A sirt5 inhibitor and preparation method and use thereof

Abstract

The application discloses a SIRT5 inhibitor and a preparation method and application thereof. A structural formula of the SIRT5 inhibitor is shown in formula I: wherein A is: B is: R is selected from Cl, CN, OCH3, CH3, COOCH3 and COOH. The compound of the application has good inhibitory activity on SIRT5, especially when R is COOH, the compound has the best activity, and is significantly superior to some currently reported small-molecule SIRT5 inhibitors. The substituted 4-benzylidene-5-methyl-2-(4-phenylthiazole-2-yl)-pyrazole-3-ketone derivative provided by the application has clear drug efficacy, and provides a new drug selection for clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a substituted 4-benzylidene-5-methyl-2-(4-phenylthiazole-2-yl)-pyrazole-3-ketone derivative, and a preparation method and use thereof. BACKGROUND

[0002] Sirtuins are a class of nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacetylases and mono-ADP-ribosyltransferases. In bacteria and humans, the amino acid sequences of Sirtuin proteins are highly conserved, and are expressed in various tissues and organs, such as liver, heart, brain and pancreas. Currently, seven kinds of human Sirtuin proteins have been found, namely SIRT1-SIRT7, which are divided into four categories according to their functions and subcellular localization. Among them, SIRT1-SIRT3 belong to the first category, have strong deacetylation effect, and also have certain de-long-chain fatty acylation effect; SIRT4 and SIRT5 belong to the second and third categories, respectively; SIRT6-SIRT7 belong to the fourth category together, and they almost have no deacetylation effect or only weak deacetylation effect. However, in recent years, a large number of studies have shown that SIRT5 among them has strong de-succinyl, de-malonyl and de-glutaryl effects, and thus has attracted widespread attention from researchers.

[0003] Because SIRT5 is mainly located in mitochondria, it can regulate the deacylation of various mitochondrial proteins and plays an important role in metabolism. For example, it can regulate the degradation of ammonia in the body by desuccinylating carbamoyl phosphate synthase 1 (CPSI), regulate active oxygen (ROS) enzymes by desuccinylating copper-zinc superoxide dismutase (SODI) to eliminate active oxygen, and regulate glycolysis by desuccinylating 3-phosphoglyceraldehyde dehydrogenase (GAPDH). In addition, the acyl donor molecules of SIRT5, including acetyl coenzyme A, malonyl coenzyme A and succinyl coenzyme A, are important components in the process of energy metabolism, and the regulation of the amount of acyl donor can also directly affect the metabolic process. In view of its wide physiological role, more and more studies in recent years have shown that SIRT5 is highly expressed in pathological tissues including tumors, metabolic diseases, central nervous system diseases, and its expression level is related to the poor prognosis of related diseases. Therefore, SRT5 is considered as a potential new target for the treatment of various diseases, and the development of its inhibitors has been highly valued by many pharmaceutical companies and research institutions. At present, the reported SIRT5 inhibitors mainly include substrate analogs and small molecule inhibitors. The substrate analog inhibitors have the shortcomings of poor selectivity and poor cell permeability, which limits their further development as drugs. On the contrary, small molecule inhibitors may have better development potential, but the reported SIRT5 small molecule inhibitors are very limited, and the activity and selectivity of only a few inhibitors are poor, so it is urgent to develop new high-activity SIRT5 small molecule inhibitors to provide candidate compounds for the development of SIRT5-targeted drugs. SUMMARY

[0004] In view of the above prior art, the purpose of the present application is to provide a substituted 4-benzylidene-5-methyl-2-(4-phenylthiazole-2-yl)-pyrazole-3-ketone derivative and its preparation method and use. The compound prepared by the present application has good inhibitory activity on SIRT5, especially the inhibitory activity of compound Y-17-4 is the best, which is significantly better than some currently reported SIRT5 small molecule inhibitors, providing a new drug selection for clinical use.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect of the present application, a substituted 4-benzylidene-5-methyl-2-(4-phenylthiazole-2-yl)-pyrazole-3-ketone derivative is provided, and its structural formula is shown as formula I:

[0007]

[0008] wherein A is:

[0009]

[0010] B is:

[0011] Preferably, R is selected from the group consisting of Cl, CN, OCH3, CH3, COOCH3, COOH.

[0012] Preferably, R is COOH.

[0013] Preferably, the substituted 4-benzylidene-5-methyl-2-(4-phenylthiazol-2-yl)-pyrazol-3-one derivative has the following structure:

[0014]

[0015] In a second aspect of the present application, a method for preparing a substituted 4-benzylidene-5-methyl-2-(4-phenylthiazol-2-yl)-pyrazol-3-one derivative is provided, comprising the following steps:

[0016] (1) Synthesis of compound 2-hydrazino-4-phenylthiazole derivative: slowly add aminothiourea and different substituted 2-bromoacetophenone derivative into an aqueous solution of potassium acetate in ethanol, then react at room temperature, wash and dry the filtered residue to obtain a substituted 2-hydrazino-4-phenylthiazole derivative;

[0017] (2) Synthesis of compound 3-methyl-l-(4-phenyl-2-thiazolyl)-lH-pyrazol-5-ol derivative: slowly add the substituted 2-hydrazino-4-phenylthiazole derivative obtained in step (1) into acetic acid, stir and react at 80°C, remove the organic solvent after the reaction is completed, treat the residue with saturated sodium bicarbonate solution, extract with ethyl acetate, combine the organic phases, rinse with saturated brine, dry with anhydrous sodium sulfate, and finally separate by column chromatography to obtain a 3-methyl-l-(4-phenyl-2-thiazolyl)-lH-pyrazol-5-ol derivative;

[0018] (3) Synthesis of 4-benzylidene-5-methyl-2-(4-phenylthiazol-2-yl)-pyrazol-3-one derivative: add the 3-methyl-l-(4-phenyl-2-thiazolyl)-lH-pyrazol-5-ol derivative obtained in step (2) and different substituted benzaldehyde into anhydrous ethanol, reflux and react, and purify to obtain a 4-benzylidene-5-methyl-2-(4-phenylthiazol-2-yl)-pyrazol-3-one derivative.

[0019] The synthesis route is as follows:

[0020]

[0021] Preferably, the preparation method further comprises a hydrolysis reaction of step (4): dissolving the substituted 4-benzylidene-5-methyl-2-(4-benzothiazole-2-yl)-pyrazole-3-ketone derivative obtained in step (3) in an ethanol aqueous solution containing potassium carbonate, refluxing the reaction, cooling to room temperature after the reaction is completed, removing the organic solvent, adjusting the solution to be acidic with hydrochloric acid, collecting the filter cake by filtration, and vacuum drying to obtain the target compound with R being a carboxylic acid.

[0022] Preferably, the ethanol aqueous solution is obtained by mixing ethanol and water at a volume ratio of 10:1; and the concentration of the hydrochloric acid is 1M.

[0023] In a third aspect of the present application, there is provided a use of a substituted 4-benzylidene-5-methyl-2-(4-benzothiazole-2-yl)-pyrazole-3-ketone derivative in the preparation of a SIRT5 protein inhibitor.

[0024] In a third aspect of the present application, there is provided a SIRT5 protein inhibitor, which is a compound as shown in formula I; and the SIRT5 protein inhibitor further comprises one or more pharmaceutically acceptable carriers or excipients.

[0025] Preferably, the SIRT5 protein inhibitor is an oral preparation or an injection preparation.

[0026] Advantages of the present application:

[0027] The compound of the present application has good inhibitory activity on SIRT5, and in particular, the inhibitory activity of compound Y-17-4 is the best, which is significantly better than some currently reported SIRT5 small molecule inhibitors such as nicotinamide, and the substituted 4-benzylidene-5-methyl-2-(4-benzothiazole-2-yl)-pyrazole-3-ketone derivative of the present application has clear drug efficacy, and provides a new drug selection for clinical use. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed description is exemplary and is intended to further explain the present application. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0029] As introduced in the background section, the reported SIRT5 inhibitors mainly include substrate analogs and small molecule inhibitors. The substrate analog inhibitors have the disadvantages of poor selectivity and poor cell permeability, which limits their further development as drugs. In contrast, the small molecule inhibitors may have better development potential, but the reported SIRT5 small molecule inhibitors are very limited, and the activity and selectivity of only a few inhibitors are poor, and therefore it is urgent to develop new SIRT5 small molecule inhibitors with high activity.

[0030] Based on this, the purpose of the present application is to provide a substituted 4-benzylidene-5-methyl-2-(4-phenylthiazole-2-yl)-pyrazole-3-ketone derivative, a preparation method and use thereof. Although there are similar compounds in the prior art, the inventors have found that the A and B moieties must be phenyl groups, and the functional groups carried thereby affect whether they can be used to prepare SIRT5 protein inhibitors. For example, Pharmacological folding chaperones act as allosteric ligands of Frizzled4 (Serena F. Generoso et al.) discloses a compound with the following structural formula:

[0031]

[0032] The A and B moieties at both ends of the structural formula are also benzene ring-containing groups, but this compound does not have the effect of inhibiting SIRT5. Through the research of the inventors, it has been found that when the group R of the B moiety is a carboxyl group, the effect of inhibiting SIRT5 is best.

[0033] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0034] The test materials used in the embodiments of the present application are all conventional test materials in the art and can be purchased through commercial channels.

[0035] Example 1: Preparation of compound Y-3 and derivatives thereof

[0036] (1) Synthesis of compound 4-(4-methoxyphenyl)-2-hydrazinylthiazole (Y-3)

[0037]

[0038] Aminothiourea (0.91 g, 10 mmol) and p-methoxy-2-bromoacetophenone (2.28 g, 10 mmol) were slowly added to an aqueous solution of potassium acetate (0.98 g, 10 mmol) in ethanol (38 mL, EtOH / H2O = 1:1, V:V), and the reaction was stirred at room temperature for 3 h. After the reaction was completed, the mixture was filtered, the filter cake was washed with 10 mL of ice ethanol, and vacuum dried to obtain white solid compound 4-(4-methoxyphenyl)-2-hydrazinylthiazole (1.67 g). No further treatment was required, and the yield was about 75.44%.

[0039] (2) Synthesis of compound 1-(4-(4-methoxyphenyl)thiazol-2-yl)-3-methyl-1H-pyrazol-5-ol (Y-3-1)

[0040]

[0041] Compound Y-3 (1.5 g, 6.78 mmol) was added slowly to ethyl acetoacetate (0.9 mL, 6.78 mmol) in acetic acid (15 mL) and stirred at 80 °C for 3 h, TLC monitoring, after completion of the reaction, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally isolated by column chromatography to obtain compound 1-(4-(4-methoxyphenyl)thiazol-2-yl)-3-methyl-1H-pyrazol-5-ol (Y-3-1) as a light yellow solid, 1.45 g, yield about 66.18 %).

[0042] (3) Synthesis of compound 4-(4-chlorobenzylidene)-5-methyl-2-[4-(4-methoxyphenyl)thiazol-2-yl]-pyrazol-3-one (Y-3-2)

[0043]

[0044] Compound Y-3-1 (200 mg, 0.7 mmol) was added to p-chlorobenzaldehyde (98 mg, 0.7 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (35 uL, 0.35 mmol) was added. The reaction was refluxed, TLC monitoring, after completion of the reaction, the crude was isolated by column chromatography, then ethanol was added and refluxed for 1 h, then cooled to room temperature naturally, filtered to obtain high purity light yellow solid Y-3-2 (80 mg), yield about 25.32 %. Purity 96.40 %. Mp. 286-287 °C. 1 H NMR (600 MHz, DMSO-d6) δ 7.93-7.91 (d, J = 12 Hz, 3H), 7.58 (s, 1H), 7.38-7.29 (m, 3H), 7.02-7.00 (m, 3H), 3.79 (s, 3H), 2.11 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 161.22, 159.20, 152.67, 149.91, 149.00, 139.75, 130.79, 129.84, 128.16, 127.35, 126.72, 114.06, 106.74, 104.16, 55.22, 11.66. LCMS: m / z [M+H] + calculated for C 21 H 16CIN3O2S, 410.0725; found, 410.0723. HPLC purity at 254 nm, 96.40%.

[0045] (4) Synthesis of compound Y-3-5

[0046]

[0047] Compound Y-3-1 (200 mg, 0.7 mmol) was dissolved in anhydrous ethanol (1.4 mL, 0.5 M) with p-formylbenzaldehyde (115 mg, 0.7 mmol) and a catalytic amount of piperidine (35 uL, 0.35 mmol) was added. The reaction was monitored by TLC and after completion of the reaction, the crude was isolated by column chromatography to get a light yellow solid. Further, ethanol was added and refluxed for 1 h and allowed to cool to room temperature naturally. The light yellow solid was filtered to get high purity Y-3-5 (92 mg) with a yield of about 35.21%. Mp. 218-219 °C. 1 H NMR (400 MHz, DMSO-d6) d 7.93-7.91 (m, 5H), 7.58 (s, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) d 166.17, 161.64, 159.20, 152.77, 149.96, 149.02, 146.52, 129.19, 128.28, 127.62, 127.33, 126.72, 114.04, 106.70, 103.94, 55.19, 52.03, 11.64. LCMS: m / z [M+H] + calculated for C 23 H 19 N3O4S, 434.1169;

[0048] found, 434.1164. HPLC purity at 254 nm, 97.31%.

[0049] (6) Synthesis of compound Y-3-4

[0050]

[0051] Compound Y-3-5 (130 mg, 0.3 mmol) was dissolved in 16 mL of ethanol aqueous solution (EtOH / H2O = 1 : 1, v:v) containing potassium carbonate (373 mg, 2.7 mmol) and refluxed overnight, TLC monitoring, after the reaction was completed, cooled to room temperature, then removed organic solution, adjusted the solution to be acidic with 1 N HC1, filtered to collect the filter, recrystallized with ethanol, vacuum dried to obtain a light yellow solid compound Y-3-4, 100 mg, yield about 86.21%. Mp. 256-257 °C. 1 H NMR (600 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.93 (d, J = 6.0 Hz, 3H), 7.90 (d, J = 6.0 Hz, 2H), 7.59 (s, 1H), 7.40 (d, J = 6.0 Hz, 2H), 7.02-7.00 (m, 2H), 3.80 (s, 3H), 2.10 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 167.29, 161.30, 159.19, 152.60, 150.05, 148.89, 145.96, 129.37, 128.73, 128.19, 127.34, 126.74, 114.06, 106.61, 104.05, 55.21, 11.64. LCMS: m / z [M+H] + calculated for C 22 H 17 N3O4S, 420.1013; found, 420.1008. HPLC purity at 254 nm, 97.92%.

[0052] (7) Synthesis of compound Y-3-6

[0053]

[0054] Compound Y-3-1 (287 mg, 1 mmol) and p-tolualdehyde (120 mg, 1 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M), and a catalytic amount of piperidine (35 uL, 0.35 mmol) was added. The reaction was refluxed, TLC monitoring, after the reaction was completed, column chromatography was used to separate the crude product to obtain a light yellow solid, then ethanol was added to reflux for 1 h, and then cooled to room temperature naturally, filtered to obtain a light yellow solid Y-3-6 (92 mg) with high purity, yield about 24.21%. Mp. 270-271 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.93 - 7.91 ppm (m, 3H), 7.57 (s, 1H), 7.16 - 7.10 (q, J = 8.0 Hz, 3H), 7.01 (d, J = 8.0 Hz, 3H), 3.79 (s, 3H), 2.28 (s, 3H), 2.08 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 161.81, 159.18, 153.11, 150.06, 148.89, 135.11, 128.80, 127.80, 127.32, 126.74, 114.04, 106.51, 104.77, 55.19, 20.60, 11.64. LCMS: m / z [M+H] + calculated for C 22 H 19 N3O2S, 390.1271; found, 390.1271. HPLC purity at 254 nm, 96.37%.

[0055] Example 2: Preparation of compound Y-4 and its derivatives

[0056] (1) Synthesis of compound 4-(2-fluorophenyl)-2-hydrazinylthiazole (Y-4)

[0057]

[0058] Amino thioamide (0.46 g, 5.07 mmol) and 2-fluoro-2-bromoacetophenone (1.00 g, 4.61 mmol) were slowly added to an aqueous solution of potassium acetate (0.45 g, 4.61 mmol) in ethanol (15 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After the reaction was completed, it was filtered, washed with 10 mL of ice ethanol and dried under vacuum to obtain white solid compound 4-(4-fluorophenyl)-2-hydrazinylthiazole (0.76 g) without further treatment, with a yield of about 78.92%.

[0059] (2) Synthesis of compound 1-[4-(2-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-4-1).

[0060]

[0061] Compound Y-4 (0.63 g, 3 mmol) was added slowly to ethyl acetoacetate (0.40 mL, 3 mmol) in acetic acid (6 mL) and stirred at 80 °C for 3 h, monitored by TLC, after completion of the reaction, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain compound 1-[4-(2-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-4-1) as a light yellow solid (0.5 g, yield about 69.52 %).

[0062] (3) Synthesis of compound Y-4-3

[0063]

[0064] Compound Y-4-1 (287 mg, 1 mmol) was added to p-cyanobenzaldehyde (132 mg, 1 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed, monitored by TLC, after completion of the reaction, the crude was purified by column chromatography to obtain a light yellow solid, which was further refluxed in ethanol for 1 h, cooled to room temperature naturally and filtered to obtain a light yellow solid Y-4-3 (80 mg) with high purity, yield about 22.21 %. Mp. 293-294 °C. 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (t, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.66 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.44 - 7.38 (m, 1H), 7.35 (s, 1H), 7.33 - 7.30 (m, 2H), 2.13 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 161.29, 159.56 (d, J = 247.6 Hz), 152.20, 150.15, 147.48, 142.63, 132.15, 129.83, 129.73 (d, J = 3.2 Hz), 129.12, 124.68 (d, J = 2.7 Hz), 121.52 (d, J = 11.1 Hz), 119.00, 116.14 (d, J = 21.7 Hz), 113.14 (d, J = 9.6 Hz), 109.01, 103.47, 11.62. LCMS: m / z [M+Na] + calculated for C 21 H 13FN4OS, 411.0686; found, 411.0680. HPLC purity at 254 nm, 96.24%.

[0065] (4) Synthesis of compound Y-4-5

[0066]

[0067] Compound Y-4-1 (194 mg, 0.7 mmol) was dissolved in anhydrous ethanol (1.4 mL, 0.5 M) with p-formylbenzaldehyde (115 mg, 0.7 mmol) and a catalytic amount of piperidine (35 uL, 0.35 mmol) was added. The reaction was monitored by TLC and after completion of the reaction, the crude was isolated by column chromatography to get a light yellow solid. Further, ethanol was added and refluxed for 1 h and allowed to cool to room temperature naturally. The light yellow solid was filtered to get high purity of Y-4-5 (75 mg) with a yield of about 25.42%. Mp. 289-290 °C. 1 H NMR (400 MHz, DMSO-d6) δ 8.24 - 8.20 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 7.66 (s, 1H), 7.44 - 7.38 (m, 3H), 7.35 (s, 1H), 7.33 - 7.30 (m, 2H), 3.84 (s, 3H), 2.13 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 166.18, 161.36, 159.57 (d, J = 247.6 Hz), 152.31, 150.23, 146.47, 142.71, 129.83, 129.75 (d, J = 3.3 Hz), 129.21, 128.33, 127.64, 124.69 (d, J = 2.7 Hz), 121.54 (d, J = 10.9 Hz), 116.15 (d, J = 21.7 Hz), 113.14 (d, J = 16.8 Hz), 103.86, 52.05, 11.67. LCMS: m / z [M+H] + calculated for C 22 H 16 FN3O3S, 422.0969; found, 422.0968. HPLC purity at 254 nm, 96.63%.

[0068] (5) Synthesis of compound Y-4-4

[0069]

[0070] Compound Y-4-5 (127 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous solution of ethanol containing potassium carbonate (373 mg, 2.7 mmol) (EtOH / H2O = 1:1, v:v), refluxed overnight, monitored by TLC, cooled to room temperature after completion of the reaction, then the organic solvent was removed, the solution was adjusted to be acidic with 1 N HCI, the filtrate was collected by filtration, recrystallized from ethanol, and dried under vacuum to obtain compound Y-4-4 as a light yellow solid, 100 mg, with a yield of about 88.92%. Mp. 285-286 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.25-8.20 (m, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.66 (s, 1H), 7.44-7.38 (m, 3H), 7.35 (s, 1H), 7.33-7.30 (m, 2H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.28, 161.37, 159.58 (d, J = 247.5 Hz), 152.25, 150.27, 142.65, 129.84, 129.76 (d, J = 3.5 Hz), 129.39, 128.79, 128.17, 124.70 (d, J = 2.8 Hz), 121.55 (d, J = 11.2 Hz), 116.15 (d, J = 21.7 Hz), 113.16 (d, J = 11.2 Hz) 103.95, 11.67. LCMS: m / z [M+H] + calculated for C 21 H 14 FN3O3S, 408.0813; found, 408.0813. HPLC purity at 254 nm, 97.82%.

[0071] Example 3: Preparation of compound Y-5 and its derivatives

[0072] (1) Synthesis of compound 4-(4-fluorophenyl)-2-hydrazinylthiazole (Y-5)

[0073]

[0074] Amino thiosemicarbazide (0.46 g, 5.07 mmol) and 4-fluoro-2-bromoacetophenone (1.00 g, 4.61 mmol) were slowly added to an aqueous solution of potassium acetate (0.45 g, 4.61 mmol) in ethanol (15 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain compound 4-(4-fluorophenyl)-2-hydrazinylthiazole (0.76 g) as a white solid, which was used without further purification, in about 78.92% yield.

[0075] (2) Synthesis of compound 1-[4-(4-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-5-1).

[0076]

[0077] Compound Y-5 (0.63 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were slowly added to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed and the residue was treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain compound 1-[4-(4-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-5-1) as a pale yellow solid (0.5 g, in about 69.52% yield).

[0078] (3) Synthesis of compound Y-5-2

[0079]

[0080] Compound Y-5-1 (287 mg, 1 mmol) and p-chlorobenzaldehyde (120 mg, 1 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction mixture was refluxed and monitored by TLC. After completion of the reaction, the reaction mixture was purified by column chromatography to obtain a pale yellow solid. The pale yellow solid was further refluxed in ethanol for 1 h, cooled to room temperature and filtered to obtain high purity pale yellow solid Y-5-2 (90 mg) in about 32.51% yield. Mp. 289-290 °C. 1 H NMR (400 MHz, DMSO-d6) δ 8.05 - 8.01 (m, 3H), 7.73 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.31 - 7.26 (t, J = 8.0 Hz, 4H), 2.12 (s, 3H). 13CNMR (100 MHz, DMSO-d6) δ 161.97 (d, J = 243.6 Hz), 161.28, 153.20, 150.09, 148.24, 139.98, 130.82, 130.53 (d, J = 3.0 Hz), 129.84, 128.16, 128.08 (d, J = 8.2 Hz), 115.60 (d, J = 21.5 Hz), 108.52, 104.13, 11.66. LCMS: m / z [M+H] 398.0525; found, 398.0523. HPLC purity at 254 nm, 95.11%. + calculated for C 20 H 13 ClFN3OS, 398.0525; found, 398.0523. HPLC purity at 254 nm, 95.11%.

[0081] (4) Synthesis of compound Y-5-5

[0082]

[0083] Compound Y-5-1 (287 mg, 1 mmol) was added to p-formylbenzaldehyde (159 mg, 1 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) with a catalytic amount of piperidine (50 uL, 0.5 mmol) and refluxed. TLC was used to monitor the reaction, and after the reaction was completed, column chromatography was used to isolate the crude product to obtain a light yellow solid. After adding ethanol and refluxing for 1 h, the reaction was naturally cooled to room temperature, and a light yellow solid Y-5-5 (121 mg) with high purity was obtained by filtration, with a yield of about 31.51%. Mp. 284-285 °C. 1 H NMR (400 MHz, DMSO-d6) δ 8.05 - 8.01 (m, 3H), 7.92 (d, J = 8.0 Hz, 2H), 7.73 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 8.0 Hz, 2H), 3.84 (s, 3H), 2.10 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 166.18, 161.94 (d, J = 243.8 Hz), 161.31, 152.79, 150.17, 147.97, 130.54 (d, J = 2.7 Hz), 129.19, 129.00, 128.35, 128.01 (d, J = 8.4 Hz), 127.61, 115.57 (d, J = 21.5 Hz), 108.45, 103.92, 52.03, 11.63. LCMS: m / z [M+H] 398.0525; found, 398.0523. HPLC purity at 254 nm, 95.11%. + calculated for C 22 H 16FN3O3S, 422.0969; found, 422.0969. HPLC purity at 254 nm, 96.75%.

[0084] (4) Synthesis of compound Y-5-4

[0085]

[0086] Y-5-5 (130 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous solution of ethanol containing potassium carbonate (373 mg, 2.7 mmol) (EtOH / H2O = 1:1, v:v), refluxed overnight, TLC monitoring, after the reaction was completed, cooled to room temperature, then removed the organic solution, adjusted the solution to be acidic with 1 N HCl, filtered to collect the filtrate, recrystallized with ethanol, vacuum dried to obtain compound Y-5-4 as a light yellow solid, 115 mg, yield about 94.51%. Mp. 278-279 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 8.05 - 8.02 (m, 3H), 7.90 (d, J = 8.0 Hz, 2H), 7.74 (s, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 8.0 Hz, 2H), 2.11 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.27, 161.95 (d, J = 243.7 Hz), 161.33, 152.80, 150.19, 147.98, 146.40, 130.52 (d, J = 2.7 Hz), 129.37, 128.78, 128.01 (d, J = 8.9 Hz), 127.97, 115.58 (d, J = 21.6 Hz), 108.45, 104.00, 11.65. LCMS: m / z [M+H] + calculated for C 21 H 14 FN3O3S, 408.0813; found, 408.0815. HPLC purity at 254 nm, 97.57%

[0087] Example 4: Preparation of compound Y-6 and its derivatives

[0088] (1) Synthesis of compound 4-(3-fluorophenyl)-2-hydrazinylthiazole (Y-6)

[0089]

[0090] Compound 4-(4-fluorophenyl)-2-hydrazinylthiazole (0.78 g) was obtained as a white solid by slow addition of aminothiourea (0.46 g, 5.07 mmol) and m-fluoro-2-bromoacetophenone (1.00 g, 4.61 mmol) to an aqueous solution of potassium acetate (0.45 g, 4.61 mmol) in ethanol (15 mL, EtOH / H2O = 1:1, V:V) and stirring the reaction at room temperature for 3 h. After completion of the reaction, the reaction mixture was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain the product as a white solid in about 79.92% yield without further purification.

[0091] (2) Synthesis of compound 1-[4-(3-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-6-1).

[0092]

[0093] Compound Y-6 (0.63 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were added slowly to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed and the residue was treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain compound 1-[4-(3-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-6-1) as a pale yellow solid (0.53 g, 70.01% yield).

[0094] (3) Synthesis of compound Y-6-3

[0095]

[0096] Compound Y-6-1 (287 mg, 1 mmol) and p-formylbenzaldehyde (159 mg, 1 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed and monitored by TLC. After completion of the reaction, the reaction mixture was purified by column chromatography to obtain a pale yellow solid. The pale yellow solid was further refluxed in ethanol for 1 h, cooled to room temperature and filtered to obtain high purity pale yellow solid Y-6-3 (93 mg) in about 25.51% yield. Mp. 284-285 °C. 1 H NMR (600 MHz, DMSO-d6) δ 7.92 (t, J = 12.0 Hz, 2H), 7.89 (s, 1H) 7.85-7.83 (m, 3H), 7.52-7.42 (m, 3H), 7.20-7.17 (m, 1H), 3.84 (s, 3H), 2.11 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 160.10 (d, J = 320.3 Hz), 159.20, 152.54, 149.94, 148.89, 146.75, 132.33 (d, J = 51.1 Hz), 129.75, 129.06 (d, J = 29.8 Hz), 127.34, 126.71, 118.90 (d, J = 42.4 Hz), 113.91 (d, J = 45.1 Hz), 109.34 (d, J = 107.1 Hz) 106.65, 103.59, 55.21, 11.60. LCMS: m / z [M+H] 422.0969; found, 422.0970. HPLC purity at 254 nm, 98.12%. + calculated for C 22 H 16 FN3O3S, 422.0969; found, 422.0970. HPLC purity at 254 nm, 98.12%.

[0097] (4) Synthesis of compound Y-6-2

[0098]

[0099] Y-6-3 (130 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H2O = 1:1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, monitored by TLC, cooled to room temperature after the reaction was completed, then the organic solvent was removed, the solution was adjusted to be acidic with 1 N HC1, the filtrate was collected by filtration, recrystallized with ethanol, and dried in vacuum to obtain compound Y-6-2 as a light yellow solid, 120 mg, with a yield of about 95.51%. Mp. 212-213 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 7.92 (s, 1H), 7.90 (t, J = 8 Hz 2H), 7.85-7.82 (m, 3H), 7.52-7.40 (m, 3H), 7.20-7.15 (m, 1H), 2.13 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.29, 162.63 (d, J = 241.4 Hz), 152.73, 152.17 150.16, 147.68, 146.04, 136.28 (d, J = 8.3 Hz), 130.76 (d, J = 8.3 Hz), 129.39, 128.80, 128.14, 121.92 (d, J = 2.6 Hz), 114.73 (d, J = 20.9 Hz), 112.60 (d, J = 22.8 Hz), 110.12, 103.90, 11.67. LCMS: m / z [M+H] + C 21 H 14 FN3O3S, 408.0813; found, 408.0815. HPLC purity at 254 nm, 99.12%.

[0100] Example 5: Preparation of compound Y-7 and its derivatives

[0101] (1) Synthesis of compound 4-(4-methylphenyl)-2-hydrazinylthiazole (Y-7)

[0102]

[0103] Amino thiosemicarbazide (0.46 g, 5.07 mmol) and p-methyl-2-bromoacetophenone (1.00 g, 4.61 mmol) were slowly added to an aqueous solution of potassium acetate (0.45 g, 4.61 mmol) in ethanol (15 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After the reaction was completed, it was filtered, washed with 10 mL of ice ethanol and dried under vacuum to obtain white solid compound 4-(4-methylphenyl)-2-hydrazinylthiazole (0.76 g), which was used without further treatment, with a yield of about 75.92%.

[0104] (2) Synthesis of compound 1-[4-(4-fluorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-7-1).

[0105]

[0106] Compound Y-7 (0.51 g, 3 mmol) was added slowly to ethyl acetoacetate (0.40 mL, 3 mmol) in acetic acid (6 mL) and stirred at 80 °C for 3 h, monitored by TLC, after completion of the reaction, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally isolated by column chromatography to obtain compound 1-[4-(4-methylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-7-1) as a light yellow solid (0.42 g, yield about 66.52 %).

[0107] (3) Synthesis of compound Y-7-3

[0108]

[0109] Compound Y-7-1 (272 mg, 1 mmol) was added to p-formylbenzaldehyde (159 mg, 1 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed, monitored by TLC, after completion of the reaction, it was isolated by column chromatography to obtain a light yellow solid, which was further refluxed in ethanol for 1 h, cooled to room temperature naturally and filtered to obtain a light yellow solid Y-7-3 (105 mg) with high purity, yield about 29.51 %. Mp. 268-269 °C. 1 H NMR (400 MHz, DMSO-d6) δ 7.93 (s, 1H), 7.91-7.88 (m, 4H), 7.68 (s, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 3.84 (s, 3H), 2.33 (s, 3H), 2.13 (s, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 166.17, 161.48, 152.72, 150.03, 149.11, 146.51, 137.40, 131.21, 129.24, 129.19, 128.29, 127.61, 125.88, 107.86, 103.93, 52.04, 20.84, 11.63. LCMS: m / z [M+H] + calculated for C 23 H 19 N3O3S, 418.1220; found, 418.1219. HPLC purity at 254 nm, 97.32 %.

[0110] (4) Synthesis of compound Y-7-2

[0111]

[0112] Y-7-3 (120 mg, 0.3 mmol) was dissolved in 16 mL of an aqueous ethanol solution containing potassium carbonate (373 mg, 2.7 mmol) (EtOH / H2O = 1:1, v:v). The mixture was refluxed overnight under TLC monitoring. After the reaction was complete, the mixture was cooled to room temperature, the organic solvent was removed, and the solution was adjusted to acidity with 1 N HCl. The solution was collected by filtration, recrystallized from ethanol, and dried under vacuum to give 98 mg of a pale yellow solid compound Y-7-2, with a yield of approximately 89.92%. Mp. 274–275 °C. 1 H NMR (400MHz, DMSO-d6) δ12.87(s,1H),7.92–7.88(m,5H),7.68(s,1H),7.40(d,J=8.0Hz,2H),7.25(d,J=8.0Hz,2H),2.33(s,3H),2.13(s,6H). 13 C NMR(100MHz,DMSO-d6)δ167.28,160.91,153.02,150.06,149.11,145.99,137.41,131.22 ,129.37,129.26,128.77,128.11,125.89,107.85,104.02,20.86,11.64.LCMS:m / z[M+H] + Calculated for C 22 H 17 N3O3S,404.1063; found,404.1059.HPLCpurity at 254nm,98.20%.

[0113] Example 6: Preparation of compound Y-8 and its derivatives

[0114] (1) Synthesis of compound 4-(2-fluorophenyl)-2-hydrazinothiazole (Y-8)

[0115]

[0116] Aminothiourea (0.50 g, 6.6 mmol) and 2-methoxy-2-bromoacetophenone (1.38 g, 6.0 mmol) were slowly added to an ethanol-water solution (20 mL, EtOH / H2O = 1:1, V:V) containing potassium acetate (0.91 g, 6.6 mmol). The mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was filtered, washed with 10 mL of ice-cold ethanol, and dried under vacuum to give a white solid compound 4-(2-methoxyphenyl)-2-hydrazinothiazole (1.21 g). No further processing was required, and the yield was approximately 82.31%.

[0117] (2) Synthesis of compound 1-[4-(2-methoxyphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-8-1).

[0118]

[0119] Compound Y-8 (0.63 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were added slowly to acetic acid (6 mL) and stirred at 80 °C for 3 h, TLC monitoring, after the reaction was completed, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, and finally separated by column chromatography to obtain a light yellow solid compound 1-[4-(2-methoxyphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-8-1) 0.52 g, yield about 62.52 %).

[0120] (3) Synthesis of compound Y-8-3

[0121]

[0122] Compound Y-8-1 (194 mg, 0.7 mmol) and p-formylbenzaldehyde (115 mg, 0.7 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M), and a catalytic amount of piperidine (35 uL, 0.35 mmol) was added, and the reaction was refluxed, TLC monitoring, after the reaction was completed, the crude was separated by column chromatography to obtain a light yellow solid, and then ethanol was added and refluxed for 1 h, and then cooled to room temperature naturally, and filtered to obtain a light yellow solid Y-8-3 (100 mg) with high purity, yield about 32.92 %. Mp. 277-278 °C. 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.79 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.35 - 7.31 (m, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 8.0 Hz, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 2.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.19, 161.86, 156.65, 151.17, 149.83, 146.54, 144.90, 129.45, 129.20, 129.11, 128.34, 127.60, 122.12, 120.34, 112.30, 111.61, 103.93, 55.47, 52.06, 11.62. LCMS: m / z [M+Na] 456.0988; found, 456.0984. HPLC purity at 254 nm, 96.15%. + calculated for C 23 H 19 N3O4S, 456.0988; found, 456.0984. HPLC purity at 254 nm, 96.15%.

[0123] (4) Synthesis of compound Y-8-2

[0124]

[0125] Y-8-3 (130 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H2O = 1:1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, TLC monitoring, after the reaction was completed, cooled to room temperature, then removed the organic solution, adjusted the solution to be acidic with 1 N HCl, filtered to collect the filter, recrystallized with ethanol, vacuum dried to obtain compound Y-8-2 as a light yellow solid, 110 mg, yield about 87.31%. Mp. 214-215 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.79 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.36 - 7.31 (m, 2H), 7.13 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 8.0 Hz, 2H), 3.93 (s, 3H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.30, 161.80, 156.66, 151.14, 149.84, 146.00, 145.12, 129.45, 129.38, 129.13, 128.75, 128.16, 122.12, 120.35, 112.66, 111.62, 104.54, 55.48, 11.63. LCMS: m / z [M+H] 456.0988; found, 456.0984. HPLC purity at 254 nm, 96.15%. + calculated for C 22 H 17N3O4S, 420.1013; found, 420.1008. HPLC purity at 254 nm, 98.67%.

[0126] Example 7: Preparation of compound Y-9 and its derivatives

[0127] (1) Synthesis of compound 4-(3-methoxyphenyl)-2-hydrazinylthiazole (Y-9)

[0128]

[0129] Amino thiourea (0.71 g, 7.7 mmol) and 3-methoxy-2-bromoacetophenone (1.60 g, 7.0 mmol) were slowly added to an aqueous solution of potassium acetate (1.07 g, 7.7 mmol) in ethanol (24 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain compound 4-(4-methoxyphenyl)-2-hydrazinylthiazole (1.32 g) as a white solid, which was used as such without further purification, in about 84.95% yield.

[0130] (2) Synthesis of compound l-[4-(3-methoxyphenyl)thiazol-2-yl]-3-methyl-lH- pyrazol-5-ol (Y-9-1).

[0131]

[0132] Compound Y-9 (0.67 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were slowly added to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed and the residue was treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain compound l-[4-(3-methoxyphenyl)thiazol-2-yl]-3-methyl-lH-pyrazol-5-ol (Y-9-1) as a pale yellow solid, 0.60 g, in about 69.45% yield.

[0133] (3) Synthesis of compound Y-9-3

[0134]

[0135] Compound Y-9-1 (287 mg, 1 mmol) was taken in anhydrous ethanol (1.4 mL, 0.5 M) along with p-formylbenzaldehyde (165 mg, 1 mmol) and catalytic amount of piperidine (50 uL, 0.5 mmol) was added and the reaction was refluxed, monitored by TLC and upon completion of the reaction, the crude was isolated by column chromatography to get a pale yellow solid, which was again refluxed in ethanol for 1 h and allowed to cool to room temperature naturally, filtered to get a pale yellow solid of high purity, Y-9-3 (101 mg) with a yield of about 23.28%. Mp. 250-251 °C. 1 H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 8.0 Hz, 2H), 7.79 (s, 1H), 7.58 (d, J = 8.0 Hz, 3H), 7.44 - 7.34 (m, 3H), 6.94 - 6.91 (m, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 166.18, 161.32, 159.66, 152.59, 150.11, 148.87, 146.51, 135.24, 129.79, 129.21, 128.33, 127.63, 118.37, 113.46, 111.70, 109.10, 103.91, 55.22, 52.05, 11.66. LCMS: m / z [M+Na] 456.0988; found, 456.0986. HPLC purity at 254 nm, 96.89%. + calculated for C 23 H 19 N3O4S, 456.0988; found, 456.0986. HPLC purity at 254 nm, 96.89%.

[0136] (4) Synthesis of compound Y-9-2

[0137]

[0138] Y-9-3 (130 mg, 0.3 mmol) was dissolved in 16 mL of an aqueous ethanol solution (EtOH / H2O = 1:1, v:v) containing potassium carbonate (373 mg, 2.7 mmol) and refluxed overnight, monitored by TLC and upon completion of the reaction, cooled to room temperature and the organic solvent was removed, the solution was made acidic with 1 N HCI, the filtrate was collected, recrystallized from ethanol and dried under vacuum to get a pale yellow solid of compound Y-9-2, 110 mg with a yield of about 92.51%. Mp. 203-204 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.79 (s, 1H), 7.58 (d, J = 8.0 Hz, 3H), 7.41 - 7.34 (m, 3H), 6.94 - 6.91 (m, 1H), 3.82 (s, 3H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.28, 161.50, 159.66, 152.60, 150.10, 148.90, 146.00, 135.25, 129.80, 129.37, 128.78, 128.13, 118.38, 113.48, 111.69, 109.09, 103.99, 55.23, 11.67. LCMS: m / z [M+H] + C 22 H 17 N3O4S, 420.1013; found, 420.1008. HPLC purity at 254 nm, 97.43%.

[0139] Example 8: Preparation of compound Y-10 and its derivatives

[0140] (1) Synthesis of compound 4-(4-cyanophenyl)-2-hydrazinylthiazole (Y-10)

[0141]

[0142] Amino thioamide (0.51 g, 5.5 mmol) and p-methyl-2-bromoacetophenone (1.13 g, 5 mmol) were slowly added to an aqueous solution of potassium acetate (0.80 g, 5.5 mmol) in ethanol (20 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After the reaction was completed, it was filtered, washed with 10 mL of ice ethanol and vacuum dried to obtain white solid compound 4-(4-cyanophenyl)-2-hydrazinylthiazole (0.90 g) without further treatment, with a yield of about 83.34%.

[0143] (2) Synthesis of compound 1-[4-(4-cyanophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-10-1).

[0144]

[0145] Compound Y-10 (0.65 g, 3 mmol) was added slowly to ethyl acetoacetate (0.40 mL, 3 mmol) in acetic acid (6 mL) and stirred at 80 °C for 3 h, monitored by TLC, after completion of the reaction the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to get compound 1-[4-(4-cyanophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-10-1) as a light yellow solid (0.61 g, yield ~ 72.11 %).

[0146] (3) Synthesis of compound Y-10-3

[0147]

[0148] Compound Y-10-1 (283 mg, 1 mmol) was taken in anhydrous ethanol (1.4 mL, 0.5 M) and p-formylbenzaldehyde (164 mg, 1 mmol) was added to it followed by addition of catalytic amount of piperidine (50 uL, 0.5 mmol) and refluxed, monitored by TLC, after completion of the reaction, it was purified by column chromatography to get a light yellow solid, which was further refluxed in ethanol for 1 h, cooled to room temperature naturally and filtered to get high purity light yellow solid Y-10-3 (150 mg) with a yield of ~ 34.97 %. Mp. 257-258 °C. 1 H NMR (400 MHz, DMSO-d6) d 8.18 (d, J = 8.0 Hz, 4H), 8.04 (s, 1H), 7.92 (d, J = 8.0 Hz, 4H), 7.43 (d, J = 8.0 Hz, 1H), 3.83 (s, 3H), 2.11 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) d 166.17, 161.38, 152.03, 150.43, 147.05, 146.17, 138.01, 132.80, 129.20, 128.35, 127.69, 126.53, 118.92, 112.29, 110.15, 103.82, 52.04, 11.66. LCMS: m / z [M+H] + calculated for C 23 H 16 N4O3S, 429.1016; found, 429.1012. HPLC purity at 254 nm, 95.71 %.

[0149] (4) Synthesis of compound Y-10-2

[0150]

[0151] Y-10-3 (129 mg, 0.3 mmol) was dissolved in 16 mL of an aqueous ethanol solution (EtOH / H2O = 1 : 1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, monitored by TLC, cooled to room temperature after completion of the reaction, then the organic solvent was removed, the solution was adjusted to be acidic with 1 N HCI, the filtrate was collected by filtration, recrystallized from ethanol, and dried under vacuum to obtain compound Y-10-2 as a light yellow solid, 112 mg, with a yield of about 89.96%. Mp. 250-251 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.18 (d, J = 8.0 Hz, 2H), 8.07 (t, J = 8.0 Hz, 2H), 7.98 - 7.90 (m, 4H), 7.41 (d, J = 8.0 Hz, 2H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.54, 167.30, 161.42, 156.77, 153.06, 150.47, 147.19, 138.01, 136.37, 133.49, 132.82, 129.41, 128.80, 128.19, 128.04, 126.56, 125.66, 118.94, 112.28, 110.19, 103.94, 11.69. LCMS: m / z [M+H] + calculated for C 22 H 14 N4O3S, 418.0859; found, 415.0855. HPLC purity at 254 nm, 96.56%.

[0152] Example 9: Preparation of compound Y-11 and its derivatives

[0153] (1) Synthesis of compound 4-(3-trifluoromethylphenyl)-2-hydrazinylthiazole (Y-11)

[0154]

[0155] Compound 4-(3-trifluoromethylphenyl)-2-hydrazinylthiazole (1.34 g) was obtained as a white solid by slow addition of thiosemicarbazide (0.61 g, 6.6 mmol) and 3-trifluoromethyl-2-bromoacetophenone (1.56 g, 6.0 mmol) to an aqueous solution of potassium acetate (0.91 g, 6.6 mmol) in ethanol (15 mL, EtOH / H2O = 1:1, V:V) and stirring the reaction at room temperature for 3 h. After completion of the reaction, the reaction mixture was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain the compound as a white solid. The compound was used without further purification, and the yield was about 85.89%.

[0156] (2) Synthesis of compound 1-[4-(3-trifluoromethylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-11-1).

[0157]

[0158] Compound Y-11 (0.78 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were added slowly to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and finally isolated as a pale yellow solid by column chromatography to obtain compound 1-[4-(3-trifluoromethylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-11-1) (0.45 g, yield about 54.35%).

[0159] (3) Synthesis of compound Y-11-3

[0160]

[0161] Compound Y-11-1 (287 mg, 1 mmol) and p-formylbenzaldehyde (159 mg, 1 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed, and TLC was used to monitor the reaction. After completion of the reaction, the reaction mixture was separated by column chromatography to obtain a pale yellow solid. The pale yellow solid was refluxed in ethanol for 1 h, cooled to room temperature, and filtered to obtain a high-purity pale yellow solid Y-11-3 (60 mg) with a yield of about 18.31%. Mp. 281-282 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.30 (d, J = 6.0 Hz, 2H), 8.02 (s, 1H), 7.93 (d, J = 6.0 Hz, 2H), 7.72 - 7.68 (m, 3H), 7.44 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H), 2.12 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 166.18, 161.41, 153.03, 150.44, 147.29, 146.39, 134.84, 129.80 (q, J = 31.5 Hz), 129.36, 129.23, 128.36, 127.65, 126.08 (q, J = 270.8 Hz), 124.46 (q, J = 4.0 Hz), 122.35 (q, J = 4.0 Hz), 121.57, 110.66, 103.89, 52.08, 11.69. LCMS: m / z [M+H] + calculated for C 23 H 16 F3N3O3S, 472.0937; found, 472.0930. HPLC purity at 254 nm, 95.47%.

[0162] Example 10: Preparation of compound Y-12 and its derivatives

[0163] (1) Synthesis of compound 4-(4-hydroxyphenyl)-2-hydrazinylthiazole (Y-12)

[0164]

[0165] Amino thioamide (0.51 g, 5.5 mmol) and 4-hydroxy-2-bromoacetophenone (1.08 g, 5 mmol) were slowly added to an aqueous solution of potassium acetate (0.80 g, 5.5 mmol) in ethanol (20 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After the reaction was completed, it was filtered, washed with 10 mL of ice ethanol and vacuum dried to obtain white solid compound 4-(4-hydroxyphenyl)-2-hydrazinylthiazole (0.90 g) without further treatment, with a yield of about 86.54%.

[0166] (2) Synthesis of compound 1-[4-(4-hydroxyphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-12-1).

[0167]

[0168] Compound Y-12 (0.65 g, 3 mmol) was added slowly to ethyl acetoacetate (0.40 mL, 3 mmol) in acetic acid (6 mL) and stirred at 80 °C for 3 h, monitored by TLC, after completion of the reaction, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally isolated by column chromatography to obtain compound 1-[4-(4-hydroxyphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-12-1) as a light yellow solid (0.61 g, yield about 72.11 %).

[0169] (3) Synthesis of compound Y-12-3

[0170]

[0171] Compound Y-12-1 (283 mg, 1 mmol) was added to p-formylbenzaldehyde (164 mg, 1 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed, monitored by TLC, after completion of the reaction, it was isolated by column chromatography to obtain a light yellow solid, which was further refluxed in ethanol for 1 h, cooled to room temperature naturally and filtered to obtain a light yellow solid Y-12-3 (150 mg) with high purity, yield about 33.97 %. Mp. 232-233 °C. 1 H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 2H), 7.92 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.48 (s, 2H), 7.42 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 2H), 3.84 (s, 3H), 2.10 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 166.18, 161.25, 157.49, 152.48, 149.96, 149.33, 146.50, 129.20, 128.36, 127.60, 127.42, 125.25, 115.39, 105.74, 103.99, 52.05, 11.62. LCMS: m / z [M+H] + calculated for C 22 H 17 N3O4S, 420.1013; found, 420.1023. HPLC purity at 254 nm, 98.08 %.

[0172] (4) Synthesis of compound Y-12-2

[0173]

[0174] Y-12-3 (130 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H2O = 1 : 1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, monitored by TLC, cooled to room temperature after completion of the reaction, then the organic solvent was removed, the solution was adjusted to be acidic with 1 N HCI, the filtrate was collected by filtration, recrystallized from ethanol, and dried under vacuum to obtain compound Y-12-2 as a light yellow solid, 110 mg, with a yield of about 88.96%. Mp. 218-219 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 9.63 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.49 (s, 2H), 7.39 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 2H), 2.12 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.31, 162.44, 157.52, 152.71, 149.95, 149.55, 146.06, 129.40, 128.78, 128.13, 127.45, 125.27, 115.42, 106.10, 104.08, 11.66. LCMS: m / z [M+H] + calculated for C 21 H 15 N3O4S, 406.0856; found, 406.0857. HPLC purity at 254 nm, 99.01%.

[0175] Example 11: Preparation of compound Y-13 and its derivatives

[0176] (1) Synthesis of compound 4-(3-cyanophenyl)-2-hydrazinylthiazole (Y-13)

[0177]

[0178] Compound 4-(3-cyanophenyl)-2-hydrazinylthiazole (1.24 g) was obtained as a white solid by slow addition of aminothiourea (0.71 g, 7.7 mmol) and 3-cyano-2-bromoacetophenone (1.60 g, 7.0 mmol) to an aqueous solution of potassium acetate (1.07 g, 7.7 mmol) in ethanol (24 mL, EtOH / H2O = 1:1, V:V) and stirring the reaction at room temperature for 3 h. After the reaction was completed, the reaction mixture was filtered, washed with 10 mL of ice ethanol and dried under vacuum to obtain the white solid. The product was used without further purification, with a yield of about 85.95%.

[0179] (2) Synthesis of compound 1-[4-(3-cyanophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-13-1).

[0180]

[0181] Compound Y-13 (0.67 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were added slowly to acetic acid (6 mL) and stirred at 80 °C for 3 h. After the reaction was completed, the organic solvent was removed and the residue was treated with a saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally separated by column chromatography to obtain the compound 1-[4-(3-cyanophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-13-1) as a yellowish solid, 0.60 g, with a yield of about 72.45%.

[0182] (3) Synthesis of compound Y-13-3

[0183]

[0184] Compound Y-13-1 (287 mg, 1 mmol) and p-formylbenzaldehyde (165 mg, 1 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed, monitored by TLC, and after the reaction was completed, the product was separated by column chromatography as a yellowish solid. The yellowish solid was refluxed in ethanol for 1 h, cooled to room temperature and filtered to obtain the compound Y-13-3 (111 mg) as a yellowish solid with a high purity, with a yield of about 29.28%. Mp. 255-256 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.33 (d, J = 6.0 Hz, 1H), 8.00 (s, 1H), 7.93 (d, J = 6.0 Hz, 2H), 7.82 (d, J = 6.0 Hz, 1H), 7.68 (t, J = 6.0, 12.0 Hz, 2H), 7.43 (d, J = 12.0 Hz, 2H), 3.84 (s, 3H), 2.12 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 166.18, 161.26, 152.93, 150.19, 146.74, 146.40, 134.93, 131.43, 130.33, 130.08, 129.35, 129.23, 128.36, 127.65, 118.78, 111.95, 110.87, 103.72, 52.08, 11.67. LCMS: m / z [M+H] + calculated for C 23 H 16 N4O3S, 429.1016; found, 429.1012. HPLC purity at 254 nm, 95.96%.

[0185] (4) Synthesis of compound Y-13-2

[0186]

[0187] Y-13-3 (130 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H2O = 1 : 1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, TLC monitoring, after the reaction was completed, cooled to room temperature, then removed the organic solution, adjusted the solution to be acidic with 1 N HC1, filtered to collect the filtrate, recrystallized with ethanol, vacuum dried to obtain compound Y-13-2 as a light yellow solid, 115 mg, yield about 93.51%. Mp. 254-255 °C. 1 H NMR (600 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.33 (d, J = 6.0 Hz, 1H), 8.00 (s, 1H), 7.93 (d, J = 6.0 Hz, 2H), 7.82 (d, J = 6.0 Hz, 1H), 7.68 (t, J = 6.0, 12.0 Hz, 2H), 7.43 (d, J = 12.0 Hz, 2H), 3.84 (s, 3H), 2.12 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 167.29, 161.29, 152.95, 150.24, 146.74, 145.86, 134.96, 131.45, 130.36, 130.11, 129.40, 129.35, 128.78, 128.19, 118.79, 111.96, 110.87, 103.81, 11.67. LCMS: m / z [M+H] 415.0859; found, 415.0856. HPLC purity at 254 nm, 97.84%. + C 22 H 14 N4O3S, 415.0859; found, 415.0856. HPLC purity at 254 nm, 97.84%.

[0188] Example 12: Preparation of compound Y-14 and its derivatives

[0189] (1) Synthesis of compound 4-(3-methylphenyl)-2-hydrazinylthiazole (Y-14)

[0190]

[0191] To a solution of 3-methyl-2-bromoacetophenone (1.60 g, 7.0 mmol) and aminothiourea (0.78 g, 7.7 mmol) in ethanol (24 mL) was added potassium acetate (1.17 g, 7.7 mmol) in water (24 mL) at room temperature. The reaction mixture was stirred for 3 h at room temperature. After the reaction was completed, the mixture was filtered and washed with 10 mL of ice ethanol. The white solid was dried under vacuum to give compound 4-(3-methylphenyl)-2-hydrazinylthiazole (1.25 g) in about 86.98% yield without further purification.

[0192] (2) Synthesis of compound 1-[4-(3-methylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-14-1).

[0193]

[0194] Compound Y-14 (0.67 g, 3 mmol) was added slowly to ethyl acetoacetate (0.40 mL, 3 mmol) in acetic acid (6 mL) and stirred at 80 °C for 3 h, monitored by TLC, after completion of the reaction the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 times), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to get compound 1-[4-(3-methylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-14-1) as a light yellow solid (0.60 g, yield ~ 73.45 %).

[0195] (3) Synthesis of compound Y-14-3

[0196]

[0197] Compound Y-14-1 (287 mg, 1 mmol) was added to p-formylbenzaldehyde (165 mg, 1 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed, monitored by TLC, after completion of the reaction, the crude was purified by column chromatography to get a light yellow solid, which was further refluxed in ethanol for 1 h, cooled to room temperature and filtered to get high purity light yellow solid Y-14-3 (156 mg) with a yield of ~ 36.82 %. Mp. 293-294 °C. 1 H NMR (600 MHz, DMSO-d6) δ 7.93 (d, J = 6.0 Hz, 2H), 7.64 (d, J = 6.0 Hz, 1H), 7.43 (d, J = 12.0 Hz, 2H), 7.39 (s, 1H), 7.29 - 7.26 (m, 4H), 3.84 (s, 3H), 2.43 (s, 3H), 2.08 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 166.19, 161.22, 151.88, 149.91, 149.22, 146.50, 135.72, 134.19, 130.70, 129.62, 129.21, 128.37, 128.07, 127.60, 125.81, 111.51, 103.85, 52.08, 21.01, 11.60. LCMS: m / z [M+H] + calculated for C 23 H 19 N3O3S, 418.1220; found, 418.1225. HPLC purity at 254 nm, 96.22 %.

[0198] (4) Synthesis of compound Y-14-2

[0199]

[0200] Y-14-3 (150 mg, 0.3 mmol) was dissolved in 16 mL of an aqueous ethanol solution (EtOH / H2O = 1 : 1, v:v) containing potassium carbonate (396 mg, 2.7 mmol), refluxed overnight, monitored by TLC, cooled to room temperature after completion of the reaction, then the organic solvent was removed, the solution was adjusted to be acidic with 1 N HCI, the filtrate was collected by filtration, recrystallized from ethanol, and dried under vacuum to obtain compound Y-14-2 as a light yellow solid, 140 mg, with a yield of about 96.51%. Mp. 216-217 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 7.91 (d, J = 4.0 Hz, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 4.0 Hz, 4H), 7.31 - 7.28 (m, 3H), 7.28 - 7.24 (m, 1H), 2.43 (s, 3H), 2.10 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 167.30, 162.11, 152.13, 149.91, 149.29, 146.01, 135.73, 134.19, 130.71, 129.62, 129.38, 129.00, 128.76, 128.54, 125.81, 111.69, 103.94, 21.02, 11.63. LCMS: m / z [M+H] 404.1063; found, 404.1060. HPLC purity at 254 nm, 98.18%. + calculated for C 22 H 17 N3O3S, 404.1063; found, 404.1060. HPLC purity at 254 nm, 98.18%.

[0201] Example 13: Preparation of compound Y-16 and its derivatives

[0202] (1) Synthesis of compound 4-(4-trifluoromethylphenyl)-2-hydrazinylthiazole (Y-16)

[0203]

[0204] Compound 4-(4-trifluoromethylphenyl)-2-hydrazinylthiazole (0.90 g) was obtained as a white solid by slow addition of aminothiourea (0.51 g, 5.5 mmol) and 3-trifluoromethyl-2-bromoacetophenone (1.08 g, 5 mmol) to an aqueous solution of potassium acetate (0.80 g, 5.5 mmol) in ethanol (20 mL, EtOH / H2O = 1:1, V:V) and stirring the reaction at room temperature for 3 h. After completion of the reaction, the reaction mixture was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain the product as a white solid in about 86.54% yield without further purification.

[0205] (2) Synthesis of compound 1-[4-(4-trifluoromethylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-16-1).

[0206]

[0207] Compound Y-16 (0.85 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were added slowly to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed and the residue was treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain compound 1-[4-(4-trifluoromethylphenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-16-1) as a pale yellow solid (0.72 g, 79.11% yield).

[0208] (3) Synthesis of compound Y-16-3

[0209]

[0210] Compound Y-16-1 (284 mg, 1 mmol) and p-formylbenzaldehyde (164 mg, 1 mmol) were added to anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was refluxed and monitored by TLC. After completion of the reaction, the reaction mixture was purified by column chromatography to obtain a pale yellow solid. The pale yellow solid was refluxed in ethanol for 1 h, cooled to room temperature and filtered to obtain high purity pale yellow solid Y-16-3 (120 mg) in about 23.97% yield. Mp. 248-249 °C. 1 H NMR (600 MHz, DMSO-d6) δ 8.87 (s, 1H), 8.01 (s, 1H), 7.93 (d, J = 6.0 Hz, 2H), 7.84-7.82 (d, J = 12.0 Hz, 3H), 7.43 (d, J = 12.0 Hz, 2H), 3.84 (s, 3H), 2.11 (s, 3H).13 C NMR (150 MHz, DMSO-d6) δ 166.18, 161.44, 153.07, 150.49, 147.38, 146.41, 137.59, 129.23, 128.05 (q, J = 31.7 Hz), 127.04, 126.48, 126.11, 125.68 (q, J = 3.9 Hz), 124.33 (q, J = 270.4 Hz), 111.44, 103.90, 33.30, 11.68. LCMS: m / z [M+H] 472.0937; found, 472.0935. HPLC purity at 254 nm, 98.79%. + calculated for C 23 H 16 F3N3O3S, 472.0937; found, 472.0935. HPLC purity at 254 nm, 98.79%.

[0211] (4) Synthesis of compound Y-16-2

[0212]

[0213] Y-16-3 (160 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H2O = 1 : 1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, TLC monitoring, after the reaction was completed, cooled to room temperature, then removed the organic solution, adjusted the solution to be acidic with 1 N HC1, filtered to collect the filtrate, recrystallized with ethanol, vacuum dried to obtain compound Y-16-2 as a light yellow solid, 110 mg, yield about 80.96%. Mp. 259-260 °C. 1 H NMR (600 MHz, DMSO-d6) δ 12.28 (s, 1H), 8.22 (d, J = 12 Hz, 2H), 8.01 (s, 1H), 7.91 (d, J = 6.0 Hz, 2H), 7.82 (d, J = 12 Hz, 3H), 7.42 (d, J = 6.0 Hz, 2H), 2.10 (s, 3H). 13 C NMR (150 MHz, DMSO-d6) δ 167.28, 161.46, 153.08, 150.50, 147.38, 145.85, 137.59, 129.39, 128.80, 128.09 (q, J = 31.9 Hz), 127.04, 126.48, 126.11, 125.75 (q, J = 3.8 Hz), 124.34 (q, J = 270.3 Hz), 111.42, 103.99, 11.68. LCMS: m / z [M+H] 472.0937; found, 472.0935. HPLC purity at 254 nm, 98.79%. + calculated for C22 H 14 F3N3O3S, 458.0781 ; found, 458.0780. HPLC purity at 254 nm, 99.76 %.

[0214] Example 14: Preparation of compound Y-17 and its derivatives

[0215] (1) Synthesis of compound 4-(4-chlorophenyl)-2-hydrazinylthiazole (Y-17)

[0216]

[0217] Amino thioamide (0.61 g, 6.6 mmol) and 3-chloro-2-bromoacetophenone (1.41 g, 6 mmol) were slowly added to an aqueous solution of potassium acetate (0.91 g, 6.6 mmol) in ethanol (25 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain white solid compound 4-(4-chlorophenyl)-2-hydrazinylthiazole (1.24 g) without further purification with an approximate yield of 91.17 %.

[0218] (2) Synthesis of compound 1-[4-(4-chlorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-17-1)

[0219]

[0220] Compound Y-17 (0.68 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were slowly added to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed and the residue was treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain yellowish solid compound 1-[4-(4-chlorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-17-1) 0.68 g with an approximate yield of 77.63 %.

[0221] (3) Synthesis of compound Y-17-2

[0222]

[0223] Compound Y-17-1 (284 mg, 1 mmol) was taken in anhydrous ethanol (1.4 mL, 0.5 M) and p-chlorobenzaldehyde (141 mg, 1 mmol) was added to it followed by addition of catalytic amount of piperidine (50 uL, 0.5 mmol) and the reaction was carried out under reflux. TLC monitoring was done and upon completion of the reaction, the crude was isolated by column chromatography to get a pale yellow solid. Further, ethanol was added to it and refluxed for 1 h, allowed to cool to room temperature naturally and filtered to get a pale yellow solid of high purity, Y-17-2 (120 mg) in about 24.10% yield. Mp. 249-250 °C. 1 H NMR (400 MHz, DMSO) δ 8.02 (d, J = 8.8 Hz, 3H), 7.82 (s, 1H), 7.5 (d, J = 8.6 Hz, 3H), 7.38-7.28 (m, 3H), 2.11 (s, 3H). 13 C NMR (100 MHz, DMSO) δ 161.35, 152.85, 150.18, 147.72, 138.88, 132.74, 132.53, 130.79, 129.87, 128.74, 128.14, 127.64, 109.43, 104.11, 11.65. LCMS: m / z [M+H] + C 20 H 13 Cl2N3OS, 414.0229; found, 414.0225. HPLC purity at 254 nm, 95.06%.

[0224] (4) Synthesis of compound Y-17-3

[0225]

[0226] Compound Y-17-1 (284 mg, 1 mmol) was taken in anhydrous ethanol (1.4 mL, 0.5 M) and p-chlorobenzaldehyde (141 mg, 1 mmol) was added to it followed by addition of catalytic amount of piperidine (50 uL, 0.5 mmol) and the reaction was carried out under reflux. TLC monitoring was done and upon completion of the reaction, the crude was isolated by column chromatography to get a pale yellow solid. Further, ethanol was added to it and refluxed for 1 h, allowed to cool to room temperature naturally and filtered to get a pale yellow solid of high purity, Y-17-2 (120 mg) in about 24.10% yield. Mp. 249-250 °C. 1 H NMR (400 MHz, DMSO) δ 8.02 (d, J = 8.8 Hz, 3H), 7.82 (s, 1H), 7.5 (d, J = 8.6 Hz, 3H), 7.38-7.28 (m, 3H), 2.11 (s, 3H). 13C NMR (101 MHz, DMSO) δ 166.16, 161.38, 152.11, 150.23, 147.76, 146.43, 132.74, 132.54, 129.20, 128.74, 128.31, 128.30, 127.65, 109.53, 103.88, 52.05, 11.64. LCMS: m / z [M+H] 438.0674; found, 438.0673. HPLC purity at 254 nm, 98.08%. + calculated for C 22 H 16 ClN3O3S, 424.0517; found, 424.0516. HPLC purity at 254 nm, 98.01%.

[0227] (5) Synthesis of compound Y-17-4

[0228]

[0229] Y-17-3 (132 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H20 = 1:1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), refluxed overnight, TLC monitored, cooled to room temperature after the reaction was completed, then the organic solution was removed, the solution was adjusted to be acidic with IN HCl, the filtrate was collected by filtration, recrystallized with ethanol, and dried in vacuum to obtain compound Y-17-4 as a light yellow solid, 120 mg, yield about 94.34%. Mp. 262-263 °C. 1 H NMR (400 MHz, DMSO) δ 12.86 (s, 1H), 8.02 (d, J = 8.6 Hz, 3H), 7.90 (d, J = 8.2 Hz, 2H), 7.83 (s, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 2.11 (s, 3H). 13 C NMR (100 MHz, DMSO) δ 167.27, 161.90, 152.67, 150.26, 147.74, 132.75, 132.54, 129.37, 128.75, 128.24, 128.15, 127.65, 109.57, 103.96, 11.65. LCMS: m / z [M+H] 424.0517; found, 424.0516. HPLC purity at 254 nm, 98.01%. + calculated for C 21 H 14 ClN3O3S, 424.0517; found, 424.0516. HPLC purity at 254 nm, 98.01%.

[0230] Example 15: Preparation of compound Y-18 and its derivatives

[0231] (1) Synthesis of compound 4-(4-bromophenyl)-2-hydrazinylthiazole (Y-18)

[0232]

[0233] Amino thiourea (0.61 g, 6 mmol) and 3-bromo-2-bromoacetophenone (1.83 g, 6 mmol) were slowly added to an aqueous solution of potassium acetate (0.91 g, 6.6 mmol) in ethanol (25 mL, EtOH / H2O = 1:1, V:V) and stirred at room temperature for 3 h. After completion of the reaction, it was filtered, washed with 10 mL of ice-cold ethanol and dried under vacuum to obtain white solid compound 4-(4-bromophenyl)-2-hydrazinylthiazole (1.54 g) without further purification, with an approximate yield of 89.17%.

[0234] (2) Synthesis of compound 1-[4-(4-bromophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-18-1).

[0235]

[0236] Compound Y-18 (0.75 g, 3 mmol) and ethyl acetoacetate (0.40 mL, 3 mmol) were slowly added to acetic acid (6 mL) and stirred at 80 °C for 3 h. After completion of the reaction, the organic solvent was removed and the residue was treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate and finally purified by column chromatography to obtain pale yellow solid compound 1-[4-(4-bromophenyl)thiazol-2-yl]-3-methyl-1H-pyrazol-5-ol (Y-18-1) 0.75 g, with an approximate yield of 72.63%.

[0237] (3) Synthesis of compound Y-18-2

[0238]

[0239] Compound Y-18-1 (337 mg, 1 mmol) and p-chlorobenzaldehyde (141 mg, 1 mmol) were taken in anhydrous ethanol (1.4 mL, 0.5 M) and a catalytic amount of piperidine (50 uL, 0.5 mmol) was added. The reaction was carried out under reflux, monitored by TLC and after completion of the reaction, it was purified by column chromatography to obtain pale yellow solid. Further, ethanol was added and refluxed for 1 h, cooled to room temperature and filtered to obtain high purity pale yellow solid Y-18-2 (180 mg) with an approximate yield of 35.53%. Mp. 254-255 °C. 1H NMR (400 MHz, DMSO) δ 7.96 (d, J = 8.6 Hz, 3H), 7.83 (s, 1H), 7.65 (d, J = 8.6 Hz, 3H), 7.38 - 7.28 (m, 3H), 2.11 (s, 3H). 13 C NMR (100 MHz, DMSO) δ 161.35, 152.86, 150.22, 147.75, 139.62, 133.08, 131.65, 130.78, 129.88, 128.14, 127.93, 121.17, 109.52, 104.10, 11.64. LCMS: m / z [M+H] + calculated for C 20 H 13 BrClN3OS, 457.9724; found, 457.9719. HPLC purity at 254 nm, 95.68 %.

[0240] (4) Synthesis of compound Y-18-3

[0241]

[0242] Compound Y-18-1 (334 mg, 1 mmol) was added to p-formylbenzaldehyde (159 mg, 1 mmol) in anhydrous ethanol (1.4 mL, 0.5 M) with a catalytic amount of piperidine (50 uL, 0.5 mmol) and refluxed. TLC was used to monitor the reaction. After the reaction was completed, the crude product was isolated by column chromatography to obtain a light yellow solid. After adding ethanol and refluxing for 1 h, the reaction was naturally cooled to room temperature, and a light yellow solid Y-18-3 (210 mg) with high purity was obtained by filtration. The yield was about 64.32 %. Mp. 271-272 °C. 1 H NMR (400 MHz, DMSO) δ 7.96 (d, J = 8.6 Hz, 3H), 7.83 (s, 1H), 7.65 (d, J = 8.6 Hz, 3H), 7.38 - 7.28 (m, 3H), 2.11 (s, 3H). 13 C NMR (100 MHz, DMSO) δ 166.16, 150.22, 148.02, 146.43, 133.08, 131.65, 130.22, 129.71, 129.20, 128.31, 127.94, 127.63, 121.19, 109.65, 103.87, 52.06, 11.65. LCMS: m / z [M+Na] + calculated for C 22 H 16BrN3O3S, 503.9988; found, 503.9979. HPLC purity at 254 nm, 97.20%.

[0243] (5) Synthesis of compound Y-18-4

[0244]

[0245] Y-18-3 (160 mg, 0.3 mmol) was dissolved into 16 mL of an aqueous ethanol solution (EtOH / H2O = 1:1, v:v) containing potassium carbonate (373 mg, 2.7 mmol), and refluxed overnight, and TLC was monitored until the reaction was completed. After cooling to room temperature, the organic solvent was removed, the solution was adjusted to be acidic with 1 N HCl, and the filtrate was collected by filtration, recrystallized with ethanol, and dried in vacuo to obtain compound Y-18-4 as a light yellow solid, 148 mg, at a yield of about 89.24%. Mp. 249-250 °C 1 H NMR (400 MHz, DMSO) δ 12.87 (s, 1H), 7.95 (d, J = 8.6 Hz, 3H), 7.90 (d, J = 8.2 Hz, 2H), 7.84 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.39 ((d, J = 8.2 Hz, 2H), 2.11 (s, 3H). 13 C NMR (100 MHz, DMSO) δ 167.28, 161.35, 153.61, 150.28, 147.80, 146.16, 133.09, 131.66, 129.38, 128.78, 128.14, 127.95, 121.20, 109.57, 103.98, 11.67. LCMS: m / z [M+H] + calculated for C 21 H 15 BrN3O3S, 468.0012; found, 468.0012. HPLC purity at 254 nm, 99.87%.

[0246] Test Example

[0247] (1) Experimental materials: SIRT5 (Supplier: Novus) Product name: Recombinant Human Sirtuin 5 / SIRT5 His Protein; Catalog No: NBP2-22807. Suramin of Sigma was used as a positive control, and the target compounds synthesized in Examples 1 to 15.

[0248] (2) Experimental Methods: A pH 8.0 assay buffer was prepared: 50 mM Tris, 137 mM NaCl, and 2.7 mM KCl. The reaction was carried out in a 96-well black microplate (Corning No. 3603). 50 μL of buffer was added to each well to dissolve the enzyme and substrate. The reaction wells were then filled with buffer, SIRT5 protein (0.2 μM), AC-K(Succ)-AMC (10 μM), and NAD+. + (200 μM) and different concentrations of compounds prepared in Examples 1-15 or positive controls. Buffer AC-K(Succ)-AMC (10 μM) and NAD+ were added to the control wells. + (200 μM) and the compounds prepared in Examples 1-15 or positive controls (same concentration as in the reaction wells). The reaction was incubated at 37°C and 140 rpm for 1.5 hours, then 50 μl containing The reaction was terminated with a solution of trypsin (Sigma-Aldrich No. T8003) and 8 mM nicotinamide, and incubated at 37°C and 140 rpm for 30 minutes. The reaction was then analyzed using a BioTek Cytation 3 microplate reader. ex =370nm, λ em Fluorescence intensity was measured at 460 nm. The compound concentration was double-diluted, ranging from 100 μM to 0.1 μM. The inhibition rate was calculated using the following formula:

[0249]

[0250] Among them, F ac F represents the fluorescence in the reaction well containing the compound. a F represents the fluorescence in reaction wells that do not contain the compound. c F0 represents the fluorescence in the control well containing the compound, while F0 represents the fluorescence in the control well without the compound.

[0251] Specific operation method:

[0252] The inhibition rate was used to fit the corresponding half-maximal inhibitory concentration (IC50) using GraphPad.

[0253] (3) Experimental results:

[0254] The inhibitory activity of the compounds of this invention against SIRT5 was tested using the above experimental methods. The specific half-maximal inhibitory concentrations (IC50) of the compounds against SIRT5 are shown in Table 1.

[0255] Table 1

[0256]

[0257]

[0258]

[0259] As can be seen from Table 1, the compounds prepared in the present application have the best effect on inhibiting SIRT5 activity, Y-5-4, Y-6-2, Y-3-4, Y-7-2, Y-10-2, Y-16-2, Y-17-4, Y-18-4, it can be seen that the above compounds are all compounds with R as COOH. Especially Y-17-4 has the best effect, which is significantly higher than the SIRT5 inhibitor in the prior art.

[0260] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A SIRT5 protein inhibitor, characterized in that, The active ingredient of the SIRT5 protein inhibitor is a substituted 4-benzyl-5-methyl-2-(4-benzothiazol-2-yl)-pyrazole-3-one derivative; the structural formula of the substituted 4-benzyl-5-methyl-2-(4-benzothiazol-2-yl)-pyrazole-3-one derivative is as follows: ， The SIRT5 protein inhibitor also includes one or more pharmaceutically acceptable carriers or excipients; The substituted 4-benzyl-5-methyl-2-(4-benzothiazo-2-yl)-pyrazole-3-one derivative was prepared by the following method: (1) Add aminothiourea and 3-chloro-2-bromoacetophenone to an aqueous ethanol solution containing potassium acetate, stir and react at room temperature for 3 h, filter after the reaction is complete, wash the filter with ice-cold ethanol and dry under vacuum to obtain a white solid compound 4-(4-chlorophenyl)-2-hydrazylthiazole; (2) 4-(4-chlorophenyl)-2-hydrazylthiazole and ethyl acetoacetate were added to acetic acid and stirred at 80°C for 3 h. The reaction was monitored by TLC. After the reaction was completed, the organic solvent was removed, the residue was treated with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and finally separated by column chromatography to obtain the pale yellow solid compound 1-[4-(4-chlorophenyl)thiazole-2-yl]-3-methyl-1H-pyrazole-5-ol; (3) 1-[4-(4-chlorophenyl)thiazol-2-yl]-3-methyl-1H-pyrazole-5-ol and p-formylbenzaldehyde were added to anhydrous ethanol, and then a catalytic amount of piperidine was added and refluxed. The reaction was monitored by TLC. After the reaction was completed, the solid was crudely separated by column chromatography to obtain a pale yellow solid. Ethanol was added and refluxed for 1 h. The solid was then naturally cooled to room temperature and filtered to obtain a high-purity pale yellow solid Y-17-3. (4) Dissolve Y-17-3 in an ethanol aqueous solution containing potassium carbonate, reflux overnight, monitor by TLC, cool to room temperature after the reaction is complete, remove organic solvent, adjust the acidity of the solution with 1N HCl, filter and collect the filtrate, recrystallize from ethanol, and dry under vacuum to obtain a pale yellow solid compound, which is the substituted 4-benzylidene-5-methyl-2-(4-benzothiazolyl-2-yl)pyrazol-3-one derivative.

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