CN114671900A - 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 preparing an NLRP3 inflammasome inhibitor. The compounds can selectively inhibit the activation of the NLRP3 inflammasome, so that the diseases related to the NLRP3 inflammasome, such as colitis, can be treated or ameliorated, and thus can be used to prepare a therapeutic drug for the diseases related to the NLRP3 inflammasome.

Description

Boric acid compounds and their applications

technical field

The invention relates to the field of medicinal chemistry, in particular to a class of boronic acid compounds and applications thereof.

Background technique

The NLRP3 inflammasome is a NOD-like receptor composed of three parts: the inflammasome sensor molecule (NLRP3 protein), the adaptor protein ASC, and the effector molecule caspase-1 precursor protein (Pro-caspase-1). A multiprotein complex in the cytoplasm. After NLRP3 inflammasome activation, Pro-caspase-1 self-cleaves into active caspase-1, which further cleaves pro-IL-1β and Pro-IL-18 into active interleukin-1β (IL-1β) and Interleukin-18 (IL-18), which ultimately leads to inflammation and pyroptosis. A large body of evidence indicates that many human diseases, such as Alzheimer's disease, gout, multiple sclerosis, type II diabetes, inflammatory bowel disease, etc., are closely related to the NLRP3 inflammasome. So far, a variety of NLRP3 inflammasome inhibitors have been discovered, but none of them are available for clinical use. Therefore, the discovery of new NLRP3 inflammasome inhibitors is of great significance for the treatment of NLRP3-related diseases.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention provides a boronic acid derivative, which can selectively inhibit the activation of NLRP3 inflammasome, so as to treat or improve diseases related to NLRP3 inflammasome, such as colitis.

The present invention includes the following technical solutions:

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

where X is selected from:

R ₁ is selected from: C ₆ -C ₁₀ aryl, R ₅ substituted C ₆ -C ₁₀ aryl, 6-10-membered heteroaryl, R ₅ substituted 6-10-membered heteroaryl;

R ₂ is selected from: H, C ₁ -C ₆ alkyl;

R ₃ is selected from: H, C ₁ -C ₆ alkyl, R ₆ substituted C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, R ₅ substituted C ₆ -C ₁₀ aryl;

R ₅ is selected from: hydroxyl, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy;

R ₆ is selected from: C ₆ -C ₁₀ aryl, hydroxyl, C ₆ -C ₁₀ aryl substituted by R ₇ ;

R ₇ is selected from: hydroxy, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy.

In some embodiments, the boronic acid compound has the structure shown in the following formula (II):

In some of these 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 of these embodiments, R ₁ is selected from: phenyl, R substituted phenyl, naphthyl, R ₅ substituted naphthyl, pyridyl, R ₅ substituted pyridyl, _{quinoxalinyl, R 5 substituted} quinoxalinyl, pyrazinyl, R ₅ substituted pyrazinyl.

In some of these embodiments, R ₁ is selected from: R ₅ substituted phenyl, pyridyl, R ₅ substituted pyridyl; wherein, R ₅ is selected from: halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy.

In some of these embodiments, 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 -C ₃ alkyl substituted pyridyl, 3,6-dichloropyridyl, quinoxalinyl, Pyrazinyl, C ₁ -C ₃ alkyl substituted pyrazinyl.

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

In some of these embodiments, R ₃ is selected from the group consisting of: benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4-hydroxybenzyl, naphthylmethyl.

In some of these embodiments, when _R3 is benzyl, R1 is not 2,5 _- dichlorophenyl, pyridyl, and quinoxalinyl.

In some of these 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 -C ₃ alkyl substituted pyridyl, 3,6-dichloropyridyl, pyrazinyl, C ₁ -C ₃ alkyl Substituted pyrazinyl.

In some of these 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-methoxy Phenyl, 2,6-difluorophenyl, 2,4-difluorophenyl, 2,3-difluorophenyl, 2,5-difluorophenyl, 2-fluoro-5-chlorophenyl, 5 -Fluoro-2-methoxyphenyl, pyridyl, C ₁ -C ₃ alkyl substituted pyridyl, 3,6-dichloropyridyl, quinoxalinyl, pyrazinyl, C ₁ -C ₃ alkane substituted pyrazinyl.

In some of these embodiments, the boronic acid compound is selected from the following compounds:

Use of the boric acid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating NLRP3 inflammasome-related diseases.

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

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

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

A pharmaceutical composition for preventing and treating diseases related to NLRP3 inflammasome, prepared from active ingredients and pharmaceutically acceptable excipients, the active ingredients include the above-mentioned boric acid compounds or pharmaceutically acceptable compounds thereof. Salt.

The boronic acid derivatives or pharmaceutically acceptable salts thereof provided by the present invention can selectively inhibit the activation of NLRP3 inflammasome, so as to treat or improve diseases related to NLRP3 inflammasome, such as colitis, and thus can be used for preparing Therapeutic drugs for diseases associated with the NLRP3 inflammasome.

Description of drawings

Figure 1 is a graph showing the results of compound 25 specifically inhibiting the activation of NLRP3 inflammasome in vitro.

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

Figure 3 is a graph showing the results of compound 25 improving dextran sulfate sodium (DSS)-induced colitis.

Detailed ways

^In the compounds of the present invention, when any variable (eg, R5, etc.) occurs more than once in any component, its definition at each occurrence is independent of the definition at each other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations stabilize the compound. A line drawn into a ring system from a substituent indicates that the indicated bond 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 atoms adjacent to the ring. It will be appreciated that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the present invention to provide compounds that are chemically stable and readily synthesized from readily available starting materials by the skill in the art and the methods set forth below. 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 stabilized.

The term "alkyl" as used herein is meant to include branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, the definition of " _C1 - _C6 " in " _C1 - _C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched chain arrangement. For example, " _C1 - _C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.

The term "alkoxy" as used herein refers to groups having the structure -O _- alkyl, such as _-OCH3 , _-OCH2CH3 , -OCH2CH2CH3, _{-O -} CH2CH _{( CH3 ) 2} , -OCH ₂ CH ₂ CH ₂ CH ₃ , -O-CH(CH ₃ ) ₂ , etc.

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

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

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention. Methods and materials for preferred embodiments described herein are provided for illustrative purposes only.

The starting materials in the following examples can 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 present invention is as follows:

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

(1) Dissolve 2-carboxylate pyrazine (300mg, 2.4mmol), L-phenylalanine methyl ester (400mg, 2.42mmol) and HATU (920mg, 2.42mmol) in dichloromethane, stir, after 10 minutes N,N-diisopropylethylamine (DIPEA, 3.0 equiv.) was added, and the reaction was carried out at room temperature for 3 hours. The reaction was monitored by TLC. The reaction solution was washed with 10% hydrochloric acid solution, 5% NaHCO ₃ and saturated brine, and anhydrous sulfuric acid. Dry over sodium, filter, evaporate the solvent under reduced pressure, and separate the residue by column chromatography to give the corresponding ester.

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

萃取，有机相依次用10％柠檬酸、5％NaHCO₃和饱和食盐水洗涤，无水Na₂SO₄干燥，减压去除溶剂得到油状物。(3) The above carboxylic acid (product of step 2) and HOBT (306 mg, 2.27 mmol) were added to dichloromethane, the mixture was cooled to -10°C, and EDCI (435 mg, 2.27 mmol) and L-leucine were added respectively. acid methyl ester (270 mg, 2.06 mmol), stirred for 15 minutes, DIPEA was added dropwise to the reaction solution, and the stirring was continued for 1 hour, and the reaction was carried out at room temperature for 3 hours. with dichloromethane

After extraction, the organic phase was washed successively with 10% citric acid, 5% NaHCO ₃ and saturated brine, dried over anhydrous Na ₂ SO ₄ , and the solvent was removed under reduced pressure to obtain an oil.

(4) The above 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. Monitored by TLC until the reaction of the raw materials was completed, the reaction solution 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 a white solid 130 mg with a yield of 47%. ¹ H NMR(400MHz,Methanol-d ₄ )δ9.10(s,1H),8.72(d,J=2.6Hz,1H),8.64-8.55(m,1H),7.24(d,J=7.5Hz, 2H), 7.20–7.10 (m, 3H), 4.46 (t, J=7.3Hz, 1H), 3.07 (dd, J=13.9, 8.7Hz, 2H), 1.75–1.65 (m, 1H), 1.63 (t , J=7.2Hz, 2H), 1.34–1.20 (m, 1H), 0.91 (d, J=6.0Hz, 3H), 0.88 (d, J=6.0Hz, 3H); ¹³ C NMR (100MHz, Methanol- d ₄ ) 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, 20.49calc; ₂ N ₂ O ₄ [M+H] ⁺ 385.1870, found385.1967.

Example 2: Preparation of (2,5-dichlorobenzoyl)-L-phenylalanyl-L-leucine (compound 2)

Referring to the method of Example 1, a white solid was obtained with a yield of 39%. ¹ HNMR(400MHz,Methanol-d ₄ )δ7.41-7.38(m,2H),7.33-7.23(m,5H),7.17(d,J=2.3Hz,1H),4.50(t,J=7.5Hz ,1H),3.27(d,J＝4.7Hz,2H),2.94(dd,J＝14.0,10.4Hz,1H),1.84–1.74(m,1H),1.67(t,J＝7.2Hz,2H) ,0.98(d,J=6.3Hz,3H),0.95(d,J=6.6Hz,3H); ¹³ C NMR(100MHz,Methanol-d ₄ )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 forC ₁₄ H ₁₉ BCl ₂ N ₂ O ₄ [M+H] ⁺ 451.0186, found

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

萃取，有机相依次用10％柠檬酸、5％NaHCO₃和饱和食盐水洗涤，无水Na₂SO₄干燥，减压去除溶剂得到600mg油状物。将所得油状物溶解在甲醇中，加入异丁基硼酸(175mg，1.71mmol)、正己烷(4mL)和1M HCl(2mL)，反应过夜。TCL监测反应，用分液漏斗将甲醇相和正己烷相分液。正己烷相用甲醇萃取两次，减压除去甲醇，加入适量的水，水相用二氯甲烷萃取两次，无水硫酸钠除水。减压蒸发除去溶剂，再通过柱色谱分离，得到白色固体化合物3，产率为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.Referring to the method of Example 1, the intermediate carboxylic acid compound was obtained, the obtained carboxylic acid (300 mg, 0.89 mmol) and HOBT (132 mg, 0.98 mmol) were added to dichloromethane, the mixture was cooled to -10 ° C, EDCI (188 mg, 0.98 mmol) was added. 0.98 mmol) and stirred for 10 minutes, then (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 (3eq.) was added dropwise to the reaction solution, stirred at -10°C for 1 hour, and reacted at room temperature for 5 hours. use

After extraction, the organic phase was washed successively with 10% citric acid, 5% NaHCO ₃ and saturated brine, dried over anhydrous Na ₂ SO ₄ , and the solvent was removed under reduced pressure to obtain 600 mg of oil. The obtained oil was dissolved in methanol, and isobutylboronic acid (175 mg, 1.71 mmol), n-hexane (4 mL) and 1M HCl (2 mL) were added and reacted overnight. The reaction was monitored by TCL, and the methanol and n-hexane phases were separated with 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 evaporated under reduced pressure and separated by column chromatography to obtain compound 3 as a white solid in 36% yield. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.42(s,2H),7.34-7.26(m,6H),4.94(t,J=8.1Hz,1H),3.12(d,J=7.9Hz, 2H), 2.66 (t, J=7.8Hz, 1H), 1.38–1.28 (m, 1H), 1.12 (t, J=7.5Hz, 2H), 0.84–0.79 (m, 6H); ¹³ C NMR (100MHz) ,Methanol-d ₄ )δ175.62,166.83,136.83,135.62,132.52,131.22,130.97,129.31,129.24,129.11,128.55,128.41,126.99,51.66,39.54,37.20,25.2; C ₂₁ H ₂₅ BCl ₂ N ₂ O ₄ [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)

Referring to the method of Example 3, a white solid was obtained with a yield of 25%. ¹ HNMR(400MHz,Methanol-d ₄ )δ7.98(d,J=8.4Hz,2H),7.57(d,J=8.1Hz,2H),7.49-7.43(m,3H),4.61(s,2H) ), 2.83 (t, J=7.5Hz, 1H), 1.80–1.69 (m, 1H), 1.42 (t, J=7.3Hz, 2H), 0.95–0.93 (m, 6H); ¹³ C NMR (100MHz, _{Methanol -} d ₄ ) _{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} ; ₂ O ₄ [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)

Referring to the method of Example 3, a white solid was obtained with a yield of 13%. ¹ H NMR(400MHz,Methanol-d ₄ ,rotamers)δ7.37-7.26(m,3H),4.40-3.85(m,2H),2.91(two single peaks,3H),2.80-2.64(m,1H) ,1.62–1.42(m,1H),1.25–1.12(m,2H),0.76–0.73(m,6H); ¹³ C NMR(100MHz,Methanol-d ₄ )δ173.27,168.30,136.33,133.10,130.97,130.71 , 128.38, 127.59, 46.11, 39.48, 36.77, 25.57, 22.33, 20.88; HRMS(ESI) calcd for C ₁₅ H ₂₁ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 397.0864, found 397.0853.

Example 6: ((R)-1-((S)-2-((2,5-dichlorophenyl)sulfonamido)-3-phenylpropionamido)-3-methylbutyl) Preparation of Boric Acid (Compound 6)

Referring to the method of Example 3, a white solid was obtained with a yield of 41%. ¹ H NMR(400MHz,Methanol-d ₄ )δ ¹ HNMR(400MHz,Methanol-d ₄ )δ7.88(d,J=2.5Hz,1H),7.50(dd,J=8.5,2.5Hz,1H), 7.39(d,J＝8.5Hz,1H),7.15-7.04(m,6H),4.23(t,J＝7.8Hz,1H),3.04-2.86(m,2H),2.56(t,J＝7.7Hz ,1H),1.45–1.34(m,1H),1.13(t,J=7.4Hz,2H),0.84–0.81(m,6H); ¹³ C NMR(100MHz,Methanol-d ₄ )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 forC ₁₄ H ₁₉ BCl ₂ N ₂ O ₄ [M ⁺ Na] 509.0864, found 509.0840.

Example 7: Preparation of (R)-(1-(2,5-dichlorobenzamide)-3-methylbutyl)boronic acid (compound 7)

萃取，有机相依次用10％柠檬酸、5％NaHCO₃和饱和食盐水洗涤，无水Na₂SO₄干燥，减压去除溶剂得到油状物。将上述化合物溶解在甲醇中，加入异丁基硼酸(348mg，3.42mmol)、正己烷(6mL)和1M HCl(3mL)，反应过夜。TCL监测反应，用分液漏斗将甲醇相和正己烷相分液。正己烷相用甲醇萃取两次，减压除去甲醇，水相用二氯甲烷萃取两次，无水硫酸钠除水。减压蒸发除去溶剂，再通过柱色谱分离，得到白色固体化合物8，产率为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 forC₁₄H₁₉BCl₂N₂O₄[M+Na]⁺326.0493,found 326.0491.2,5-Dichlorobenzoyl chloride (300 mg, 144 mmol) and HOBT (254 mg, 1.88 mmol) were added to the dichloromethane solvent, and the reaction was stirred at -10 °C, EDCI (360 mg, 1.88 mmol) was added, and the mixture was stirred for 10 minutes. Then add (R)-1-amino-3-methylbutyl borate pinanediol trifluoroacetate (540mg, 1.42mmol) and continue to stir for 15 minutes, and finally DIPEA is added dropwise to the reaction solution, in- The mixture was stirred at 10°C for 1 hour, and then reacted at room temperature for 5 hours. with DCM

After extraction, the organic phase was washed successively with 10% citric acid, 5% NaHCO ₃ and saturated brine, dried over anhydrous Na ₂ SO ₄ , and the solvent was removed under reduced pressure to obtain an oil. The above compound was dissolved in methanol, isobutylboronic acid (348 mg, 3.42 mmol), n-hexane (6 mL) and 1M HCl (3 mL) were added, and the reaction was carried out overnight. The reaction was monitored by TCL, and the methanol and n-hexane phases were separated with 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 the water was removed with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and separated by column chromatography to obtain compound 8 as a white solid in 64% yield. ¹ H NMR (400MHz, Methanol-d ₄ )δ ¹ H NMR (400MHz, Methanol-d ₄ )δ7.60-7.51(m, 3H), 2.96(t, J=7.8Hz, 1H), 1.74-1.67( m, 1H), 1.45–1.38 (m, 2H), 0.93 (d, J=6.6Hz, 6H); ¹³ C NMR (100MHz, Methanol-d ₄ )δ175.73,132.56,131.75,129.53,128.30,39.60,25.73 , 22.35, 21.54, 21.13; HRMS(ESI) calcd for C ₁₄ H ₁₉ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 326.0493, found 326.0491.

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

Referring to the method of Example 3, a white solid was obtained with a yield of 42%. ¹ HNMR(400MHz,Methanol-d ₄ )δ7.46-7.43(m,3H),4.45(d,J=8.6Hz,1H),2.75(dd,J=9.2,6.2Hz,1H),2.15(dd , J=7.8, 5.9Hz, 1H), 1.65 (dd, J=12.4, 5.9Hz, 1H), 1.39–1.30 (m, 2H), 1.07 (d, J=6.8Hz, 3H), 1.01 (d, J=6.7Hz, 3H), 0.93 (d, J=1.8Hz, 3H), 0.91 (d, J=2.1Hz, 3H); ¹³ C NMR (100MHz, Methanol-d ₄ )δ176.04, 167.25, 132.60, 131.20 , 130.92, 129.21, 128.49, 55.96, 39.86, 30.20, 25.69, 22.53, 20.87, 18.08; HRMS(ESI) calcd for C ₁₇ H ₂₅ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 425.1177, found 425.1171.

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

Referring to the method of Example 3, a white solid was obtained with a yield of 38%. ¹ HNMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR (100MHz, Methanol-d ₄ )δ176.06,167.11,137.03,132.48,131.09,130.81,129.07,128.33,54.24,39.73 , 35.81, 25.56, 24.87, 22.46, 20.73, 14.19, 9.34; HRMS(ESI) calcd for C ₁₈ H ₂₇ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 439.1333, found439.1326.

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

Referring to the method of Example 3, a white solid was obtained with a yield of 47%. ¹ HNMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR (100MHz, Methanol-d ₄ )δ175.12,174.96,166.95 , _{136.74,134.84,132.55,131.20,131.01,129.41,128.82,128.74,128.69,127.70,54.60,39.46,25.62,22.47,20.96} ; HRMS(ESI)calcd for C ₂₀ H ₂₃ BCl ₂ N ₂ O Na] ⁺ 459.1020, found 459.1018.

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

Referring to the method of Example 3, a white solid was obtained with a yield of 34%. ¹ HNMR(400MHz,Methanol-d ₄ )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=6.6 Hz, 6H); ¹³ C NMR (100 MHz, Methanol-d ₄ ) 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, , 21.00, 20.51; HRMS(ESI) calcd for C ₁₈ H ₂₇ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 439.1333, found 439.1326.

Example 13: ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-hydroxypropionamide)-3-methylbutyl)boronic acid (Compound 13) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 35%. ¹ HNMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR (100MHz, Methanol-d4) δ175.18, 166.97, 136.57, 132.50, 131.18, 130.97, 129.28, 128.64, 60.69, 52.62, 39.56, 25.57, 22.35, 20.91); HRMS (ESI calcd for C ₁₅ H ₂₁ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 413.0813, found 413.0809.

Example 14: ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-(4-hydroxyphenyl)propionamide)-3-methylbutyl) Preparation of boronic acid (compound 14)

Referring to the method of Example 3, a white solid was obtained with a yield of 40%. ¹ HNMR(400MHz,Methanol-d ₄ )δ7.35-7.29(m,2H),7.27-7.17(m,1H),7.00-6.92(m,2H),6.63-6.56(m,2H),4.73( t, J=7.7Hz, 1H), 2.95–2.80 (m, 2H), 2.51 (dd, J=9.5, 4.4Hz, 1H), 1.20–1.13 (m, 1H), 1.07–0.85 (m, 2H) ,0.71–0.62(m,6H); ¹³ C NMR(100MHz,Methanol-d ₄ )δ175.72,166.72,156.38,136.73,132.40,131.10,130.84,130.16,129.20,128.46,125.81,114.,9,3.51. , 25.18, 22.60, 20.30; HRMS(ESI) calcd for C ₂₁ H ₂₅ BCl ₂ N ₂ O ₅ [M+Na] ⁺ 489.1226, found489.1119.

Example 15: ((R)-1-((S)-2-(2,5-dichlorobenzamide)-3-(naphthalen-1-yl)propionamide)-3-methylbutyl) Preparation of boronic acid (compound 15)

Referring to the method of Example 3, a white solid was obtained with a yield of 47%. ¹ H NMR(400MHz,Methanol-d ₄ )δ7.80-7.76(m,3H),7.70(d,J=1.6Hz,1H),7.42-7.37(m,5H),7.28(t,J=1.2 Hz, 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=6.4 Hz, 3H), 0.44 (d, J=6.4 Hz, 3H); ¹³ C NMR (100 MHz, Methanol-d ₄ ) δ175.68,166.89,136.79,133.57,132.84,132.77,132.51,131.24,131.00,129.30,128.56,128.25,128.18,127.52,127.38,127.00,125.98,125.65,51.54,39.36,37.38,25.10,22.36,20.29；HRMS( ESI) calcd for C ₂₅ H ₂₇ BCl ₂ N ₂ O ₄ [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)

Referring to the method of Example 3, a white solid was obtained with a yield of 46%. ¹ HNMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR(100MHz,Methanol-d ₄ )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 forC ₂₀ H ₂₆ BN ₃ O ₄ [M+H] ⁺ 38 found 384.2089.

Example 17: ((R)-3-Methyl-1-((S)-2-(6-methylpyrazine-2-carboxamido)-3-phenylpropionamido)butyl)boronic acid Preparation of (Compound 17)

Referring to the method of Example 3, a white solid was obtained with a yield of 42%. ¹ H NMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR (100MHz, Methanol-d ₄ )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 C ₂₀ H ₂₇ BN ₄ O ₄ [M+H] ⁺ 399.2198, found 399.2385.

Example 18: ((R)-3-Methyl-1-((S)-3-phenyl-2-(quinoxaline-2-carboxamido)propionamido)butyl)boronic acid (Compound 18 ) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 35%. ¹ H NMR(400MHz,Methanol-d ₄ )δ9.36(s,1H),8.15(d,J=9.8Hz,2H),7.95-7.83(m,2H),7.33-7.25(m,4H), 7.19(s, 1H), 5.08(t, J=7.3Hz, 1H), 3.30-3.22(m, 2H), 2.69(t, J=6.6Hz, 1H), 1.46-1.34(m, 1H), 1.19 (t, J=6.5Hz, 2H), 0.82 (d, J=4.5Hz, 3H), 0.81 (d, J=4.6Hz, 3H); ¹³ C NMR (100MHz, Methanol-d ₄ )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 ; _ _} [M+H] ⁺ 435.2198, found 435.2410.

Example 19: ((R)-1-((S)-2-(3,6-dichloropyridineamide)-3-phenylpropionamide)-3-methylbutyl)boronic acid (Compound 19) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 39%. ¹ HNMR(400MHz,Methanol-d ₄ )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).; ¹³ C NMR (100MHz, Methanol- d ₄ ) 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; N ₃ O ₄ [M+H] ⁺ 432.1310, found 452.1318.

Example 20: ((R)-1-((S)-2-(2,3-dichlorobenzamido)-3-phenylpropionamido)-3-methylbutyl)boronic acid (compound 20) Preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 36%. ¹ HNMR(400MHz,Methanol-d ₄ )δ7.59(dd,J=8.0,1.6Hz,1H),7.33-7.23(m,7H),4.96(t,J=8.1Hz,1H),3.12(dd , J=8.2, 2.4Hz, 2H), 2.67 (t, J=7.6Hz, 1H), 1.36–1.30 (m, 1H), 1.13 (t, J=7.4Hz, 2H), 0.84–0.80 (m, 6H); ¹³ C NMR (100MHz, Methanol-d ₄ )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, 47.31, 6.8.9, 47.31, 4.8.9 47.24, 47.03, 39.54, 37.23, 25.33, 22.63, 20.53; HRMS(ESI) calcd for C ₂₁ H ₂₅ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 473.1177, found 473.1168.

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

Referring to the method of Example 3, a white solid was obtained with a yield of 47%. ¹ H NMR(400MHz,Methanol-d ₄ )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=7.4 Hz, 2H), 0.83–0.79 (m, 6H); ¹³ C NMR (100MHz, Methanol-d ₄ )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. , 37.46, 25.34, 22.66, 20.50; HRMS(ESI) calcd for C ₂₁ H ₂₅ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 473.1177, found 473.1169.

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

Referring to the method of Example 3, a white solid was obtained with a yield of 48%. ¹ HNMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR(100MHz,Methanol-d ₄ )δ175.67,167.38,136.25,135.61,134.06,131.90,129.88,129.47,129.23,128.42,127.08,126.898,5 , 37.24, 25.32, 22.63, 20.54; HRMS(ESI) calcd for C ₂₁ H ₂₅ BCl ₂ N ₂ O ₄ [M+Na] ⁺ 473.1177, found473.1169.

Example 23: ((R)-1-((S)-2-(5-Chloro-2-methoxybenzamido)-3-phenylpropionamido)-3-methylbutyl) Preparation of boronic acid (compound 23)

Referring to the method of Example 3, a white solid was obtained with a yield of 46%. ¹ H NMR(400MHz,Methanol-d ₄ )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＝7.7 Hz,1H),1.43–1.37(m,1H),1.19(t,J=7.4Hz,2H),0.84(d,J=2.2Hz,3H),0.82(d,J=2.1Hz,3H); ¹³ C NMR(100MHz,Methanol-d ₄ )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 C ₂₂ H ₂₈ BClN ₂ O ₅ [M+Na] ⁺ 469.1672, found 469.1664.

Example 24: ((R)-1-((S)-2-(5-fluoro-2-methoxybenzamido)-3-phenylpropionamido)-3-methylbutyl) Preparation of boronic acid (compound 24)

Referring to the method of Example 3, a white solid was obtained with a yield of 52%. ¹ HNMR(400MHz,Methanol-d ₄ )δ7.56(dd,J=9.2,3.3Hz,1H),7.34-7.23(m,6H),7.11(dd,J=9.2,4.2Hz,1H),4.97 (t, J＝7.3Hz, 1H), 3.87(s, 3H), 3.17(dd, J＝7.3, 3.2Hz, 2H), 2.67(t, J＝7.7Hz, 1H), 1.42–1.36(m, 1H), 1.19(t, J=7.4Hz, 2H), 0.84(d, J=2.6Hz, 3H), 0.82(d, J=2.7Hz, 3H); ¹³ C NMR (100MHz, Methanol-d ₄ ) 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), HRMS(ESI)calcd for C ₂₂ H ₂₈ BClN ₂ O ₅ [M+Na] ⁺ 453.1968, found 453.1953.

Example 25: ((R)-1-((S)-2-(2,6-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boronic acid (Compound 25) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 48%. ¹ H NMR(400MHz,Methanol-d4)δ7.50-7.43(m,1H),7.30-7.24(m,5H),7.02(t,J=8.1Hz,2H),4.94(t,J=8.0Hz ,1H),3.11(d,J＝8.2Hz,2H),2.63(t,J＝7.7Hz,1H),1.30–1.25(m,1H),1.08(t,J＝7.4Hz,2H),0.81 (d, J=6.4Hz, 3H), 0.79 (d, J=6.7Hz, 3H); ¹³ C NMR (100MHz, Methanol-d ₄ ) δ 176.84, 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 C ₂₁ H ₂₅ BF ₂ N ₂ O ₄ [M+Na] ⁺ 441.1768, found 441.1757.

Example 26: ((R)-1-((S)-2-(2,4-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boronic acid (Compound 26) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 43%. ¹ HNMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR (100 MHz, Methanol-d ₄ ) δ 177.22, 165.84 (d, J=2.0 Hz), 163.58 (dd, J=260.0, 12.5 Hz), 162.06 (dd, J=252.3, 12.5 Hz) ), 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 C ₂₁ H ₂₅ BF ₂ N ₂ O ₄ [M+Na] ⁺ 441.1768, found 441.1759.

Example 27: ((R)-1-((S)-2-(2,3-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boronic acid (Compound 27) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 38%. ¹ H NMR(400MHz,Methanol-d ₄ )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); ¹³ C NMR (101 MHz, Methanol-d4) δ 175.76, 164.45 (d, J=2.9 Hz), 150.49 (dd, J=260.0 Hz, 13.0 Hz), 148.11 (dd, J=251.6, 11.0 Hz),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=1.4Hz) =17.5Hz), 51.97, 39.52, 37.15, 25.30, 22.60, 20.56; HRMS(ESI) calcd for C ₂₁ H ₂₅ BF ₂ N ₂ O ₄ [M+Na] ⁺ 441.1768, found 441.1756.

Example 28: ((R)-1-((S)-2-(2,5-difluorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boronic acid (Compound 28) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 39%. ¹ HNMR(400MHz,Methanol-d ₄ )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＝24.5,9.2 Hz), 119.06 (dd, J=26.1, 8.3 Hz), 117.52 (dd, J=26.0, 3.0 Hz), 53.31, 40.86, 38.51, 26.64, 23.92, 21.92; HRMS(ESI) calcd for C ₂₁ H ₂₅ BF ₂ N ₂ O ₄ [M+Na] ⁺ 441.1768, found 441.1755.

Example 29: ((R)-1-((S)-2-(2-Fluoro-5-chlorobenzamide)-3-phenylpropionamide)-3-methylbutyl)boronic acid (Compound 29 ) preparation

Referring to the method of Example 3, a white solid was obtained with a yield of 35%. ¹ HNMR(400MHz,Methanol-d4)δ7.58-7.53(m,1H),7.53-7.48(m,1H),7.30-7.19(m,6H),4.93(t,J=7.9Hz,1H), 3.15(d,J＝7.9Hz,2H),2.65(t,J＝7.7Hz,1H),1.37-1.29(m,1H),1.12(t,J＝7.4Hz,2H),0.83-0.79(m , 6H).; ¹³ C NMR (100MHz, Methanol-d ₄ )δ177.02,165.36(d,J=1.8Hz),159.88(d,J=250.8Hz),136.92,134.05(d,J=9.0Hz), 130.97(d,J＝2.9Hz),130.68(d,J＝3.4Hz),130.55,129.76,128.34,125.30(d,J＝16.0Hz),119.14(d,J＝24.8Hz),53.31,40.86, 38.48, 26.64, 23.92, 21.91; HRMS(ESI) calcd for C ₂₁ H ₂₅ BClFN ₂ O ₄ [M+Na] ⁺ 457.1472, found457.1462.

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

Divide J774A.1 cells into 96-well plates, about 5×10 ⁵ cells per well, seed the plates overnight, discard the supernatant, and add 100 μL of bacterial lipopolysaccharide (LPS) (1 μg/ml) containing 10 to each well. DMEM medium with % serum, then add different concentrations (10μM, 5μM, 1μM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125nM) boric acid derivatives for 1 hour, and then add nigericin (Nigericin, 10 μM) was treated for 1 hour, and then the cell supernatant was collected, and the IL-1β content was measured by Mouse IL-1β ELISA kit, and the inhibitory effect of the compounds of the present invention on NLRP3 inflammasome was calculated. The results are shown in Table 1.

Table 1

Example 31: Compound 25 specifically inhibits NLRP3 inflammasome activation in vitro

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

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

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

The results are shown in Figure 1. Compound 25 can concentration-dependently inhibit the secretion of IL-1β in the NLRP3 inflammasome-activated J774A.1 and BMDMs cell models (A and B in Figure 1). Western blot experiments showed that compound 25 inhibited caspase-1 (p20) maturation and IL-1β secretion in a dose-dependent manner, but did not affect pro-IL-1β, pro-caspase-1, NLRP3 or ASC in cell lysates (C in Figure 1). At the same time, compound 25 had no inhibitory effect on AIM2 or NLRC4 inflammasome activation (D, E in Figure 1). The above results indicated that compound 25 could specifically inhibit NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion.

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

1. Detection of ASC oligomerization by chemical cross-linking method: Spread J774A.1 cells on a 6-well plate, add 1 mL of DMEM medium containing bacterial lipopolysaccharide (LPS) (1 μg/ml) containing 10% serum, and then add Different concentrations (15nM, 60nM) of compound 25 were treated for 1 h, then Nigericin (10 μM) was added for 1 h, the supernatant was discarded, washed twice with pre-cooled D-PBS, and 500 μL of NP-40 was added to each well for lysis. solution and lysed on ice for 30 min. The cells from each well were scraped and pipetted into a 1.5 mL EP tube. Preparation of Input group: 4°C, 12000rpm, centrifugation for 15min, pipette the supernatant into a new EP tube and quantify the protein concentration of each tube by BCA. Store in -20°C refrigerator after quantification. The precipitated protein was washed twice with PBS, and then 500 μL of disuccinimidyl suberate (DSS) solution prepared in PBS was added to each tube at a concentration of 2 mM, incubated at room temperature for 30 min by rotation, centrifuged at 5000 rpm for 15 min, and the supernatant was discarded. 500 μL of Ttis solution prepared with PBS was added at a concentration of 2 mM to quench unreacted DSS, and incubated at room temperature with rotation for 15 min. Centrifuge at 5000 rpm for 15 min, discard the supernatant, add 1× Loading Buffer to the pellet, vortex carefully, boil it in a metal bath at 100 °C for 10 min, put it in a -20 °C refrigerator for later use, and analyze ASC protein oligomerization by western blotting The content of the state, the results are shown in A in Figure 2.

2. Co-immunoprecipitation to detect the interaction between NLRP3 and ASC: cells were plated in five large dishes, 5 mL of DMEM medium containing bacterial lipopolysaccharide (LPS) (1 μg/ml) containing 10% serum was added, and then different concentrations were added (15nM, 60nM) of compound 25 was treated for 1h, then Nigericin (10μM) was added for 1h, the supernatant was discarded, washed twice with pre-cooled D-PBS, and 500μL of NP-40 lysis solution was added to each large dish , lysed on ice for 30 min. The cells from each well were scraped and pipetted into a 1.5 mL EP tube. Preparation of Input group: 4°C, 12000rpm, centrifugation for 15min, aspirate part of the supernatant into a new EP tube, and determine the protein concentration of each tube by BCA quantification. Pipette the remaining supernatant into a new 1.5mL EP tube with a volume of cell lysate corresponding to 500 μg of protein. NP40 lysate was added to make up the system. Pre-purification of cell lysate: add 20 μL of ProteinA/G magnetic beads to each tube, and incubate at -4°C with rotation for 6 hours. 4°C, 2500rpm, 5min, centrifuge, carefully pipette the supernatant into a new EP tube. Immunoprecipitation: add IgG antibody to the IgG sample, add the remaining antibody required for IP, and incubate overnight at 4°C with rotation. Add 20 μL of ProteinA/G magnetic beads to each tube and incubate at -4°C with rotation for 4 h. 4°C, 2500 rpm, centrifugation for 5 min, and washed 5 times with pre-cooled D-PBS buffer. During washing, the samples were kept on ice for the whole process, and the supernatant was carefully aspirated so as not to touch the precipitate. Sample analysis: Add 20 μL of 1× Loading Buffer to each tube and vortex carefully. Centrifuge at 12000 rpm for 1 min. The samples were boiled in a metal bath at 100°C for 10 min, and then loaded by microcentrifugation. The contents of different proteins were analyzed by western blotting. The results are shown in B in Figure 2.

The results are shown in FIG. 2 , in the J774A.1 cell model of NLRP3 inflammasome activation, compound 25 inhibited ASC oligomerization in a concentration-dependent manner (A in FIG. 2 ). Significant interaction between NLRP3 and ASC was seen in the nigericin-treated group, while the interaction between NLRP3 and ASC was significantly attenuated in the high-dose group. This indicated that compound 25 inhibited the interaction between NLRP3 and ASC after inhibiting ASC oligomerization (B in Figure 2), which indicated that compound 25 inhibited the assembly and activation of the NLRP3 inflammasome.

Example 33: Compound 25 ameliorates dextran sulfate sodium (DSS)-induced colitis

1. Female C57BL/6 mice at 6-8 weeks were divided into 5 groups, 6 mice in each group. The specific grouping treatment is as follows:

Group 1: Distilled water was given in the diet on days 1-10, while the vehicle was gavaged daily;

Group 2: Distilled water was given in the diet on days 1-3, and distilled water with 2.5% DSS was given in the diet every day from the fourth day, while the vehicle was given daily by gavage;

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

Group 4: Distilled water was given in the diet on days 1-3, and 2.5% DSS of distilled water was given in the diet every day from the fourth day, and compound 25 (0.25 mg/kg) was administered by gavage every three days;

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

2. The blood in the stool and body weight of each group of mice were monitored every day from the day before DSS administration, and on the 11th day, the colons of the mice were taken to measure the length and the content of IL-1β in the colon.

The results are shown in Figure 3. From the results of Figure 3, after 2.5% DSS in the diet, the severity of blood in the stool of the mice increased, and the length of the colon in the mice was shortened, and the IL-1β in the colon was significantly increased. Compound 25 can dose-dependently improve blood in the stool of mice, shorten the length of colon, and reduce the level of IL-1β in the colon.

The activity test results show that the boronic acid compounds of the present invention show the activity of inhibiting the NLRP3 inflammasome on the test cell J774A.1 cells. At the same time, the representative compound 25 can selectively inhibit the activation of NLRP3 inflammasome and improve DSS-induced colitis. It can be seen that the boronic acid compounds of the present invention can be used for the treatment of NLRP3 inflammasome-related diseases.

The technical features of the above-described embodiments can be combined arbitrarily. 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 these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the patent of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (17)

1. the use of a boric acid compound having a structure shown in formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an NLRP3 inflammasome inhibitor,

where X is selected from:

R ₁ is selected from: C ₆ -C ₁₀ aryl, R ₅ substituted C ₆ -C ₁₀ aryl, 6-10-membered heteroaryl, R ₅ substituted 6-10-membered heteroaryl; R ₂ is selected from: H, C ₁ -C ₆ alkyl; R ₃ is selected from: H, C ₁ -C ₆ alkyl, R ₆ substituted C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, R ₅ substituted C ₆ -C ₁₀ aryl; R ₅ is selected from: hydroxyl, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy; R ₆ is selected from: C ₆ -C ₁₀ aryl, hydroxyl, C ₆ -C ₁₀ aryl substituted by R ₇ ; R ₇ is selected from: hydroxy, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy. 2. application according to claim 1, is characterized in that, described boric acid compound has the structure shown in following formula (II):

3. The application according to claim ₁ or ₂ , wherein R is selected from: phenyl, R substituted phenyl, naphthyl, R substituted naphthyl, 6-10 yuan nitrogen _- containing heteroaromatic base, 6-10-membered nitrogen-containing heteroaryl substituted by R ₅ . 4. application according to claim 3 is characterized in that, 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. 5. application according to claim 4 is characterized in that, R ₁ is selected from: the phenyl that R ₅ replaces, pyridyl, the pyridyl group that R ₅ replaces; Wherein, R ₅ is selected from: halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy. 6. The application according to claim 4, wherein 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, pyridyl, C ₁ -C ₃ alkyl substituted pyridyl, 3,6-dichloro Pyridyl, quinoxalinyl, pyrazinyl, _{C1 -} C3 alkyl substituted pyrazinyl. 7. The application according to claim 1 or 2, wherein _R is selected from: C ₁ -C ₄ alkyl, phenyl, naphthyl, hydroxyl substituted C ₁ -C ₃ alkyl, phenyl substituted C ₁ -C ₃ alkyl, naphthyl substituted C ₁ -C ₃ alkyl, 4-hydroxyphenyl substituted C ₁ -C ₃ alkyl. 8. The application according to claim 7, wherein _R is selected from: benzyl, isopropyl, 2-methylpropyl, phenyl, 1-methylpropyl, hydroxymethyl, 4- Hydroxybenzyl, naphthylmethyl. 9. application according to claim 1, is characterized in that, described boric acid compound is selected from following compound:

10. Use of the boric acid compound described in any one of claims 1-9 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating a disease associated with NLRP3 inflammasome. 11. The use according to claim 10, wherein the disease associated with the NLRP3 inflammasome is Alzheimer's disease, gout, multiple sclerosis, type II diabetes, and inflammatory bowel disease. 12. The use according to claim 11, wherein the diseases associated with the NLRP3 inflammasome are peritonitis and colitis. 13. The boric acid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-9. 14. The boric acid compound according to claim 13 or a pharmaceutically acceptable salt thereof, wherein when R ₃ is benzyl, R ₁ is not 2,5-dichlorophenyl, pyridyl, and Quinoxalinyl. 15. The boric acid compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein 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 -C ₃ alkyl substituted pyridyl, 3, 6-Dichloropyridyl, pyrazinyl, C ₁ -C ₃ alkyl substituted pyrazinyl. 16. The boric acid compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein _R is selected from the group consisting of: 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, pyridyl, C ₁ -C ₃ alkyl substituted pyridyl, 3,6-dichloro Pyridyl, quinoxalinyl, pyrazinyl, _{C1 -} C3 alkyl substituted pyrazinyl. 17. A pharmaceutical composition for preventing and treating diseases related to NLRP3 inflammasome, characterized in that, it is prepared from an active ingredient and a pharmaceutically acceptable adjuvant, and the active ingredient comprises any of claims 1-9. The boric acid compound described in one item or a pharmaceutically acceptable salt thereof.

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