in the low-dose group pretreated with compound 3, indicating that the dose has an effect on alleviating renal damage.

[0127] In addition, if Figure 4 As shown in the figure, the results of pathological examination showed that the damage to the outer medullary region of the kidneys of mice in the compound 3 pretreatment group was significantly reduced, and the degree of tubular epithelial cell necrosis and tubular lumen deformation was significantly reduced. In contrast, the mice in the ischemia-reperfusion group had severe kidney damage, with obvious tubular necrosis and renal tissue edema.

[0128] like Figure 5 As shown, in terms of ferroptosis markers, immunohistochemistry results showed that compound 3 significantly inhibited the accumulation of iron ions and the increase of lipid peroxidation products (4-HNE) after ischemia-reperfusion injury, indicating that compound 3 alleviated kidney damage by inhibiting ferroptosis. In summary, this example shows that compound 3 has a significant kidney protective effect by alleviating acute kidney injury in mice caused by ischemia-reperfusion, improving kidney function, and inhibiting the occurrence of ferroptosis, suggesting that it has broad application prospects in the prevention and treatment of acute kidney injury.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A thiophenol compound or a pharmaceutically acceptable salt thereof, characterized in that: The thiophenol compound has a structure shown in Formula I: Formula I; Wherein, R is independently hydrogen, C1~C2 alkyl, , , , , , , , , .

2. The thiophenol compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The thiophenol compound is one of the following compounds: 。 3. A method for preparing thiophenol compounds as claimed in claim 2, characterized in that: The following steps are involved: S1, mixing methyl 3-amino-4-mercaptobenzoate and hydrazine hydrate and dissolving them in an organic solvent, heating the mixture to obtain 3-amino-4-mercaptobenzoyl hydrazide, mixing the mixture with KOH and CS2 to obtain 2-(3-amino-4-mercaptobenzoyl)hydrazine-1-aminodithioic acid potassium, and mixing the mixture with hydrazine hydrate to obtain compound 1, wherein compound 1 is ; S2. Mix compound 1, KOH and compound R to react, wherein the compound R is selected from the group consisting of benzyl chloride, 4-methylbenzyl chloride, 4-methoxybenzyl chloride, 4-cyanobenzyl chloride, 4-fluorobenzyl chloride, 2-(chloromethyl)naphthalene, methyl chloride, ethyl chloride, 1-(chloromethyl)-4-nitrobenzene, 4-(chloromethyl)-N,N-diethylimine, and 4-(trifluoromethyl)benzyl chloride.

4. The preparation method according to claim 3, characterized in that: In step S1, the molar ratio of methyl 3-amino-4-mercaptobenzoate to hydrazine hydrate is 1-1.5:1; the organic solvent is selected from one or more of anhydrous ethanol and methanol; the temperature of the heating reaction is 80-90°C, and the time is 5-10 h; the molar ratio of 3-amino-4-mercaptobenzoyl hydrazide, KOH and CS2 is 1:1-2:1-2; the molar ratio of 2-(3-amino-4-mercaptobenzoyl)hydrazine-1-aminodithioic acid potassium and hydrazine hydrate is 1:1.5-3.5; the temperature of the mixed reaction is 100-110°C, and the time is 20-40 min; In step S2, the molar ratio of the compound 1, KOH and compound R is 1~1.5:1~1.5:1; the reaction temperature is 0°C and the reaction time is 0.5~24 h; the crude product obtained by the reaction needs to be further purified, specifically: adding 98% concentrated sulfuric acid by mass to the crude product for purification, and then filtering, washing and recrystallizing in sequence.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an active dose of the thiophenol compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2 as a main component, and is prepared by adding auxiliary components acceptable to biopharmaceuticals.

6. Use of the thiophenol compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 and / or the pharmaceutical composition according to claim 5 in the preparation of a medicament for preventing and / or treating renal injury caused by ischemia-reperfusion.

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