Boric acid compounds and their applications

Abstract

The present invention provides a boric acid compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and use thereof as an active ingredient in the preparation of an NLRP3 inflammasome inhibitor. The compound can selectively inhibit the activation of the NLRP3 inflammasome, thereby treating or improving diseases associated with the NLRP3 inflammasome, such as colitis, and can be used to prepare therapeutic drugs for diseases associated with the NLRP3 inflammasome.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a class of boric acid compounds and applications thereof. Background Art

[0002] The NLRP3 inflammasome is a NOD-like receptor, which consists of three parts: the inflammasome sensor molecule (NLRP3 protein), the adaptor protein ASC, and the effector molecule caspase-1 precursor protein (Pro-caspase-1). It is a multi-protein complex present in the cytoplasm. After the NLRP3 inflammasome is activated, Pro-caspase-1 is self-cleaved into active caspase-1, which further cleaves pro-IL-1β and Pro-IL-18 into active interleukin-1β (IL-1β) and interleukin-18 (IL-18), ultimately leading to inflammatory response and cell pyroptosis. A large amount of evidence shows that many human diseases, such as Alzheimer's disease, gout, multiple sclerosis, type II diabetes, inflammatory bowel disease, etc., are closely related to the NLRP3 inflammasome. So far, a variety of NLRP3 inflammasome inhibitors have been discovered, but none of them can be used clinically. Therefore, the discovery of new NLRP3 inflammasome inhibitors is of great significance for the treatment of NLRP3-related diseases. Summary of the invention

[0003] In view of the above problems, the present invention provides a boric acid derivative, which can selectively inhibit the activation of NLRP3 inflammasome, thereby treating or improving diseases related to NLRP3 inflammasome, such as colitis.

[0004] The present invention includes the following technical solutions:

[0005] Use of a boric acid compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an NLRP3 inflammasome inhibitor,

[0006]

[0007] Wherein, X is selected from:

[0008] R1 is selected from: C6-C 10 Aryl, R5 substituted C6-C 10 Aryl, 6-10 membered heteroaryl, 6-10 membered heteroaryl substituted by R5;

[0009] R2 is selected from: H, C1-C6 alkyl;

[0010] R3 is selected from the group consisting of: H, C1-C6 alkyl, R6 substituted C1-C6 alkyl, C6-C 10Aryl, R5 substituted C6-C 10 Aryl;

[0011] R5 is selected from: hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy;

[0012] R6 is selected from: C6-C 10 Aryl, hydroxyl, R7 substituted C6-C 10 Aryl;

[0013] R7 is selected from the group consisting of hydroxy, halogen, C1-C6 alkyl, and C1-C6 alkoxy.

[0014] In some embodiments, the boric acid compound has a structure shown in the following formula (II):

[0015]

[0016] In some embodiments, R1 is selected from: phenyl, phenyl substituted with R5, naphthyl, naphthyl substituted with R5, 6-10 membered nitrogen-containing heteroaryl, and 6-10 membered nitrogen-containing heteroaryl substituted with R5.

[0017] In some embodiments, R1 is selected from: phenyl, R5 substituted phenyl, naphthyl, R5 substituted naphthyl, pyridinyl, R5 substituted pyridinyl, quinoxalinyl, R5 substituted quinoxalinyl, pyrazinyl, R5 substituted pyrazinyl.

[0018] In some embodiments, R1 is selected from: phenyl substituted with R5, pyridyl, pyridyl substituted with R5; wherein R5 is selected from: halogen, C1-C3 alkyl, C1-C3 alkoxy.

[0019] In some embodiments, R1 is selected from: phenyl, 2,5-dichlorophenyl, 2,3-dichlorophenyl, 2,6-dichlorophenyl, 2,4-dichlorophenyl, 5-chloro-2-methoxyphenyl, 2,6-difluorophenyl, 2,4-difluorophenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, 2-fluoro-5-chlorophenyl, 5-fluoro-2-methoxyphenyl, pyridyl, C1-C3 alkyl-substituted pyridyl, 3,6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3 alkyl-substituted pyrazinyl.

[0020] In some embodiments, R3 is selected from: C1-C4 alkyl, phenyl, naphthyl, hydroxy-substituted C1-C3 alkyl, phenyl-substituted C1-C3 alkyl, naphthyl-substituted C1-C3 alkyl, 4-hydroxyphenyl-substituted C1-C3 alkyl.

[0021] In some embodiments, R3 is selected from the group consisting of benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, and naphthylmethyl.

[0022] In some embodiments, when R3 is benzyl, R1 is not 2,5-dichlorophenyl, pyridyl, and quinoxalinyl.

[0023] In some embodiments, R3 is benzyl; R1 is selected from: phenyl, 2,3-dichlorophenyl, 2,6-dichlorophenyl, 2,4-dichlorophenyl, 5-chloro-2-methoxyphenyl, 2,6-difluorophenyl, 2,4-difluorophenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, 2-fluoro-5-chlorophenyl, 5-fluoro-2-methoxyphenyl, C1-C3 alkyl-substituted pyridyl, 3,6-dichloropyridyl, pyrazinyl, C1-C3 alkyl-substituted pyrazinyl.

[0024] In some embodiments, R3 is selected from: isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl; R1 is selected from: phenyl, 2,5-dichlorophenyl, 2,3-dichlorophenyl, 2,6-dichlorophenyl, 2,4-dichlorophenyl, 5-chloro-2-methoxyphenyl, 2,6-difluorophenyl, 2,4-difluorophenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, 2-fluoro-5-chlorophenyl, 5-fluoro-2-methoxyphenyl, pyridyl, C1-C3 alkyl substituted pyridyl, 3,6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3 alkyl substituted pyrazinyl.

[0025] In some embodiments, the boronic acid compound is selected from the following compounds:

[0026]

[0027]

[0028] Use of the boric acid compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating diseases associated with NLRP3 inflammasome.

[0029] In some of the embodiments, the disease associated with NLRP3 inflammasome is Alzheimer's disease, gout, multiple sclerosis, type II diabetes, and inflammatory bowel disease.

[0030] In some embodiments, the disease associated with NLRP3 inflammasome is peritonitis and colitis.

[0031] In some embodiments, the disease associated with NLRP3 inflammasome is acute peritonitis and colitis.

[0032] A pharmaceutical composition for preventing and treating diseases associated with NLRP3 inflammasomes, prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the above-mentioned boric acid compounds or pharmaceutically acceptable salts thereof.

[0033] The boric acid derivatives or pharmaceutically acceptable salts thereof provided by the present invention can selectively inhibit the activation of NLRP3 inflammasomes, thereby treating or improving diseases associated with NLRP3 inflammasomes, such as colitis, and can be used to prepare therapeutic drugs for diseases associated with NLRP3 inflammasomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure shows the result that compound 25 specifically inhibits the activation of NLRP3 inflammasome in vitro.

[0035] Figure 2 This is a diagram showing the results of compound 25 inhibiting ASC oligomerization and ASC-NLRP3 interaction.

[0036] Figure 3 The graph shows the results of compound 25 improving dextran sulfate sodium (DSS)-induced colitis. DETAILED DESCRIPTION

[0037] In the compounds described in the present invention, when any variable (such as R 5 If a (, etc.) compound occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permitted so long as such combinations result in stable compounds. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds are attached only to any suitable carbon atom of an adjacent ring. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the invention to provide compounds that are chemically stable and that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0038] As used herein, the term "alkyl" is intended to include branched and straight chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0039] The term "alkoxy" used herein refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0040] The term "heteroaryl" as used herein refers to an aromatic ring containing one or more heteroatoms selected from O, N or S, which may be monocyclic, bicyclic or polycyclic, including but not limited to: quinolyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, etc.; "heteroaryl" is also understood to include any N-oxide derivatives of heteroaryl containing nitrogen. The connection of the heteroaryl group can be achieved through a carbon atom or through a heteroatom.

[0041] As will be appreciated by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0042] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0043] The starting materials in the following examples can be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein.

[0044] The synthetic route of the compound of the present invention is as follows:

[0045]

[0046] Example 1: Preparation of (pyrazine-2-carbonyl)-L-phenylalanyl-L-leucine (Compound 1):

[0047]

[0048] (1) 2-Pyrazine carboxylate (300 mg, 2.4 mmol), L-phenylalanine methyl ester (400 mg, 2.42 mmol) and HATU (920 mg, 2.42 mmol) were dissolved in dichloromethane and stirred. After 10 minutes, N,N-diisopropylethylamine (DIPEA, 3.0 equiv.) was added and reacted at room temperature for 3 hours. The reaction was monitored by TLC. The reaction solution was washed with 10% hydrochloric acid solution, 5% NaHCO3 and saturated brine, respectively, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography to obtain the corresponding ester.

[0049] (2) The above ester (product of step 1) was added to a solution of LiOH (3.0 equiv.) in methanol-water (3:1, 4 mL), stirred for 2 hours, and the reaction was monitored by TLC until the starting material disappeared. The product was acidified with 6M hydrochloric acid, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 512 mg of the hydrolyzed carboxylic acid (the yield of the two steps was 63.4%).

[0050] (3) The above carboxylic acid (product of step 2) and HOBT (306 mg, 2.27 mmol) were added to dichloromethane, the mixture was cooled to -10°C, EDCI (435 mg, 2.27 mmol) and L-leucine methyl ester (270 mg, 2.06 mmol) were added respectively, and stirred for 15 minutes. DIPEA was then added dropwise to the reaction solution and stirred for 1 hour. The reaction was allowed to proceed at room temperature for 3 hours. After extraction, the organic phase was washed with 10% citric acid, 5% NaHCO3 and saturated brine in sequence, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure to obtain an oil.

[0051] (4) The above oil (the product of step 3) was added to a solution of LiOH (3.0 equiv.) in methanol-water (3:1, 4 mL) and stirred for 2 hours. TLC monitoring was performed until the reaction of the raw material was complete. The reaction solution was acidified with 6M hydrochloric acid, extracted with dichloromethane three times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to precipitate a white solid of 130 mg, with a yield of 47%. 1 H NMR(400MHz, Methanol-d4)δ9.10(s,1H),8.72(d,J=2.6Hz,1H),8.64–8.55(m,1H),7.24(d,J=7.5Hz,2H),7.20–7.10(m,3H),4.46(t,J=7.3Hz ,1H),3.07(dd,J=13.9,8.7Hz,2H),1.75–1.65(m,1H),1.63(t,J=7.2Hz,2H),1.34–1.20(m,1H),0.91(d,J=6.0Hz,3H),0.88(d,J=6.0Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ174.37,171.81,163.31,147.40,144.32,143.48,143.29,136.6 1,129.19,128.10,126.54,54.24,50.80,40.32,37.87,24.63,22.08,20.49; HRMS(ESI)calcd for C 14 H 19BCl2N2O4[M+H] + 385.1870, found 385.1967.

[0052] Example 2: Preparation of (2,5-dichlorobenzoyl)-L-phenylalanyl-L-leucine (Compound 2)

[0053]

[0054] Referring to the method of Example 1, a white solid was obtained with a yield of 39%. 1 HNMR (400MHz, Methanol-d4) δ7.41–7.38(m,2H),7.33–7.23(m,5H),7.17(d,J=2.3Hz,1H),4.50(t,J=7.5Hz,1H),3.27(d,J=4. 7Hz,2H),2.94(dd,J=14.0,10.4Hz,1H),1.84–1.74(m,1H),1.67(t,J=7.2Hz,2H),0.98(d,J=6.3Hz,3H),0.95(d,J=6.6Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ174.56,171.86,166.79,137.21,137.12,132.38,131.06,130.71,129.2 1,129.14128.50,128.12,126.48,54.83,50.86,40.50,37.25,24.60,22.15,20.52; HRMS(ESI)calcd forC 14 H 19 BCl2N2O4[M+H] + 451.1186, found 451.1190.

[0055] Example 3: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 3)

[0056]

[0057] The intermediate carboxylic acid compound was obtained by referring to the method of Example 1. The obtained carboxylic acid (300 mg, 0.89 mmol) and HOBT (132 mg, 0.98 mmol) were added to dichloromethane, the mixture was cooled to -10°C, EDCI (188 mg, 0.98 mmol) was added and stirred for 10 minutes, and (R)-1-amino-3-methylbutyl boric acid pinanediol ester trifluoroacetate (372 mg, 0.98 mmol) was added and stirred for 15 minutes. Finally, DIPEA (3 eq.) was added dropwise to the reaction solution, stirred at -10°C for 1 hour, and reacted at room temperature for 5 hours. Extraction, the organic phase was washed with 10% citric acid, 5% NaHCO3 and saturated brine in turn, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure to obtain 600mg of an oily substance. The obtained oily substance was dissolved in methanol, isobutylboric acid (175mg, 1.71mmol), n-hexane (4mL) and 1M HCl (2mL) were added, and the reaction was overnight. TCL monitored the reaction, and the methanol phase and the n-hexane phase were separated with a separatory funnel. The n-hexane phase was extracted twice with methanol, methanol was removed under reduced pressure, an appropriate amount of water was added, and the aqueous phase was extracted twice with dichloromethane, and anhydrous sodium sulfate was used to remove water. The solvent was removed by evaporation under reduced pressure, and then separated by column chromatography to obtain white solid compound 3 with a yield of 36%. 1 H NMR(400MHz, Methanol-d4)δ7.42(s,2H),7.34–7.26(m,6H),4.94(t,J=8.1Hz,1H),3.12(d,J= 7.9Hz,2H),2.66(t,J=7.8Hz,1H),1.38–1.28(m,1H),1.12(t,J=7.5Hz,2H),0.84–0.79(m,6H); 13 C NMR(100MHz,Methanol-d4)δ175.62,166.83,136.83,135.62,132.52,131.22,130.97,129.31,12 9.24,129.11,128.55,128.41,126.99,51.66,39.54,37.20,25.32,22.61,20.54; HRMS(ESI)calcd for C 21 H 25 BCl2N2O4[M+Na] + 473.1177, found 473.1170.

[0058] Example 4: Preparation of (R)-(1-(4-((2,5-dichlorobenzamido)methyl)benzamido)-3-methylbutyl)boric acid (Compound 4)

[0059]

[0060] Referring to the method of Example 3, a white solid was obtained with a yield of 25%. 1 HNMR(400MHz,Methanol-d4)δ7.98(d,J=8.4Hz,2H),7.57(d,J=8.1Hz,2H),7.49–7.43(m,3H),4 .61(s,2H),2.83(t,J=7.5Hz,1H),1.80–1.69(m,1H),1.42(t,J=7.3Hz,2H),0.95–0.93(m,6H); 13 C NMR(100MHz,Methanol-d4)δ171.50,167.27,145.02,137.48,132.72,131.29,130.88 ,129.22,128.54,128.43,127.89,42.83,39.97,25.72,22.34,21.46; HRMS(ESI)calcd for C 20 H 23 BCl2N2O4[M+Na] + 459.1020, found 459.1013.

[0061] Example 5: Preparation of (R)-(1-(2-(2,5-dichloro-N-methylbenzamide)acetamide)-3-methylbutyl)boric acid (Compound 5)

[0062]

[0063] Referring to the method of Example 3, a white solid was obtained with a yield of 13%. 1 H NMR(400MHz,Methanol-d4,rotamers)δ7.37–7.26(m,3H),4.40–3.85(m,2H),2.91(two single peaks,3H),2.80–2.64(m,1H),1.62–1.42(m,1H),1.25–1.12(m,2H),0.76–0.73(m,6H); 13 C NMR (100MHz, Methanol-d4) δ173.27,168.30,136.33,133.10,130.97,130.71,128.38,127.59,46.11,39.48,36.77,25.57,22.33,20.88; HRMS(ESI)calcd for C 15 H21 BCl2N2O4[M+Na] + 397.0864, ​​found 397.0853.

[0064] Example 6: Preparation of ((R)-1-((S)-2-((2,5-dichlorophenyl)sulfonamido)-3-phenylpropionamido)-3-methylbutyl)boric acid (Compound 6)

[0065]

[0066] Referring to the method of Example 3, a white solid was obtained with a yield of 41%. 1 H NMR (400MHz, Methanol-d4)δ 1 HNMR(400MHz, Methanol-d4)δ7.88(d,J=2.5Hz,1H),7.50(dd,J=8.5,2.5Hz,1H),7.39(d,J=8.5Hz,1H),7.15–7.04(m,6H),4 .23(t,J=7.8Hz,1H),3.04–2.86(m,2H),2.56(t,J=7.7Hz,1H),1.45–1.34(m,1H),1.13(t,J=7.4Hz,2H),0.84–0.81(m,6H); 13 C NMR(100MHz,Methanol-d4)δ175.46,139.17,135.24,133.41,133.35,132.57,130.18,12 9.98,128.96,128.27,126.95,55.20,39.35,38.01,25.42,22.52,20.70; HRMS(ESI)calcd forC 14 H 19 BCl2N2O4[M+Na] + 509.0864, ​​found 509.0840.

[0067] Example 7: Preparation of (R)-(1-(2,5-dichlorobenzamide)-3-methylbutyl)boric acid (Compound 7)

[0068]

[0069] 2,5-Dichlorobenzoyl chloride (300 mg, 144 mmol) and HOBT (254 mg, 1.88 mmol) were added to dichloromethane solvent, stirred at -10 °C, EDCI (360 mg, 1.88 mmol) was added, stirred for 10 minutes, (R)-1-amino-3-methylbutyl boric acid pinanediol ester trifluoroacetate (540 mg, 1.42 mmol) was added and stirred for 15 minutes, and finally DIPEA was added dropwise to the reaction solution, stirred at -10 °C for 1 hour, and then reacted at room temperature for 5 hours. Extraction, the organic phase was washed with 10% citric acid, 5% NaHCO3 and saturated brine in sequence, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure to obtain an oil. The above compound was dissolved in methanol, isobutylboric acid (348 mg, 3.42 mmol), n-hexane (6 mL) and 1M HCl (3 mL) were added, and the reaction was overnight. TCL monitored the reaction, and the methanol phase and the n-hexane phase were separated with a separatory funnel. The n-hexane phase was extracted twice with methanol, and the methanol was removed under reduced pressure. The aqueous phase was extracted twice with dichloromethane, and water was removed with anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure, and then separated by column chromatography to obtain white solid compound 8 with a yield of 64%. 1 H NMR (400MHz, Methanol-d4)δ 1 H NMR (400MHz, Methanol-d4) δ7.60–7.51(m,3H),2.96(t,J=7.8Hz,1H),1.74–1.67(m,1H),1.45–1.38(m,2H),0.93(d,J=6.6Hz,6H); 13 C NMR(100MHz,Methanol-d4)δ175.73,132.56,131.75,129.53,128.30,39.60,25.73,22.35,21.54,21.13; HRMS(ESI)calcd forC 14 H 19 BCl2N2O4[M+Na] + 326.0493, found 326.0491.

[0070] Example 9: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-methylbutanamide)-3-methylbutyl)boric acid (Compound 9)

[0071]

[0072] Referring to the method of Example 3, a white solid was obtained with a yield of 42%. 1HNMR (400MHz, Methanol-d4) δ7.46–7.43(m,3H),4.45(d,J=8.6Hz,1H),2.75(dd,J=9.2,6.2Hz,1H),2.15(dd,J=7.8,5.9Hz,1H),1.65 (dd,J=12.4,5.9Hz,1H),1.39–1.30(m,2H),1.07(d,J=6.8Hz,3H),1.01(d,J=6.7Hz,3H),0.93(d,J=1.8Hz,3H),0.91(d,J=2.1Hz,3H); 13 C NMR (100MHz, Methanol-d4) δ176.04,167.25,132.60,131.20,130.92,129.21,128.49,55.96,39.86,30.20,25.69,22.53,20.87,18.08; HRMS(ESI)calcd for C 17 H 25 BCl2N2O4[M+Na] + 425.1177, found 425.1171.

[0073] Example 10: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-methylbutanamide)-3-methylbutyl)boric acid (Compound 10)

[0074]

[0075] Referring to the method of Example 3, a white solid was obtained with a yield of 38%. 1 HNMR(400MHz, Methanol-d4)δ7.33(dd,J=2.7,1.2Hz,2H),7.31–7.30(m,1H),4.41(dd,J=9.0,3.3Hz,1H),2.74–2.46(m,1H),1 .92–1.81(m,1H),1.57–1.50(m,2H),1.25(dd,J=8.4,4.2Hz,1H),1.18(d,J=2.3Hz,2H),0.88–0.81(m,6H),0.80–0.78(m,6H); 13C NMR(100MHz,Methanol-d4)δ176.06,167.11,137.03,132.48,131.09,130.81,129.07, 128.33,54.24,39.73,35.81,25.56,24.87,22.46,20.73,14.19,9.34; HRMS(ESI)calcd for C 18 H 27 BCl2N2O4[M+Na] + 439.1333, found 439.1326.

[0076] Example 11: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-2-phenylacetamide)-3-methylbutyl)boric acid (Compound 11)

[0077]

[0078] Referring to the method of Example 3, a white solid was obtained with a yield of 47%. 1 HNMR(400MHz,Methanol-d4)δ7.51(d,J=7.8Hz,4H),7.41(d,J=20.5Hz,4H),5.84(d,J=1 0.8Hz,1H),2.94–2.55(m,1H),1.69–1.52(m,1H),1.36–1.24(m,2H),0.89–0.83(m,6H); 13 C NMR(100MHz,Methanol-d4)δ175.12,174.96,166.95,136.74,134.84,132.55,131.20,131.01 ,129.41,128.82,128.74,128.69,127.70,54.60,39.46,25.62,22.47,20.96; HRMS(ESI)calcd for C 20 H 23 BCl2N2O4[M+Na] + 459.1020, found 459.1018.

[0079] Example 12: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-4-methylpentanamide)-3-methylbutyl)boric acid (Compound 12)

[0080]

[0081] Referring to the method of Example 3, a white solid was obtained with a yield of 34%.1 HNMR (400MHz, Methanol-d4) δ7.45–7.43(m,3H),4.75(dd,J=9.4,5.6Hz,1H),2.73(dd,J=8.6,6.6Hz,1H),1.80–1.7 1(m,2H),1.66–1.56(m,2H),1.35–1.28(m,2H),0.97(d,J=2.2Hz,3H),0.96(d,J=2.3Hz,3H),0.90(d,J=6.6Hz,6H); 13 C NMR(100MHz,Methanol-d4)δ177.20,167.16,137.02,132.62,131.24,130.97,129.2 4,128.50,48.56,39.87,25.72,24.55,22.49,21.71,21.00,20.51; HRMS(ESI)calcd for C 18 H 27 BCl2N2O4[M+Na] + 439.1333, found 439.1326.

[0082] Example 13: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-hydroxypropionamide)-3-methylbutyl)boric acid (Compound 13)

[0083]

[0084] Referring to the method of Example 3, a white solid was obtained with a yield of 35%. 1 HNMR (400MHz, Methanol-d4) δ7.47–7.42(m,1H),7.35–7.31(m,2H),4.64(t,J=5.6Hz,1H),3.74(d,J=5.6Hz,2 H),2.64(t,J=7.8Hz,1H),1.60–1.43(m,1H),1.30–1.17(m,2H),0.79(d,J=2.2Hz,3H),0.77(d,J=2.2Hz,3H); 13 C NMR (100MHz, Methanol-d4) δ175.18,166.97,136.57,132.50,131.18,130.97,129.28,128.64,60.69,52.62,39.56,25.57,22.35,20.91; HRMS(ESI)calcd forC 15 H 21BCl2N2O4[M+Na] + 413.0813, found 413.0809.

[0085] Example 14: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-(4-hydroxyphenyl)propionamide)-3-methylbutyl)boric acid (Compound 14)

[0086]

[0087] Referring to the method of Example 3, a white solid was obtained with a yield of 40%. 1 HNMR(400MHz, Methanol-d4)δ7.35–7.29(m,2H),7.27–7.17(m,1H),7.00–6.92(m,2H),6.63–6.56(m,2H),4.73(t,J= 7.7Hz,1H),2.95–2.80(m,2H),2.51(dd,J=9.5,4.4Hz,1H),1.20–1.13(m,1H),1.07–0.85(m,2H),0.71–0.62(m,6H); 13 C NMR(100MHz,Methanol-d4)δ175.72,166.72,156.38,136.73,132.40,131.10,130.84,130.16 ,129.20,128.46,125.81,114.97,51.76,39.42,36.37,25.18,22.60,20.30; HRMS(ESI)calcd for C 21 H 25 BCl2N2O5[M+Na] + 489.1226, found 489.1119.

[0088] Example 15: Preparation of ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-(naphthalen-1-yl)propionamide)-3-methylbutyl)boric acid (Compound 15)

[0089]

[0090] Referring to the method of Example 3, a white solid was obtained with a yield of 47%. 1H NMR(400MHz, Methanol-d4)δ7.80–7.76(m,3H),7.70(d,J=1.6Hz,1H),7.42–7.37(m,5H),7.28(t,J=1.2Hz,1H),5.01(t,J=8.2Hz,1H),3.25 (s,2H),2.57(dd,J=10.1,5.2Hz,1H),1.34–1.16(m,1H),1.08–0.96(m,1H),0.90–0.80(m,2H),0.68(d,J=6.4Hz,3H),0.44(d,J=6.4Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ175.68,166.89,136.79,133.57,132.84,132.77,132.51,131.24,131.00,129.30,128.56 ,128.25,128.18,127.52,127.38,127.00,125.98,125.65,51.54,39.36,37.38,25.10,22.36,20.29; HRMS(ESI)calcd for C 25 H 27 BCl2N2O4[M+Na] + 523.1325, found 523.1333.

[0091] Example 16: Preparation of ((R)-3-methyl-1-((S)-3-phenyl-2-(pyridinylamido)propionamido)butyl)boric acid (Compound 16)

[0092]

[0093] Referring to the method of Example 3, a white solid was obtained with a yield of 46%. 1 HNMR (400MHz, Methanol-d4) δ8.67–8.55(m,1H),8.01(d,J=7.8Hz,1H),7.96–7.88(m,1H),7.54(dd,J=8.8,4.8Hz,1H),7.27(d,J=4.8H z,4H),4.99(t,J=7.6Hz,1H),3.22(d,J=7.8Hz,2H),2.65(t,J=8.0Hz,1H),1.44–1.32(m,1H),1.16(t,J=7.4Hz,2H),0.84–0.79(m,6H); 13C NMR(100MHz,Methanol-d4)δ175.80,165.09,148.90,148.55,137.52,135.69,129.22,12 8.41,126.96,126.81,121.96,51.51,39.55,37.55,25.35,22.51,20.74; HRMS(ESI)calcd forC 20 H 26 BN3O4[M+H] + 384.2089, found 384.2089.

[0094] Example 17: Preparation of ((R)-3-methyl-1-((S)-2-(6-methylpyrazine-2-carboxamido)-3-phenylpropionamido)butyl)boric acid (Compound 17)

[0095]

[0096] Referring to the method of Example 3, a white solid was obtained with a yield of 42%. 1 H NMR(400MHz, Methanol-d4)δ8.93(s,1H),8.66(s,1H),7.29–7.19(m,5H),5.05–4.97(m,1H),3.24(d,J=7.5Hz,2H),2.6 6(t,J=7.6Hz,1H),2.62(s,3H),1.43–1.34(m,1H),1.17(t,J=7.2Hz,2H),0.83(d,J=4.1Hz,3H),0.81(d,J=4.0Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ175.54,163.96,153.46,147.41,143.09,140.16,135.66,129.2 4,128.42,126.98,51.42,39.54,37.33,26.64,25.36,22.47,20.79,20.06; HRMS(ESI)calcd for C 20 H 27 BN4O4[M+H] + 399.2198, found 399.2385.

[0097] Example 18: Preparation of ((R)-3-methyl-1-((S)-3-phenyl-2-(quinoxaline-2-carboxamido)propionamido)butyl)boric acid (Compound 18)

[0098]

[0099] Referring to the method of Example 3, a white solid was obtained with a yield of 35%. 1 H NMR(400MHz, Methanol-d4)δ9.36(s,1H),8.15(d,J=9.8Hz,2H),7.95–7.83(m,2H),7.33–7.25(m,4H),7.19(s,1H),5.08(t,J=7.3Hz, 1H),3.30–3.22(m,2H),2.69(t,J=6.6Hz,1H),1.46–1.34(m,1H),1.19(t,J=6.5Hz,2H),0.82(d,J=4.5Hz,3H),0.81(d,J=4.6Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ175.62,163.95,143.42,143.36,143.11,140.41,135.77,132.08,131.15 ,129.64,129.30,128.74,128.45,127.00,51.62,39.57,37.36,25.37,22.49,20.86; HRMS(ESI)calcd for C 23 H 27 BN4O4[M+H] + 435.2198, found 435.2410.

[0100] Example 19: Preparation of ((R)-1-((S)-2-(3,6-dichloropyridinecarboxamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 19)

[0101]

[0102] Referring to the method of Example 3, a white solid was obtained with a yield of 39%. 1 HNMR (400MHz, Methanol-d4) δ7.93(d,J=1.4Hz,1H),7.54(d,J=8.5Hz,1H),7.29(d,J=4.8Hz,4H),7.23(d,J=9.2Hz,1H),4.95(t,J=7.8Hz, 1H),3.17(d,J=7.8Hz,2H),2.65(t,J=7.7Hz,1H),1.40–1.28(m,1H),1.13(t,J=7.3Hz,2H),0.82(d,J=5.9Hz,3H),0.81(d,J=5.7Hz,3H).; 13C NMR(100MHz,Methanol-d4)δ175.46,164.14,148.83,148.34,141.94,135.51,129.27,129.19 ,128.98,128.43,127.23,126.97,51.56,39.53,37.25,25.30,22.57,20.63; HRMS(ESI)calcd forC 20 H 24 BCl2N3O4[M+H] + 432.1310, found 452.1318.

[0103] Example 20: Preparation of ((R)-1-((S)-2-(2,3-dichlorobenzamido)-3-phenylpropionamido)-3-methylbutyl)boric acid (Compound 20)

[0104]

[0105] Referring to the method of Example 3, a white solid was obtained with a yield of 36%. 1 HNMR(400MHz, Methanol-d4)δ7.59(dd,J=8.0,1.6Hz,1H),7.33–7.23(m,7H),4.96(t,J=8.1Hz,1H),3.12( dd,J=8.2,2.4Hz,2H),2.67(t,J=7.6Hz,1H),1.36–1.30(m,1H),1.13(t,J=7.4Hz,2H),0.84–0.80(m,6H); 13 C NMR(100MHz,Methanol-d4)δ175.66,167.60,137.79,135.62,133.21,131.52,129.23,128.99,128.42,127.84,126.98 ,126.82,51.63,48.31,48.09,47.88,47.67,47.46,47.24,47.03,39.54,37.23,25.33,22.63,20.53; HRMS(ESI)calcd forC 21 H 25 BCl2N2O4[M+Na] + 473.1177, found 473.1168.

[0106] Example 21: Preparation of ((R)-1-((S)-2-(2,6-dichlorobenzamide)-3-methylbutanamide)-3-methylbutyl)boric acid (Compound 21)

[0107]

[0108] Referring to the method of Example 3, a white solid was obtained with a yield of 47%. 1 H NMR(400MHz, Methanol-d4)δ7.37(d,J=2.9Hz,3H),7.29(d,J=4.2Hz,4H),7.24–7.20(m,1H),5.05(t,J=8.0Hz,1H ),3.12(dd,J=8.0,3.2Hz,2H),2.67(t,J=7.7Hz,1H),1.38–1.29(m,1H),1.12(t,J=7.4Hz,2H),0.83–0.79(m,6H); 13 C NMR(100MHz,Methanol-d4)δ175.34,165.56,135.50,135.35,131.91,131.03,129.25,12 8.42,127.85,127.85,126.93,51.29,39.52,37.46,25.34,22.66,20.50; HRMS(ESI)calcd for C 21 H 25 BCl2N2O4[M+Na] + 473.1177, found 473.1169.

[0109] Example 22: Preparation of ((R)-1-((S)-2-(2,4-dichlorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 22)

[0110]

[0111] Referring to the method of Example 3, a white solid was obtained with a yield of 48%. 1 HNMR(400MHz, Methanol-d4)δ7.52–7.47(m,1H),7.37(dd,J=8.3,1.9Hz,1H),7.31–7.23(m,6H),4.95(t,J=8.1Hz,1 H),3.12(dd,J=8.2,1.7Hz,2H),2.66(t,J=7.7Hz,1H),1.36–1.30(m,1H),1.12(t,J=7.4Hz,2H),0.84–0.77(m,6H); 13C NMR(100MHz,Methanol-d4)δ175.67,167.38,136.25,135.61,134.06,131.90,129.88,129.47 ,129.23,128.42,127.08,126.98,51.68,39.53,37.24,25.32,22.63,20.54; HRMS(ESI)calcd for C 21 H 25 BCl2N2O4[M+Na] + 473.1177, found 473.1169.

[0112] Example 23: Preparation of ((R)-1-((S)-2-(5-chloro-2-methoxybenzamido)-3-phenylpropionamido)-3-methylbutyl)boric acid (Compound 23)

[0113]

[0114] Referring to the method of Example 3, a white solid was obtained with a yield of 46%. 1 H NMR (400MHz, Methanol-d4) δ7.78(d,J=2.7Hz,1H),7.45(dd,J=8.9,2.8Hz,1H),7.32–7.25(m,5H),7.10(d,J=8.9Hz,1H),4.97(t,J=7.3Hz,1H),3.8 7(s,3H),3.17(dd,J=7.4,2.3Hz,2H),2.67(t,J=7.7Hz,1H),1.43–1.37(m ,1H),1.19(t,J=7.4Hz,2H),0.84(d,J=2.2Hz,3H),0.82(d,J=2.1Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ175.90,164.96,156.55,135.57,132.74,130.29,129.27,128.50,12 7.09,125.81,122.29,113.57,55.72,51.84,39.58,37.46,25.40,22.54,20.76; HRMS(ESI)calcd for C 22 H 28 BClN2O5[M+Na] + 469.1672, found 469.1664.

[0115] Example 24: Preparation of ((R)-1-((S)-2-(5-fluoro-2-methoxybenzamido)-3-phenylpropionamido)-3-methylbutyl)boric acid (Compound 24)

[0116]

[0117] Referring to the method of Example 3, a white solid was obtained with a yield of 52%. 1 HNMR (400MHz, Methanol-d4) δ7.56(dd,J=9.2,3.3Hz,1H),7.34–7.23(m,6H),7.11(dd,J=9.2,4.2Hz,1H),4.97(t,J=7.3Hz,1H),3.87(s,3H ),3.17(dd,J=7.3,3.2Hz,2H),2.67(t,J=7.7Hz,1H),1.42–1.36(m,1H),1.19(t,J=7.4Hz,2H),0.84(d,J=2.6Hz,3H),0.82(d,J=2.7Hz,3H); 13 C NMR (100MHz, Methanol-d4) δ177.24, 166.28 (d, J = 1.8Hz), 158.20 (d, J = 238.5Hz), 155.55 (d, J = 2.2Hz), 136.90, 130.60, 129.83, 128.43, 123.24 (d ,J=6.6Hz),120.82(d,J=23.7Hz),118.16(d,J=25.3Hz),114.80(d,J=7.8Hz),57.22,53.14,40.91,38.83,26.73,23.86,22.08.; HRMS(ESI)calcd for C 22 H 28 BClN2O5[M+Na] + 453.1968, found 453.1953.

[0118] Example 25: Preparation of ((R)-1-((S)-2-(2,6-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 25)

[0119]

[0120] Referring to the method of Example 3, a white solid was obtained with a yield of 48%. 1H NMR (400MHz, Methanol-d4) δ7.50–7.43(m,1H),7.30–7.24(m,5H),7.02(t,J=8.1Hz,2H),4.94(t,J=8.0Hz,1H),3.11(d,J =8.2Hz,2H),2.63(t,J=7.7Hz,1H),1.30–1.25(m,1H),1.08(t,J=7.4Hz,2H),0.81(d,J=6.4Hz,3H),0.79(d,J=6.7Hz,3H); 13 C NMR(100MHz,Methanol-d4)δ176.84,162.99,160.96(dd,J=250.9,7.1Hz),136.74,133.39(t,J=10.1Hz),130.54,129.7 3,128.30,112.83(dd,J=17.8,3.4Hz).112.82(d,J=25.3Hz),53.07,40.83,38.60,26.57,24.01,21.76; HRMS(ESI)calcd for C 21 H 25 BF2N2O4[M+Na] + 441.1768, found 441.1757.

[0121] Example 26: Preparation of ((R)-1-((S)-2-(2,4-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 26)

[0122]

[0123] Referring to the method of Example 3, a white solid was obtained with a yield of 43%. 1 HNMR(400MHz, Methanol-d4)δ7.70–7.63(m,1H),7.30–7.24(m,5H),7.06–7.00(m,2H),4.93(t,J=7.9Hz,1H ),3.15(d,J=7.9Hz,2H),2.64(t,J=7.7Hz,1H),1.35–1.30(m,1H),1.12(t,J=7.4Hz,2H),0.83–0.79(m,6H); 13C NMR (100MHz, Methanol-d4) δ177.22, 165.84 (d, J = 2.0Hz), 163.58 (dd, J = 260.0, 12.5Hz),162.06(dd,J=252.3,12.5Hz),136.95,133.27(dd,J=10.6,3.9Hz),130 .55,129.76,128.32,120.19(dd,J=13.8,3.4Hz),112.91(dd,J=21.8,3.5Hz),1 05.51(t,J=26.6Hz),53.28,40.85,38.50,26.63,23.94,21.90; HRMS(ESI)calcd forC 21 H 25 BF2N2O4[M+Na] + 441.1768, found 441.1759.

[0124] Example 27: Preparation of ((R)-1-((S)-2-(2,3-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 27)

[0125]

[0126] Referring to the method of Example 3, a white solid was obtained with a yield of 38%. 1 H NMR (400MHz, Methanol-d4) δ7.42–7.37(m,1H),7.31–7.27(m,5H),7.25–7.19(m,2H),4.94(t,J=8.0Hz,1H) ,3.15(d,J=8.0Hz,2H),2.65(t,J=7.7Hz,1H),1.35–1.29(m,1H),1.12(t,J=7.4Hz,2H),0.83–0.79(m,6H); 13C NMR (101MHz, Methanol-d4) δ175.76, 164.45 (d, J = 2.9Hz), 150.49 (dd, J = 260.0 Hz,13.0Hz),148.11(dd,J=251.6,11.0Hz),135.60,129.21,128.42,126.99,12 4.78(dd,J=26.2,11.0Hz),124.66(d,J=1.4Hz),124.61(dd,J=11.6,4.6Hz),11 9.67(d,J=17.5Hz),51.97,39.52,37.15,25.30,22.60,20.56; HRMS(ESI)calcd for C 21 H 25 BF2N2O4[M+Na] + 441.1768, found 441.1756.

[0127] Example 28: Preparation of ((R)-1-((S)-2-(2,5-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 28)

[0128]

[0129] Referring to the method of Example 3, a white solid was obtained with a yield of 39%. 1 HNMR(400MHz, Methanol-d4)δ7.31–7.22(m,8H),4.94(t,J=7.9Hz,1H),3.15(d,J=7.9Hz ,2H),2.65(t,J=7.7Hz,1H),1.35–1.30(m,1H),1.13(t,J=7.4Hz,2H),0.83–0.79(m,6H); 13 C NMR (100MHz, Methanol-d4) δ177.03,165.38,159.86(d,J=240.0Hz),157.43(d,J=249.2Hz),136.92,130.55,129.76,128.33,124.95(dd,J=24.3,7.6 HRMS (ESI)calcd for C 21 H 25BF2N2O4[M+Na] + 441.1768, found 441.1755.

[0130] Example 29: Preparation of ((R)-1-((S)-2-(2-fluoro-5-chlorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boric acid (Compound 29)

[0131]

[0132] Referring to the method of Example 3, a white solid was obtained with a yield of 35%. 1 HNMR(400MHz, Methanol-d4)δ7.58–7.53(m,1H),7.53–7.48(m,1H),7.30–7.19(m,6H),4.93(t,J=7.9Hz,1H) ,3.15(d,J=7.9Hz,2H),2.65(t,J=7.7Hz,1H),1.37–1.29(m,1H),1.12(t,J=7.4Hz,2H),0.83–0.79(m,6H).; 13 C NMR (100MHz, Methanol-d4) δ177.02, 165.36 (d, J = 1.8Hz), 159.88 (d, J = 250.8Hz), 136.92, 134.05 (d, J = 9.0Hz), 130.97 (d, J = 2.9Hz), 130.68 ( d,J=3.4Hz),130.55,129.76,128.34,125.30(d,J=16.0Hz),119.14(d,J=24.8Hz),53.31,40.86,38.48,26.64,23.92,21.91; HRMS(ESI)calcd for C 21 H 25 BClFN2O4[M+Na] + 457.1472, found 457.1462.

[0133] Example 30: In vitro study of the inhibitory effect of boronic acid derivatives on NLRP3 inflammasome

[0134] J774A.1 cells were plated in 96-well plates, with approximately 5 × 10 cells per well. 5cells, inoculated overnight, the supernatant was discarded, 100 μL of DMEM medium containing 10% serum containing bacterial lipopolysaccharide (LPS) (1 μg / ml) was added to each well, and then different concentrations of (10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM) of boric acid derivatives were added for treatment for 1 hour, and then nigericin (Nigericin, 10 μM) was added for treatment for 1 hour, and then the cell supernatant was collected, and the IL-1β content was determined by Mouse IL-1β ELISA kit, and the inhibitory effect of the compound of the present invention on NLRP3 inflammasome was calculated. The results are shown in Table 1.

[0135] Table 1

[0136]

[0137]

[0138]

[0139]

[0140] Example 31: Compound 25 specifically inhibits the activation of NLRP3 inflammasome in vitro

[0141] 1. NLRP3 inflammasome activation and IL-1β detection: J774A.1 cells or mouse bone marrow-derived macrophages (BMDMs) were divided into 96-well plates, with 5×10 5 cells, inoculated overnight, discarded the supernatant, added 100 μL of DMEM medium containing 10% serum and bacterial lipopolysaccharide (LPS) (1 μg / ml) to each well, then added different concentrations (7.5 nM, 15 nM, 30 nM, 60 nM) of compound 25 for 1 h, then added nigericin (Nigericin, 10 μM) for 1 h, then collected the cell supernatant, and used Mouse IL-1β ELISA kit to determine IL-1β content.

[0142] 2. Protein immunoblotting analysis: The J774A.1 cells or BMDMs cell samples treated in step 1 were lysed in RIPA lysis buffer with protease inhibitors at 4°C for 30 min. The proteins in the lysate or supernatant were separated by 12% SDS-polyacrylamide gel, transferred to PVDF membrane, and subjected to protein immunoblotting analysis with anti-mouse IL-1β antibody, anti-ASC antibody, anti-casepase-1 antibody, anti-NLRP3 antibody, and anti-β-actin antibody.

[0143] 3. NLRC4 and AIM2 inflammasome activation and IL-1β detection: For NLRC4 or AIM2 inflammasome activation, J774A.1 cells were stimulated with 1 μg / mL LPS for 5 h, and then treated with different concentrations (1 μM, 2 μM, 5 μM) of compound 25 for 1 h. The cells were then infected with bacterial flagellin (FLA-ST Ultrapure) (2.5 μg / mL) for 4 h, or transfected with poly(dA:dT) (0.25 μg / mL) for 4 h. The cell supernatant was then collected and the IL-1β content was determined using a Mouse IL-1β ELISA kit.

[0144] The results are as follows Figure 1 As shown in Figure 2, in the J774A.1 and BMDMs cell models of NLRP3 inflammasome activation, compound 25 could inhibit the secretion of IL-1β in a concentration-dependent manner ( Figure 1 Western blotting showed that compound 25 inhibited caspase-1 (p20) maturation and IL-1β secretion in a dose-dependent manner, but did not affect pro-IL-1β, pro-caspase-1, NLRP3 or ASC in cell lysates ( Figure 1 Meanwhile, compound 25 had no inhibitory effect on the activation of AIM2 or NLRC4 inflammasomes ( Figure 1 The above results indicate that compound 25 can specifically inhibit NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion.

[0145] Example 32: Compound 25 inhibits the assembly and activation of NLRP3 inflammasome

[0146] 1. Chemical cross-linking method to detect ASC oligomerization: J774A.1 cells were plated on a 6-well plate, 1 mL of DMEM medium containing bacterial lipopolysaccharide (LPS) (1 μg / ml) and 10% serum was added, and then different concentrations (15 nM, 60 nM) of compound 25 were added for 1 h, and then nigericin (10 μM) was added for 1 h, the supernatant was discarded, and the cells were washed twice with pre-cooled D-PBS, and 500 μL of NP-40 lysis buffer was added to each well, and lysed on ice for 30 min. The cells in each well were scraped and aspirated into a 1.5 mL EP tube. Preparation of the input group: 4 ° C, 12000 rpm, centrifuged for 15 min, aspirated the supernatant into a new EP tube, and the concentration of each tube protein was determined by BCA quantification. After quantification, it was stored in a -20 ° C refrigerator. Wash the precipitated protein twice with PBS, then add 500 μL of 2 mM disuccinimidyl suberate (DSS) solution prepared with PBS to each tube, incubate at room temperature for 30 min, centrifuge at 5000 rpm for 15 min, and discard the supernatant. Add 500 μL of 2 mM Ttis solution prepared with PBS to quench the unreacted DSS, and incubate at room temperature for 15 min. Centrifuge at 5000 rpm for 15 min, discard the supernatant, add 1× Loading Buffer to the precipitate, vortex carefully, boil in a 100°C metal bath for 10 min, and put in a -20°C refrigerator for use. Use protein immunoblotting to analyze the oligomeric state of ASC protein. The results are shown in Figure 2 A in.

[0147] 2. Immunoprecipitation to detect the interaction between NLRP3 and ASC: The cells were plated in five large dishes, 5 mL of DMEM medium containing bacterial lipopolysaccharide (LPS) (1 μg / ml) and 10% serum was added, and then different concentrations (15 nM, 60 nM) of compound 25 were added for 1 h, and then nigericin (10 μM) was added for 1 h, the supernatant was discarded, and the cells were washed twice with pre-cooled D-PBS. 500 μL of NP-40 lysis buffer was added to each large dish, and lysed on ice for 30 min. The cells in each well were scraped and aspirated into a 1.5 mL EP tube. Preparation of the input group: 4 ° C, 12000 rpm, centrifuged for 15 min, aspirated part of the supernatant into a new EP tube, and the protein concentration of each tube was determined by BCA quantification. The remaining supernatant was aspirated into a new 1.5 mL EP tube with a volume of cell lysate corresponding to 500 μg of protein. NP40 lysis buffer was added to complete the system. Pre-purification of cell lysate: Add 20μL of ProteinA / G magnetic beads to each tube and rotate and incubate in a -4℃ refrigerator for 6h. Centrifuge at 4℃, 2500rpm, 5min, and carefully aspirate the supernatant into a new EP tube. Immunoprecipitation: Add IgG antibody to the IgG sample, and add the remaining antibodies required for the corresponding IP, and incubate at 4℃ overnight. Add 20μL of ProteinA / G magnetic beads to each tube and rotate and incubate in a -4℃ refrigerator for 4h. Centrifuge at 4℃, 2500rpm for 5min, and wash 5 times with pre-cooled D-PBS buffer. During the washing process, keep the sample on ice throughout the process, and carefully aspirate the supernatant without touching the precipitate. Sample analysis: Add 20μL of 1×Loading Buffer to each tube and vortex carefully. Centrifuge at 12000rpm for 1min. Boil the sample in a 100℃ metal bath for 10min, and load it after microcentrifugation. Use protein immunoblotting to analyze the content of different proteins. The results are shown in the figure. Figure 2 B in.

[0148] The results are as follows Figure 2 As shown, in the J774A.1 cell model of NLRP3 inflammasome activation, compound 25 could inhibit ASC oligomerization in a concentration-dependent manner ( Figure 2 A in the figure). In the nigericin-treated group, a significant interaction between NLRP3 and ASC was observed, while in the high-dose group, the interaction between NLRP3 and ASC was significantly weakened. This indicates that compound 25 inhibits the interaction between NLRP3 and ASC after inhibiting ASC oligomerization ( Figure 2 B), indicating that compound 25 inhibited the assembly and activation of NLRP3 inflammasome.

[0149] Example 33: Compound 25 improves colitis induced by dextran sulfate sodium (DSS)

[0150] 1. Female C57BL / 6 mice aged 6-8 weeks were divided into 5 groups, with 6 mice in each group. The specific grouping and treatment were as follows:

[0151] Group 1: distilled water was given in the diet from day 1 to day 10, and vehicle was gavaged daily;

[0152] Group 2: Distilled water was given in the diet on days 1-3, and 2.5% DSS in distilled water was given in the diet starting from day 4, and the vehicle was gavaged daily;

[0153] Group 3: Distilled water was given in the diet on days 1-3, and 2.5% DSS in distilled water was given in the diet every day starting from the fourth day, and compound 25 (0.125 mg / kg) was gavaged every three days;

[0154] Group 4: distilled water was given in the diet on days 1-3, and 2.5% DSS in distilled water was given in the diet every day starting from the fourth day, and compound 25 (0.25 mg / kg) was gavaged every three days;

[0155] Group 5: Distilled water was given in the diet on days 1-3, and 2.5% DSS in distilled water was given in the diet every day starting from the fourth day, and compound 25 (0.5 mg / kg) was gavaged every three days;

[0156] 2. The degree of bloody stool and body weight of each group of mice were monitored daily starting from the day before DSS administration. On the 11th day, the length of the mouse colon was measured and the IL-1β content in the colon was determined.

[0157] The results are as follows Figure 3 As shown, from Figure 3 According to the results, after giving 2.5% DSS in the diet, the severity of bloody stools in mice increased, the length of their colon shortened, and the IL-1β level in the colon increased significantly. Oral administration of compound 25 can dose-dependently improve bloody stools in mice, improve the shortening of colon length, and reduce the level of IL-1β in the colon.

[0158] The activity test results show that the boric acid compounds of the present invention show the activity of inhibiting NLRP3 inflammasome in the test cell J774A.1. At the same time, the representative compound 25 can selectively inhibit the activation of NLRP3 inflammasome and improve DSS-induced colitis. It can be seen that the boric acid compounds of the present invention are useful for treating NLRP3 inflammasome-related diseases.

[0159] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0160] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a boric acid compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a drug for preventing and / or treating a disease associated with NLRP3 inflammasome, （I） in, X is selected from: , , ; R1 is selected from: C6-C 10 Aryl, R5 substituted C6-C 10 Aryl, 6-10 membered heteroaryl, 6-10 membered heteroaryl substituted by R5; R2 is selected from: H; R3 is selected from: C1-C6 alkyl, R6 substituted C1-C6 alkyl, C6-C 10 Aryl, R5 substituted C6-C 10 Aryl; R5 is selected from: hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy; R6 is selected from: C6-C 10 Aryl, hydroxyl, R7 substituted C6-C 10 Aryl; R7 is selected from: hydroxy, halogen, C1-C6 alkyl, C1-C6 alkoxy; The disease associated with NLRP3 inflammasome is colitis.

2. The use according to claim 1, characterized in that: The boric acid compound has a structure shown in the following formula (II): (II).

3. The use according to claim 1 or 2, characterized in that: R1 is selected from the group consisting of phenyl, phenyl substituted with R5, naphthyl, naphthyl substituted with R5, 6-10 membered nitrogen-containing heteroaryl, and 6-10 membered nitrogen-containing heteroaryl substituted with R5.

4. The use according to claim 3, characterized in that: R1 is selected from the group consisting of phenyl, R5-substituted phenyl, naphthyl, R5-substituted naphthyl, pyridyl, R5-substituted pyridyl, quinoxalinyl, R5-substituted quinoxalinyl, pyrazinyl, and R5-substituted pyrazinyl.

5. The use according to claim 4, characterized in that: R1 is selected from: phenyl substituted by R5, pyridyl, pyridyl substituted by R5; wherein R5 is selected from: halogen, C1-C3 alkyl, C1-C3 alkoxy.

6. The use according to claim 4, characterized in that: R1 is selected from: phenyl, 2,5-dichlorophenyl, 2,3-dichlorophenyl, 2,6-dichlorophenyl, 2,4-dichlorophenyl, 5-chloro-2-methoxyphenyl, 2,6-difluorophenyl, 2,4-difluorophenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, 2-fluoro-5-chlorophenyl, 5-fluoro-2-methoxyphenyl, pyridyl, C1-C3 alkyl-substituted pyridyl, 3,6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3 alkyl-substituted pyrazinyl.

7. The use according to claim 1 or 2, characterized in that: R3 is selected from the group consisting of: C1-C4 alkyl, phenyl, naphthyl, hydroxy-substituted C1-C3 alkyl, phenyl-substituted C1-C3 alkyl, naphthyl-substituted C1-C3 alkyl, and 4-hydroxyphenyl-substituted C1-C3 alkyl.

8. The use according to claim 7, characterized in that: R3 is selected from the group consisting of benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, and naphthylmethyl.

9. The use according to claim 1, characterized in that: The boric acid compound is selected from the following compounds: 。 10. A boric acid compound, characterized in that: The boric acid compound is selected from the following compounds: 。 11. A pharmaceutical composition for preventing and treating diseases associated with NLRP3 inflammasome, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the boric acid compound or a pharmaceutically acceptable salt thereof according to claim 10; and the disease associated with NLRP3 inflammasome is colitis.

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