CN114671900A - Boric acid compound and application thereof - Google Patents

Boric acid compound and application thereof Download PDF

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CN114671900A
CN114671900A CN202210451312.9A CN202210451312A CN114671900A CN 114671900 A CN114671900 A CN 114671900A CN 202210451312 A CN202210451312 A CN 202210451312A CN 114671900 A CN114671900 A CN 114671900A
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substituted
alkyl
dichlorophenyl
difluorophenyl
phenyl
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胡文辉
杨忠金
孙平
吴欣忆
陈秀会
熊兮
华磊
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Guangzhou Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Abstract

The invention provides a boric acid compound shown in formula (I) or a pharmaceutically acceptable salt thereof, and application of the boric acid compound as an active ingredient in preparation of an NLRP3 inflammation corpuscle inhibitor. The compounds can selectively inhibit the activation of NLRP3 inflammasome, thereby treating or improving diseases related to NLRP3 inflammasome, such as: colitis and thus can be used to prepare an inflammasome phase with NLRP3A medicine for treating diseases.

Description

Boric acid compound and application thereof
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a boric acid compound and application thereof.
Background
The NLRP3 inflammasome is an NOD-like receptor, consists of an inflammasome sensor molecule (NLRP3 protein), a linker protein ASC and an effector molecule caspase-1 precursor protein (Pro-caspase-1), and is a multi-protein complex existing in cytoplasm. Upon activation of the NLRP3 inflammasome, Pro-caspase-1 self-cleaves to active caspase-1, further cleaving Pro-IL-1 β and Pro-IL-18 to active interleukin-1 β (IL-1 β) and interleukin-18 (IL-18), ultimately leading to an inflammatory response and apoptosis of cells. There is a great deal of evidence that many human diseases, such as alzheimer's disease, gout, multiple sclerosis, type II diabetes, inflammatory bowel disease, etc., are closely associated with the NLRP3 inflammasome. To date, several NLRP3 inflammasome inhibitors have been discovered, but none have been clinically useful. Therefore, the discovery of a novel inhibitor of NLRP3 inflammasome is of great significance for the treatment of NLRP 3-related diseases.
Disclosure of Invention
In view of the above problems, the present invention provides boronic acid derivatives capable of selectively inhibiting the activation of NLRP3 inflammasome, thereby treating or ameliorating diseases associated with NLRP3 inflammasome, such as: colitis (colitis).
The invention comprises the following technical scheme:
the application of the boric acid compound with the structure shown in the formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an NLRP3 inflammation corpuscle inhibitor,
Figure BDA0003617276860000011
wherein X is selected from:
Figure BDA0003617276860000012
R1selected from: c6-C10Aryl radical, R5Substituted C6-C10Aryl, 6-10 membered heteroaryl, R5Substituted 6-10 membered heteroaryl;
R2selected from: H. c1-C6An alkyl group;
R3selected from: H. c1-C6Alkyl radical, R6Substituted C1-C6Alkyl radical, C6-C10Aryl, R5Substituted C6-C10An aryl group;
R5selected from: hydroxy, halogen, C1-C6Alkyl radical, C1-C6An alkoxy group;
R6selected from: c6-C10Aryl, hydroxy, R7Substituted C6-C10An aryl group;
R7selected from: hydroxy, halogen, C1-C6Alkyl radical, C1-C6An alkoxy group.
In some of these embodiments, the boronic acid compound has the structure shown in formula (II) below:
Figure BDA0003617276860000021
in some of these embodiments, R1Selected from: phenyl, R5Substituted phenyl, naphthyl, R5Substituted naphthyl, 6-10 membered nitrogen containing heteroaryl, R5Substituted 6-10 membered nitrogen containing heteroaryl.
In some of these embodiments, R1Selected from: phenyl, R5Substituted phenyl, naphthyl, R5Substituted naphthyl, pyridyl, R5Substituted pyridyl, quinoxalinyl, R5Substituted quinoxalinyl, pyrazinyl, R5A substituted pyrazinyl group.
In some of these embodiments, R1Selected from: r5Substituted phenyl, pyridyl, R5A substituted pyridyl group; wherein R is5Selected from: halogen, C1-C3Alkyl radical, C1-C3An alkoxy group.
In some of these embodiments, R1Selected 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-C3Alkyl-substituted pyridyl, 3, 6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3An alkyl-substituted pyrazinyl group.
In some of these embodiments, R3Selected from: c1-C4Alkyl, phenyl, naphthyl, hydroxy-substituted C1-C3Alkyl, phenyl substituted C1-C3Alkyl, naphthyl substituted C1-C3Alkyl, 4-hydroxyphenyl substituted C1-C3An alkyl group.
In some of these embodiments, R3Selected from: benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl.
In some of these embodiments, when R3When it is benzyl, R1Other than 2, 5-dichlorophenyl, pyridinyl, and quinoxalinyl.
In some of these embodiments, R3Is benzyl; r1Selected 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-C3Alkyl substituted pyridyl, 3, 6-dichloropyridyl, pyrazinyl, C1-C3An alkyl-substituted pyrazinyl group.
In some of these embodiments, R3Selected from: isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl; r1Selected 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-C3Alkyl-substituted pyridyl, 3, 6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3An alkyl-substituted pyrazinyl group.
In some of these embodiments, the boronic acid compound is selected from the following:
Figure BDA0003617276860000031
Figure BDA0003617276860000041
the application of the boric acid compound or the pharmaceutically acceptable salt thereof in preparing the medicine for preventing and/or treating the diseases related to the NLRP3 inflammasome.
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 inflammasome is peritonitis and colitis.
In some of these embodiments, the disease associated with NLRP3 inflammasome is acute peritonitis and colitis.
A pharmaceutical composition for preventing and treating diseases related to NLRP3 inflammasome is prepared from active ingredients and pharmaceutically acceptable auxiliary materials, wherein the active ingredients comprise the boric acid compounds or pharmaceutically acceptable salts thereof.
The boronic acid derivative or the pharmaceutically acceptable salt thereof provided by the invention can selectively inhibit the activation of NLRP3 inflammasome, so that diseases related to NLRP3 inflammasome can be treated or improved, such as: colitis, thus can be used for preparing the therapeutic drug for the diseases related to NLRP3 inflammasome.
Drawings
Figure 1 is a graph of the results of compound 25 specifically inhibiting activation of NLRP3 inflammasome in vitro.
FIG. 2 is a graph of the results of compound 25 inhibiting ASC oligomerization and ASC-NLRP3 interaction.
Figure 3 is a graph of the results of compound 25 in ameliorating Dextran Sodium Sulfate (DSS) -induced colitis.
Detailed Description
In the compounds of the invention, when any variable (e.g. R)5Etc.) occur more than one time in any constituent, then the definition of each occurrence is independent of the definition of each other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds. The line drawn from a substituent into the ring system indicates that the indicated bond can be attached to any ring atom that can be substituted. If the ring system is polycyclic, it means that such a bond is only attached to any suitable carbon atom of the adjacent ring. It is to be understood that substituents and substitution patterns on the compounds of the present invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by those skilled in the art and by the methods set forth below from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stable.
The term "alkyl" as used herein is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "C1-C6Alkyl radical "middle" C1-C6The definition of "includes groups having 1, 2,3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. For example, "C1-C6Alkyl "specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.
The term "alkoxy" as used herein refers to a group having the structure-O-alkyl, such as-OCH3、-OCH2CH3、-OCH2CH2CH3、-O-CH2CH(CH3)2、-OCH2CH2CH2CH3、-O-CH(CH3)2And 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 aromatic ring may be monocyclic, bicyclic or polycyclic, including, for example, but not limited to: quinolyl, pyrazolyl, pyrrolyl, thienyl, furyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, and the like; "heteroaryl" is also understood to include any N-oxide derivative of a nitrogen-containing heteroaryl group. 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 "halo" as used herein means chloro, fluoro, bromo, and iodo.
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 embodiments and materials described herein are intended to be exemplary only.
The starting materials in the following examples are commercially available or prepared by methods known in the art or according to the methods described herein.
The synthetic route of the compounds of the invention is as follows:
Figure BDA0003617276860000061
example 1: preparation of (pyrazine-2-carbonyl) -L-phenylalanyl-L-leucine (Compound 1):
Figure BDA0003617276860000062
(1) pyrazine-2-carboxylate (300mg,2.4mmol), methyl-L-phenylalanine (400mg,2.42mmol) and HATU (920mg, 2.42 mm)ol), dissolving in dichloromethane, stirring, adding N, N-diisopropylethylamine (DIPEA, 3.0equiv.) after 10min, reacting at room temperature for 3 h, monitoring the reaction by TLC, and using 10% hydrochloric acid solution and 5% NaHCO solution respectively3Washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, evaporating solvent under reduced pressure, and separating the residue by column chromatography to obtain corresponding ester.
(2) The above ester (product of step 1) was added to a solution of LiOH (3.0equiv.) in methanol-water (3: 1,4mL), the reaction was stirred for 2 hours and monitored by TLC until the starting material disappeared. Acidification with 6M hydrochloric acid, extraction with dichloromethane, drying over anhydrous sodium sulfate and removal of the solvent under reduced pressure gave 512mg (63.4% yield over two steps) of the hydrolyzed carboxylic acid.
(3) The above carboxylic acid (product of step 2) and HOBT (306mg,2.27mmol) were added to dichloromethane, the mixture was cooled to-10 deg.C, EDCI (435mg,2.27mmol) and L-leucine methyl ester (270mg,2.06mmol) were added thereto, respectively, stirring was carried out for 15 minutes, DIPEA was added dropwise to the reaction solution and stirring was continued for 1 hour and the reaction was carried out at room temperature for 3 hours. With dichloromethane
Figure BDA0003617276860000071
Extraction, organic phase using 10% citric acid, 5% NaHCO3Washed with saturated brine and anhydrous Na2SO4Drying and removal of the solvent under reduced pressure gave an oil.
(4) The oil (product of step 3) was added to a methanol-water (3: 1,4mL) solution of LiOH (3.0equiv.) and stirred for 2 hours. TLC was monitored until the reaction was complete, the reaction was acidified with 6M hydrochloric acid, extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to precipitate 130mg of a white solid in 47% yield.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)
Figure BDA0003617276860000072
Referring to the procedure of example 1, a white solid was obtained in 39% yield.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)
Figure BDA0003617276860000073
Referring to the carboxylic acid compound of the intermediate obtained in the procedure of example 1, the resulting carboxylic acid (300mg, 0.89mmol) and HOBT (132mg, 0.98mmol) were added to methylene chloride, the mixture was cooled to-10 deg.C, EDCI (188mg, 0.98mmol) was added and stirred for 10 minutes, and (R) -1-amino-3-methylbutylboronic acid pinanediol ester trifluoroacetate (372mg, 0.98mmol) was added and stirring was continued for 15 minutes. Finally, DIPEA (3eq.) was added dropwise to the reaction solution, and the mixture was stirred at-10 ℃ for 1 hour and reacted at room temperature for 5 hours. By using
Figure BDA0003617276860000081
Figure BDA0003617276860000082
Extraction, organic phase using 10% citric acid, 5% NaHCO3Washing with saturated brine, anhydrous Na2SO4Drying and removal of the solvent under reduced pressure gave 600mg of oil. The resulting oil was dissolved in methanol, and isobutylboronic acid (175mg, 1.71mmol), n-hexane (4mL) and 1M HCl (2mL) were added and reacted overnight. The reaction was monitored by TCL, and the methanol phase and the n-hexane phase were separated by a separatory 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 water was removed with anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure and subjected to column chromatography to give compound 3 as a white solid in a yield of 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-dichlorobenzamido) methyl) benzamido) -3-methylbutyl) boronic acid (Compound 4)
Figure BDA0003617276860000083
Referring to the procedure of example 3, a white solid was obtained with a yield of 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- (2, 5-dichloro-N-methylbenzamide) acetamide) -3-methylbutyl) boronic acid (Compound 5)
Figure BDA0003617276860000084
Referring to the procedure of example 3, a white solid was obtained with a yield of 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) sulfonylamino) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 6)
Figure BDA0003617276860000091
Referring to the procedure of example 3, a white solid was obtained in 41% yield.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)
Figure BDA0003617276860000092
2, 5-Dichlorobenzoyl chloride (300mg,144mmol) and HOBT (254mg,1.88mmol) were added to a dichloromethane solvent, reacted at-10 ℃ with stirring, EDCI (360mg,1.88mmol) was added, stirred for 10 minutes, then (R) -1-amino-3-methylbutylboronic acid pinanediol ester trifluoroacetate (540mg,1.42mmol) was added and stirred for 15 minutes, finally DIPEA was added dropwise to the reaction solution, stirred at-10 ℃ for 1 hour, and then reacted at room temperature for 5 hours. Using DCM
Figure BDA0003617276860000093
Extraction, organic phase using 10% citric acid, 5% NaHCO3Washed with saturated brine and anhydrous Na2SO4Drying and removal of the solvent under reduced pressure gave an oil. The above compound was dissolved in methanol, and isobutylboronic acid (348mg, 3.42mmol), n-hexane (6mL) and 1M HCl (3mL) were added to react overnight. The reaction was monitored by TCL, and the methanol phase and the n-hexane phase were separated by a separatory 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 water was removed with anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure, and separation by column chromatography gave compound 8 as a white solid in 64% yield.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-methylbutylamide) -3-methylbutyl) boronic acid (Compound 9)
Figure BDA0003617276860000101
Referring to the procedure of example 3, a white solid was obtained in 42% yield.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-methylbutylamide) -3-methylbutyl) boronic acid (Compound 10)
Figure BDA0003617276860000102
Referring to the procedure of example 3, a white solid was obtained in 38% yield.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)
Figure BDA0003617276860000111
Referring to the procedure of example 3, a white solid was obtained in 47% yield.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)
Figure BDA0003617276860000112
Referring to the procedure of example 3, a white solid was obtained in 34% yield.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-hydroxypropionamide) -3-methylbutyl) boronic acid (Compound 13)
Figure BDA0003617276860000113
Referring to the procedure of example 3, a white solid was obtained with a yield of 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)
Figure BDA0003617276860000121
Referring to the procedure of example 3, a white solid was obtained with a yield of 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)
Figure BDA0003617276860000122
Reference example3 to give a white solid in 47% yield.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) propionamido) butyl) boronic acid (Compound 16)
Figure BDA0003617276860000131
Referring to the procedure of example 3, a white solid was obtained in 46% yield.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-carboxamido) -3-phenylpropionamido) butyl) boronic acid (Compound 17)
Figure BDA0003617276860000132
Referring to the procedure of example 3, a white solid was obtained in 42% yield.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) propionamido) butyl) boronic acid (compound 18)
Figure BDA0003617276860000133
Referring to the procedure of example 3, a white solid was obtained with a yield of 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)
Figure BDA0003617276860000141
Referring to the procedure of example 3, a white solid was obtained,the yield was 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)
Figure BDA0003617276860000142
Referring to the procedure of example 3, a white solid was obtained with a yield of 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-methylbutylamide) -3-methylbutyl) boronic acid (Compound 21)
Figure BDA0003617276860000151
Referring to the procedure of example 3, a white solid was obtained in 47% yield.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)
Figure BDA0003617276860000152
Referring to the procedure of example 3, a white solid was obtained in 48% yield.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-methoxybenzamido) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 23)
Figure BDA0003617276860000161
Referring to the procedure of example 3, a white solid was obtained in 46% yield.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-methoxybenzamido) -3-phenylpropionamido) -3-methylbutyl) boronic acid (Compound 24)
Figure BDA0003617276860000162
Referring to the procedure of example 3, a white solid was obtained in 52% yield.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)
Figure BDA0003617276860000171
By the method of reference example 3, obtainedWhite solid, 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)
Figure BDA0003617276860000172
Referring to the procedure of example 3, a white solid was obtained in 43% yield.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)
Figure BDA0003617276860000181
Referring to the procedure of example 3, a white solid was obtained in 38% yield.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)
Figure BDA0003617276860000182
Referring to the procedure of example 3, a white solid was obtained in 39% yield.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)
Figure BDA0003617276860000191
Referring to the procedure of example 3, a white solid was obtained with a yield of 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 on inhibitory effect of boronic acid derivatives on NLRP3 inflammatory bodies
J774A.1 cells were plated onto 96-well plates at approximately 5X 10 per well5The cells were plated overnight, the supernatant was discarded, 100. mu.L of DMEM medium containing 10% serum containing bacterial Lipopolysaccharide (LPS) (1. mu.g/ml) was added to each well, followed by treatment with different concentrations (10. mu.M, 5. mu.M, 1. mu.M, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125nM) of the boronic acid derivative for 1 hour, followed by treatment with Nigericin (10. mu.M) for 1 hour, and cell supernatants were collected and assayed for IL-1. beta. content using a Mouse IL-1. beta. ELISA kit to calculate the inhibitory effect of the compounds of the present invention on NLRP3 inflammasome, the results of which are shown in Table 1.
TABLE 1
Figure BDA0003617276860000192
Figure BDA0003617276860000193
Figure BDA0003617276860000201
Figure BDA0003617276860000211
Example 31 Compound 25 specifically inhibits activation of NLRP3 inflammasome in vitro
1. NLRP3 inflammasome activation and IL-1 β detection: J774A.1 cells or mouse bone marrow-derived macrophages (BMDMs) were plated onto 96-well plates at 5X 10 wells per well5Cells were plated overnight, supernatants were discarded, and 100. mu.L of 10% serum-containing DMEM medium containing bacterial Lipopolysaccharide (LPS) (1. mu.g/ml) was added to each well, followed by treatment with different concentrations (7.5nM, 15nM, 30nM, 60nM) of compound 25 for 1h, followed by treatment with Nigericin (10. mu.M) for 1h, after which cell supernatants were collected and assayed for IL-1. beta. content using the Mouse IL-1. beta. ELISA kit.
2. Western blot analysis: the J774A.1 cell or BMDMs cell sample treated in step 1 is lysed in RIPA lysis buffer with protease inhibitor at 4 ℃ for 30 min. Proteins in the lysate or supernatant were separated on a 12% SDS-polyacrylamide gel, transferred to a PVDF membrane, and subjected to Western blot analysis with anti-murine IL-1. beta. antibody, anti-ASC antibody, anti-caspase-1 antibody, anti-NLRP 3 antibody, anti-beta-actin antibody.
3. Activation of NLRC4 and AIM2 inflammasome and IL-1 β assay for NLRC4 or AIM2 inflammasome activation J774A.1 cells were stimulated with 1 μ g/mL LPS for 5h, then treated with varying concentrations of (1 μ M, 2 μ M, 5 μ M) compound 25 for 1h, and then cells were infected with bacterial flagellin (FLA-ST Ultrapure) (2.5 μ g/mL) for 4h, or transfected with poly (dA: dT) (0.25 μ g/mL) for 4h, after which cell supernatants were collected and assayed for IL-1 β content using the Mouse IL-1 β ELISA kit.
The results are shown in figure 1, compound 25 can inhibit IL-1 β secretion concentration-dependently in NLRP3 inflammasome-activated j774a.1 and BMDMs cell models (a and B in figure 1). Western blot experiments showed that the effect of compound 25 in inhibiting caspase-1(p20) maturation and IL-1 β secretion was dose-dependent, but did not affect pro-IL-1 β, pro-caspase-1, NLRP3 or ASC (C in FIG. 1) in the cell lysates. At the same time, compound 25 had no inhibitory effect on the activation of AIM2 or NLRC4 inflammasome (D, E in fig. 1). The above results indicate that compound 25 can specifically inhibit NLRP3 inflammasome-dependent caspase-1 activation and IL-1 β secretion.
Example 32: compound 25 inhibits the assembly and activation of NLRP3 inflammatory bodies
1. The method for detecting ASC oligomerization by chemical cross-linking comprises the steps of spreading J774A.1 cells on a 6-well plate, adding 1mL of DMEM medium containing 10% serum and bacterial Lipopolysaccharide (LPS) (1 mu g/mL), adding compounds 25 with different concentrations (15nM and 60nM) for treatment for 1h, adding Nigericin (Nigericin and 10 mu M) for treatment for 1h, removing supernatant, washing twice by precooled D-PBS, adding 500 mu L of NP-40 lysate into each well, and lysing for 30min on ice. Cells from each well were scraped and pipetted into a 1.5mL EP tube. Preparation of Input group: the supernatant was aspirated at 12000rpm for 15min at 4 ℃ and the protein concentration per tube was quantified by BCA in fresh EP tubes. And (4) after quantification, storing in a refrigerator at the temperature of-20 ℃. The precipitated proteins were washed twice with PBS, after which 500. mu.L of disuccinimidyl suberate (DSS) solution in PBS at a concentration of 2mM was added to each tube, incubated at room temperature for 30min with rotation, centrifuged at 5000rpm for 15min, and the supernatant was discarded. mu.L of Ttis solution in PBS at a concentration of 2mM was added to quench unreacted DSS and incubated at room temperature for 15min with rotation. Centrifuging at 5000rpm for 15min, discarding supernatant, adding 1 × Loading Buffer into the precipitate, carefully vortexing, boiling in 100 deg.C metal bath for 10min, placing in-20 deg.C refrigerator for use, and analyzing the content of ASC protein in oligomeric state by Western blotting, the result is shown in A in FIG. 2.
2. Co-immunoprecipitation detection of NLRP3 interaction with ASC: cells were plated on five large dishes and lysed for 30min on ice by adding 5mL of 10% serum in DMEM medium containing bacterial Lipopolysaccharide (LPS) (1. mu.g/mL), then adding 25 compounds at different concentrations (15nM, 60nM) for 1h, then adding Nigericin (10. mu.M) for 1h, discarding the supernatant, washing twice with pre-cooled D-PBS, and adding 500. mu.L of NP-40 lysate per dish. Cells from each well were scraped and pipetted into a 1.5mL EP tube. Preparation of Input group: the supernatant was centrifuged at 12000rpm for 15min at 4 ℃ and the protein concentration per tube was quantified by BCA in fresh EP tubes. The remaining supernatant was pipetted with a corresponding volume of cell lysate to a protein amount of 500. mu.g into a new 1.5mLEP tube. NP40 lysate was added separately to complete the system. Pre-purifying cell lysate: mu.L of ProteinA/G magnetic beads were added to each tube and incubated at-4 ℃ for 6h with rotation. 4 ℃, 2500rpm, 5min, centrifuged and the supernatant carefully pipetted into a new EP tube. And (3) immunoprecipitation: IgG antibodies were added to the IgG samples, the remaining antibodies required for the corresponding IP were added, and the incubation was performed overnight at 4 ℃ with rotation. mu.L of ProteinA/G magnetic beads were added to each tube and incubated at-4 ℃ for 4h with rotation in a refrigerator. Centrifuge at 2500rpm for 5min at 4 ℃, wash 5 times with pre-cooled D-PBS buffer, place the sample on ice all the way through while washing, and carefully aspirate the supernatant without colliding with the pellet. And (3) sample analysis: add 20. mu.L of 1 × Loading Buffer to each tube and vortex carefully. Centrifuge at 12000rpm for 1 min. The samples were boiled in a metal bath at 100 ℃ for 10min, and then subjected to microcentrifugation, and the contents of different proteins were analyzed by western blotting, and the results are shown in B in FIG. 2.
The results are shown in figure 2, compound 25 can inhibit ASC oligomerization concentration-dependently in NLRP3 inflammasome-activated j774a.1 cell model (a in figure 2). A clear interaction between NLRP3 and ASC was seen in the nigericin treated group, whereas in the high dose group, the interaction between NLRP3 and ASC was significantly reduced. Indicating that compound 25 inhibits the interaction of NLRP3 and ASC (B in figure 2) after inhibiting ASC oligomerization, indicating that compound 25 inhibits the assembly and activation of NLRP3 inflammasome.
Example 33: compound 25 ameliorates Dextran Sodium Sulfate (DSS) -induced colitis
1. Female C57BL/6 mice at 6-8 weeks were divided into 5 groups of 6 mice each, and the specific groups were treated as follows:
a first group: administering distilled water to the diet on days 1-10, with daily gavage of vehicle;
second group: diet with distilled water on days 1-3, diet with 2.5% DSS distilled water daily beginning on day four with daily gavage of vehicle;
third group: distilled water was given to the diet on days 1-3, 2.5% DSS distilled water was given to the diet daily beginning on day four, and compound 25(0.125mg/kg) was gavaged every three days;
fourth group, distilled water was given on days 1-3, 2.5% DSS on a daily basis beginning on day four, and compound 25(0.25mg/kg) was gavaged every third day;
a fifth group, on days 1-3, with distilled water on the diet, and on day four with 2.5% DSS on a daily basis, and compound 25(0.5mg/kg) was gavaged every three days;
2. the degree of hematochezia and body weight of each group of mice were monitored daily starting the day before 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.
As shown in FIG. 3, from the results of FIG. 3, it can be seen that after 2.5% DSS administration in the diet, the mice had increased fecal blood severity and had shortened colon length and significantly increased IL-1 β in the colon, and that gavage compound 25 can dose-dependently improve fecal blood, improve shortened colon length, and reduce IL-1 β levels in the colon.
The activity test result shows that the boric acid compound of the invention has the activity of inhibiting NLRP3 inflammatory bodies on tested J774A.1 cells. While representative compound 25 can selectively inhibit activation of NLRP3 inflammasome while ameliorating DSS-induced colitis, it can be seen that the boronic acid compounds of the present invention have utility for the treatment of NLRP3 inflammasome-related diseases.
The technical features of the above-mentioned embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the following embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the present description.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (17)

1. The application of the boric acid compound with the structure shown in the formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an NLRP3 inflammation corpuscle inhibitor,
Figure FDA0003617276850000011
wherein X is selected from:
Figure FDA0003617276850000012
R1selected from: c6-C10Aryl radical, R5Substituted C6-C10Aryl, 6-10 membered heteroaryl, R5Substituted 6-10 membered heteroaryl;
R2selected from: H. c1-C6An alkyl group;
R3selected from: H. c1-C6Alkyl, R6Substituted C1-C6Alkyl radical, C6-C10Aryl radical, R5Substituted C6-C10An aryl group;
R5selected from: hydroxy, halogen, C1-C6Alkyl radical, C1-C6An alkoxy group;
R6selected from: c6-C10Aryl, hydroxy, R7Substituted C6-C10An aryl group;
R7selected from: hydroxy, halogen, C1-C6Alkyl radical, C1-C6An alkoxy group.
2. The use according to claim 1, wherein the boronic acid compound has the structure shown in formula (II) below:
Figure FDA0003617276850000013
3. use according to claim 1 or 2, wherein R is1Selected from: phenyl, R5Substituted phenyl, naphthyl, R5Substituted naphthyl, 6-10 membered nitrogen containing heteroaryl, R5Substituted 6-10 membered nitrogen containing heteroaryl.
4. Use according to claim 3, wherein R is1Selected from: phenyl, R5Substituted phenyl, naphthyl, R5Substituted naphthyl, pyridyl, R5Substituted pyridyl, quinoxalinyl, R5Substituted quinoxalinyl, pyrazinyl, R5A substituted pyrazinyl group.
5. Use according to claim 4, wherein R is1Selected from: r5Substituted phenyl, pyridyl, R5A substituted pyridyl group; wherein R is5Selected from: halogen, C1-C3Alkyl radical, C1-C3An alkoxy group.
6. Use according to claim 4, wherein R is1Selected 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-C3Alkyl-substituted pyridyl, 3, 6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3An alkyl-substituted pyrazinyl group.
7. Use according to claim 1 or 2, wherein R is3Selected from: c1-C4Alkyl, phenyl, naphthyl, hydroxy-substituted C1-C3Alkyl, phenyl substituted C1-C3Alkyl, naphthyl substituted C1-C3Alkyl, 4-hydroxyphenyl substituted C1-C3An alkyl group.
8. Use according to claim 7, wherein R is3Selected from: benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl.
9. Use according to claim 1, characterized in that the boronic acid compound is selected from the following compounds:
Figure FDA0003617276850000021
Figure FDA0003617276850000031
10. use of a boronic acid compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention and/or treatment of a disease associated with the NLRP3 inflammasome.
11. The use according to claim 10, wherein the disease associated with NLRP3 inflammasome is alzheimer's disease, gout, multiple sclerosis, type II diabetes, inflammatory bowel disease.
12. The use according to claim 11, wherein the diseases associated with NLRP3 inflammasome are peritonitis and colitis.
13. A boronic acid compound as claimed in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof.
14. The boronic acid compound or pharmaceutically acceptable salt thereof according to claim 13, wherein when R is3When it is benzyl, R1Other than 2, 5-dichlorophenyl, pyridyl, and quinoxalinyl.
15. The boronic acid compound or pharmaceutically acceptable salt thereof according to claim 14, wherein R is3Is benzyl; r1Selected 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-C3Alkyl substituted pyridyl, 3, 6-dichloropyridyl, pyrazinyl, C1-C3An alkyl-substituted pyrazinyl group.
16. The boronic acid compound or pharmaceutically acceptable salt thereof according to claim 14, wherein R is3Selected from: isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl; r1Selected 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-C3Alkyl-substituted pyridyl, 3, 6-dichloropyridyl, quinoxalinyl, pyrazinyl, C1-C3An alkyl-substituted pyrazinyl group.
17. A pharmaceutical composition for the prevention and treatment of NLRP3 inflammasome-related diseases, which is prepared from an active ingredient comprising a boronic acid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient.
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