A serine hydrolase inhibitor and its preparation method and application

Abstract

The present invention discloses a serine hydrolase inhibitor and a preparation method and application thereof, belonging to the field of biomedical technology. The technical solution of the present invention provides a serine hydrolase inhibitor, wherein the inhibitor has one or more of the structural formulas proposed in the present invention. In addition, the present invention also proposes a preparation method of the above-mentioned serine hydrolase inhibitor, comprising the following steps: mixing warhead (1), a benzaldehyde sulfonyl compound (2) and sodium sulfate to obtain an imine intermediate; adding the imine intermediate to palladium carbon under the action of a reducing agent to obtain the serine hydrolase inhibitor. In addition, the present invention also proposes an application of the above-mentioned serine hydrolase inhibitor in the preparation of a drug for inhibiting the expression of PNPLA6 protein. The inhibitor proposed in the present invention can specifically inhibit PNPLA6 protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a serine hydrolase inhibitor and a preparation method and application thereof. Background Art

[0002] In the field of biomedicine, serine hydrolase PNPLA6 (Patatin-like phospholipase domain-containing protein 6) plays an important role in intracellular lipid metabolism and signal transduction as a key enzyme. It has important functions in regulating intracellular lipid metabolism balance, cell membrane structure stability, and cell signal transduction pathways.

[0003] However, the current research on inhibitors of serine hydrolase PNPLA6 still faces some challenges, and its potential for application in the treatment of specific diseases needs to be further explored. Therefore, the research on seeking new serine hydrolase PNPLA6 inhibitors has important scientific significance and clinical application value. Summary of the invention

[0004] The purpose of the present invention is to overcome the above technical deficiencies, provide a serine hydrolase inhibitor and a preparation method and application thereof, and solve the technical problem of how to inhibit the serine hydrolase PNPLA6 in the prior art.

[0005] In order to achieve the above technical objectives, the technical solution of the present invention provides a serine hydrolase inhibitor, the structural formula of the inhibitor is as follows:

[0006] or or

[0007] In the formula, R 1 represents any one of H, OH, Cl, Br, F, trifluoromethyl, trifluoromethoxy, C1-C6 alkyl, and C1-C6 alkoxy;

[0008] R2 represents any one of C1-C6 alkyl, C1-C6 alkoxy, C3-C10 cycloalkyl, heteroaryl, aryl, and aryl substituted with one or more halogen substituents; n represents any integer from 0 to 10; m represents any integer from 0 to 10; and P represents any integer from 1 to 10.

[0009] In any embodiment, m represents any integer from 0 to 3; and P represents any integer from 1 to 3.

[0010] In any embodiment, n represents any integer from 0 to 3.

[0011] In any embodiment, said R2 represents an aryl group, , , C1-C2 alkyl or C3-C6 cycloalkyl. The inhibitory factor of these inhibitors on PNPLA6 protein can be as low as 0.5 or less.

[0012] In any embodiment, the inhibitor is one or more of the compounds having the following structural formula:

[0013] , , , , , , .

[0014] In any embodiment, the inhibitor is one or more of the compounds having the following structural formula:

[0015] , , , , , , , , , , , , .

[0016] In addition, the present invention also provides a method for preparing the above-mentioned serine hydrolase inhibitor, comprising the following steps:

[0017] Warhead(1) , a benzaldehyde sulfonyl compound (2) and sodium sulfate are mixed to obtain an imine intermediate;

[0018] The imine intermediate is added to palladium carbon under the action of a reducing agent to obtain the serine hydrolase inhibitor.

[0019] In any embodiment, the reducing agent is hydrogen, and the imine intermediate dissolved in a solvent is added to the palladium carbon under a hydrogen atmosphere to obtain the serine hydrolase inhibitor.

[0020] In addition, the present invention also provides a use of the serine hydrolase inhibitor or the serine hydrolase inhibitor prepared by the above preparation method in the preparation of a drug for inhibiting the expression of PNPLA6 protein.

[0021] In addition, the present invention also provides a drug comprising the above serine hydrolase inhibitor, wherein the drug is in the form of injection, powder injection, oral preparation, spray, capsule, suppository or any pharmaceutically acceptable excipient.

[0022] Compared with the prior art, the beneficial effects of the present invention include: the inhibitor proposed in the present invention can specifically inhibit the expression of PNPLA6 protein, and can be used to prepare drugs for inhibiting the expression of PNPLA6 protein, thereby achieving the treatment of PNPLA6 protein-related diseases.

[0023] In some embodiments, the present invention provides an effective chemical tool for studying the function of PNPLA6 protein and the mechanism of related neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The inhibitory effect of compound 3a at different concentration gradients in Example 1 of the present invention on the activity of PNPLA6 protein in the overexpression lysate.

[0025] Figure 2 In the in vitro experiment of Example 1 of the present invention, the IC of compound 3a against PNPLA6 protein is 50 Value analysis curve.

[0026] Figure 3 The inhibitory effect of compound 3a at different concentration gradients in Example 1 of the present invention on the activity of PNPLA6 protein in 293T living cells overexpressing PNPLA6.

[0027] Figure 4 In the in vivo experiment of Example 1 of the present invention, the IC of compound 3a against PNPLA6 protein is 50 Value analysis curve.

[0028] Figure 5 This is a schematic diagram of the process of PNPLA6 protein purification in Example 1 of the present invention.

[0029] Figure 6 This is the protein object covalently bound by 15.4 μM compound 3a in Example 1 of the present invention in the whole proteome.

[0030] Figure 7 This is a schematic diagram of the specific inhibitory effect of 15.4 μM compound 3a on PNPLA6 protein in the whole proteome in Example 1 of the present invention.

[0031] Figure 8This is the inhibitory analysis curve of compound 3a on PNPLA6 protein activity using chemical substrate characterization in Example 1 of the present invention.

[0032] Figure 9 This is the inhibitory analysis curve of compound 3a on PNPLA6 protein activity using biological substrate characterization in Example 1 of the present invention.

[0033] Figure 10 The growth activity curves of HeLa cells after being treated with different concentrations of compound 3a in Example 1 of the present invention for 2 h or 24 h.

[0034] Figure 11 This is Example 1 of the present invention, the cell proliferation ability of 293T cells stably overexpressing PNPLA6 protein and conventional 293T cells under 72 h, and the restoration of proliferation ability under the inhibitory effect of compound 3a.

[0035] Figure 12 This is the inhibitory effect of compound 3a and its derivatives in Example 1 of the present invention on PNPLA6 protein in the lysate at 770 nM.

[0036] Figure 13 The statistical results of the inhibitory effect of compound 3a and its derivatives in Example 1 of the present invention on PNPLA6 protein in the lysate at 770 nM. DETAILED DESCRIPTION

[0037] "Scope" disclosed in the present application is limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values ​​or not including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following scope can be all expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0038] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0039] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0040] This specific embodiment provides a serine hydrolase inhibitor, the structural formula of the inhibitor is as follows:

[0041] , or

[0042] In the formula, R 1 represents any one of H, OH, Cl, Br, F, trifluoromethyl, trifluoromethoxy, C1-C6 alkyl, and C1-C6 alkoxy;

[0043] R2 represents any one of C1-C6 alkyl, C1-C6 alkoxy, C3-C10 cycloalkyl, heteroaryl, aryl, and aryl substituted with one or more halogen substituents; n represents any integer from 0 to 10; m represents any integer from 0 to 10; and P represents any integer from 1 to 10.

[0044] In some embodiments, m represents any integer from 0 to 3; P represents any integer from 1 to 3.

[0045] In some embodiments, n represents any integer from 0 to 3.

[0046] In some embodiments, said R2 represents an aryl group, , , C1-C2 alkyl or C3-C6 cycloalkyl. The inhibitory factor of these inhibitors on PNPLA6 protein can be as low as 0.5 or less.

[0047] In some embodiments, the inhibitor is one or more of the compounds having the following structural formula:

[0048] , , , , , , .

[0049] In some embodiments, the inhibitor is one or more of the compounds having the following structural formula:

[0050] , , , , , , , , , , , , .

[0051] In addition, this specific embodiment also proposes a method for preparing the above-mentioned serine hydrolase inhibitor, comprising the following steps:

[0052] Warhead(1) , a benzaldehyde sulfonyl compound (2) and sodium sulfate are mixed to obtain an imine intermediate;

[0053] The imine intermediate dissolved in methanol solvent is added to palladium carbon under a hydrogen atmosphere to obtain the serine hydrolase inhibitor.

[0054] In some embodiments, in the above preparation method, warhead (1) , benzaldehyde sulfonyl compound (2) and sodium sulfate are mixed, and N,N-dimethylformamide (DMF) is added to obtain an imine intermediate;

[0055] In some embodiments, the imine intermediate is dissolved in a methanol solvent, and the imine intermediate dissolved in methanol is added to palladium carbon under a hydrogen atmosphere to obtain the serine hydrolase inhibitor.

[0056] In some embodiments, the benzaldehyde sulfonyl compound (2) is prepared by the following two methods:

[0057] Method 1:

[0058]

[0059] Add benzyl mercaptan (22 mmol, 1.1 eq.) to a 100 mL round-bottom flask, add 10 mL of anhydrous dimethyl sulfoxide (DMSO) to dissolve, then add 2-fluorobenzaldehyde or 4-fluorobenzaldehyde (20 mmol, 1 eq.) and anhydrous potassium carbonate (40mmol, 2 eq.), heat to 100 °C, and stir for 4 h. Monitor the reaction progress by thin layer chromatography (TLC). After the reaction is completed, cool to room temperature. Add 20 mL of water to dilute, extract with ethyl acetate (20 mL*3), wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify with a silica gel column. Obtain 2-(benzylthio)benzaldehyde (light yellow solid, 2.27 g, 49.8 %) or 4-(benzylthio)benzaldehyde (light yellow solid, 3.74 g, 81.9 %).

[0060] In a 100 mL round-bottom flask, add 2-(benzylthio)benzaldehyde or 4-(benzylthio)benzaldehyde (10mmol, 1 eq.), add 20 mL of anhydrous methanol and stir to dissolve, then add trimethyl orthoformate (11 mmol, 1.1 eq.) and p-toluenesulfonic acid (1.25 mmol, 0.125 eq.), and stir at room temperature for 18 h. Then add a methanol solution of sodium methoxide (25% w / w, 0.5 mL), remove the solvent by vacuum distillation, add 20 mL of ethyl acetate to dilute, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude benzyl (2- (dimethoxymethyl) phenyl) sulfone or benzyl (4- (dimethoxymethyl) phenyl) sulfone, which can be directly used for the next step without further purification.

[0061] Add benzyl (2- (dimethoxymethyl) phenyl) sulfane or benzyl (4- (dimethoxymethyl) phenyl) sulfane (10 mmol, 1 eq., crude product from the previous step, converted to 100% yield) to a 100 mL round-bottom flask, add 20 mL of anhydrous dichloromethane and stir to dissolve, then add m-chloroperbenzoic acid (37.5 mmol, 3.75 eq.) at 0°C, and then stir at room temperature for 6 h. After the reaction is complete, add saturated sodium sulfite solution, stir vigorously for 2 h, and then wash with saturated sodium bicarbonate solution. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude 1- (benzylsulfonyl) -2- (dimethoxymethyl) benzene or 1- (benzylsulfonyl) -4- (dimethoxymethyl) benzene, which can be directly used for the next step without further purification.

[0062] Add 1-(benzylsulfonyl)-2-(dimethoxymethyl)benzene or 1-(benzylsulfonyl)-4-(dimethoxymethyl)benzene (10 mmol, crude product from the previous step, converted to 100% yield) to a 100 mL round-bottom flask, add 35 mL of tetrahydrofuran and stir until completely dissolved, slowly add sulfuric acid solution (2% v / v, 40 mL), and stir at room temperature for 18 h. Add excess potassium carbonate to the reaction solution to neutralize the solution, and extract the aqueous phase with dichloromethane (30 mL*3). Collect the organic phase, dry it with anhydrous sodium sulfate, filter it, concentrate it, and purify it on a silica gel column to obtain 2-(benzylsulfonyl)benzaldehyde (colorless liquid, 1.62 g, 62%) or 4-(benzylsulfonyl)benzaldehyde (white solid, 1.99 g, 76%).

[0063] Method 2:

[0064]

[0065] Add bromomethylbenzonitrile compounds (1.0 eq.) to a 250 mL Schlenk flask, add 50 mL of anhydrous dichloromethane under an argon atmosphere, cool to 0°C, slowly drop 1.5 mol / L diisobutylaluminum hydride n-hexane solution (2.5 eq.), continue stirring for 2 h after the addition, and monitor the reaction progress by TLC. After the reaction is complete, add saturated ammonium chloride solution to quench the reaction, filter and spin dry the organic solution. After separation and purification by silica gel column chromatography, bromomethylbenzaldehyde compounds are obtained.

[0066] Add bromomethylbenzaldehyde compounds (1.0 eq.) and sodium sulfite compounds (1.2 eq.) to anhydrous N,N-dimethylformamide in a 100 mL round-bottom flask, stir at 80 °C for 2 h, and monitor the reaction progress by TLC. After the reaction is complete, filter, concentrate under reduced pressure, purify by silica gel column chromatography, and evaporate to obtain benzaldehyde sulfonyl compounds.

[0067] In some embodiments, the warhead (1) Prepared by the following steps:

[0068]

[0069] In any embodiment, benzyl carbamate, triphenyl phosphite, n-butyraldehyde and acetic acid are mixed and reacted at 85-95° C. to obtain a diphenyl phosphate compound protected by benzyl formate (Cbz);

[0070] The Cbz-protected diphenyl phosphate compound and HBr-AcOH solution are mixed to react to obtain the warhead (1) .

[0071] In addition, the present invention also provides a use of the serine hydrolase inhibitor or the serine hydrolase inhibitor prepared by the above preparation method in the preparation of a drug for inhibiting the expression of PNPLA6 protein.

[0072] The drug prepared by the present invention for inhibiting the expression of PNPLA6 protein may be only the serine hydrolase inhibitor of the present application, or the inhibitor of the present application may be used in combination with other existing known drugs to inhibit the expression of PNPLA6 protein.

[0073] In addition, the present invention also provides a drug comprising the above serine hydrolase inhibitor, wherein the drug is in the form of injection, powder injection, oral preparation, spray, capsule, suppository or any pharmaceutically acceptable excipient.

[0074] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0075] In the present invention, reference is made to “some embodiments”, “this embodiment”, examples, etc., which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0076] In this embodiment, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, object A and / or object B may represent three situations: object A exists alone, object A and object B exist at the same time, and object B exists alone.

[0077] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0078] In the following examples, the preparation method of the serine hydrolase inhibitor is as follows:

[0079] 1) Synthesis of warhead(1)

[0080]

[0081] After adding benzyl carbamate (2 mmol, 1.0 eq), triphenyl phosphite (2 mmol, 1.0 eq), n-butyraldehyde (3 mmol, 1.5 eq) and acetic acid (0.6 mL) to a dry reaction tube, it was placed in an oil bath at 90 °C for 2 h. The reaction was monitored by TLC until the benzyl carbamate was consumed. After the reaction was completed, the resulting solution was concentrated in vacuo, and the crude product was purified by silica gel column chromatography using a mixture of ethyl acetate and n-hexane as the eluent to obtain a Cbz-protected diphenyl phosphate compound (400 mg, 46% yield).

[0082] The Cbz-protected diphenyl phosphate compound (1.0 mmol) was weighed into a dry reaction tube, and a 33% HBr-AcOH solution (5 mL) was added to react at room temperature for 2 hours until the Cbz-protected diphenyl phosphate compound was consumed. The resulting solution was rotary evaporated to obtain warhead (1).

[0083] 2) Synthesis of benzaldehyde sulfonyl compounds (2)

[0084] Method 1:

[0085]

[0086] Add benzyl mercaptan (22 mmol, 1.1 eq.) to a 100 mL round-bottom flask, add 10 mL of anhydrous dimethyl sulfoxide (DMSO) to dissolve, then add 2-fluorobenzaldehyde or 4-fluorobenzaldehyde (20 mmol, 1 eq.) and anhydrous potassium carbonate (40mmol, 2 eq.), heat to 100 °C, and stir for 4 h. Monitor the reaction progress by thin layer chromatography (TLC). After the reaction is completed, cool to room temperature. Add 20 mL of water to dilute, extract with ethyl acetate (20 mL*3), wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify with a silica gel column. Obtain 2-(benzylthio)benzaldehyde (light yellow solid, 2.27 g, 49.8 %) or 4-(benzylthio)benzaldehyde (light yellow solid, 3.74 g, 81.9 %).

[0087] In a 100 mL round-bottom flask, add 2-(benzylthio)benzaldehyde or 4-(benzylthio)benzaldehyde (10mmol, 1 eq.), add 20 mL of anhydrous methanol and stir to dissolve, then add trimethyl orthoformate (11 mmol, 1.1 eq.) and p-toluenesulfonic acid (1.25 mmol, 0.125 eq.), and stir at room temperature for 18 h. Then add a methanol solution of sodium methoxide (25% w / w, 0.5 mL), remove the solvent by vacuum distillation, add 20 mL of ethyl acetate to dilute, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude benzyl (2- (dimethoxymethyl) phenyl) sulfone or benzyl (4- (dimethoxymethyl) phenyl) sulfone, which can be directly used for the next step without further purification.

[0088] Add benzyl (2- (dimethoxymethyl) phenyl) sulfane or benzyl (4- (dimethoxymethyl) phenyl) sulfane (10 mmol, 1 eq., crude product from the previous step, converted to 100% yield) to a 100 mL round-bottom flask, add 20 mL of anhydrous dichloromethane and stir to dissolve, then add m-chloroperbenzoic acid (37.5 mmol, 3.75 eq.) at 0°C, and then stir at room temperature for 6 h. After the reaction is complete, add saturated sodium sulfite solution, stir vigorously for 2 h, and then wash with saturated sodium bicarbonate solution. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude 1- (benzylsulfonyl) -2- (dimethoxymethyl) benzene or 1- (benzylsulfonyl) -4- (dimethoxymethyl) benzene, which can be directly used for the next step without further purification.

[0089] Add 1-(benzylsulfonyl)-2-(dimethoxymethyl)benzene or 1-(benzylsulfonyl)-4-(dimethoxymethyl)benzene (10 mmol, crude product from the previous step, converted to 100% yield) to a 100 mL round-bottom flask, add 35 mL of tetrahydrofuran and stir until completely dissolved, slowly add sulfuric acid solution (2% v / v, 40 mL), and stir at room temperature for 18 h. Add excess potassium carbonate to the reaction solution to neutralize the solution, and extract the aqueous phase with dichloromethane (30 mL*3). Collect the organic phase, dry it with anhydrous sodium sulfate, filter it, concentrate it, and purify it on a silica gel column to obtain 2-(benzylsulfonyl)benzaldehyde (colorless liquid, 1.62 g, 62%) or 4-(benzylsulfonyl)benzaldehyde (white solid, 1.99 g, 76%).

[0090] Method 2:

[0091]

[0092] Add bromomethylbenzonitrile compounds (1.0 eq.) to a 250 mL Schlenk flask, add 50 mL of anhydrous dichloromethane under an argon atmosphere, cool to 0°C, slowly drop 1.5 mol / L diisobutylaluminum hydride n-hexane solution (2.5 eq.), continue stirring for 2 h after the addition, and monitor the reaction progress by TLC. After the reaction is complete, add saturated ammonium chloride solution to quench the reaction, filter and spin dry the organic solution. After separation and purification by silica gel column chromatography, bromomethylbenzaldehyde compounds are obtained.

[0093] Add bromomethylbenzaldehyde compounds (1.0 eq.) and sodium sulfite compounds (1.2 eq.) to anhydrous N,N-dimethylformamide in a 100 mL round-bottom flask, stir at 80 °C for 2 h, and monitor the reaction progress by TLC. After the reaction is complete, filter, concentrate under reduced pressure, purify by silica gel column chromatography, and evaporate to obtain benzaldehyde sulfonyl compounds.

[0094] 3) Synthesis of serine hydrolase inhibitors

[0095]

[0096] It should be noted that, in the reaction formula, R represents a benzaldehydesulfonyl compound (2).

[0097] Add warhead (1) (0.5 mmol, 1.0 eq.), benzaldehyde sulfonyl compound (2) (0.5 mmol, 1.0 eq.) and excess anhydrous sodium sulfate to the Schlenk tube, and then add 5 mL of anhydrous DMF to the Schlenk tube. Stir at room temperature overnight, and monitor the reaction progress by TLC and LC-MS. After the reaction is complete, filter, concentrate under reduced pressure, and purify by preparative HPLC to obtain the imine intermediate.

[0098] 10% palladium on carbon was added to the Schlenk tube, and the imine intermediate (1.0 eq.) dissolved in 5 mL methanol was injected into the reaction tube under a hydrogen atmosphere, and stirred at room temperature overnight. The reaction progress was monitored by LC-MS. After the reaction was complete, the reaction solution was filtered with diatomaceous earth, concentrated under reduced pressure, and purified by preparative HPLC.

[0099] The 20 serine hydrolase inhibitors in the following examples are all synthesized by the above method. Those skilled in the art can synthesize the serine hydrolase inhibitors proposed in this application according to the above preparation method.

[0100] The structural characterization data of the obtained partial products are shown below:

[0101] 二苯基(1 - ((2 - ((苯基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3a)

[0102]

[0103] 1 H NMR (600 MHz, MeOD) δ 7.71–7.64 (m, 3H), 7.49 (t, J = 7.8 Hz, 2H),7.36–7.26 (m, 6H), 7.23–7.10 (m, 7H), 7.09–7.07 (m, 1H), 4.73–4.67 (m, 2H),3.97–3.90 (m, 2H), 3.26–3.21 (m, 1H), 1.98–1.90 (m, 1H), 1.82–1.74 (m, 1H),1.68–1.61 (m, 1H), 1.50–1.43 (m, 1H), 0.91 (t, J = 7.4 Hz, 3H).

[0104] 13 C NMR (151 MHz, MeOD) δ 150.42, 150.37, 150.35, 150.30, 139.83,138.64, 133.65, 132.50, 130.43, 129.52, 129.49, 128.85, 128.63, 128.24,127.41, 127.12, 125.07, 125.04, 120.44, 120.41, 120.39, 58.34, 54.63, 53.64,49.54, 49.51, 31.68, 31.66, 19.13, 19.05, 12.69.

[0105] 二苯基(1 - ((2 - (苄基磺酰基)苄基)氨基)丁基)膦酸酯 (3b)

[0106]

[0107] 1 H NMR (600 MHz, CDCl3) δ 7.59 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 4.3 Hz,2H), 7.22–7.21 (m, 1H), 7.21–7.17 (m, 5H), 7.13–7.08 (m, 6H), 7.08–7.04 (m,2H), 6.97 (d, J= 7.5 Hz, 2H), 4.45–4.37 (m, 2H), 4.17–4.08 (m, 2H), 3.24–3.19(m, 1H), 1.94–1.87 (m, 1H), 1.76–1.69 (m, 1H), 1.60–1.54 (m, 1H), 1.40–1.34(m, 1H), 0.83 (t, J = 7.3 Hz, 3H).

[0108] 13 C NMR (151 MHz, CDCl3) δ 150.61, 140.23, 136.31, 133.93, 132.26,131.37, 130.92, 129.74, 129.69, 128.78, 128.54, 128.01, 127.80, 125.09,125.04, 120.73, 120.70, 120.68, 63.06, 55.52, 54.50, 49.68, 49.64, 31.96,19.42, 19.33, 13.86.

[0109] 二苯基(1 - ((4 - (苄基磺酰基)苄基)氨基)丁基)膦酸酯 (3c)

[0110]

[0111] 1 H NMR (400 MHz, DMSO- d 6) δ 7.62 (d, J = 8.2 Hz, 2H), 7.50 (d, J = 8.0 Hz,2H), 7.44–7.35 (m, 4H), 7.34–7.24 (m, 4H), 7.23–7.18 (m, 5H), 7.16–7.11 (m,2H), 4.64 (s, 2H), 4.03 (s, 2H), 3.26–3.17 (m, 1H), 2.98–2.75 (m, 1H), 1.89–1.78 (m, 1H), 1.77–1.67 (m, 1H), 1.64–1.55 (m, 1H), 1.46–1.39 (m, 1H), 0.86(t, J = 7.3 Hz, 3H).

[0112] 13 C NMR (101 MHz, DMSO- d6) δ 150.83, 150.80, 150.73, 150.70, 147.44,137.18, 131.43, 130.30, 130.28, 129.23, 129.00, 128.76, 128.65, 128.37,125.46, 121.01, 120.97, 120.94, 61.22, 55.15, 53.65, 51.22, 51.17, 32.03,32.00, 19.38, 19.25, 14.19.

[0113] 二苯基(1 - ((3 - 氟 - 2 - ((苯基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3d)

[0114]

[0115] 1 H NMR (600 MHz, CDCl3) δ 7.64 (d, J = 7.6 Hz, 2H), 7.53 (t, J = 7.4 Hz,1H), 7.37 (t, J = 7.6 Hz, 2H), 7.24–7.19 (m, 4H), 7.18–7.14 (m, 1H), 7.14–7.04(m, 7H), 6.77 (t, J = 8.7 Hz, 1H), 4.68 (s, 2H), 4.22 (d, J = 12.2 Hz, 1H), 4.13(d, J = 12.4 Hz, 1H), 3.24–3.11 (m, 1H), 1.97–1.91 (m, 1H), 1.78–1.70 (m, 1H),1.63–1.57 (m, 1H), 1.45–1.38 (m, 1H), 0.86 (t, J = 7.2 Hz, 3H).

[0116] 13C NMR (151 MHz, CDCl3) δ 162.61, 160.95, 150.49, 150.42, 150.35,138.61, 133.83, 130.45, 130.39, 129.82, 129.78, 128.96, 128.53, 126.63,125.18, 125.15, 120.56, 120.54, 115.64, 115.54, 114.78, 114.63, 55.14, 54.17,52.69, 52.67, 49.81, 32.07, 29.72, 19.57, 19.49, 13.85.

[0117] 19 F NMR (565 MHz, CDCl3) δ -114.34.

[0118] 二苯基(1 - ((4 - 氟 - 2 - ((苯基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3e)

[0119]

[0120] 1 H NMR (600 MHz, DMSO- d 6) δ 7.74–7.70 (m, 3H), 7.60–7.57 (m, 2H),7.39–7.31 (m, 5H), 7.22–7.14 (m, 6H), 6.95–6.90 (m, 2H), 4.71 (s, 2H), 3.99–3.91 (m, 2H), 3.23–3.18 (m, 1H), 2.67–2.56 (m, 1H), 1.84–1.77 (m, 1H), 1.73–1.66 (m, 1H), 1.57–1.49 (m, 1H), 1.39–1.32 (m, 1H), 0.83 (t, J = 7.3 Hz, 3H).

[0121] 13 C NMR (151 MHz, DMSO- d6) δ 161.09, 159.47, 150.79, 150.76, 150.73,150.70, 138.71, 134.37, 130.75, 130.72, 130.28, 130.24, 130.02, 129.96,129.62, 128.50, 128.47, 127.83, 127.73, 127.26, 127.24, 125.46, 125.43,120.99, 120.97, 120.94, 117.90, 117.75, 60.28, 54.89, 53.88, 44.67, 31.91,31.89, 19.24, 19.16, 14.12.

[0122] 19 F NMR (376 MHz, DMSO) δ -119.35, -119.37, -119.37, -119.40.

[0123] 二苯基(1 - ((2 - ((((4 - 氟苯基)磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3f)

[0124]

[0125] 1 H NMR (600 MHz, CDCl3) δ 7.66–7.58 (m, 2H), 7.33–7.25 (m, 1H), 7.24–7.20 (m, 4H), 7.20–7.18 (m, 1H), 7.15–7.12 (m, 1H), 7.11–7.06 (m, 6H), 7.00(t, J = 8.1 Hz, 2H), 6.97 (d, J = 7.6 Hz, 1H), 4.60–4.49 (m, 2H), 4.15–4.01 (m,1H), 3.98–3.85 (m, 1H), 3.25–3.10 (m, 1H), 1.97–1.92 (m, 1H), 1.81–1.72 (m,1H), 1.61–1.55 (m, 1H), 1.45–1.38 (m, 1H), 0.86 (t, J = 7.2 Hz, 3H).

[0126] 13C NMR (151 MHz, CDCl3) δ 166.69, 164.99, 150.33, 150.27, 134.45,132.74, 131.62, 131.56, 129.85, 129.79, 129.31, 128.06, 127.33, 125.26,125.21, 120.60, 120.57, 120.54, 116.31, 116.16, 59.29, 55.20, 54.22, 50.10,31.88, 29.72, 19.54, 19.47, 13.83.

[0127] 二苯基(1 - ((2 - ((((4 - 甲氧基苯基)磺酰基)甲基)苄基)氨基) 丁基)膦酸酯 (3g)

[0128]

[0129] 1 H NMR (400 MHz, DMSO- d 6) δ 7.69–7.59 (m, 2H), 7.41–7.26 (m, 6H),7.23–7.16 (m, 6H), 7.14–7.10 (m, 1H), 7.10–6.99 (m, 2H), 4.82–4.66 (m, 2H),3.99–3.87 (m, 2H), 3.82 (s, 3H), 2.59–2.50 (m, 1H), 1.92–1.77 (m, 1H), 1.75–1.62 (m, 1H), 1.60–1.49 (m, 1H), 1.44–1.32 (m, 1H), 0.81 (t, J = 7.3 Hz, 3H).

[0130] 13 C NMR (101 MHz, DMSO- d6) δ 163.75, 150.78, 150.74, 150.68, 150.64,140.56, 132.94, 130.83, 130.77, 130.71, 130.26, 130.24, 128.91, 128.09,127.43, 125.47, 125.44, 121.04, 121.00, 120.97, 114.76, 58.50, 56.22, 55.04,53.56, 49.46, 49.40, 31.91, 31.89, 19.37, 19.24, 14.05.

[0131] 环己 - 2,4 - 二烯 - 1 - 基苯基(1 - ((2 - (([1,1'-联苯] - 4 - 基磺酰基) 甲基)苄基)氨基)丁基)膦酸酯(3h)

[0132]

[0133] 1 H NMR (400 MHz, DMSO- d 6) δ 7.90–7.79 (m, 4H), 7.78–7.71 (m, 2H),7.56–7.50 (m, 2H), 7.48–7.42 (m, 1H), 7.41–7.28 (m, 6H), 7.27–7.10 (m, 8H),4.92–4.76 (m, 2H), 4.09–3.83 (m, 2H), 3.23–3.13 (m, 1H), 2.68–2.52 (m, 1H),1.88–1.75 (m, 1H), 1.74–1.61 (m, 1H), 1.59–1.47 (m, 1H), 1.44–1.32 (m, 1H),0.80 (t, J = 7.3 Hz, 3H).

[0134] 13 C NMR (101 MHz, DMSO- d6) δ 150.78, 150.73, 150.68, 150.63, 145.73,140.70, 138.69, 138.08, 133.08, 130.78, 130.26, 130.24, 129.65, 129.24,129.21, 129.04, 127.73, 127.70, 127.64, 127.50, 125.46, 125.44, 121.03,121.00, 120.96, 58.22, 55.07, 53.59, 49.52, 49.46, 31.91, 31.88, 19.37,19.24, 14.05.

[0135] 二苯基(1 - ((2 - ((萘 - 2 - 基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3i)

[0136]

[0137] 1 H NMR (400 MHz, DMSO- d 6) δ 8.46 (d, J = 1.8 Hz, 1H), 8.11–8.05 (m, 3H),7.81–7.73 (m, 2H), 7.70–7.66 (m, 1H), 7.36–7.27 (m, 6H), 7.24–7.12 (m, 8H),4.92 (d, J = 13.9 Hz, 1H), 4.82 (d, J = 13.9 Hz, 1H), 4.00–3.86 (m, 2H), 3.20–3.13 (m, 1H), 2.58–2.51 (m, 1H), 1.87–1.75 (m, 1H), 1.72–1.61 (m, 1H), 1.56–1.46 (m, 1H), 1.41–1.31 (m, 1H), 0.79 (t, J = 7.3 Hz, 3H).

[0138] 13 C NMR (101 MHz, DMSO- d6) δ 150.76, 150.70, 150.66, 150.60, 140.69,136.51, 135.25, 133.02, 132.96, 132.06, 130.85, 130.23, 130.18, 130.11,129.87, 129.82, 129.71, 129.62, 129.17, 129.02, 128.39, 128.23, 128.16,127.74, 127.51, 125.45, 125.40, 123.49, 123.42, 121.02, 120.99, 120.95,119.23, 115.72, 58.16, 55.08, 53.59, 49.65, 49.59, 31.88, 31.86, 19.36,19.23, 14.04.

[0139] 二苯基(1 - ((2 - ((嘧啶 - 2 - 基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3j)

[0140]

[0141] 1 H NMR (400 MHz, DMSO- d 6) δ 9.06 (d, J = 4.9 Hz, 2H), 7.84 (t, J = 4.9 Hz,1H), 7.41–7.33 (m, 6H), 7.29–7.23 (m, 2H), 7.21–7.14 (m, 6H), 5.16 (s, 2H),4.20–4.09 (m, 2H), 3.27–3.21 (m, 1H), 1.90–1.80 (m, 1H), 1.77–1.66 (m, 1H),1.62–1.52 (m, 1H), 1.47–1.38 (m, 1H), 0.81 (t, J = 7.3 Hz, 3H).

[0142] 13 C NMR (101 MHz, DMSO- d6) δ 165.65, 159.56, 150.73, 150.64, 141.17,133.17, 130.98, 130.24, 129.18, 127.65, 126.48, 125.44, 125.20, 121.03,121.00, 120.96, 55.09, 53.74, 53.61, 49.77, 49.71, 31.94, 31.91, 19.39,19.26, 14.10.

[0143] 二苯基(1 - ((2 - (甲苯磺酰基甲基)苄基)氨基)丁基)膦酸酯 (3k)

[0144]

[0145] 1 H NMR (600 MHz, DMSO- d 6) δ 7.60–7.58 (m, 2H), 7.41–7.33 (m, 6H),7.32–7.27 (m, 2H), 7.23–7.19 (m, 3H), 7.18–7.13 (m, 4H), 7.12–7.10 (m, 1H),4.76 (d, J = 14.0 Hz, 1H), 4.70 (d, J = 13.9 Hz, 1H), 3.92–3.81 (m, 2H), 3.19–3.11 (m, 1H), 2.38 (s, 3H), 1.82–1.75 (m, 1H), 1.70–1.63 (m, 1H), 1.55–1.49(m, 1H), 1.38–1.34 (m, 1H), 0.80 (t, J = 7.3 Hz, 3H).

[0146] 13 C NMR (151 MHz, DMSO- d6) δ 150.74, 150.70, 150.68, 150.63, 144.83,140.61, 136.49, 132.98, 132.96, 130.72, 130.26, 130.24, 130.17, 130.07,128.94, 128.50, 128.47, 128.46, 127.85, 127.45, 127.43, 125.47, 125.44,121.02, 120.99, 120.96, 60.55, 58.23, 58.11, 57.33, 54.77, 53.78, 49.43,49.39, 42.10, 31.89, 31.87, 29.35, 21.54, 20.48, 19.33, 19.24, 14.05.

[0147] 二苯基(1 - ((2 - ((噻吩 - 2 - 基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3l)

[0148]

[0149] 1 H NMR (400 MHz, DMSO- d 6) δ 8.08–8.04 (m, 1H), 7.58–7.55 (m, 1H),7.39–7.35 (m, 4H), 7.33–7.28 (m, 2H), 7.25–7.20 (m, 4H), 7.19–7.13 (m, 5H),7.13–7.10 (m, 1H), 4.92–4.82 (m, 2H), 3.91–3.83 (m, 2H), 3.18–3.12 (m, 1H),2.59–2.53 (m, 1H), 1.85–1.76 (m, 1H), 1.71–1.62 (m, 1H), 1.57–1.49 (m, 1H),1.41–1.34 (m, 1H), 0.81 (t, J = 7.3 Hz, 3H).

[0150] 13 C NMR (101 MHz, DMSO- d6) δ 150.74, 150.70, 150.64, 150.60, 140.64,139.70, 136.05, 135.15, 132.82, 130.74, 130.36, 130.29, 130.27, 129.13,128.76, 127.88, 127.53, 125.50, 125.47, 121.02, 120.99, 120.95, 59.68, 54.97,53.49, 49.34, 49.29, 31.88, 31.85, 19.35, 19.22, 14.06.

[0151] 二苯基(1 - ((2 - ((乙基磺酰基)甲基)苄基)氨基)丁基)膦酸酯 (3n)

[0152]

[0153] 1 H NMR (400 MHz, DMSO- d 6) δ 7.42–7.34 (m, 6H), 7.33–7.28 (m, 2H),7.25–7.16 (m, 6H), 4.68 (d, J = 13.6 Hz, 1H), 4.54 (d, J = 13.7 Hz, 1H), 4.12–4.00 (m, 2H), 3.11–3.03 (m, 2H), 2.62–2.52 (m, 1H), 1.91–1.80 (m, 1H), 1.76–1.67 (m, 1H), 1.62–1.54 (m, 1H), 1.47–1.38 (m, 1H), 1.20 (t, J = 7.4 Hz, 3H),0.84 (t, J = 7.3 Hz, 3H).

[0154] 13 C NMR (101 MHz, DMSO- d6) δ 150.81, 150.71, 150.62, 140.76, 132.83,130.90, 130.32, 130.25, 128.82, 127.68, 127.64, 125.52, 125.47, 121.10,121.06, 121.03, 120.99, 55.24, 54.16, 53.74, 50.00, 49.94, 46.75, 32.01,31.99, 19.37, 19.25, 14.11, 6.49.

[0155] 二苯基(1 - ((2 - ((甲基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3p)

[0156]

[0157] 1 H NMR (400 MHz, DMSO- d 6) δ 7.44–7.35 (m, 6H), 7.34–7.28 (m, 2H),7.27–7.08 (m, 6H), 4.74 (d, J = 13.7 Hz, 1H), 4.56 (d, J = 13.8 Hz, 1H), 4.13–4.08 (m, 1H), 4.05–3.94 (m, 1H), 2.93 (s, 3H), 2.62–2.52 (m, 1H), 1.93–1.80(m, 1H), 1.77–1.66 (m, 1H), 1.62–1.52 (m, 1H), 1.47–1.38 (m, 1H), 0.84 (t, J =7.3 Hz, 3H).

[0158] 13 C NMR (101 MHz, DMSO- d 6) δ 150.80, 150.70, 150.61, 140.64, 132.68,130.93, 130.32, 130.26, 128.86, 128.15, 127.70, 125.52, 125.49, 121.11,121.07, 121.04, 121.00, 56.42, 55.27, 53.77, 50.07, 50.01, 31.99, 19.36,19.24, 14.11.

[0159] 二苯基(1 - ((2 - ((环丙基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3q)

[0160]

[0161] 1 H NMR (400 MHz, DMSO- d 6) δ 7.41–7.35 (m, 6H), 7.32–7.27 (m, 2H),7.23–7.16 (m, 6H), 4.72 (d, J = 13.3 Hz, 2H), 4.59 (d, J = 13.9 Hz, 1H), 2.75–2.59 (m, 2H), 1.87–1.79 (m, 1H), 1.75–1.68 (m, 1H), 1.60–1.54 (m, 1H), 1.45–1.38 (m, 1H), 1.06–0.95 (m, 2H), 0.93–0.87 (m, 4H), 0.85–0.81 (m, 3H).

[0162] 13 C NMR (101 MHz, DMSO- d 6) δ 150.80, 150.72, 150.70, 150.62, 140.54,133.49, 132.95, 130.81, 130.31, 130.26, 130.13, 129.82, 129.27, 128.80,128.47, 128.02, 127.79, 127.55, 125.51, 125.47, 121.09, 121.05, 121.03,120.99, 120.91, 115.70, 56.16, 55.73, 55.12, 53.63, 49.84, 31.97, 31.94,29.29, 29.18, 19.36, 19.24, 14.09, 4.82, 4.77, 4.65.

[0163] 二苯基(1 - ((2 - ((环丁基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3r)

[0164]

[0165] 1 H NMR (400 MHz, DMSO- d6) δ 7.41–7.35 (m, 6H), 7.32–7.29 (m, 2H),7.23–7.15 (m, 6H), 4.53 (d, J = 13.4 Hz, 1H), 4.43 (d, J = 13.7 Hz, 1H), 4.02–3.88 (m, 2H), 2.57 (s, 1H), 2.41–2.23 (m, 4H), 2.23–2.04 (m, 4H), 2.00–1.82(m, 4H), 1.75–1.67 (m, 1H), 1.59–1.51 (m, 1H), 1.46–1.37 (m, 1H), 0.83 (t, J =7.3 Hz, 3H).

[0166] 13 C NMR (101 MHz, DMSO- d 6) δ 157.92, 150.79, 150.73, 150.69, 150.63,140.76, 140.67, 136.59, 133.37, 132.84, 132.80, 130.83, 130.33, 130.31,130.26, 130.23, 130.20, 129.79, 129.30, 128.81, 128.71, 127.61, 127.57,127.45, 127.34, 125.51, 125.47, 125.15, 121.07, 121.03, 120.98, 120.90,120.86, 119.15, 115.71, 55.15, 53.66, 53.61, 53.57, 53.54, 53.52, 53.33,52.78, 50.02, 49.97, 49.80, 49.74, 31.99, 31.96, 22.33, 22.29, 22.14, 19.37,19.25, 17.19, 17.14, 14.10.

[0167] 二苯基(1 - ((2 - ((环戊基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3s)

[0168]

[0169] 1 H NMR (400 MHz, DMSO- d6) δ 7.43–7.30 (m, 8H), 7.27–7.14 (m, 6H),4.66–4.53 (m, 2H), 4.12–4.01 (m, 2H), 2.64–2.54 (m, 1H), 2.04–1.95 (m, 1H),1.95–1.82 (m, 5H), 1.78–1.66 (m, 2H), 1.64–1.60 (m, 2H), 1.58–1.52 (m, 2H),1.46–1.38 (m, 1H), 0.85 (t, J = 7.3 Hz, 3H).

[0170] 13 C NMR (101 MHz, DMSO- d 6) δ 150.78, 150.73, 150.69, 150.63, 140.80,132.98, 130.83, 130.30, 130.26, 129.82, 128.80, 127.56, 127.42, 125.51,125.47, 121.07, 121.03, 120.99, 115.69, 60.86, 55.18, 53.69, 53.59, 49.83,49.78, 32.00, 31.98, 26.92, 26.85, 26.68, 26.12, 26.07, 26.05, 19.37, 19.25,14.11.

[0171] 二苯基(1 - ((2 - ((环己基磺酰基)甲基)苄基)氨基)丁基) 膦酸酯 (3t)

[0172]

[0173] 1 H NMR (400 MHz, DMSO- d 6) δ 7.41–7.35 (m, 5H), 7.35–7.26 (m, 4H),7.24–7.17 (m, 5H), 4.59 (d, J = 13.4 Hz, 1H), 4.50 (d, J= 13.4 Hz, 1H), 4.08–3.98 (m, 2H), 2.59–2.53 (m, 1H), 2.20–2.10 (m, 1H), 2.09–2.01 (m, 2H), 1.92–1.81 (m, 2H), 1.80–1.74 (m, 2H), 1.64–1.56 (m, 2H), 1.43–1.34 (m, 3H), 1.23–1.11 (m, 3H), 0.84 (t, J = 7.3 Hz, 3H).

[0174] 13 C NMR (101 MHz, DMSO- d 6) δ 150.78, 150.73, 150.68, 150.64, 140.95,133.08, 130.83, 130.29, 130.25, 128.77, 127.53, 127.16, 125.52, 125.49,121.09, 121.05, 121.04, 121.00, 60.56, 55.25, 53.75, 51.69, 49.91, 49.85,31.99, 25.33, 25.27, 25.22, 25.19, 25.00, 24.96, 24.93, 19.39, 19.26, 14.12.

[0175] Example 1

[0176] Combination Figures 1 - 13 This embodiment provides an inhibitor of serine hydrolase PNPLA6, including the following characteristics:

[0177] 1. In in vitro experiments, the structural formula is The inhibitory effect of compound 3a reached the nanomolar level.

[0178] The product compound 3a was synthesized by organic synthesis. In order to explain the inhibitory effect of the molecules screened by proteomics on PNPLA6 protein, we chose to confirm the effect in the cell lysate overexpressing PNPLA6 protein. Lipo8000, psPAX2 pMD2.G plasmid and pLenti-PNPLA6-3×flag plasmid were mixed and transfected in 293T cells. The virus liquid was collected after 48 hours, and (culture medium: virus liquid = 3:1) was infected, and Blasticidin was used for three generations of screening and verification. After washing 293T cells stably expressing PNPLA6 with PBS twice, the cells were lysed with 4×PBS. After BCA quantification, the inhibitor was added with a final concentration gradient starting from 5 nM and increasing by three times to 100 μM (a total of 10 concentrations), and incubated at room temperature for 2 hours. Then, the broad-spectrum labeled probe FP-Rhodamine (commercial FP probe labeled serine hydrolase active pocket) was used at a final concentration of 2 μM for competitive labeling, and the reaction was protected from light for 1 hour. Denature at 95°C for 10 min and analyze by SDS-PAGE.

[0179] Combination Figure 1 The results showed that under the condition of treatment concentration of 1.2 μM, the intensity of the protein band was obviously weakened in the fluorescent gel image, and the fluorescence intensity of the protein was almost undetectable under the condition of 100 μM, indicating that the activity of PNPLA6 protein was almost completely inhibited. The gel results were quantified using ImageJ software. Figure 1 It can be seen that in the cell lysate, the inhibitory effect of compound 3a on PNPLA6 protein at a concentration of 0-100 μM gradually increased from 400 nM. Figure 2 , based on quantitative information, IC 50 Curve fitting, in the treatment of cell lysate, the inhibitory effect of inhibitor compound 3a on PNPLA6 protein is IC 50 Value = 770 nM. For the first time, a small molecule inhibitor structure that can selectively inhibit PNPLA6 protein in whole proteome lysate was discovered, and the effect reached the nanomolar level. This molecular structure is expected to be further optimized and play an important role in the functional study of PNPLA6 protein.

[0180] 2. In vivo experiments, the inhibitory effect of compound 3a was IC 50 is 1.0 μM.

[0181] The in vivo inhibitory effect of compound 3a was detected by treating 293T living cells overexpressing PNPLA6 protein with inhibitor molecules at different concentration gradients.

[0182] Inhibitor molecules were added to 293T cells stably expressing PNPLA6 protein. The final concentration gradient started at 5 nM and increased threefold to 100 μM (a total of 10 concentrations). The DMSO content was 0.5% and incubated at 37 degrees for 4 h. The cells were removed, washed twice with PBS, and lysed with 4×PBS. BCA was quantified to 1 mg / ml, and the FP-Rhodamine probe was used at a final concentration of 2 μM. The cells were labeled in the dark for 1 h. Denatured at 95 °C for 10 min and tested by SDS-PAGE.

[0183] from Figure 3 It can be seen that in the treatment of living cells, compound 3a has an inhibitory effect on PNPLA6 protein at concentrations of 0~100 μM, and PNPLA6 is completely inhibited starting from 11 μM.

[0184] Combination Figure 4 ,from Figure 4 It can be seen that in the treatment of living cells, the inhibitory effect of compound 3a on PNPLA6 protein was obtained by fitting the IC after grayscale analysis. 50 The value is 1 μM. By treating 293T living cells overexpressing PNPLA6 protein with inhibitor molecules at different concentration gradients, it was found that the inhibitory effect in vivo can still reach 1.0 μM. This is expected to be used as a lead inhibitor molecule for PNPLA6 protein for further research.

[0185] 3. Compound 3a has specificity for endogenous protein inhibition.

[0186] Combination Figure 5 , Schematic diagram of the process of purifying PNPLA6 protein. First, the plasmid expressing 3×flag-PNPLA6 was transfected into the cells. After the successful expression of PNPLA6 protein was detected, the lysate was prepared using IP buffer. Magnetic beads connected with anti-DYKDDDDK antibody were used for protein enrichment, and finally the PNPLA6 protein was eluted with the help of 3×flag peptide.

[0187] The SILAC quantitative proteomics experiment was used to perform both positive and negative label analysis to detect the specific inhibition of compound 3a on endogenous PNPLA6 protein. 50The positive and negative label analysis was performed simultaneously by SILAC quantitative proteomics experiment under the condition of 20 times the value. After that, the HeLa cell lysate (Heavy / Light, Heavy metabolic labeling rate 99.4%) obtained by SILAC medium with 4×PBS (pH=6.2) was prepared, 2 mg / mL. Heavy: Light was mixed at 1:4 or 4:1. Sample: 945 μL lysate + 10 μL inhibitor molecule (final concentration 15.4 μM) + 45 μL DMSO. Control: 945 μL lysate + 55 μL DMSO. React at room temperature for 2 h.

[0188] Mix the Heavy and Light samples in a 1:1 ratio, then shake in the dark for 30 minutes with FP-Alkyne (final concentration 100 μM). Click reaction: add 24 μL 5 mM Biotin-PEG-Picolyl-Azide, 24 μL 50 mM TCEP (prepared and used immediately), 70 μL 1.7 mM TBTA, 24 μL 50 mM CuSO4, and shake at room temperature for 1 hour. Chloroform-methanol precipitation: add 2 mL of protein sample to the premixed solution (1 mL CHCl3+4 mL MeOH+2 mL H2O) and centrifuge at 3000 rpm for 10 minutes. Take out the protein precipitate in the middle part, disperse it with methanol, and transfer it to a 1.5 mL ep tube. Redissolve the precipitated protein with 1 mL of freshly prepared 8 M Urea in 25 mM NH4HCO3. Add DTT at a final concentration of 10 mM and shake at 55℃ for 40 minutes. After cooling to room temperature, add IAA at a final concentration of 15 mM and shake for 30 minutes in the dark. Add DTT at a final concentration of 5 mM to quench the IAA reaction. Then add the protein sample to 50 μL High Capacity Neutravidin Beads (in 5mL PBS) that have been washed in advance and enrich at room temperature for 4 hours. Then wash three times with 5 mL 0.2% SDS in PBS, 5 mL 1 M Urea in PBS, and 5 mL PBS, each for 5 minutes. Resuspend the beads in enzyme digestion buffer (freshly prepared 1 M Urea in 25 mM NH4HCO3), add 2.5 μg Trypsin and shake overnight at 1050 rpm. Centrifuge at 8000 rpm for 5 minutes, take the supernatant, and adjust the pH of the peptide solution to 2~3 with 20% FA to terminate enzyme digestion. Desalting: 200 μL ACN activation and 200 μL 0.1% FA / H2O were added to the SOLAµ™ SPE plate, and the sample was loaded into the desalting column. Centrifugation was repeated twice at 50 xg for 2 min, and the desalting column was washed three times with 200 μL 0.1% FA / H2O, each time at 50 xg for 2 min. Finally, the peptides adsorbed on the desalting column were eluted with 100 μL 75% ACN / H2O. The peptide solution was placed in a vacuum concentrator and spun dry at 37 °C. The peptides were re-dissolved with 10 μL 0.1% FA / H2O, and the samples were quantified to 500 ng / μL using Nanodrop. LC-MS / MS analysis was performed using a ThermoFisher Scientific Q-Exactive Plus quadrupole-orbital ion trap high-resolution mass spectrometer.

[0189] All raw data obtained in LC-MS / MS were analyzed in MsFragger software. The specific steps of searching the database are as follows:

[0190] Download the general process of "SILAC" from the official website of Fragpipe and load it into the software. Import the original data file. Load the human protein database downloaded from the official website of UniProt and establish the Decoy library. Define the variable modifications M (15.9949Da), K (8.01419 Da), R (10.00827 Da); define the fixed modification C (57.02146 Da); define "PercusorMass Tolerance" as -20 ppm~20 ppm. (5) Execute the Labeling mode of "IonQuant" in "MS1 Quantification". Other parameters not mentioned are executed according to the default values. Perform subsequent result analysis on the "Protein_Label_Quant.tsv" file of the software processing result. From Figure 6 It can be seen that among the endogenous proteins in the cells, the general inhibition multiple of 15.4 μM compound 3a on serine hydrolase proteins (compared with the DMSO control group) is that only PNPLA6 protein has an inhibitory effect of more than 10 times, while other proteins have no inhibitory effect (inhibition multiple > 1).

[0191] Combination Figure 7 Compound 3a can produce a specific inhibitory effect on the endogenous PNPLA6 protein of 293T cells. At a concentration of 15.4 μM, the difference is 11.1 times, and no other off-target proteins exist. Among the protein objects covalently bound by 15.4 μM compound 3a in the whole proteome, PNPLA6 was significantly enriched in the experimental group.

[0192] 4. The inhibition of compound 3a on the purified PNPLA6 protein in vitro was consistent with that in the lysate, which was characterized by the chemical substrates of PNPLA6 protein, phenyl valerate and lysophosphatidylcholine LPC, respectively.

[0193] PNPLA6, or Neuropathy Target Esterase (NTE), is a membrane protein found in human neurons and other cells, including lymphocytes. Nanostructured biosensor interfaces based on the catalytically active fragment of NTE (NEST) are associated with severe neurological diseases such as motor neuron disease. During the development of the biosensor, it was found that among the esterases involved in neurological processes, phenyl valerate is the preferred substrate of NEST. In addition, the product of phenyl valerate hydrolysis by NTE is phenol, so the NTE hydrolase activity is directly measured by colorimetry.

[0194] First, 10 μl of phenyl valerate (0.5 mM, 0.03% w / v Tx-100, 0.06% DMF) was incubated with 10 μl of PNPLA6 (0.4 μg) at 37 °C for 20 minutes. Then, 10 μl of 1.23 mM 4-aminoantipyrine (50 mM Tris, 20 mM EDTA, 9 mg / ml SDS, pH = 9.0) was added to stop the reaction. Finally, 12.1 mM (4 mg / ml) potassium ferrocyanide was used for colorimetric determination, and the absorbance at 486 nm was measured. 0.4, 0.8, 1.2, 1.6, and 2.0 μg of phenol were used as the standard curve for quantitative calculation.

[0195] In addition, the serine hydrolase PNPLA6 mainly hydrolyzes phosphatidylcholine (PC) and lysophosphatidylcholine (LPC), among which lysophosphatidylcholine (LPC) is the preferred substrate of PNPLA6. Figures 8 - 9 , a decrease in PNPLA6 protein levels will lead to an increase in LPC at the cellular level, while the background concentration of cells is usually quite low, and increased LPC levels are associated with various diseases, including demyelination and neuropathic pain. Therefore, among the substrates of PNPLA6, LPC is important for the study of disease models. PNPLA6 hydrolyzes LPC to produce free fatty acids FFA, which can be quantitatively detected by enzyme-linked immunosorbent assay (ELISA). Figure 8 It can be seen that the inhibitory effect of compound 3a on pure PNPLA6 protein was obtained by characterizing the chemical substrate of PNPLA6 protein, phenyl valerate, and the absorbance measured by colorimetry was converted to IC 50 The fitted value is 1.7 μM. Figure 9 It can be seen that the inhibitory effect of compound 3a on pure PNPLA6 protein was obtained by characterizing the biological substrate of PNPLA6 protein, lysophosphatidylcholine (LPC). The absorbance measured by colorimetry was converted to IC 50 The fitted value is 1.55 μM.

[0196] from Figure 10 It can be seen that the effect of 10 μM compound 3a on cell growth rate shows that compound 3a 72 hours of treatment has a 50% inhibitory effect on the growth ability of normal cells. Cells overexpressing PNPLA6 will increase their growth ability, and the addition of compound 3a can inhibit their growth rate to be consistent with that of normal cells.

[0197] First, PNPLA6 and LPC were incubated in 50 μL PEN buffer at 37 °C, and the free fatty acid FFA produced was detected by ELISA kit. The process of detecting the content of free fatty acid (FFA) by ELISA kit is a highly specific immunological analysis method.

[0198] The detection process includes: adding samples and standards to pre-coated microplates, the well walls of which are fixed with specific antibodies against FFA; free fatty acids bind to these antibodies to form antigen-antibody complexes; adding biotin-labeled secondary antibodies, which specifically bind to FFA to form a more stable complex; adding enzyme-labeled secondary antibodies, which recognize and bind to biotin to form enzyme-labeled antigen-antibody-biotin complexes.

[0199] After washing to remove unbound substances, the substrate solution is added. The substrate changes color under the action of the enzyme, and the color depth is proportional to the content of FFA in the sample. Finally, the absorbance value is read using an enzyme reader, and the specific concentration of FFA in the sample is calculated using the standard curve. The entire detection process relies on the specific binding principle of antigen-antibody, and the enzyme amplification reaction improves the sensitivity and accuracy of the detection.

[0200] 5. Cytocompatibility and potential applications of PNPLA6 protein inhibitors

[0201] In the process of cytotoxicity analysis of compound 3a, a new inhibitor molecule of PNPLA6 protein, we used HeLa cells as a model and evaluated the effect of compound 3a on cell proliferation activity by setting a series of final concentration gradients from 0.2 μM to 50 μM. In the experiment, the cells were treated with compound 3a under conditions of 6 hours and 24 hours, and then the cell proliferation activity was tested using the CCK8 kit. The results showed that within the 6-hour treatment time, the cells at all tested concentrations did not show significant growth inhibition. This result shows that compound 3a has little effect on the proliferation of living cells within the effective concentration range and has no obvious toxic side effects. In a further 24-hour treatment experiment, when the concentration of compound 3a was maintained below 20 μM, the cell growth rate was not significantly different from that of the blank control group, indicating that within this concentration range, compound 3a has little effect on the proliferation activity of cells. However, when the concentration of compound 3a was increased to 50 μM, the cell growth rate dropped to 85%, indicating that at higher concentrations, compound 3a began to have a certain inhibitory effect on cell growth. From Figure 11 It can be seen that the effects of different concentrations of compound 3a on cell viability at 2 hours and 24 hours indicate that the toxicity of the inhibitor to cells is negligible.

[0202] Combining these experimental data, we can conclude that compound 3a has limited toxic side effects on cell growth at a reasonable working concentration, which provides the possibility of using it as a chemical tool to study the function of PNPLA6 protein at the living cell level.

[0203] from Figure 12It can be seen that compound 3a and its derivatives have an inhibitory effect on the activity of PNPLA6 protein in cell lysate at 770 nM, among which compounds 3a and 3j have better inhibitory effects. Figure 13 It can be seen that compound 3a and its derivatives have an inhibitory effect on the activity of PNPLA6 protein in cell lysate under the condition of 770 nM. The inhibition multiple of each molecule on PNPLA6 protein was confirmed by grayscale analysis, among which the inhibition multiples of compounds 3a, 3j, 3m, 3n, 3p and 3s were below 0.5.

[0204] Other beneficial effects:

[0205] 1) The present invention proposes a molecule that specifically inhibits PNPLA6 protein, designs and synthesizes 19 new derivatives, and optimizes the specific binding and activity inhibition of the serine hydrolase PNPLA6 protein based on the core characteristics of the original inhibitor.

[0206] 2) Specificity discovered for the first time: This is the first discovered inhibitor that can specifically inhibit PNPLA6 protein, providing an effective chemical tool for studying the function of PNPLA6 protein and the mechanism of related neurological diseases.

[0207] 3) Structural composition: The inhibitor consists of two parts. One is a warhead group with a broad spectrum of targeting serine hydrolases, which has reactivity, selectivity and preference for the serine hydrolase family; the other is an aldehyde ligand that enhances the inhibitor's specificity for a certain serine hydrolase, which combines with the warhead group to produce highly specific recognition of a serine hydrolase protein.

[0208] 4) In vitro inhibitory effect: In vitro experiments verified that the inhibitor had a significant inhibitory effect on 293T cell lysate overexpressing PNPLA6 protein. 50 The value is 770 nM.

[0209] 5) In vivo inhibitory effect: In vivo, by treating 293T living cells overexpressing PNPLA6 protein with inhibitor molecules at different concentration gradients, the inhibitory effect IC 50 The concentration of 1.0 μM showed good inhibitory activity in vivo.

[0210] 6) Specific inhibition of the endogenous whole proteome: The inhibitor molecule can produce a specific inhibitory effect on the endogenous PNPLA6 protein of 293T cells. When its concentration is 15.4 μM, the difference is 11.1 times, and there are no other off-target proteins.

[0211] 7) Inhibitory effect on purified protein: The inhibitor showed good inhibitory effect on purified PNPLA6 protein, which was characterized by chemical substrates and biological substrates of PNPLA6.

[0212] 8) Influence on cell proliferation ability: By inhibiting the activity of PNPLA6 protein, the inhibitor can affect the proliferation ability of cells. In 293T cells overexpressing PNPLA6 protein, the proliferation activity of cells was significantly reduced after adding the inhibitor.

[0213] 9) Safety: Compound 3a does not have obvious toxic side effects on cell growth within a reasonable working concentration range, which proves that the inhibitor has good safety at the concentration where it works.

[0214] 10) In addition, the inhibitor had an 11.1-fold specific inhibitory effect on endogenous PNPLA6 protein in 293T cells at a concentration of 15.4 μM, and no off-target phenomenon was found. At the purified protein level, the inhibitor had a highly effective inhibitory effect on both chemical and biological substrates of PNPLA6, with an IC 50 The values ​​were 1.7 μM and 1.5 μM, respectively.

[0215] Application potential: Due to its high efficiency and specificity, this inhibitor is expected to become a new chemical tool to help study the function of this protein in living cells and may play an important role in the treatment of related diseases (e.g., neurological diseases).

[0216] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A serine hydrolase inhibitor, characterized in that The inhibitor has one or more of the following structural formulas:

2. A method for preparing the serine hydrolase inhibitor according to claim 1, characterized in that: The following steps are involved: The Warhead The benzaldehyde sulfonyl compound and sodium sulfate are mixed to obtain an imine intermediate; The imine intermediate is added to palladium carbon under the action of a reducing agent to obtain the serine hydrolase inhibitor.

3. The method for preparing a serine hydrolase inhibitor according to claim 2, characterized in that: The reducing agent is hydrogen, and the imine intermediate dissolved in a solvent is added to the palladium carbon under a hydrogen atmosphere to obtain the serine hydrolase inhibitor.

4. Use of the serine hydrolase inhibitor according to claim 1 or the serine hydrolase inhibitor prepared by the preparation method according to any one of claims 2 to 3 in the preparation of a drug for inhibiting PNPLA6 protein expression.

5. A drug comprising the serine hydrolase inhibitor according to claim 1, characterized in that: The drug is in the form of injection, oral administration, spray or suppository.

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