CN114671900B - Boric acid compounds and their applications - Google Patents

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

Boric acid compound and application thereof

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to boric acid compounds and application thereof.

Background

NLRP3 inflammatory corpuscles are NOD-like receptors, consisting of three parts, namely an inflammatory corpuscle sensor molecule (NLRP 3 protein), a linker protein ASC, and an effector molecule caspase-1 precursor protein (Pro-caspase-1), and are a multiprotein complex present in the cytosol. After activation of NLRP3 inflammatory corpuscles, pro-caspase-1 self-cleaves into active caspase-1, further cleavage of Pro-IL-1β and Pro-IL-18 into active interleukin-1β (IL-1β) and interleukin-18 (IL-18), ultimately leading to inflammatory response and apoptosis. Much evidence suggests that many human diseases, such as Alzheimer's disease, gout, multiple sclerosis, type II diabetes, inflammatory bowel disease, etc., are closely associated with NLRP3 inflammatory corpuscles. To date, a variety of NLRP3 inflammatory body inhibitors have been found, but none are clinically useful. Thus, the discovery of new inhibitors of NLRP3 inflammatory bodies is of great importance in the treatment of NLRP3 related diseases.

Disclosure of Invention

In view of the above problems, the invention provides boric acid derivatives which can selectively inhibit activation of NLRP3 inflammatory corpuscles, so that diseases related to NLRP3 inflammatory corpuscles, such as colonitis, can be treated or improved.

The invention comprises the following technical scheme:

The application of boric acid compounds with a structure shown in a formula (I) or pharmaceutically acceptable salts thereof as active ingredients in the preparation of NLRP3 inflammation small body inhibitors,

Wherein X is selected from:

R ₁ is selected from the group consisting of C ₆-C₁₀ aryl, R ₅ substituted C ₆-C₁₀ aryl, 6-10 membered heteroaryl, R ₅ substituted 6-10 membered heteroaryl;

r ₂ is selected from H, C ₁-C₆ alkyl;

R ₃ is selected from H, C ₁-C₆ alkyl, R ₆ substituted C ₁-C₆ alkyl, C ₆-C₁₀ aryl, R ₅ substituted C ₆-C₁₀ aryl;

R ₅ is selected from hydroxy, halogen, C ₁-C₆ alkyl, C ₁-C₆ alkoxy;

R ₆ is selected from C ₆-C₁₀ aryl, hydroxy, R ₇ substituted C ₆-C₁₀ aryl;

R ₇ is selected from hydroxy, halogen, C ₁-C₆ alkyl, C ₁-C₆ alkoxy.

In some embodiments, the boronic acid compound has a structure according to the following formula (II):

In some embodiments, R ₁ is selected from phenyl, R ₅ substituted phenyl, naphthyl, R ₅ substituted naphthyl, 6-10 membered nitrogen containing heteroaryl, R ₅ substituted 6-10 membered nitrogen containing heteroaryl.

In some embodiments, R ₁ is selected from phenyl, R ₅ substituted phenyl, naphthyl, R ₅ substituted naphthyl, pyridyl, R ₅ substituted pyridyl, quinoxalinyl, R ₅ substituted quinoxalinyl, pyrazinyl, R ₅ substituted pyrazinyl.

In some embodiments, R ₁ is selected from the group consisting of R ₅ substituted phenyl, pyridyl, R ₅ substituted pyridyl, wherein R ₅ is selected from the group consisting of halogen, C ₁-C₃ alkyl, C ₁-C₃ alkoxy.

In some embodiments, R ₁ 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, pyridinyl, C ₁-C₃ alkyl-substituted pyridinyl, 3, 6-dichloropyridinyl, quinoxalinyl, pyrazinyl, C ₁-C₃ alkyl-substituted pyrazinyl.

In some embodiments, R ₃ is selected from the group consisting of C ₁-C₄ alkyl, phenyl, naphthyl, hydroxy-substituted C ₁-C₃ alkyl, phenyl-substituted C ₁-C₃ alkyl, naphthyl-substituted C ₁-C₃ alkyl, 4-hydroxyphenyl-substituted C ₁-C₃ alkyl.

In some embodiments, R ₃ is selected from benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl.

In some of these embodiments, when R ₃ is benzyl, R ₁ is not 2, 5-dichlorophenyl, pyridinyl, or quinoxalinyl.

In some embodiments, R ₃ is benzyl, R ₁ 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, C ₁-C₃ alkyl-substituted pyridinyl, 3, 6-dichloropyridyl, pyrazinyl, C ₁-C₃ alkyl-substituted pyrazinyl.

In some embodiments, R ₃ is selected from isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl, R ₁ 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, pyridinyl, C ₁-C₃ alkyl-substituted pyridinyl, 3, 6-dichloropyridinyl, quinoxalinyl, pyrazinyl, C ₁-C₃ alkyl-substituted pyrazinyl.

In some of these embodiments, the boronic acid compound is selected from the group consisting of:

The boric acid compound or the pharmaceutically acceptable salt thereof is applied to the preparation of medicines for preventing and/or treating diseases related to NLRP3 inflammation small body.

In some of these embodiments, the disease associated with NLRP3 inflammasome is alzheimer's disease, gout, multiple sclerosis, type II diabetes, inflammatory bowel disease.

In some of these embodiments, the disease associated with NLRP3 inflammatory bodies is peritonitis and colitis.

In some of these embodiments, the disease associated with NLRP3 inflammatory bodies is acute peritonitis and colitis.

A pharmaceutical composition for preventing and treating diseases related to NLRP3 inflammation small body is prepared from active ingredients and pharmaceutically acceptable auxiliary materials, wherein the active ingredients comprise the boric acid compound or pharmaceutically acceptable salts thereof.

The boric acid derivative or the pharmaceutically acceptable salt thereof provided by the invention can selectively inhibit the activation of NLRP3 inflammatory small bodies, so that diseases related to the NLRP3 inflammatory small bodies, such as colonitis, can be treated or improved, and can be used for preparing therapeutic drugs for diseases related to the NLRP3 inflammatory small bodies.

Drawings

FIG. 1 is a graph showing the results of specific inhibition of NLRP3 inflammatory activation in vitro by compound 25.

FIG. 2 is a graph showing the results of compound 25 inhibiting ASC oligomerization and ASC-NLRP3 interaction.

Fig. 3 is a graph showing the results of compound 25 in improving Dextran Sodium Sulfate (DSS) induced colitis.

Detailed Description

In the compounds of the present invention, when any variable (e.g., R ⁵, etc.) occurs more than once in any component, the definition of each occurrence is independent of the definition of each other occurrence. Also, combinations of substituents and variables are permissible provided that such combinations stabilize the compounds. The lines drawn from the substituents into the ring system indicate that the bond referred to may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any suitable carbon atom adjacent to the ring. It is to be understood that substituents and substitution patterns of the compounds of this invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that may be readily synthesized from readily available starting materials by techniques in the art and methods set forth below. If the substituent itself is substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, as long as the structure is stabilized.

The term "alkyl" as used herein is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, the definition of "C ₁-C₆" in "C ₁-C₆ alkyl" includes groups having 1,2,3, 4, 5, or 6 carbon atoms in a straight or branched chain arrangement. For example, "C ₁-C₆ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl.

The term "alkoxy" as used herein refers to a group having the structure of an-O-alkyl group, such as -OCH₃、-OCH₂CH₃、-OCH₂CH₂CH₃、-O-CH₂CH(CH₃)₂、-OCH₂CH₂CH₂CH₃、-O-CH(CH₃)₂ and the like.

The term "heteroaryl" as used herein refers to an aromatic ring containing 1 or more heteroatoms selected from O, N or S, which may be monocyclic, bicyclic or polycyclic, including, for example, but not limited to, quinolinyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, and the like, and "heteroaryl" is also understood to include any N-oxide derivative of a heteroaryl containing nitrogen. The attachment of the heteroaryl group may be through a carbon atom or through a heteroatom.

As understood by those skilled in the art, "halo" or "halogen" as used herein means chlorine, fluorine, bromine and iodine.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The starting materials in the following examples may be obtained commercially, or prepared by methods known in the art, or prepared according to the methods described herein.

The synthetic route of the compound of the invention is as follows:

Example 1 preparation of (pyrazine-2-carbonyl) -L-phenylalanyl-L-leucine (compound 1):

(1) 2-Carboxylic acid pyrazine (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, stirred, after 10 minutes N, N-diisopropylethylamine (DIPEA, 3.0 equiv.) was added and reacted for 3 hours at room temperature, TLC monitored the reaction solution was washed with 10% hydrochloric acid solution, 5% NaHCO ₃ and saturated saline respectively, dried over anhydrous sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and the residue was separated by column chromatography to give the corresponding ester.

(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) and the reaction was stirred for 2 hours, monitored by TLC until the starting material disappeared. Acidification with 6M hydrochloric acid, extraction with dichloromethane, drying over anhydrous sodium sulphate and removal of the solvent under reduced pressure gives 512mg of carboxylic acid after hydrolysis (63.4% yield in two steps).

(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℃and EDCI (435 mg,2.27 mmol) and L-leucine methyl ester (270 mg,2.06 mmol) were added, respectively, stirred for 15 minutes, and DIPEA was added dropwise to the reaction solution and stirred for further 1 hour, and reacted at room temperature for 3 hours. With dichloromethaneThe organic phase was washed with 10% citric acid, 5% nahco ₃, and saturated brine, dried over anhydrous Na ₂SO₄, and the solvent was removed under reduced pressure to give an oil.

(4) The oil (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 until the starting material was complete, acidifying the reaction solution with 6M hydrochloric acid, extracting three times with dichloromethane, combining the organic phases, drying over anhydrous sodium sulfate, removing the solvent under reduced pressure, precipitating a white solid 130mg, yield 47％.¹H 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);¹³C NMR(100MHz,Methanol-d₄)δ174.37,171.81,163.31,147.40,144.32,143.48,143.29,136.61,129.19,128.10,126.54,54.24,50.80,40.32,37.87,24.63,22.08,20.49;HRMS(ESI)calcd for C₁₄H₁₉BCl₂N₂O₄[M+H]⁺385.1870,found 385.1967.

EXAMPLE 2 preparation of (2, 5-dichlorobenzoyl) -L-phenylalanyl-L-leucine (Compound 2)

Referring to the procedure of example 1, a white solid was obtained in yield 39％.¹HNMR(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);¹³C NMR(100MHz,Methanol-d₄)δ174.56,171.86,166.79,137.21,137.12,132.38,131.06,130.71,129.21,129.14128.50,128.12,126.48,54.83,50.86,40.50,37.25,24.60,22.15,20.52;HRMS(ESI)calcd for C₁₄H₁₉BCl₂N₂O₄[M+H]⁺451.1186,found 451.1190.

EXAMPLE 3 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (Compound 3)

The intermediate carboxylic acid compound was obtained by the method of example 1, the resulting carboxylic acid (300 mg,0.89 mmol) and HOBT (132 mg,0.98 mmol) were added to dichloromethane, the mixture was cooled to-10 ℃, EDCI (188 mg,0.98 mmol) was added and stirred for 10 minutes, and further (R) -1-amino-3-methylbutylboronic acid pinanediol ester trifluoroacetate (372 mg,0.98 mmol) was added and stirring was continued for 15 minutes. Finally, DIPEA (3 eq.) was added dropwise to the reaction solution, stirred at-10 ℃ for 1 hour, and reacted at room temperature for 5 hours. By using The organic phase was washed with 10% citric acid, 5% nahco ₃, and saturated brine, dried over anhydrous Na ₂SO₄, and the solvent was removed under reduced pressure to give 600mg of an oil. The resulting oil was dissolved in methanol, and isobutyl boronic acid (175 mg,1.71 mmol), n-hexane (4 mL) and 1M HCl (2 mL) were added and reacted overnight. The TCL monitors the reaction and the methanol phase and the n-hexane phase are separated using a separating funnel. The n-hexane phase was extracted twice with methanol, the methanol was removed under reduced pressure, an appropriate amount of water was added, 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, followed by column chromatography to give compound 3 as a white solid in the yield 36％.¹H 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);¹³C NMR(100MHz,Methanol-d₄)δ175.62,166.83,136.83,135.62,132.52,131.22,130.97,129.31,129.24,129.11,128.55,128.41,126.99,51.66,39.54,37.20,25.32,22.61,20.54;HRMS(ESI)calcd for C₂₁H₂₅BCl₂N₂O₄[M+Na]⁺473.1177,found 473.1170.

EXAMPLE 4 preparation of (R) - (1- (4- ((2, 5-dichlorobenzoylamino) methyl) benzoylamino) -3-methylbutyl) boronic acid (Compound 4)

Referring to the procedure of example 3, a white solid was obtained in yield 25％.¹HNMR(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);¹³C 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 C₂₀H₂₃BCl₂N₂O₄[M+Na]⁺459.1020,found 459.1013.

EXAMPLE 5 preparation of (R) - (1- (2, 5-dichloro-N-methylbenzamide) acetamide) -3-methylbutyl) boronic acid (Compound 5)

Referring to the procedure of example 3, a white solid was obtained in yield 13％.¹H 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);¹³C 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 C₁₅H₂₁BCl₂N₂O₄[M+Na]⁺397.0864,found 397.0853.

EXAMPLE 6 preparation of((R) -1- ((S) -2- ((2, 5-dichlorophenyl) sulfonamide) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 6)

Referring to the procedure of example 3, a white solid was obtained in yield 41％.¹H NMR(400MHz,Methanol-d₄)δ¹H NMR(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);¹³C NMR(100MHz,Methanol-d₄)δ175.46,139.17,135.24,133.41,133.35,132.57,130.18,129.98,128.96,128.27,126.95,55.20,39.35,38.01,25.42,22.52,20.70;HRMS(ESI)calcd for C₁₄H₁₉BCl₂N₂O₄[M+Na]⁺509.0864,found 509.0840.

EXAMPLE 7 preparation of (R) - (1- (2, 5-dichlorobenzamide) -3-methylbutyl) boronic acid (Compound 7)

2, 5-Dichlorobenzoyl chloride (300 mg,144 mmol) and HOBT (254 mg,1.88 mmol) were added to a dichloromethane solvent, stirred at-10℃and EDCI (360 mg,1.88 mmol) was added thereto, stirred for 10 minutes, further (R) -1-amino-3-methylbutylboronic acid pinanediol ester trifluoroacetate (540 mg,1.42 mmol) was added thereto and stirred for 15 minutes, and finally DIPEA was added dropwise to the reaction solution, stirred at-10℃for 1 hour, and then reacted at room temperature for 5 hours. With DCMThe organic phase was washed with 10% citric acid, 5% nahco ₃, and saturated brine, dried over anhydrous Na ₂SO₄, and the solvent was removed under reduced pressure to give an oil. The above compound was dissolved in methanol, and isobutyl boric acid (348 mg,3.42 mmol), n-hexane (6 mL) and 1M HCl (3 mL) were added and reacted overnight. The TCL monitors the reaction and the methanol phase and the n-hexane phase are separated using a separating funnel. The n-hexane phase was extracted twice with methanol, the methanol was removed under reduced pressure, 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, followed by column chromatography to give compound 8 as a white solid in the yield 64％.¹H NMR(400MHz,Methanol-d₄)δ¹H 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);¹³C 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 for C₁₄H₁₉BCl₂N₂O₄[M+Na]⁺326.0493,found 326.0491.

EXAMPLE 9 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -3-methylbutanamide) -3-methylbutyl) boronic acid (Compound 9)

Referring to the procedure of example 3, a white solid was obtained in yield 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);¹³C 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 C₁₇H₂₅BCl₂N₂O₄[M+Na]⁺425.1177,found 425.1171.

EXAMPLE 10 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -3-methylbutanamide) -3-methylbutyl) boronic acid (Compound 10)

Referring to the procedure of example 3, a white solid was obtained in yield 38％.¹HNMR(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 C₁₈H₂₇BCl₂N₂O₄[M+Na]⁺439.1333,found 439.1326.

EXAMPLE 11 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -2-phenylacetamide) -3-methylbutyl) boronic acid (Compound 11)

Referring to the procedure of example 3, a white solid was obtained in yield 47％.¹HNMR(400MHz,Methanol-d₄)δ7.51(d,J＝7.8Hz,4H),7.41(d,J＝20.5Hz,4H),5.84(d,J＝10.8Hz,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);¹³C 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 C₂₀H₂₃BCl₂N₂O₄[M+Na]⁺459.1020,found 459.1018.

EXAMPLE 12 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -4-methylpentanamide) -3-methylbutyl) boronic acid (Compound 12)

Referring to the procedure of example 3, a white solid was obtained in yield 34％.¹HNMR(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),1.80–1.71(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);¹³C NMR(100MHz,Methanol-d₄)δ177.20,167.16,137.02,132.62,131.24,130.97,129.24,128.50,48.56,39.87,25.72,24.55,22.49,21.71,21.00,20.51;HRMS(ESI)calcd for C₁₈H₂₇BCl₂N₂O₄[M+Na]⁺439.1333,found 439.1326.

EXAMPLE 13 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -3-hydroxypropyl) -3-methylbutyl) boronic acid (Compound 13)

Referring to the procedure of example 3, a white solid was obtained in yield 35％.¹HNMR(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,2H),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);¹³C 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 for C₁₅H₂₁BCl₂N₂O₄[M+Na]⁺413.0813,found 413.0809.

EXAMPLE 14 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -3- (4-hydroxyphenyl) propionamide) -3-methylbutyl) boronic acid (Compound 14)

Referring to the procedure of example 3, a white solid was obtained in yield 40％.¹HNMR(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);¹³C 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 C₂₁H₂₅BCl₂N₂O₅[M+Na]⁺489.1226,found 489.1119.

EXAMPLE 15 preparation of((R) -1- ((S) -2- (2, 5-dichlorobenzamide) -3- (naphthalen-1-yl) propanamide) -3-methylbutyl) boronic acid (Compound 15)

Referring to the procedure of example 3, a white solid was obtained in yield 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);¹³C 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 C₂₅H₂₇BCl₂N₂O₄[M+Na]⁺523.1325,found 523.1333.

EXAMPLE 16 preparation of((R) -3-methyl-1- ((S) -3-phenyl-2- (pyridylamido) propanamido) butyl) boronic acid (Compound 16)

Referring to the procedure of example 3, a white solid was obtained in yield 46％.¹HNMR(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＝4.8Hz,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)δ175.80,165.09,148.90,148.55,137.52,135.69,129.22,128.41,126.96,126.81,121.96,51.51,39.55,37.55,25.35,22.51,20.74;HRMS(ESI)calcd for C₂₀H₂₆BN₃O₄[M+H]⁺384.2089,found 384.2089.

EXAMPLE 17 preparation of((R) -3-methyl-1- ((S) -2- (6-methylpyrazine-2-carboxamide) -3-phenylpropionamido) butyl) boronic acid (Compound 17)

Referring to the procedure of example 3, a white solid was obtained in yield 42％.¹H 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),2.66(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);¹³C NMR(100MHz,Methanol-d₄)δ175.54,163.96,153.46,147.41,143.09,140.16,135.66,129.24,128.42,126.98,51.42,39.54,37.33,26.64,25.36,22.47,20.79,20.06;HRMS(ESI)calcd for C₂₀H₂₇BN₄O₄[M+H]⁺399.2198,found 399.2385.

EXAMPLE 18 preparation of((R) -3-methyl-1- ((S) -3-phenyl-2- (quinoxaline-2-carboxamido) propanamido) butyl) boronic acid (Compound 18)

Referring to the procedure of example 3, a white solid was obtained in yield 35％.¹H 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);¹³C 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 C₂₃H₂₇BN₄O₄[M+H]⁺435.2198,found 435.2410.

EXAMPLE 19 preparation of((R) -1- ((S) -2- (3, 6-dichloropyridinamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (Compound 19)

Referring to the procedure of example 3, a white solid was obtained in yield 39％.¹HNMR(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 for C₂₀H₂₄BCl₂N₃O₄[M+H]⁺432.1310,found 452.1318.

EXAMPLE 20 preparation of((R) -1- ((S) -2- (2, 3-dichlorobenzamido) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 20)

Referring to the procedure of example 3, a white solid was obtained in yield 36％.¹HNMR(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);¹³C 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 for C₂₁H₂₅BCl₂N₂O₄[M+Na]⁺473.1177,found 473.1168.

EXAMPLE 21 preparation of((R) -1- ((S) -2- (2, 6-dichlorobenzamide) -3-methylbutanamide) -3-methylbutyl) boronic acid (Compound 21)

Referring to the procedure of example 3, a white solid was obtained in yield 47％.¹H 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);¹³C NMR(100MHz,Methanol-d₄)δ175.34,165.56,135.50,135.35,131.91,131.03,129.25,128.42,127.85,127.85,126.93,51.29,39.52,37.46,25.34,22.66,20.50;HRMS(ESI)calcd for C₂₁H₂₅BCl₂N₂O₄[M+Na]⁺473.1177,found 473.1169.

EXAMPLE 22 preparation of((R) -1- ((S) -2- (2, 4-dichlorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (Compound 22)

Referring to the procedure of example 3, a white solid was obtained in yield 48％.¹HNMR(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,1H),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 C₂₁H₂₅BCl₂N₂O₄[M+Na]⁺473.1177,found 473.1169.

EXAMPLE 23 preparation of((R) -1- ((S) -2- (5-chloro-2-methoxybenzamide) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 23)

Referring to the procedure of example 3, a white solid was obtained in yield 46％.¹H 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),3.87(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);¹³C NMR(100MHz,Methanol-d₄)δ175.90,164.96,156.55,135.57,132.74,130.29,129.27,128.50,127.09,125.81,122.29,113.57,55.72,51.84,39.58,37.46,25.40,22.54,20.76;HRMS(ESI)calcd for C₂₂H₂₈BClN₂O₅[M+Na]⁺469.1672,found 469.1664.

EXAMPLE 24 preparation of((R) -1- ((S) -2- (5-fluoro-2-methoxybenzamide) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 24)

Referring to the procedure of example 3, a white solid was obtained in yield 52％.¹HNMR(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);¹³C 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 C₂₂H₂₈BClN₂O₅[M+Na]⁺453.1968,found 453.1953.

EXAMPLE 25 preparation of((R) -1- ((S) -2- (2, 6-difluorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (compound 25)

Referring to the procedure of example 3, a white solid was obtained in yield 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);¹³C NMR(100MHz,Methanol-d₄)δ176.84,162.99,160.96(dd,J＝250.9,7.1Hz),136.74,133.39(t,J＝10.1Hz),130.54,129.73,128.30,112.83(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 C₂₁H₂₅BF₂N₂O₄[M+Na]⁺441.1768,found 441.1757.

EXAMPLE 26 preparation of((R) -1- ((S) -2- (2, 4-difluorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (compound 26)

Referring to the procedure of example 3, a white solid was obtained in yield 43％.¹HNMR(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),105.51(t,J＝26.6Hz),53.28,40.85,38.50,26.63,23.94,21.90;HRMS(ESI)calcd for C₂₁H₂₅BF₂N₂O₄[M+Na]⁺441.1768,found 441.1759.

EXAMPLE 27 preparation of((R) -1- ((S) -2- (2, 3-difluorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (compound 27)

Referring to the procedure of example 3, a white solid was obtained in yield 38％.¹H 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＝260.0Hz,13.0Hz),148.11(dd,J＝251.6,11.0Hz),135.60,129.21,128.42,126.99,124.78(dd,J＝26.2,11.0Hz),124.66(d,J＝1.4Hz),124.61(dd,J＝11.6,4.6Hz),119.67(d,J＝17.5Hz),51.97,39.52,37.15,25.30,22.60,20.56;HRMS(ESI)calcd for C₂₁H₂₅BF₂N₂O₄[M+Na]⁺441.1768,found 441.1756.

EXAMPLE 28 preparation of((R) -1- ((S) -2- (2, 5-difluorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (compound 28)

Referring to the procedure of example 3, a white solid was obtained in yield 39％.¹HNMR(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);¹³C 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＝24.3,7.6Hz),120.83(dd,J＝24.5,9.2Hz),119.06(dd,J＝26.1,8.3Hz),117.52(dd,J＝26.0,3.0Hz),53.31,40.86,38.51,26.64,23.92,21.92;HRMS(ESI)calcd for C₂₁H₂₅BF₂N₂O₄[M+Na]⁺441.1768,found 441.1755.

EXAMPLE 29 preparation of((R) -1- ((S) -2- (2-fluoro-5-chlorobenzamide) -3-phenylpropionamide) -3-methylbutyl) boronic acid (Compound 29)

Referring to the procedure of example 3, a white solid was obtained in yield 35％.¹HNMR(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).;¹³C 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 C₂₁H₂₅BClFN₂O₄[M+Na]⁺457.1472,found 457.1462.

EXAMPLE 30 in vitro study of the inhibition of NLRP3 inflammatory corpuscles by boronic acid derivatives

J774a.1 cells were plated on 96-well plates of about 5×10 ⁵ cells each, plated 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, then boric acid derivatives were added at different concentrations (10 μΜ, 5 μΜ,1 μΜ, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125 nM) for 1 hour, nigericin (NIGERICIN, 10 μΜ) was added for 1 hour, and then the cell supernatant was collected, and the IL-1 β content was measured using the Mouse IL-1 β ELISA kit to calculate the inhibition of the present compounds on NLRP3 inflammatory bodies, as shown in table 1.

TABLE 1

Example 31 specific inhibition of activation of NLRP3 inflammasome by Compound 25 in vitro

1. NLRP3 inflammatory body activation and IL-1β detection by dividing J774A.1 cells or Mouse Bone Marrow Derived Macrophages (BMDMs) into 96 well plates, 5×10 ⁵ cells per well, plating overnight, discarding the supernatant, adding 100 μl of DMEM medium containing bacterial Lipopolysaccharide (LPS) (1 μg/ml) containing 10% serum per well, then adding different concentrations (7.5 nM, 15nM, 30nM, 60 nM) of compound 25 for 1h, then adding nigericin (NIGERICIN, 10 μM) for 1h, then collecting cell supernatant, and measuring IL-1β content using Mouse IL-1β ELISA kit.

2. Western immunoblot analysis J774A.1 or BMDMs cell samples treated in step 1 were lysed in RIPA lysis buffer with protease inhibitor at 4℃for 30min. Proteins in the lysate or supernatant were separated with a 12% SDS-polyacrylamide gel, transferred to PVDF membrane, and subjected to Western blot analysis with anti-murine IL-1. Beta. Antibody, anti-ASC antibody, anti-casepase-1 antibody, anti-NLRP 3 antibody, anti-beta-actin antibody.

3. NLRC4 and AIM2 inflammatory body activation and IL-1β detection for NLRC4 or AIM2 inflammatory body activation, J774A.1 cells were stimulated with 1. Mu.g/mL LPS for 5h, then treated with different concentrations of (1. Mu.M, 2. Mu.M, 5. Mu.M) compound 25 for 1h, then cells were infected with bacterial flagellin (FLA-ST Ultrapure) (2.5. Mu.g/mL) for 4h, or transfected with poly (dA: dT) (0.25. Mu.g/mL) for 4h, after which cell supernatants were collected and IL-1β content was determined using the Mouse IL-1β ELISA kit.

Results as shown in fig. 1, compound 25 can inhibit secretion of IL-1β in concentration-dependent manner in the NLRP3 inflammatory body-activated j774a.1 and BMDMs cell models (a and B in fig. 1). Western blot experiments showed that the effect of compound 25 on inhibition of caspase-1 (p 20) maturation and IL-1β secretion was dose dependent, but did not affect pro-IL-1β, pro-caspase-1, NLRP3 or ASC (C in FIG. 1) in cell lysates. At the same time, compound 25 had no inhibitory effect on activation of either AIM2 or NLRC4 inflammatory bodies (D, E in fig. 1). The above results indicate that compound 25 can specifically inhibit caspase-1 activation and IL-1β secretion that are dependent on the inflammatory bodies of NLRP 3.

EXAMPLE 32 Compound 25 inhibits the assembly and activation of NLRP3 inflammatory corpuscles

1. ASC oligomerization was detected by chemical cross-linking by plating J774A.1 cells onto 6-well plates, adding 1mL of DMEM medium containing 10% serum containing bacterial Lipopolysaccharide (LPS) (1. Mu.g/mL), then adding different concentrations of (15 nM, 60 nM) compound 25 for 1h, then adding nigericin (NIGERICIN, 10. Mu.M) for 1h, discarding the supernatant, washing twice with pre-chilled D-PBS, adding 500. Mu.L of NP-40 lysate per well, and lysing on ice for 30min. Cells from each well were scraped and aspirated into a 1.5mL EP tube. Preparation of Input group at 4℃at 12000rpm for 15min, the supernatant was aspirated into new EP tubes and the concentration of protein per tube was quantitatively determined by BCA. After quantitative determination, put-20 preserving the refrigerator with the temperature of C. The precipitated proteins were washed twice with PBS, after which 500. Mu.L of disuccinimide suberate (DSS) solution in PBS was added to each tube at a concentration of 2mM, incubated at room temperature for 30min with spin at 5000rpm for 15min, centrifuged and the supernatant was discarded. mu.L of Ttis solution in PBS at a concentration of 2mM was added to quench unreacted complete DSS and incubated for 15min at room temperature with rotation. Centrifugation was performed at 5000rpm for 15min, the supernatant was discarded, 1X Loading Buffer was added to the pellet, carefully vortexed and then boiled in a metal bath at 100℃for 10min, placed in a-20℃refrigerator for use, and the content of ASC protein in the oligomeric state was analyzed by Western blotting, as shown in FIG. 2A.

2. Co-immunoprecipitation to examine the interaction of NLRP3 with ASC cells were plated in five dishes, 5mL of DMEM medium containing 10% serum containing bacterial Lipopolysaccharide (LPS) (1. Mu.g/mL) was added, then different concentrations of (15 nM, 60 nM) compound 25 were added for 1h, then nigericin (NIGERICIN, 10. Mu.M) was added for 1h, the supernatant was discarded, washed twice with pre-chilled D-PBS, 500. Mu.L of NP-40 lysate was added per dish, and lysed on ice for 30min. Cells from each well were scraped and aspirated into a 1.5mL EP tube. Preparation of Input group at 4℃at 12000rpm, centrifugation for 15min, pipetting part of the supernatant in the new EP tube and quantifying by BCA the concentration of protein per tube. The remaining supernatant was pipetted into a new 1.5mLEP tube with a corresponding volume of cell lysate in an amount of 500 μg protein. And adding NP40 lysate replenishing system respectively. Cell lysates were prepurified by adding 20. Mu.L of ProteinA/G magnetic beads per tube and incubating for 6h in a-4℃refrigerator with rotation. At 4 ℃,2500rpm,5min, centrifuged and the supernatant carefully aspirated into a new EP tube. Immunoprecipitation by adding IgG antibodies to IgG samples, the remainder of the addition of the corresponding IP-desired antibodies, and incubation overnight at 4 ℃. mu.L of ProteinA/G beads were added to each tube and incubated for 4h with rotation in a-4℃refrigerator. Centrifugation was performed at 4℃at 2500rpm for 5min, washing with pre-chilled D-PBS buffer 5 times, and during washing the sample was placed on ice throughout the course and the supernatant was carefully aspirated without touching the pellet. Sample analysis 20. Mu.L of 1X Loading Buffer was added per tube, carefully vortexed. Centrifuge at 12000rpm for 1min. The samples were cooked in a metal bath at 100 ℃ for 10min, and were loaded by microcentrifugation, and the content of different proteins was analyzed by western blotting, as shown in fig. 2B.

As shown in fig. 2, compound 25 can inhibit ASC oligomerization in a concentration-dependent manner in the NLRP3 inflammatory body-activated j774a.1 cell model (a in fig. 2). There was a clear interaction between NLRP3 and ASC seen in the nigericin-treated group, whereas in the high dose group the interaction between NLRP3 and ASC was significantly reduced. Illustrating that compound 25 inhibits the interaction of NLRP3 and ASC after inhibiting ASC oligomerization (B in fig. 2), this suggests that compound 25 inhibits the assembly and activation of NLRP3 inflammatory bodies.

Example 33 Compound 25 improves sodium dextran sulfate (DSS) induced colitis

1. Female C57BL/6 mice from 6 to 8 weeks were divided into 5 groups of 6 mice each, and the specific grouping treatment was as follows:

A first group comprising administering distilled water in a diet on days 1-10 with daily gavage of vehicle;

a second group of distilled water administered on a 1-3 day diet, 2.5% dss of distilled water administered daily on the diet starting on day four, with daily gavage of vehicle;

a third group consisting of feeding distilled water on a diet of days 1-3, feeding distilled water of 2.5% DSS on a diet every day on a fourth day, and feeding gastric compound 25 (0.125 mg/kg) every three days;

A fourth group, distilled water was administered on the diet of days 1-3, distilled water of 2.5% DSS was administered daily on the diet beginning on day four, and the gastric lavage compound 25 (0.25 mg/kg) every three days;

A fifth group, distilled water was administered on the diet of days 1-3, distilled water of 2.5% DSS was administered daily on the diet beginning on day four, and the gastric lavage compound 25 (0.5 mg/kg) was administered every three days;

2. the hematochezia and body weight of each group of mice were monitored daily starting from the day prior to DSS administration, and the colon of the mice was taken on day 11 for length measurement and the IL-1 β content in the colon was determined.

Results as shown in fig. 3, from the results of fig. 3, the severity of hematochezia in mice increased after 2.5% dss administration in the diet, and the colon length of mice was shortened, and IL-1 beta in the colon was significantly increased, and the gastric lavage compound 25 could dose-dependently improve hematochezia in mice, improve colon length shortening, and lower IL-1 beta levels in the colon.

The activity test result shows that the boric acid compound disclosed by the invention has the activity of inhibiting NLRP3 inflammatory corpuscles on a tested cell J774A.1. Meanwhile, the representative compound 25 can selectively inhibit activation of NLRP3 inflammation small body and improve colitis induced by DSS, so that the boric acid compound has application in treating NLRP3 inflammation small body related diseases.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the following embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (11)

1. The application of boric acid compounds with a structure shown in a formula (I) or pharmaceutically acceptable salts thereof as active ingredients in preparing medicines for preventing and/or treating diseases related to NLRP3 inflammation small body,

(I)

Wherein X is selected from:、、;

R ₁ is selected from the group consisting of C ₆-C₁₀ aryl, R ₅ substituted C ₆-C₁₀ aryl, 6-10 membered heteroaryl, R ₅ substituted 6-10 membered heteroaryl;

R ₂ is selected from H;

R ₃ is selected from C ₁-C₆ alkyl, R ₆ substituted C ₁-C₆ alkyl, C ₆-C₁₀ aryl, R ₅ substituted C ₆-C₁₀ aryl;

R ₅ is selected from hydroxy, halogen, C ₁-C₆ alkyl, C ₁-C₆ alkoxy;

R ₆ is selected from C ₆-C₁₀ aryl, hydroxy, R ₇ substituted C ₆-C₁₀ aryl;

R ₇ is selected from hydroxy, halogen, C ₁-C₆ alkyl, C ₁-C₆ alkoxy;

the disease associated with NLRP3 inflammatory bodies is colitis.

2. The use according to claim 1, wherein the boric acid compound has a structure represented by the following formula (II):

(II)。

3. The method according to claim 1 or 2, wherein R ₁ is selected from the group consisting of phenyl, R ₅ substituted phenyl, naphthyl, R ₅ substituted naphthyl, 6-10 membered nitrogen containing heteroaryl, and R ₅ substituted 6-10 membered nitrogen containing heteroaryl.

4. The method of claim 3, wherein R ₁ is selected from the group consisting of phenyl, R ₅ substituted phenyl, naphthyl, R ₅ substituted naphthyl, pyridyl, R ₅ substituted pyridyl, quinoxalinyl, R ₅ substituted quinoxalinyl, pyrazinyl, R ₅ substituted pyrazinyl.

5. The method of claim 4, wherein R ₁ is selected from the group consisting of R ₅ substituted phenyl, pyridyl, R ₅ substituted pyridyl, and wherein R ₅ is selected from the group consisting of halogen, C ₁-C₃ alkyl, and C ₁-C₃ alkoxy.

6. The method according to claim 4, wherein R ₁ is selected from the group consisting of 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, C ₁-C₃ alkyl-substituted pyridyl, 3, 6-dichloropyridyl, quinoxalinyl, pyrazinyl, C ₁-C₃ alkyl-substituted pyrazinyl.

7. The method according to claim 1 or 2, wherein R ₃ is selected from the group consisting of C ₁-C₄ alkyl, phenyl, naphthyl, hydroxy-substituted C ₁-C₃ alkyl, phenyl-substituted C ₁-C₃ alkyl, naphthyl-substituted C ₁-C₃ alkyl, and 4-hydroxyphenyl-substituted C ₁-C₃ alkyl.

8. The method of claim 7, wherein R ₃ is selected from the group consisting of benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, and naphthylmethyl.

9. Use according to claim 1, characterized in that the boric acid-based compound is selected from the following compounds:

。

10. boric acid compound, characterized in that the boric acid compound is selected from the following compounds:

。

11. the pharmaceutical composition for preventing and treating diseases related to NLRP3 inflammation small body is characterized by being prepared from an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises the boric acid compound or pharmaceutically acceptable salt thereof according to claim 10, and the diseases related to NLRP3 inflammation small body are colonitis.