Substituted benzyl sulfide oxadiazole compounds, methods of making and uses thereof

Abstract

The present application relates to a kind of substituted benzyl sulfide oxadiazole compound, it has the regulation effect to PIEZO1 ion channel, can specifically agonize or inhibit PIEZO1 ion channel function, potential cardiotoxicity is lower, side effect is smaller, further perfects the structure-effect relationship of this kind of general formula applied to PIEZO1 agonist or antagonist and provides part pharmacological activity.

Description

TECHNICAL FIELD

[0001] The present application relates to a substituted benzyl sulfide oxadiazole compound, a preparation method thereof, and the use of the compound in related biology by agonizing or antagonizing the PIEZO1 protein. BACKGROUND

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

[0003] At present, there are few reports on modulators targeting PIEZO1 channels, and the effects are uneven, such as PIEZO1 specific small molecule agonists (Yoda1, Jedi1 and Jedi2), but the antagonists are non-selective, and there is no report on PIEZO1 specific antagonists.

[0004] The following is a summary of the current status of PIEZO1 small molecule agonists:

[0005] Jedi1 and Jedi2 are two small molecule PIEZO1 agonists obtained by Wang Yanfeng et al. through high-throughput screening, and the EC 50 of mPIEZO1 is 200 μM and 158 μM, respectively, and the K d of mPIEZO1 is 2754 μM and 2770 μM, respectively. Studies have found that they activate PIEZO1 through the helical blade structure of PIEZO1, and can act on PIEZO1 simultaneously with Yoda1, showing 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, with EC 50 of 17.1 μM and 26.6 μM, respectively, and the K dThe value is 45.6 μM, and it has good binding affinity. The discovery of Yoda1 provides great help for 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 region where the benzene ring and the pyrazine ring are located, and the effect of a small molecule PIEZO1 agonist can be better and the toxicity can be smaller through structure optimization.

[0008]

[0009] Evans et al. designed a series of derivatives based on Yoda1, and when other substituents (such as phenyl, 4-nitrophenyl, 4-methoxyphenyl, and phenyl containing halogen atoms at different positions) were replaced with 2,6-dichlorobenzyl, 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 of Yoda1-activated PIEZO1.

[0010] Li et al. also reported a series of Yoda1 analogs, and when the thioether bond was replaced with an ether bond or an amino group, the activity was significantly reduced, while the ether bond and 2,6-dichlorobenzyl were retained by adding substituents (such as 2,6-dichloro-3-fluorophenyl, 2,6-dichloro-3-nitrophenyl, and 2,6-dichloropyridin-3-yl, etc.), the activity was comparable to or better than Yoda1. Therefore, 2,6-dichlorobenzyl and thioether bond may be the key to Yoda1 activation, and the absence of these two parts will lead to the disappearance of Yoda1 activity. With this research idea, the benzene ring and pyrazine ring parts are explored while retaining these two parts.

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

[0012]

[0013] In summary, there are few reports on PIEZO1 modulators at present, and the agonists found 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 significance to research new PIEZO1 small molecule modulators with better effects. SUMMARY

[0014] The purpose of this invention is to provide a substituted benzyl thioether diazole compound and its application in the biological function of PIEZO1, so as to discover novel PIEZO1 regulators 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 thion diazole compound having the structure of general formula I:

[0017]

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

[0019] R 2 Selected from H and Cl;

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

[0021] A is selected from the following groups:

[0022] More preferably, the following specific compounds are preferred among the compounds with 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-l,3,4-thiadiazol-2-yl)-N-methylbenzamide;

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

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

[0033] Table 1 Preferred compound number and corresponding structural formula

[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 as follows:

[0038] 1) Compound 1 is used as raw material, esterification is carried out with methanol, then hydrazinolysis is carried out with hydrazine hydrate, and then the intermediate 4 is obtained by cyclization with carbon disulfide and concentrated sulfuric acid;

[0039] 2) Compound 5 is used as raw material, acylation is carried out under the catalysis of an organic base, then the aldehyde group is reduced, and the intermediate 8 is obtained by chlorination with thionyl chloride.

[0040] 3) The target product I is obtained by condensation reaction of intermediate 4 and intermediate 8.

[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 as follows:

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

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

[0046] 3) The target compound I is obtained by suzuki coupling of intermediate 10.

[0047] A substituted benzyl sulfide diazole compound as described above is used in the regulation of PIEZO1 protein.

[0048] A PIEZO1 protein modulator comprising the above substituted benzyl sulfide diazole compound.

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

[0050] The compound of the present application can be used for PIEZO1 biological regulation (including human) in the form of combination through oral, injection and other routes, among which oral route is the best, and the dosage is 0.0001 mg / kg-200 mg / kg body weight per day. The optimal dosage varies with individuals, and the dosage is usually small at the beginning and then gradually increased.

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

[0052] The carrier refers to the common carrier in the pharmaceutical field, such as diluent, excipient, water, etc.; binding agent such as cellulose derivative, gelatin, polyvinylpyrrolidone, etc.; filling agent such as starch, etc.; disintegrating agent such as calcium carbonate, sodium bicarbonate; in addition, other auxiliary agents such as flavoring agent and sweetening agent can also be added in the composition.

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

[0054] The above-mentioned pharmaceutical composition can be prepared into conventional solid preparations such as tablets, capsules, etc. for oral administration, and can also be prepared into injection for injection.

[0055] The present application has the following beneficial effects:

[0056] 1) The compound of the present application has PIEZO1 agonist or inhibitor effect, and has good regulation effect on various cells expressing PIEZO1 in human body, and has the characteristics of broad spectrum and high efficiency. It can also have good curative effect on diseases caused by abnormal gene expression, such as cardiovascular system diseases, immune system diseases, bone diseases and cancers.

[0057] 2) Compared with the reported PIEZO1 modulators, the compound of the present application can effectively agonize or inhibit the expression of PIEZO1.

[0058] 3) Compared with the reported PIEZO1 modulators, the compound of the present application has good safety and physicochemical properties, and has the value of further research.

[0059] In summary, the present application provides a new type of modulator acting on PIEZO1. In the structure of such compounds, compounds with agonistic effects are found to be superior to positive control compounds and compounds with inhibitory effects. In addition, the new structural type of compounds of the present application not only has strong agonistic activity, but also has better liver microsomal stability than the reported positive control compounds, and the new structural compounds have higher safety and less toxic side effects, and are more suitable for use as candidate compounds. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 Agonistic effect graph for the first group of compounds in Example 19.

[0061] Figure 2 Agonistic effect graph for the second group of compounds in Example 19.

[0062] Figure 3 Inhibitory effect graph for the second group of compounds in Example 19.

[0063] Figure 4 Agonistic effect graph for the third group of compounds in Example 19.

[0064] Figure 5 Inhibitory effect graph for the third group of compounds in Example 19. DETAILED DESCRIPTION

[0065] The present application will be further described in conjunction with examples, but the embodiments of the application are not limited thereto.

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

[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, a tail gas absorption device was connected, and thionyl chloride (9.6 mL, 119.46 mmol) was slowly added at 0°C using a constant pressure dropping funnel. After the addition was completed, the reaction was stirred at room temperature. It was observed that the reaction liquid changed from turbidity to colorless transparency. After 2 h of reaction, the reaction liquid was slowly added to ice water, and a saturated Na2CO3 solution was used to adjust to neutral. Dichloromethane (4 x 20 mL) was used for extraction, and the organic phase was combined. Saturated brine (3 x 20 mL) was used for washing, and anhydrous magnesium sulfate was used for drying, filtration, and concentration to obtain an orange solid, which was intermediate 5-methylpyrazine-2-carboxylic acid methyl ester (4.3 g, 71.5%). Without further purification, it can be directly used in the next step reaction.

[0069] Synthesis of intermediate 5-methylpyrazine-2-carboxylic acid methyl ester in Example 1

[0070]

[0071] Methyl 5-methylpyrazine-2-carboxylate (4.00 g, 26.31 mmol) was dissolved in 100 mL of anhydrous ethanol in a 250 mL single neck flask and 80% hydrazine hydrate (4.7 mL, 121.73 mmol) was added. The reaction was refluxed for 2 h. The reaction was concentrated and washed with ethyl acetate (15 mL), filtered and the filter cake was dried to give yellow solid, intermediate 5-methylpyrazine-2-carbohydrazide (2.92 g, 73.9%). It was used in the next step without further purification.

[0072] Synthesis of Example 3 intermediate 5-(5-methylpyrazin-2-yl)-1,3,4-thiadiazol-2- thiol

[0073]

[0074] Methyl 5-methylpyrazine-2-carboxylate (4.00 g, 26.31 mmol) was dissolved in 100 mL of anhydrous ethanol in a 250 mL single neck flask and 80% hydrazine hydrate (4.7 mL, 121.73 mmol) was added. The reaction was refluxed for 2 h. The reaction was concentrated and washed with ethyl acetate (15 mL), filtered and the filter cake was dried to give yellow solid, intermediate 5-methylpyrazine-2-carbohydrazide (2.92 g, 73.9%). It was used in the next step without further purification.

[0075] 1 H-NMR (500 MHz, DMSO-d6) δ 14.94 (s, 1H), 9.10 (s, 1H), 8.64 (s, 1H), 2.58 (s, 3H).

[0076] Synthesis of Example 4 intermediate 2,6-dichlorobenzaldehyde

[0077]

[0078] The starting material, 1,3-dichlorobenzene (2.00 g, 13.61 mmol), was dissolved in 40 mL of anhydrous tetrahydrofuran. Butyllithium (2.80 g, 43.55 mmol) was added at -78 °C, and the mixture was stirred for 30 min. Anhydrous N,N-dimethylformamide (3.48 g, 47.64 mmol) was then added, and the reaction was continued for another 30 min. The reaction was quenched with 30 mL of saturated NaHCO3 solution, resulting in a white precipitate. The precipitate was filtered, and the filtrate was extracted with ethyl acetate (5 × 20 mL). The combined organic phases were washed with saturated brine (3 × 20 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the crude product. The crude product 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 while stirring at room temperature. Saturated NH4Cl solution was added dropwise to the reaction mixture until neutral, and the mixture was extracted with ethyl acetate (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. This solid was then slurried with petroleum ether to give the intermediate 2,6-dichlorobenzaldehyde (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] Intermediate 2,6-dichlorobenzyl alcohol (200 mg, 1.13 mmol) was dissolved in 10 mL of dichloromethane, SOCl2(240.1 mg, 1.19 mmol) was added dropwise slowly at 0 °C, N,N-dimethylformamide (16.5 mg, 0.23 mmol) was added, stirred at room temperature for 3 h. After completion of the reaction, the reaction was quenched with 20 mL of water, extracted with dichloromethane (3 x 15 mL), combined organic layers were washed with saturated NaHC03solution (2 x 15 mL), saturated brine (2 x 15 mL), dried over anhydrous magnesium sulfate, filtered, concentrated to get the product as a crude, purified by column chromatography (petroleum ether-ethyl acetate, 0-10% gradient elution) to get the product as a white solid, intermediate 2,6-dichlorobenzyl chloride (155.7 mg, 70.5%).

[0087] 1 H-NMR (600 MHz, CDC13) 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-methylpyrazin-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 K2C03(328.94 mg, 2.38 mmol) were dissolved in 15 mL of tetrahydrofuran, the reaction was refluxed for 2 h, the reaction was concentrated to get a light yellow solid, distilled water (15 mL) was added, the crude product was filtered to get a white solid, which was 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 (500 MHz, DMSO-d6) δ 9.30 (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 (126 MHz, DMSO-d6) δ 166.22, 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] The target compound I-1 was prepared according to the preparation method of the target compound I-3 of Examples 1-7, and the yield was 64.9%.

[0097] 1 H-NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.84 (s, 1H), 8.81 (s, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 4.94 (s, 2H), 2.36 (s, 3H).

[0098] 13 C-NMR (126 MHz, DMSO-d6) δ 168.90, 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] The target compound I-2 was prepared according to the preparation method of the target compound I-3 of Examples 1-7, and the yield was 85.1%.

[0103] 1H-NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.84 (s, 1H), 8.80 (s, 1H), 7.18 (d, J = 10.2 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 5.69 (s, 2H), 4.83 (s, 2H).

[0104] 13 C-NMR (126 MHz, DMSO-d6) δ 168.73, 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 compound I-4)

[0107]

[0108] The target compound I-4 was prepared according to the procedure of Example 1-7 for the preparation of the target compound I-3, in 84.1% yield.

[0109] 1 H-NMR (500 MHz, DMSO-d6) δ 7.58 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 7.40 (t, J = 8.1 Hz, 1H), 6.65 (d, J = 8.5 Hz, 2H), 5.92 (s, 2H), 4.75 (s, 2H).

[0110] 13 C-NMR (126 MHz, DMSO-d6) δ 171.05, 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 compound I-5)

[0113]

[0114] The target compound I-5 was prepared according to the preparation method of the target compound I-3 of Examples 1 to 7, and the yield was 33.2%.

[0115] 1 H-NMR (500 MHz, DMSO-d6) δ 9.30 (s, 1H), 8.69 (s, 1H), 7.45 (dd, J = 6.2 Hz, 2H), 4.92 (s, 2H), 2.61 (s, 3H), 2.36 (s, 3H).

[0116] 13 C-NMR (126 MHz, DMSO-d6) δ 169.12, 166.45, 156.68, 156.14, 145.02, 141.42, 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 compound I-6)

[0119]

[0120] The target compound I-6 was prepared according to the preparation method of the target compound I-3 of Examples 1 to 7, and the yield was 93.1%.

[0121] 1H-NMR (500 MHz, DMSO-d6) δ 7.97 (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 (126 MHz, DMSO-d6) δ 166.50, 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] Synthesis of 4-(5-((2,6-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)aniline (Target compound I-7)

[0125]

[0126] The target compound I-7 was prepared according to the procedure of Example 1-7 for the preparation of the target compound I-3, in a yield of 57.2%.

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

[0128] 13 C-NMR (126 MHz, 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 compound I-8)

[0131]

[0132] The target compound I-8 was prepared according to the procedure of Example 1-7 for the preparation of the target compound I-3, in a yield of 67.3%.

[0133] 1 H-NMR (500 MHz, DMSO-d6) δ 14.59 (s, 1H), 7.98 (d, J = 7.1 Hz, 2H), 7.51 (d, J = 8.1 Hz, 5H), 7.37 (t, J = 8.1 Hz, 1H), 4.65 (s, 2H).

[0134] 13 C-NMR (126 MHz, DMSO-d6) δ 135.44, 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-dichlorobenzylsulfide)-1,3,4-thiadiazole

[0137]

[0138] The intermediate 2-(2,6-dichlorobenzylsulfide)-1,3,4-thiadiazole was prepared according to the procedure of Example 7 as a white solid in a yield of 86.7%.

[0139] 1 H-NMR (500 MHz, 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-dichlorobenzylsulfide)-1,3,4- thiadiazole

[0141]

[0142] Dissolve 2-(2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole (3.0 g, 10.87 mmol) and NBS (2.71 g, 15.22 mmol) in 50 mL of DCM and reflux. Monitor by TLC until no starting material is present, cool the reaction, quench with saturated Na2S203, extract with DCM (3 x 20 mL), combine the organic phases, dry over anhydrous MgS04, filter, and concentrate to give an oil 2.90 g. Purify by column chromatography (0-17% petroleum ether ethyl acetate) to give 2.75 g of white solid, 71.7% yield.

[0143] 1 H-NMR (500 MHz, CDC13) δ 7.37 (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 Na2C03(302.10 mg, 2.85 mmol) in water (3 mL) and add to a mixture of intermediate 2-bromo-5-(2,6-dichlorobenzyl sulfide)-1,3,4-thiadiazole (500 mg, 1.41 mmol) and 4-N-methylcarboxyamidobenzoic acid pinacol ester (553.57 mg, 2.12 mmol) in DME (7 mL). Stir at room temperature under N2for 10 min, add pd(dppf)2Cl2(51.58 mg, 0.07 mmol), and heat at 90 °C for 3 h. After the reaction is complete, quench the reaction with 20 mL of water, extract with ethyl acetate (3 x 15 mL), dry over anhydrous MgS04, filter, and concentrate to give a brown oil 997.1 mg. Purify by column chromatography (PE:EA = 0% to 6%) to give the target product I-9 as a white solid 75.4 mg, 12.7% yield.

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

[0148] 13C-NMR (126 MHz, 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 compound I-10)

[0151]

[0152] Target compound I-10 was prepared according to the procedure described in Example 17 for the preparation of target compound I-9, in a yield of 3.5%.

[0153] 1 H-NMR (500 MHz, DMSO-d6) δ 8.24 (d, J = 7.3 Hz, 2H), 7.97 (d, J = 7.7 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.43 (t, J = 8.0 Hz, 1H), 4.87 (s, 2H).

[0154] 13 C-NMR (126 MHz, 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.

[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 compound of the present application on PIEZO1

[0157] 1. Experimental method

[0158] Compound activity was evaluated using intracellular calcium signaling, using calcium ion imaging to measure the influx of Ca2+into cells 2+ Fluorescent labeling, after the compound acts on the cells, the fluorescence intensity was quantitatively analyzed by ImageJ software. The agonistic or inhibitory effect of the test compound was tested.

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

[0160] The human myocardial cell line AC16 cells were taken out from the -80°C refrigerator, thawed in a 37°C water bath, and when the cells were almost completely thawed, 1 mL of pipette was used to suck the cells into 3 mL of DMEM (high sugar) medium containing 10% FBS, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and 4 mL of DMEM (high sugar) medium containing 10% FBS was used to resuspend, and plated into a 10 cm culture dish. After shaking the cells, they were placed in a 37°C, 5% CO2, saturated humidity cell incubator for culture.

[0161] The growth state of the cells was observed every day, and the cells were changed every 1-2 days. Cells with good growth and a confluence rate of 90% were taken for digestion and passage. The medium was discarded, and the cells were washed twice with phosphate buffered saline (PBS), 1 mL of 37°C preheated 0.25% trypsin + 0.02% EDTA solution was added and digested for 1-2 min, the cells were placed under a microscope for observation, when more than half of the cells were shrunk and moved like sand, 3 mL of 10% FBS DMEM (high sugar) medium was added to terminate the digestion. Centrifuged at 1000 rpm for 5 min. Discard the supernatant, add DMEM (high sugar) medium (90% DMEM (high sugar) medium + 10% FBS + 1% PS) to resuspend and plate.

[0162] The composition of DMEM (high sugar) medium is: containing 4.5 g / L D-glucose, containing 580 mg / L L-glutamine, containing 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). Trypsin contains trypsin, trypsin and pancreatic lipase (Sunwei, CR25200).

[0164] Yoda1 solution: Yoda1 (15 mg) was dissolved in 10 ml of DMSO solution, aliquoted into EP tubes, stored at -80°C, and added to the cell culture medium at a ratio of 1:200 to make the final concentration 20 μM.

[0165] Lentivirus: and the metazoan pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE lentivirus.

[0166] (2) Lentivirus infection method

[0167] a) Plate the AC16 cells.

[0168] b) The next day, observe the cell state, and when the cell growth state is good and the confluence reaches 60-70%, discard the culture medium, calculate the virus addition amount per well according to the optimal MOI = 20 (add Polybrene) determined in step (2), and add DMEM (high sugar) medium containing the corresponding volume of lentivirus and Polybrene for infection. Among them, the concentration of Polybrene in the culture medium is 5 μg / mL; the virus amount added per well = MOI x cell number at the time of infection / dose (TU / mL) x 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 a small molecule compound is an agonist of the PIEZO1 channel

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

[0172] (ii) Agonist group: lentivirus infection, 16h after infection, discard the culture medium, wash with PBS for 2 times, add new DMEM (high sugar) medium for culture, until 48h after infection, add PIEZO1 agonist Yoda1 (20 μM), stimulate for 24h, and then perform fluorescence microscope observation.

[0173] (iii) Small molecule compound group: 16h after infection, discard the culture medium, wash with PBS for 2 times, add new DMEM (high sugar) medium for culture, until 48h after infection, add the small molecule compound to be tested (20 μM), stimulate for 24h, and then perform fluorescence microscope observation.

[0174] Place the cells under an inverted fluorescence microscope for observation. Randomly select 3 fields of view for photography. Apply ImageJ software for quantitative analysis of image fluorescence intensity, and count the data.

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

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

[0177] According to the results of step ①, the compounds with no obvious difference compared with the control group were selected for further inhibition detection. The AC16 cells were divided into two groups.

[0178] (i) Control group: lentivirus infection, 16h after infection, discard the culture medium, wash with PBS for 2 times, add new DMEM (high sugar) culture medium for culture, until 48h after infection, add PIEZO1 agonist Yoda1 (20μM), stimulate for 24h, then observe under fluorescence microscope.

[0179] (ii) Small molecule compound group: lentivirus infection, 16h after infection, discard the culture medium, wash with PBS for 2 times, add new DMEM (high sugar) culture medium for culture, until 48h after infection, add PIEZO1 agonist Yoda1 (20μM) and small molecule compound (20μM) at the same time, stimulate for 24h, then observe under fluorescence microscope.

[0180] The cells were observed under an inverted fluorescence microscope. Randomly select 3 fields for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and the data were statistically analyzed. The relative fluorescence intensity of the small molecules with a decrease of 20% or more compared with the control group was considered as possible inhibitors of PIEZO1 channel.

[0181] 2、Experimental results

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

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

[0184] The agonistic effect of the compounds was detected, and the results are shown in Figure 1 .

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

[0186]

[0187] In the agonistic 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 agonistic effect of the compounds was detected, and the results are shown in Figure 2 . The compounds with no obvious difference compared with the control group were selected for inhibition detection, and the results are as follows.Figure 3 Results are shown in Table 3.

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

[0191]

[0192] In the agonistic effect test of the second group of compounds, the compound with no obvious agonistic effect was I-9. The inhibitory effect of all compounds in the second group was tested, and the compound with possible inhibitory effect was I-9, whose fluorescence intensity decreased by 22.13%.

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

[0194] The agonistic effect of the compounds was tested, and the results are shown in Table 4. Figure 4 The inhibitory effect of the compounds with no obvious difference compared with the control group was tested, and the results are shown in Table 4. Figure 5

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

[0196]

[0197] In the agonistic effect test of the third group of compounds, the compounds with relatively obvious increase in fluorescence intensity were I-4 (75.72%), I-5 (39.91%) and I-7 (70.54%), but all were weaker than Yoda1 (139.79%). In addition, the compounds I-6 and I-8 with no obvious difference in agonistic effect fluorescence intensity compared with the negative control were selected to test the inhibitory effect, and the relative fluorescence intensity of both compounds changed little, i.e., no agonistic or inhibitory effect.

[0198] Based on the above results, the agonistic effect of the compounds I-1, I-2, I-4, I-5 and I-7 was better, and the agonistic effect of I-2 was better than that of Yoda1, according to the standard that the fluorescence intensity increased or decreased by 20% compared with the control group. The compound I-9 might have inhibitory effect. The preferred compound I-2 was obtained by the preliminary screening result, and in-depth exploration was carried out based on the structure.

[0199] Example 20 Mouse liver microsomal stability test of the compound of the application

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

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

[0202] (2) Experimental method:

[0203] Solution preparation: Prepare the stock solution (10 mM) of the test compound in DMSO, and then dilute the stock solution of the test compound to 200 μM with acetonitrile. ​

[0204] Microsomal incubation: Incubation mixture was prepared 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 compound (1 μM). All other components were pre-incubated at 37 °C for 5 min before addition of NADPH or buffer (negative control). Mixed by pipette to achieve uniform suspension and immediately 20 μL of the above solution was taken as 0 min sample, incubation was transferred to wells in a "quench" plate and the remaining solution was pipette mixed. At 5, 15, 30 and 60 min, the incubation was pipette mixed and 20 μL of solution was serially transferred for each time point, incubation samples were transferred to wells in a separate "quench" plate and the remaining solution was pipette mixed again. 200 μL acetonitrile with IS was added in the "quench" plate.

[0205] Sample analysis: The 96-well plate was centrifuged at 4000 rpm, 4 °C for 10 min. 50 μL of supernatant was mixed with 50 μL of ddH2O and then injected into the LC-MS / MS system for analysis.

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

[0207] Table 5 Liver microsomal stability results

[0208]

[0209] The mouse liver microsomal stability results show that the half-life of compound Yoda1 in mouse liver microsomes is 12.0 min, and the remaining amount at 60 min is 2.7%, which has poor stability. The half-life of I-2 is 51.1 min, and the remaining amount at 60 min is 41.4%, which has improved stability, which can be used as a reference to gradually improve the stability of the compound in subsequent development.

[0210] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, alternative combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the scope of the present application.

Claims

1. A substituted benzyl sulfide oxadiazole compound, characterized by, A compound having a structure of formula (I): Specifically, the compound I-2 has a structure of formula (I): I-2： I-9： 2. A method for preparing the substituted benzyl thioether diazole compound as described in claim 1, characterized in that, The synthesis route of the compound I-2 is as follows: The formula (I) is the compound I-2. The specific steps are as follows: 1) taking the compound 1 as a raw material, esterification is performed with methanol, then hydrazinolysis is performed with hydrazine hydrate, and then the intermediate 4 is obtained through cyclization of carbon bisulfide and concentrated sulfuric acid; 2) then taking the compound 5 as a raw material, acylation is performed under catalysis of an organic base, then the aldehyde group is reduced, and the intermediate 8 is obtained through chlorination of thionyl chloride; 3) the intermediate 4 and the intermediate 8 are obtained through a condensation reaction to obtain the target product I-2.

3. A method for preparing the substituted benzyl thioether diazole compound as described in claim 1, characterized in that, The synthesis route of the compound I-9 is as follows: The formula (I) is the compound I-9. The specific steps are as follows: 1) taking the intermediate 8 as a raw material, a base catalyst is added, and nucleophilic substitution is performed with 1,3,4-thiadiazole-2-thiol to obtain the intermediate 9; 2) the intermediate 9 is brominated with NBS to obtain the intermediate 10; 3) the intermediate 10 is obtained through suzuki coupling to obtain the target compound I-9.

4. Use of the substituted benzyl sulfide oxadiazole compound in claim 1 in preparation of a PI EZO1 protein modulator.

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