Substituted benzyl sulfide diazole compound as well as preparation method and application thereof

Abstract

The invention relates to a substituted benzyl sulfide diazole compound which has an adjusting effect on a PIEZO1 ion channel, can specifically excite or inhibit the function of the PIEZO1 ion channel, has low potential cardiotoxicity and small toxic and side effects, further improves the structure-function relationship of the general formula applied to a PIEZO1 agonist or antagonist, and provides partial pharmacological activity.

Description

Technical Field

[0001] The invention relates to a substituted benzyl thioether diazole compound, a preparation method thereof, and the application of the compound in related biology by stimulating or antagonizing PIEZO1 protein. Background Art

[0002] PIEZO1 is a highly conserved cation channel protein and mechanical stimulus sensor, expressed in a variety of tissues or organs, and is of great significance to the normal life activities of organisms. Its overexpression or underexpression can cause diseases, so regulating abnormally expressed PIEZO1 is a promising strategy for disease treatment. However, due to its short discovery time, the specific protein structure, mode of action and biological function are still in the exploratory stage, and it is urgent to study and discover new regulators for the biological function analysis and drug development of PIEZO1.

[0003] At present, there are few reports on modulators targeting the PIEZO1 channel, and the effects are mixed, such as PIEZO1-specific small molecule agonists (Yoda1, Jedi1 and Jedi2), but the antagonists are all non-selective, and no PIEZO1-specific antagonists have been reported.

[0004] The following is an overview of the current status of research on PIEZO1 small molecule agonists:

[0005] Jedi1 and Jedi2 are two small molecule PIEZO1 agonists obtained by high-throughput screening by Wang Yanfeng et al. 50 200 μM and 158 μM, respectively, and the K of mPIEZO1 d The values ​​were 2754μM and 2770μM respectively. The study found that it activates PIEZO1 through the spiral blade structure of PIEZO1, and can act on PIEZO1 at the same time as Yoda1, showing a synergistic effect.

[0006]

[0007] Syeda et al. obtained the first small molecule PIEZO1 agonist through high-throughput screening, named Yoda1, which can specifically activate mouse and human PIEZO1, EC 50 17.1 μM and 26.6 μM, respectively, and the K dThe value is 45.6μM, which has good binding affinity. The discovery of Yoda1 has greatly helped further research on the mechanism of action and biological function of PIEZO1 ion channels, but the low water solubility of Yoda1 affects its further clinical research. Molecular virtual docking shows that there is a large space in the area where its benzene ring and pyrazine ring are located, and structural optimization can be used to obtain a small molecule PIEZO1 agonist with better effect and less toxicity.

[0008]

[0009] Evans et al. designed a series of derivatives based on Yoda1. When the 2,6-dichlorobenzyl group was replaced by other substituents (such as phenyl, 4-nitrophenyl, 4-methoxyphenyl, and phenyl containing halogen atoms at different positions), the activity was significantly reduced. Among them, the tool compound Dooku1 was obtained, which can effectively antagonize the PIEZO1 channel activity induced by Yoda1 and can be used for biological research on Yoda1 activation of PIEZO1.

[0010] Li et al. also reported a series of Yoda1 analogs. When the thioether bond was replaced by an ether bond or an amino group, the activity was significantly reduced. However, when the ether bond and 2,6-dichlorobenzyl were retained and substituents (such as 2,6-dichloro-3-fluorophenyl, 2,6-dichloro-3-nitrophenyl and 2,6-dichloropyridin-3-yl) were added, the activity was equivalent to or better than that of Yoda1. Therefore, 2,6-dichlorobenzyl and thioether bond may be the key to Yoda1 activation, and the loss of these two parts will lead to the disappearance of Yoda1 activity. Based on this research idea, these two parts were retained, and the benzene ring and pyrazine ring parts were explored in detail.

[0011] In 2022, Parsonage et al. reported a Yoda1 analog, KC159, and its potassium salt, KC289, which replaced pyrazine with phenyl and introduced a carboxyl group at the para position. The EC of hPIEZO1 was 50 They are 2.28μM and 1.14μM respectively. The solubility is 160 times that of Yoda1, the stability is 20 times higher than that of Yoda1, and the plasma protein binding ratio is improved compared with Yoda1.

[0012]

[0013] In summary, there are few reports on PIEZO1 modulators, and the agonists that have been discovered still have shortcomings, such as low water solubility of Yoda1, poor binding of Jedi, and non-specific antagonists, which hinder the functional research and clinical application of the channel. Therefore, it is of great research significance to study and discover new PIEZO1 small molecule modulators with better effects. Summary of the invention

[0014] The purpose of the present invention is to provide a substituted benzyl thioether diazole compound and its application in the biological function of PIEZO1, so as to discover a new type of PIEZO1 regulator and meet the needs of research on the mechanism of action and biological function of PIEZO1.

[0015] The present invention adopts the following technical solution:

[0016] A substituted benzyl sulfide oxadiazole compound having a structure of general formula I:

[0017]

[0018] R 1 Selected from the following groups: H, CH3, NH2;

[0019] R 2 Selected from H, Cl;

[0020] Y is selected from S, O, NH;

[0021] A is selected from the following groups:

[0022] More preferably, the following specific compounds are preferred among the compounds of the general formula (I): I-1: 2-((2,6-dichloro-3-methylbenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadiazole;

[0023] I-2: 2,4-dichloro-3-(((5-(pyrazin-2-yl)-1,3,4-thiadiazol-2-yl)thio)methyl)aniline;

[0024] I-3: 2-(2,6-dichlorobenzyl)thio-5-(5-methylpyrazin-2-yl)-1,3,4-thiadiazole;

[0025] I-4: 4-(5-((2,6-dichlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)aniline;

[0026] I-5: 2-((2,6-dichloro-3-methylbenzyl)thio)-5-(5-methylpyrazin-2-yl)-1,3,4-thiadiazole;

[0027] I-6: 2-(2,6-dichlorobenzyl)thio-5-phenyl-1,3,4-oxadiazole;

[0028] I-7: 4-(5-((2,6-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)aniline;

[0029] I-8: 3-(2,6-dichlorobenzyl)thio-5-phenyl-4H-1,2,4-triazole;

[0030] I-9: 4-(5-(2,6-dichlorobenzyl)thio-1,3,4-thiadiazol-2-yl)-N-methylbenzamide;

[0031] I-10: 2-(2,6-dichlorobenzyl)thio-5-(3-trifluoromethyl)phenyl-1,3,4-thiadiazole;

[0032] The structures of the above compounds are shown in Table 1.

[0033] Table 1 Preferred compound numbers and corresponding structural formulas

[0034]

[0035] A method for preparing the above compounds I-1 to I-8, the synthetic route is as follows:

[0036]

[0037] The specific steps are:

[0038] 1) Compound 1 is used as a raw material, esterified with methanol, then hydrazine-hydrated, and then cyclized with carbon disulfide and concentrated sulfuric acid to obtain intermediate 4;

[0039] 2) Compound 5 is used as a raw material to undergo acylation under the catalysis of an organic base, and then the aldehyde group is reduced and chlorinated with thionyl chloride to obtain intermediate 8.

[0040] 3) Intermediate 4 and intermediate 8 are reacted by condensation to obtain target product I.

[0041] A method for preparing the above compounds I-9 and I-10, the synthetic route is as follows:

[0042]

[0043] The specific steps are:

[0044] 1) Intermediate 8 is used as a raw material, a base catalyst is added, and 1,3,4-thiadiazole-2-thiol is subjected to nucleophilic substitution to obtain intermediate 9;

[0045] 2) Intermediate 9 is brominated with NBS to obtain intermediate 10;

[0046] 3) Intermediate 10 was subjected to Suzuki coupling to obtain target compound I.

[0047] An application of the above-mentioned substituted benzyl thioether diazole compound in regulating PIEZO1 protein.

[0048] A PIEZO1 protein regulator comprises the above-mentioned substituted benzyl thioether diazole compound.

[0049] Specifically, the PIEZO1 protein modulator is a PIEZO1 agonist.

[0050] The compounds of the present invention can be used in combination for PIEZO1 biological regulation (including humans) through oral administration, injection and other routes, wherein oral administration is the best, and the dosage is 0.0001 mg / kg to 200 mg / kg body weight per day. The optimal dosage depends on the individual, usually starting with a small dosage and then gradually increasing the dosage.

[0051] A pharmaceutical composition comprising a therapeutically effective dose of a compound represented by formula (I) and a pharmaceutically acceptable carrier.

[0052] The carrier refers to a common carrier in the pharmaceutical field, for example: a diluent, an excipient such as water, etc.; a binder such as cellulose derivatives, gelatin, polyvinyl pyrrolidone, etc.; a filler such as starch, etc.; a disintegrant such as calcium carbonate, sodium bicarbonate; in addition, other auxiliary agents such as flavoring agents and sweeteners may also be added to the composition.

[0053] The above-mentioned pharmaceutical composition can be prepared into conventional solid preparations, such as tablets, capsules, etc., for oral administration, or it can be prepared into injections for injection.

[0054] The various dosage forms of the above-mentioned pharmaceutical composition can be prepared by conventional methods in the pharmaceutical field, wherein the content of the active ingredient I is 0.1% to 99.5% (by weight) of the weight of the compound.

[0055] The beneficial effects of the present invention are:

[0056] 1) The compounds of the present invention have PIEZO1 stimulatory or inhibitory effects, and have good regulatory effects on various PIEZO1-expressing cells in the human body, and have the characteristics of broad spectrum and high efficiency. They may also have good therapeutic effects on diseases caused by abnormal gene expression, such as cardiovascular system diseases, immune system diseases, bone diseases and cancer.

[0057] 2) Compared with the reported PIEZO1 regulators, the compounds of the present invention can effectively stimulate or inhibit the expression of PIEZO1.

[0058] 3) Compared with the reported PIEZO1 modulators, the compounds described in the present invention have better safety and physicochemical properties and are worthy of further research.

[0059] In summary, the present invention discloses a group of novel regulators acting on PIEZO1. In the structures of such compounds, compounds with agonistic effects superior to those of positive control compounds and compounds with inhibitory effects are found. In addition, the novel structural type compounds of the present invention not only have strong agonistic activity, but also have better liver microsomal stability than the reported positive control compounds. At the same time, the novel structural compounds have higher safety, less toxic and side effects, and are easier to use as candidate compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a diagram showing the agonistic effect of the first group of compounds in Example 19.

[0061] Figure 2 This is a graph showing the agonistic effect of the second group of compounds in Example 19.

[0062] Figure 3 This is a diagram showing the inhibitory effect of the second group of compounds in Example 19.

[0063] Figure 4 This is a diagram showing the agonistic effect of the third group of compounds in Example 19.

[0064] Figure 5 This is a diagram showing the inhibitory effect of the third group of compounds in Example 19. DETAILED DESCRIPTION

[0065] The present invention is further described in detail with reference to the embodiments, but the embodiments of the present invention are not limited thereto.

[0066] Example 1 Synthesis of intermediate 5-methylpyrazine-2-carboxylic acid methyl ester

[0067]

[0068] 5-Methyl-2-pyrazinecarboxylic acid (5.50 g, 39.82 mmol) was placed in a 250 mL three-necked flask, 150 mL of methanol was slowly added, and a tail gas absorption device was connected. Sulfoxide chloride (9.6 mL, 119.46 mmol) was slowly added dropwise with a constant pressure dropping funnel at 0°C. After the addition was completed, the reaction solution was stirred at room temperature. It was observed that the reaction solution changed from turbid to colorless and transparent. After reacting for 2 hours, the reaction solution was slowly added to ice water, adjusted to neutral with saturated Na2CO3 solution, extracted with dichloromethane (4×20 mL), the organic phases were combined, washed with saturated brine (3×20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain an orange solid, namely the intermediate 5-methylpyrazine-2-carboxylic acid methyl ester (4.3 g, 71.5%). No further purification is required and it can be directly used in the next step.

[0069] Example 2 Synthesis of Intermediate 5-Methylpyrazine-2-Carboxylic Acid Hydrazide

[0070]

[0071] Dissolve 5-methylpyrazine-2-carboxylic acid methyl ester (4.00 g, 26.31 mmol) in 100 mL of anhydrous ethanol, place in a 250 mL single-mouth bottle, add 80% hydrazine hydrate (4.7 mL, 121.73 mmol), and reflux for 2 h. After the reaction solution is concentrated, add ethyl acetate (15 mL) for washing, filter, and dry the filter cake to obtain a yellow solid, i.e., the intermediate 5-methylpyrazine-2-carboxylic acid hydrazide (2.92 g, 73.9%). No further purification is required and it can be directly used in the next step.

[0072] Example 3 Synthesis of Intermediate 5-(5-methylpyrazine-2-yl)-1,3,4-thiadiazole-2-thiol

[0073]

[0074] 5-Methylpyrazine-2-carboxylic acid hydrazide (1.00 g, 6.66 mmol), KOH (0.45 g, 7.99 mmol) and 45 mL of anhydrous ethanol were added to a 250 mL single-mouth bottle, and CS2 (0.48 mL, 7.99 mmol) was slowly added dropwise. The reaction was allowed to react overnight at room temperature. The reaction solution was filtered and the filter cake was dried to obtain 1.67 g of a bright yellow solid. The solid was dissolved in 3 mL of concentrated sulfuric acid and stirred at 0 ° C. The solid was observed to dissolve slowly. After the solid was completely dissolved, the reaction solution was slowly added to 30 mL of ice water. A light yellow solid precipitated and filtered. The filter cake was washed with distilled water to obtain a crude product. The yellow solid was obtained by methanol pulping and dichloromethane recrystallization, i.e., the intermediate 5-(5-methylpyrazine-2-yl)-1,3,4-thiadiazole-2-thiol (0.38 g, 27.2%).

[0075] 1 H-NMR (500MHz, DMSO-d6) δ14.94(s,1H),9.10(s,1H),8.64(s,1H),2.58(s,3H).

[0076] Example 4 Synthesis of Intermediate 2,6-Dichlorobenzaldehyde

[0077]

[0078] The raw material 1,3-dichlorobenzene (2.00 g, 13.61 mmol) was dissolved in 40 mL of anhydrous tetrahydrofuran, and n-butyl lithium (2.80 g, 43.55 mmol) was added at -78 ° C, stirred for 30 min, and anhydrous N, N-dimethylformamide (3.48 g, 47.64 mmol) was added, and the reaction was continued to stir for 30 min. The reaction was quenched with 30 mL of saturated NaHCO3 solution to generate a white precipitate, which was filtered and the filtrate was extracted with ethyl acetate (5×20 mL), the organic phases were combined, washed with saturated brine (3×20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether-ethyl acetate, 0-10% gradient elution) to obtain a white solid, namely the intermediate 2,6-dichlorobenzaldehyde (1.9 g, 79.0%).

[0079] 1 H-NMR (500MHz, DMSO-d6) δ: 10.37 (s, 1H), 7.62 (t, J = 2.5Hz, 3H).

[0080] Example 5 Synthesis of Intermediate 2,6-Dichlorobenzyl alcohol

[0081]

[0082] The intermediate 2,6-dichlorobenzaldehyde (600 mg, 3.43 mmol) was dissolved in 13 mL of methanol, and NaBH4 (181.6 mg, 4.8 mmol) was slowly added at 0°C, and the reaction was stirred at room temperature. Saturated NH4Cl solution was added dropwise to the reaction solution until it was neutral, and ethyl acetate was extracted (4×10 mL). The organic phases were combined, washed with saturated brine (3×20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a white solid, which was slurried with petroleum ether to obtain the intermediate 2,6-dichlorobenzyl alcohol (554.7 mg, 91.4%).

[0083] 1 H-NMR (500MHz, DMSO-d6) δ: 7.47 (d, J = 8.1 Hz, 2H), 7.34 (t, J = 8.0 Hz, 1H), 5.02 (d, J = 13.1 Hz, 1H), 4.69 (s, 2H).

[0084] Example 6 Synthesis of Intermediate 2,6-Dichlorobenzyl Chloride

[0085]

[0086] The intermediate 2,6-dichlorobenzyl alcohol (200 mg, 1.13 mmol) was dissolved in 10 mL of dichloromethane, and SOCl2 (240.1 mg, 1.19 mmol) was slowly added dropwise at 0°C, and N,N-dimethylformamide (16.5 mg, 0.23 mmol) was added, and stirred at room temperature for 3 h. After the reaction was completed, the reaction was quenched with 20 mL of water, extracted with dichloromethane (3×15 mL), and the organic phases were combined, washed with saturated NaHCO3 solution (2×15 mL), washed with saturated brine (2×15 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether-ethyl acetate, 0-10% gradient elution) to obtain a white solid, namely the intermediate 2,6-dichlorobenzyl chloride (155.7 mg, 70.5%).

[0087] 1 H-NMR (600MHz, CDCl3) δ: 7.37 (d, J = 8.1 Hz, 2H), 7.24 (t, J = 8.1 Hz, 1H), 4.91 (s, 2H).

[0088] Example 7 Synthesis of 2-(2,6-dichlorobenzyl)thio)-5-(5-methylpyrazine-2-yl)-1,3,4-thiadiazole (target product I-3)

[0089]

[0090] 2,6-Dichlorobenzyl chloride (250 mg, 1.19 mmol), 5-(5-methylpyrazin-2-yl)-1,3,4-thiadiazole-2-thiol (232.61 mg, 1.19 mmol) and K2CO3 (328.94 mg, 2.38 mmol) were dissolved in 15 mL of tetrahydrofuran and refluxed for 2 h. The reaction solution was concentrated to obtain a light yellow solid. Distilled water (15 mL) was added and the crude product was filtered. The white solid was purified by P-TLC, i.e., the target product (224.6 mg, 51.3%).

[0091] HRMS: m / z C 14 H 11 Cl2N4S2[M+H] + Calculate 368.9724, Found 368.9797.

[0092] 1 H-NMR (500MHz, DMSO-d6) δ9.30 (d, J = 1.5 Hz, 1H), 8.69 (s, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.45 (t, J = 7.9 Hz, 1H), 4.90 (s, 2H), 2.61 (s, 3H).

[0093] 13 C-NMR (126MHz, DMSO-d6) δ166.22,156.71,145.03,141.40,140.71(2C),135.67,131.89,131.40,129.40(2C),34.85,21.87.

[0094] Example 8 Synthesis of 2-((2,6-dichloro-3-methylbenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadiazole (target product I-1)

[0095]

[0096] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-1 can be prepared with a yield of 64.9%.

[0097] 1 H-NMR (500MHz, DMSO-d6) δ9.44(s,1H),8.84(s,1H),8.81(s,1H),7.47(d,J=8.3Hz,1H),7.42(d,J=8.3Hz,1H),4.94(s,2H),2.36(s,3H).

[0098] 13 C-NMR (126MHz, DMSO-d6) δ168.90,167.13,147.16,145.48,144.19,141.93,136.60,135.78,132.76,132.35,131.62,128.56,35.38,20.62.

[0099] HRMS: m / z C 14 H 11 Cl2N4S2[M+H] + Calculate 368.9724, Found 368.9805.

[0100] Example 9 Synthesis of 2,4-dichloro-3-(((5-(pyrazin-2-yl)-1,3,4-thiadiazol-2-yl)thio)methyl)aniline (target product I-2)

[0101]

[0102] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-2 can be prepared with a yield of 85.1%.

[0103] 1H-NMR (500MHz, DMSO-d6) δ9.44(s,1H),8.84(s,1H),8.80(s,1H),7.18(d,J=10.2Hz,1H),6.82(d,J=8.8Hz,1H),5.69(s,2H),4.83(s,2H).

[0104] 13 C-NMR (126MHz, DMSO-d6) δ168.73,167.43,147.10,145.45,145.26,144.21,141.90,131.07,128.76,120.48,118.60,116.33,35.45.

[0105] HRMS: m / z C 13 H 10 Cl2N5S2[M+H] + Calculate 369.9676, Found 369.9760.

[0106] Example 10 Synthesis of 4-(5-((2,6-dichlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)aniline (target product I-4)

[0107]

[0108] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-4 can be prepared with a yield of 84.1%.

[0109] 1 H-NMR (500MHz, DMSO-d6) δ7.58(d,J=8.4Hz,2H),7.54(d,J=8.1Hz,2H),7.40(t,J=8.1Hz,1H),6.65(d,J=8.5Hz,2H),5.92(s,2H),4.75(s,2H).

[0110] 13 C-NMR (126MHz, DMSO-d6) δ171.05,159.72,152.68,135.58,132.35,131.17(2C),129.58(2C),129.32(2C),116.56,114.14(2C),35.29.

[0111] HRMS: m / z C 15 H 12 Cl2N3S2[M+H] +Calculate 367.9771, Found 367.9839.

[0112] Example 11 Synthesis of 2-((2,6-dichloro-3-methylbenzyl)thio)-5-(5-methylpyrazin-2-yl)-1,3,4-thiadiazole (target product I-5)

[0113]

[0114] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-5 can be prepared with a yield of 33.2%.

[0115] 1 H-NMR (500MHz, DMSO-d6) δ9.30 (s, 1H), 8.69 (s, 1H), 7.45 (dd, J = 6.2Hz, 2H), 4.92 (s, 2H), 2.61 (s, 3H), 2.36 (s, 3H).

[0116] 13 C-NMR(126MHz,DMSO-d6)δ169.12,166.45,156.68,156.14,145.02,141.4 2,140.71,136.59,132.76,132.33,131.68,128.55,35.42,21.86,20.62.

[0117] HRMS: m / z C 15 H 13 Cl2N4S2[M+H] + Calculate 382.9880, Found 382.9946.

[0118] Example 12 Synthesis of 2-(2,6-dichlorobenzyl)thio-5-phenyl-1,3,4-oxadiazole (target product I-6)

[0119]

[0120] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-6 can be prepared with a yield of 93.1%.

[0121] 1H-NMR (500MHz, DMSO-d6) δ7.97 (d, J = 7.0 Hz, 2H), 7.66 (t, J = 7.3 Hz, 1H), 7.61 (t, J = 7.2 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.42 (t, J = 8.1 Hz, 1H), 4.75 (s, 2H).

[0122] 13 C-NMR (126MHz, DMSO-d6) δ166.50,162.32,135.47,132.75,132.29,131.35(2C),129.98(2C),129.39(2C),126.97(2C),123.41,33.72.

[0123] HRMS: m / z C 15 H 11 Cl2N2OS[M+H] + Calculate 336.9891, Found 336.9957.

[0124] Example 13 Synthesis of 4-(5-((2,6-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)aniline (target product I-7)

[0125]

[0126] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-7 can be prepared with a yield of 57.2%.

[0127] 1 H-NMR (500MHz, DMSO-d6) δ: 7.59 (d, J = 8.7Hz, 2H), 7.53 (d, J = 8.1Hz, 2H), 7.40 (t, J = 7.7Hz, 1H), 6.67 (d, J = 8.7Hz, 2H), 6.00 (s, 2H), 4.66 (s, 2H).

[0128] 13 C-NMR(126MHz, DMSO-d6)δ:167.42,159.89,153.07,135.41,132.58,131.18(2C),129.32(2C),128.53(2)C,114.02(2C),109.63,33.82.

[0129] HRMS: m / z C 15 H 12 Cl2N3OS[M+H] +Calculate 352.0000, Found 352.00656.

[0130] Example 14 Synthesis of 3-(2,6-dichlorobenzyl)thio-5-phenyl-4H-1,2,4-triazole (target product I-8)

[0131]

[0132] According to the preparation method of target compound I-3 in Examples 1 to 7, target compound I-8 can be prepared with a yield of 67.3%.

[0133] 1 H-NMR (500MHz, DMSO-d6) δ14.59(s,1H),7.98(d,J=7.1Hz,2H),7.51(d,J=8.1Hz,5H),7.37(t,J=8.1Hz,1H),4.65(s,2H).

[0134] 13 C-NMR (126MHz, DMSO-d6) δ135.44,133.43,130.71,129.53,129.38,129.21,126.48,126.47,32.78.

[0135] HRMS: m / z C 15 H 12 Cl2N3S[M+H] + Calculate 336.0051,Found 336.01205.

[0136] Example 15 Synthesis of Intermediate 2-(2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole

[0137]

[0138] According to the method of Example 7, the intermediate 2-(2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole was prepared to obtain a white solid with a yield of 86.7%.

[0139] 1 H-NMR (500MHz, DMSO-d6) δ: 9.62 (s, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.41 (t, J = 8.1 Hz, 1H), 4.82 (s, 2H).

[0140] Example 16 Synthesis of Intermediate 2-Bromo-5(2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole

[0141]

[0142] 2-(2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole (3.0 g, 10.87 mmol) and NBS (2.71 g, 15.22 mmol) were dissolved in 50 mL of DCM and refluxed. TLC monitoring showed no raw material, the reaction was cooled, quenched with saturated Na2S2O3, extracted with DCM (3×20 mL), the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain 2.90 g of an oily liquid. Column chromatography purification (0-17% petroleum ether ethyl acetate) gave 2.75 g of a white solid with a yield of 71.7%.

[0143] 1 H-NMR (500MHz, CDCl3) δ7.37 (d, J = 8.1 Hz, 2H), 7.24 (t, J = 8.1 Hz, 1H), 4.92 (s, 2H).

[0144] Example 17 Synthesis of 4-(5-(2,6-dichlorobenzyl)thio-1,3,4-thiadiazol-2-yl)-N-methylbenzamide (target product I-9)

[0145]

[0146] Dissolve Na2CO3 (302.10 mg, 2.85 mmol) in water (3 mL) and add the intermediate 2-bromo-5 (2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole (500 mg, 1.41 mmol) and 4-N-methylcarboxyamine phenylboronic acid pinacol ester (553.57 mg, 2.12 mmol) DME (7 mL) to the mixed solution. Stir at room temperature for 10 min under N2 protection, add pd(dppf)2Cl2 (51.58 mg, 0.07 mmol), and heat at 90 ° C for 3 h. After the reaction is completed, add 20 mL of water to quench the reaction, extract with ethyl acetate (3×15 mL), dry with anhydrous magnesium sulfate, filter, and concentrate to obtain 997.1 mg of brown oil. Purify by column chromatography (PE:EA=0%~6%) to obtain the target product I-9 as a white solid of 75.4 mg with a yield of 12.7%.

[0147] 1 H-NMR (500MHz, DMSO-d6) δ: 8.62 (s, 1H), 8.01 (dd, J = 8.25Hz, 4H), 7.56 (d, J = 8.1Hz, 2H), 7.42 (t, J = 8.1Hz, 1H), 4.86 (s, 2H), 2.82 (d, J = 4.2Hz, 3H).

[0148] 13C-NMR(126MHz,DMSO-d6)δ:168.97,166.09,164.31,137.31,135.65,131.97 ,131.74,131.35(2C),129.38(2C),128.65(2C),128.04(2C),35.07,26.80.

[0149] HRMS: m / z C 17 H 14 Cl2N3OS2[M+H] + Calculate 409.9877,Found 409.9926.

[0150] Example 18 Synthesis of 2-(2,6-dichlorobenzyl)thio-5-(3-trifluoromethyl)phenyl-1,3,4-thiadiazole (target product I-10)

[0151]

[0152] According to the preparation method of target compound I-9 in Example 17, target compound I-10 can be prepared with a yield of 3.5%.

[0153] 1 H-NMR (500MHz, DMSO-d6) δ8.24(d,J=7.3Hz,2H),7.97(d,J=7.7Hz,1H),7.82(t,J=7.7Hz,1H),7.56(d,J=8.1Hz,2H),7.43(t,J=8.0Hz,1H),4.87(s,2H).

[0154] 13 C-NMR(126MHz,DMSO-d6)δ168.14,164.83,135.65,132.31,131.92,131.37 ,131.34(2C),130.76,130.65,130.50,129.38(2C),128.39,124.12,35.06.

[0155] HRMS: m / z C 16 H 10 Cl2F3N3S2[M+H] + Calculate 420.9536, Found 420.9620.

[0156] Example 19 In vitro biological function test of the compounds of the present invention on PIEZO1

[0157] 1. Experimental methods

[0158] The activity of compounds was assessed using intracellular calcium signals, using calcium imaging to measure the influx of Ca into cells. 2+ Fluorescence labeling, after the compound acts on the cells, the fluorescence intensity is quantitatively analyzed using ImageJ software to test the agonist or inhibitory effect of the compound.

[0159] (1) Cell culture and solution preparation for experiments

[0160] Take out the human cardiomyocyte cell line AC16 cells from the -80℃ freezer and thaw them in a 37℃ water bath. When the cells are almost completely thawed, use a 1mL pipette to aspirate the cells in a clean bench, add them to 3mL of DMEM (high glucose) medium containing 10% FBS, and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend with 4mL of DMEM (high glucose) medium containing 10% FBS, and spread them in a 10cm culture dish. After shaking the cells, place them in a cell culture incubator at 37℃, 5% CO2, and saturated humidity for culture.

[0161] Observe the cell growth status every day, and change the cell medium every 1 to 2 days. Take cells with good growth status and a confluence rate of 90% for digestion and subculture. Discard the culture medium, wash the cells twice with phosphate buffered saline (PBS), add 1mL of 0.25% trypsin + 0.02% EDTA solution preheated at 37°C to digest for 1 to 2 minutes, place the cells under a microscope for observation, and when more than half of the cells shrink and move in a sandy state, add 3mL of 10% FBS DMEM (high glucose) culture medium to terminate digestion. Place in a centrifuge and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, add DMEM (high glucose) culture medium (90% DMEM (high glucose) culture medium + 10% FBS + 1% PS) to resuspend, and plate.

[0162] The composition of DMEM (high glucose) medium is as follows: 4.5 g / L D-glucose, 580 mg / L L-glutamine, and 110 mg / L sodium pyruvate (Gicbo, C11995500BT).

[0163] The specific components of PS are: Penicillin: 10000 U / mL, Streptomycin: 10000 μg / mL (VivaCell, C3420-0100). Pancreatic enzymes include trypsin, pancreatic amylase and pancreatic lipase (Senrui, CR25200).

[0164] Yoda1 solution: Dissolve Yoda1 (15 mg) in 10 ml DMSO solution, dispense into EP tubes, store at –80°C, and add to cell culture medium at a ratio of 1:200 to achieve a final concentration of 20 μM.

[0165] Lentivirus: Heyuan Bio pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE lentivirus.

[0166] (2) Lentiviral infection method

[0167] a) AC16 cells were plated.

[0168] b) The next day, observe the cell status. When the cell growth status is good and the confluence reaches 60-70%, discard the culture medium, calculate the amount of virus added to each well according to the optimal MOI = 20 (with Polybrene) determined in step (2), and add DMEM (high glucose) culture medium containing the corresponding volume of lentivirus and Polybrene for infection. The concentration of Polybrene in the culture medium is 5 μg / mL; the amount of virus added to each well = MOI × number of cells / titer at infection (TU / mL) × 10 3 .

[0169] (3) Method for determining whether a small molecule compound is an agonist or inhibitor of the PIEZO1 channel

[0170] ① Determine whether the small molecule compound is an agonist of the PIEZO1 channel

[0171] (i) Control group: simple lentivirus infection.

[0172] (ii) Agonist group: lentiviral infection. After 16 h of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h of infection. The PIEZO1 agonist Yoda1 (20 μM) was added, and fluorescence microscopy was performed after 24 h of stimulation.

[0173] (iii) Small molecule compound group: 16 h after infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h after infection. The small molecule compound to be tested (20 μM) was added, and fluorescence microscopy was performed after 24 h of stimulation.

[0174] The cells were observed under an inverted fluorescence microscope. Three fields of view were randomly selected for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and to generate statistical data.

[0175] Small molecules whose relative fluorescence intensity increased by 20% or more compared with the control group were listed as possible agonists of the PIEZO1 channel.

[0176] ② Determine whether the small molecule compound is an inhibitor of the PIEZO1 channel

[0177] According to the results of step ①, compounds with no significant difference compared with the control group were selected to continue the inhibition test. AC16 cells were divided into 2 groups.

[0178] (i) Control group: lentiviral infection. After 16 h of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h of infection. The PIEZO1 agonist Yoda1 (20 μM) was added, and fluorescence microscopy was performed after 24 h of stimulation.

[0179] (ii) Small molecule compound group: lentivirus infection. After 16 h of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture until 48 h of infection. PIEZO1 agonist Yoda1 (20 μM) and small molecule compounds (20 μM) were added at the same time. Fluorescence microscopy was performed after 24 h of stimulation.

[0180] The cells were observed under an inverted fluorescence microscope. Three fields of view were randomly selected for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and statistically analyze the data. Small molecules whose relative fluorescence intensity decreased by 20% or more compared with the control group were listed as possible inhibitors of the PIEZO1 channel.

[0181] 2. Experimental results

[0182] The biological functions of compounds I-1 to I-10 were tested in three batches, and the agonist and inhibitory effects of the compounds at a concentration of 20 μM on PIEZO1 were tested. Due to biological instability, positive and negative controls were added to each batch of tests, and the results of the three groups are as follows.

[0183] (1) Effect diagram of the first group of compounds

[0184] The compounds were tested for agonism, and the results were Figure 1 shown.

[0185] Table 2 Relative fluorescence intensity of the first batch of compounds

[0186]

[0187] In the agonist effect test of the first group of compounds, the fluorescence intensity of I-2 increased by 61.68%, which was better than Yoda1 (33.95%). The fluorescence intensity of I-1 increased by 31.18%, which was comparable to Yoda1.

[0188] (2) Effect diagram of the second group of compounds

[0189] The compounds were tested for agonism, and the results were Figure 2 The compounds with no significant difference compared with the control group were selected for inhibitory effect detection, and the results are shown in Figure 3 shown.

[0190] Table 3 Relative fluorescence intensity of the second group of compounds

[0191]

[0192] In the test of the agonistic effect of the second group of compounds, no compound had a better agonistic effect. All compounds in the second group were tested for inhibitory effect, and the compounds that may have inhibitory effect were: I-9 fluorescence intensity decreased by 22.13%.

[0193] (3) Effect diagram of the third group of compounds

[0194] The compounds were tested for agonism, and the results were Figure 4 The compounds with no significant difference compared with the control group were selected for inhibitory effect detection, and the results are shown in Figure 5 shown.

[0195] Table 4 Relative fluorescence intensity of the third group of compounds

[0196]

[0197] In the agonist effect test of the third group of compounds, the fluorescence intensity of compounds I-4 (75.72%), I-5 (39.91%), and I-7 (70.54%) was significantly increased, but they were weaker than Yoda1 (139.79%). Compounds I-6 and I-8, whose agonist fluorescence intensity was not significantly different from that of the negative control, were selected to test the inhibitory effect. The relative fluorescence intensity did not change much, that is, there was no agonist or inhibitory effect.

[0198] In summary, the agonist effect of compounds I-1, I-2, I-4, I-5 and I-7 was better, and the agonist effect of I-2 was better than Yoda1, with the fluorescence intensity increased or decreased by 20% compared with the control group as the standard. Compound I-9 may have an inhibitory effect. The initial screening results obtained the preferred compound I-2, and further exploration was carried out based on this structure.

[0199] Example 20 Stability test of the compounds of the present invention in mouse liver microsomes

[0200] The preferred compound I-2 was subjected to a liver microsome stability test, and the test was as follows:

[0201] (1) Test compounds: Yoda1, I-2.

[0202] (2) Experimental methods:

[0203] Preparation of solutions: Stock solutions of test compounds (10 mM) were prepared in DMSO and then diluted to 200 μM with acetonitrile.

[0204] Microsome incubation: Prepare an incubation mixture in a total volume of 200 μL with final component concentrations of 0.1 M PBS (pH 7.4), 3 mM MgCl2, NADPH (2 mM). Liver microsomes (0.2 mg / mL) and test compounds (1 μM). Pre-incubate all other components at 37°C for 5 min before adding NADPH or buffer (negative control). Mix by pipetting to achieve a uniform suspension and immediately transfer 20 μL of the above solution as the 0 min sample, incubate to the wells in the "quench" plate, and then pipette to mix the remaining solution. At 5, 15, 30 and 60 min, pipette to mix the incubation and continuously transfer 20 μL of solution for each time point, incubate the sample to the wells in a separate "quench" plate, and then pipette to mix the remaining solution again. Add 200 μL of acetonitrile with IS to the "quench" plate.

[0205] Sample analysis: Centrifuge the 96-well plate at 4000 rpm, 4°C for 10 min. Take 50 μL of the supernatant and mix with 50 μL of ddH2O, then inject into the LC-MS / MS system for analysis.

[0206] (3) Experimental results: The results are shown in Table 6

[0207] Table 5 Liver microsome stability results

[0208]

[0209] The results of mouse liver microsome stability showed that the half-life of compound Yoda1 in mouse liver microsomes was 12.0 min, and the residual amount at 60 min was 2.7%, indicating poor stability. However, the half-life of I-2 was 51.1 min, and the residual amount at 60 min was 41.4%, indicating improved stability. It can be used as a reference to gradually improve the stability of the compound in subsequent development.

[0210] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitution combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of the present invention.

Claims

1. A substituted benzyl sulfide diazole compound, characterized in that: Having the structure of general formula I: R 1 Selected from the following groups: H, CH3, NH2; R 2 Selected from H, Cl; Y is selected from S, O, NH; A is selected from an aromatic ring or an aromatic heterocyclic group.

2. The substituted benzyl sulfide diazole compound according to claim 1, characterized in that: R 1 Selected from the following groups: H, CH3, NH2; R 2 is selected from H, Cl; Y is selected from S, O, NH; A is selected from the following groups:

3. The substituted benzyl sulfide diazole compound according to claim 1, characterized in that: The R 1 The group is NH2; R 2 The group is H; the Y group is S; the A group is 4. The substituted benzyl sulfide oxadiazole compound according to claim 1, characterized in that: The R 1 The group is H; R 2 The group is H; the Y group is S; A is selected from the following groups 5. A method for preparing a substituted benzyl sulfide diazole compound as claimed in claim 2 or 3, characterized in that: Its synthetic route is as follows: The specific steps are: 1) Compound 1 is used as a raw material, esterified with methanol, then hydrazine-hydrated, and then cyclized with carbon disulfide and concentrated sulfuric acid to obtain intermediate 4; 2) Compound 5 is used as a raw material, acylated under the catalysis of an organic base, and then the aldehyde group is reduced and chlorinated with thionyl chloride to obtain intermediate 8; 3) Intermediate 4 and intermediate 8 are reacted by condensation to obtain target product I.

6. A method for preparing a substituted benzyl sulfide diazole compound as claimed in claim 4, characterized in that: Its synthetic route is as follows: The specific steps are: 1) Intermediate 8 is used as a raw material, a base catalyst is added, and the intermediate 9 is obtained by nucleophilic substitution with 1,3,4-thiadiazole-2-thiol; 2) Intermediate 9 is brominated with NBS to obtain intermediate 10; 3) Intermediate 10 was subjected to Suzuki coupling to obtain target compound I.

7. Use of the substituted benzyl thioether diazole compound according to any one of claims 1 to 4 in regulating PIEZO1 protein.

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