A 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound, and a preparation method and application thereof

Abstract

The application discloses a 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound and a preparation method and application thereof, and particularly relates to compounds as shown in general formulae I-A, I-B and I-C and a preparation method of the compounds. Researches show that the compound has significant NLRP3 inflammasome inhibitory activity, and can be used for preparing medicines for treating and / or preventing diseases related to abnormal activation of NLRP3 inflammasome, including but not limited to gouty arthritis, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, type 2 diabetes, Alzheimer's disease and other inflammation-related diseases. The compound has novel structure, good activity and good development and application prospects in medicines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and particularly relates to a 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or a pharmaceutically acceptable salt, as well as its preparation method and application. Background Technology

[0002] The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is an intracellular polyprotein complex that is mainly assembled and activated in monocytes / macrophages, neutrophils, and dendritic cells, serving as a crucial hub for the innate immune system to sense danger signals. Numerous studies have shown that abnormal activation of the NLRP3 inflammasome is closely related to the development and progression of various inflammatory diseases, including gouty arthritis, Alzheimer's disease, and inflammatory bowel disease, making it a significant therapeutic target in the development of anti-inflammatory drugs. However, to date, no NLRP3 inflammasome inhibitors have been approved for marketing. Therefore, developing safe and effective NLRP3 inflammasome inhibitors has significant scientific and clinical application value.

[0003] Tranilast, an anti-asthmatic drug, has a good clinical safety profile and well-defined pharmacokinetic characteristics. However, its current indications are mainly focused on asthma and allergic diseases. There are no reports in the clinical practice of using it or its derivatives for the treatment and / or prevention of diseases related to abnormal activation of the NLRP3 inflammasome.

[0004] Based on this, we are now adopting a "drug repurposing" R&D strategy, using Tranilast, which has a good clinical safety profile, as a lead compound to conduct structural optimization studies, in order to obtain Tranilast derivatives with novel structures and novel indications, so as to effectively treat and / or prevent diseases related to abnormal activation of NLRP3 inflammasomes. Summary of the Invention

[0005] Purpose of the invention: The first objective of this invention is to provide a 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound and its pharmaceutically acceptable salt that has NLRP3 inflammasome inhibitory activity and excellent anti-inflammatory activity.

[0006] A second objective of this invention is to provide a method for preparing the above-mentioned compound.

[0007] A third objective of this invention is the use of the above-mentioned compound as an inhibitor of the NLRP3 inflammasome.

[0008] Technical solution: The present invention relates to 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds, such as compounds represented by general formula IA, and their pharmaceutically acceptable salts:

[0009] ;

[0010] Where R1 is located at any position in the benzene ring 2, 2', 3, 3', 4;

[0011] R1 is independently selected from: hydrogen, alkoxy, hydroxyl, alkyl, haloalkyl, halogen, phenyl, -O-R2, -NH-C=O-R3;

[0012] R2 is independently selected from: substituted or unsubstituted benzyl, substituted or unsubstituted phenethyl, substituted or unsubstituted ethylpyridyl, C 1-5 Saturated or unsaturated alkyl groups;

[0013] R3 is independently selected from: substituted or unsubstituted phenyl, substituted or unsubstituted benzyl.

[0014] Further, R1 is preferably: methoxy, hydroxy, methyl, trifluoromethyl, fluorine, bromine, phenyl, or chlorine; at the same time, when R1 is independently selected from: methoxy, hydroxy, methyl, trifluoromethyl, fluorine, bromine, or phenyl, R1 is located at the meta or para position of the phenyl group, that is, at the 3, 3', or 4 position of the benzene ring in formula IA; when R1 is a halogenated chlorine, R1 is located at the meta, ortho, or para position of the phenyl group, that is, at the 2, 2', 3, 3', or 4 position of the benzene ring in formula IA.

[0015] When R1 is independently selected from methoxy groups, it is even more preferred that R1 is located at the 3 or 3' position of the benzene ring in formula IA.

[0016] When R1 is independently selected from hydroxyl groups, it is even more preferred that R1 is located at the 4-position of the benzene ring in formula IA.

[0017] When R1 is independently selected from methyl, it is even more preferred that R1 is located at the 4-position of the benzene ring in formula IA.

[0018] When R1 is independently selected from trifluoromethyl, it is even more preferred that R1 is located at the 3 or 3' position of the benzene ring in formula IA.

[0019] Furthermore, when R1 is -O-R2, it is as shown in general formula IB:

[0020] ;

[0021] In the formula: R2 is independently selected from: benzyl, phenethyl, 4-ethylpyridinyl, 2-methyl-2-pentenyl, 2-methylpentyl, 4-methylbenzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl.

[0022] Furthermore, when R1 is -NH-C=O-R3, it is as shown in general formula IC:

[0023] ;

[0024] In the formula: R3 is independently selected from: phenyl, 3-chlorophenyl, 4-chlorophenyl, 3-chlorobenzyl, 4-chlorobenzyl.

[0025] Furthermore, the compound is selected from any one of 1 to 35:

[0026] Compound numbering Compound Structure name 1 (E)-2-[(3-phenylprop-2-enoyl)amino]benzoic acid 2 (E)-2-[3-(3-methoxyphenyl)acryloylamino]benzoic acid 3 (E)-2-[3-(4-methoxyphenyl)acryloylamino]benzoic acid 4 (E)-2-[3-(3-hydroxyphenyl)acryloylamino]benzoic acid 5 (E)-2-[3-(4-hydroxyphenyl)acryloylamino]benzoic acid 6 (E)-2-[3-(3-methylphenyl)acryloylamino]benzoic acid 7 (E)-2-[3-(4-methylphenyl)acryloylamino]benzoic acid 8 (E)-2-[3-(3-trifluoromethylphenyl)acryloylamino]benzoic acid 9 (E)-2-[3-(4-trifluoromethylphenyl)acryloylamino]benzoic acid 10 (E)-2-[3-(3-fluorophenyl)acryloylamino]benzoic acid 11 (E)-2-[3-(4-fluorophenyl)acryloylamino]benzoic acid 12 (E)-2-[3-(3-bromophenyl)acryloylamino]benzoic acid 13 (E)-2-[3-(4-bromophenyl)acryloylamino]benzoic acid 14 (E)-2-[3-(2-chlorophenyl)acryloylamino]benzoic acid 15 (E)-2-[3-(3-chlorophenyl)acryloylamino]benzoic acid 16 (E)-2-[3-(4-chlorophenyl)acryloylamino]benzoic acid 17 (E)-2-[3-([1,1′-biphenyl]-3-yl)acryloylamino]benzoic acid 18 (E)-2-[3-([1,1′-biphenyl]-4-yl)acryloylamino]benzoic acid 19 (E)-2-[3-(3-benzyloxyphenyl)acryloylamino]benzoic acid 20 (E)-2-[3-(3-phenethoxyphenyl)acryloylamino]benzoic acid 21 (E)-2-[3-(3-(4-pyridinemethoxy)phenyl)acryloylamino]benzoic acid 22 (E)-2-[3-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)acryloylamino]benzoic acid 23 (E)-2-[3-(3-isopentoxyphenyl)acryloylamino]benzoic acid 24 (E)-2-[3-(3-((4-methylbenzyl)oxy)phenyl)acryloylamino]benzoic acid 25 (E)-2-[3-(3-((4-fluorobenzyl)oxy)phenyl)acryloylamino]benzoic acid 26 (E)-2-[3-(3-((4-chlorobenzyl)oxy)phenyl)acryloylamino]benzoic acid 27 (E)-2-[3-(3-((4-bromobenzyl)oxy)phenyl)acryloylamino]benzoic acid 28 (E)-2-[3-(3-((3-fluorobenzyl)oxy)phenyl)acryloylamino]benzoic acid 29 (E)-2-[3-(3-((3-chlorobenzyl)oxy)phenyl)acryloylamino]benzoic acid 30 (E)-2-[3-(3-((3-bromobenzyl)oxy)phenyl)acryloylamino]benzoic acid 31 (E)-2-[3-(3-benzoylaminophenyl)acryloylamino]benzoic acid 32 (E)-2-[3-(3-(3-chlorobenzoylamino)phenyl)acryloylamino]benzoic acid 33 (E)-2-[3-(3-(4-chlorobenzoylamino)phenyl)acryloylamino]benzoic acid 34 (E)-2-[3-(3-(2-(3-chlorophenyl)acetamido)phenyl)acryloylamino]benzoic acid 35 (E)-2-[3-(3-(2-(4-chlorophenyl)acetamido)phenyl)acryloylamino]benzoic acid

[0027] .

[0028] Based on the chemical structure of tranilast, this invention employs a drug design strategy combining ligand-directed and structure-directed approaches. While retaining its advantageous scaffold structure, precise structural modification and functional optimization were carried out to design and synthesize 35 novel 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds in three series (IA, IB, and IC) that were previously unreported in the literature. Compared to tranilast, the compounds described in this invention not only inherit the superior safety profile of the lead scaffold but also further enhance their regulatory activity and anti-inflammatory efficacy on the NLRP3 inflammasome-related pathway, thus obtaining novel NLRP3 inflammasome modulators with a "novel structure and novel indication" characteristic.

[0029] This invention provides a method for preparing the above-mentioned 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds. When R1 is a non-aromatic group, i.e. selected from hydrogen, alkoxy, hydroxyl, alkyl, haloalkyl, or halogen, the method includes the following steps:

[0030] Compound Ia reacts with compound IIa to obtain IIIa, which is then hydrolyzed to obtain formula IA;

[0031] In the formula: a represents the amidation reaction; b represents the hydrolysis reaction.

[0032] This invention provides a method for preparing the above-mentioned 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds, wherein when R1 is phenyl, the method includes the following steps:

[0033] Compound Ib and compound IIb were simultaneously coupled and hydrolyzed under alkaline conditions to obtain formula IA;

[0034] ;

[0035] In the formula: a represents the Suzuki-Miyaura coupling reaction.

[0036] This invention provides a method for preparing the above-mentioned 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds, wherein when R1 is -O-R2, the method includes the following steps:

[0037] Compound Ic is reacted with a haloalkane to give compound IIc, which is then hydrolyzed to give IB.

[0038]

[0039] In the formula: a represents the alkylation reaction; b represents the hydrolysis reaction.

[0040] This invention provides a method for preparing the above-mentioned 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds, wherein when R1 is -NH-C=O-R3, the method includes the following steps:

[0041] Compound Id and compound IId are condensed to obtain compound IIId, which is then reduced to obtain compound IVd. Finally, compound V is condensed with various acids to obtain compound V. Compound V is hydrolyzed to obtain formula IC.

[0042] ;

[0043] In the formula: a represents condensation reaction; b represents reduction reaction; c represents amidation reaction; and d represents hydrolysis reaction.

[0044] The present invention also provides a pharmaceutical composition wherein the active ingredient is selected from the above-mentioned 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers or excipients.

[0045] This invention also provides the use of the above-mentioned 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts, as well as pharmaceutically acceptable carriers or excipients, in the preparation of products for the prevention, relief, and / or treatment of diseases and complications related to abnormal activation of the NLRP3 inflammasome. These diseases include gouty arthritis, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, type 2 diabetes, and Alzheimer's disease. Complications related to abnormal activation of the NLRP3 inflammasome include joint erosion, intestinal mucosal barrier disruption, kidney damage, cardiovascular events, and neurodegenerative diseases.

[0046] The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts provided in this invention are selected from the inhibitory effects on the target protein NLRP3 inflammasome in the prevention, relief and / or treatment of abnormal activation of the NLRP3 inflammasome and its complications.

[0047] This invention relates to a series of 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts, prepared via artificial synthesis, and their inhibitory activity against the NLRP3 inflammasome was systematically evaluated. An NLRP3 inflammasome activation model was constructed using mouse peritoneal macrophages stimulated with LPS and ATP. The activity of the compounds was assessed by detecting the IL-1β release level in the cell supernatant using ELISA. The results showed that the compounds of this invention exhibit good inhibitory activity against the NLRP3 inflammasome. Further SPR experiments demonstrated that these compounds can directly bind to the NACHT domain of NLRP3 with high affinity. Selectivity experiments showed that they specifically inhibit NLRP3 inflammasome-mediated inflammatory responses, while having no significant effect on the AIM2 and NLRC4 inflammasomes. Western blot experiments showed that these compounds could inhibit the expression of mature IL-1β and caspase-1 proteins, while having no significant effect on the expression of ASC, NLRP3, pro-IL-1β, and pro-caspase-1 proteins. Immunofluorescence experiments showed that they could significantly inhibit the formation of ASC spots, thereby blocking the assembly and activation of NLRP3 inflammasomes, and have good anti-inflammatory application prospects.

[0048] Furthermore, in this invention, the purified form of 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds refers to 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds with a purity greater than 80%, preferably greater than 85%, particularly preferably greater than 90%, and even more preferably greater than 95%. The purity range of the purified form of 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds is 90-96%.

[0049] In this invention, "pharmaceutically acceptable" means any substance that does not interfere with the bioactivity of the active ingredient and is non-toxic to the host to which it is given.

[0050] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0051] (1) The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts of the present invention, with general formulas I-A, I-B and I-C, are novel monomeric compounds with advantages such as simple synthetic routes, readily available raw materials, simple preparation processes and good drug-like properties.

[0052] (2) The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts of the present invention have good NLRP3 inflammasome inhibitory activity and significant anti-inflammatory effects. They can be used to prevent, alleviate and / or treat gouty arthritis, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, type 2 diabetes and Alzheimer's disease and other diseases and complications related to abnormal activation of NLRP3 inflammasomes. They have good application and development prospects and can be applied to drug preparation.

[0053] (3) The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts of the present invention can directly bind to NLRP3 protein and inhibit its inflammasome assembly and activation, selectively reduce the release of inflammatory factors such as IL-1β and caspase-1, and are a new class of compounds with important medicinal value in the prevention and treatment of inflammation-related diseases, with good clinical translation potential. Attached Figure Description

[0054] Figure 1 For compound B11 1 H NMR spectrum;

[0055] Figure 2 For compound B11 13 C NMR spectrum;

[0056] Figure 3 Here is the HRMS plot of compound B11;

[0057] Figure 4 This is the HPLC chromatogram of compound B11;

[0058] Figure 5 To evaluate the cytotoxicity of compound B11 and the existing drug MCC950 in mouse primary macrophages (PMs);

[0059] Figure 6 Surface plasmon resonance (SPR) experiment of compound B11 with the NACHT domain of human NLRP3 protein;

[0060] Figure 7 Selectivity assays for compound B11 on NLRP3, NLRC4 inflammasomes and AIM2 inflammasomes;

[0061] Figure 8 A graph showing the ASC dot matrix experimental data of compound B11;

[0062] Figure 9 The image shows the Western Blot experimental data of compound B11.

[0063] Figure 10 This is a graph showing the cytotoxicity evaluation data of colchicine and B11;

[0064] Figure 11 This is a graph showing the pharmacodynamic data of MSU-induced acute gouty arthritis in rats. Detailed Implementation

[0065] The preparation process of 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their inhibitory effect on NLRP3 inflammasome protein activity are further explained below with reference to the accompanying drawings and examples.

[0066] The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds of the present invention and their pharmaceutically acceptable salts involve mixtures of two or more salts. When a mixture of multiple salts is present, if a single salt compound is required, it can be prepared by separation or recrystallization according to conventional methods.

[0067] In addition, all starting materials and reagents were purchased commercially available (analytical grade unless otherwise specified, and used directly); anhydrous solvents were prepared according to standard methods. Reaction monitoring was performed using TLC (GF254, 0.20–0.25 nm, Qingdao Ocean Chemical Co., Ltd.) at 254 / 365 nm UV. Melting point determination was performed using a WRS-1A melting point apparatus. Mass spectrometry (MS) was performed using an Agilent 1100 LC-MSD Trap (ESI); high-resolution mass spectrometry (HRMS) was performed using an Agilent 6530 Q-TOF LC-MS (ESI). HPLC (Agilent G1314F) was used for purity detection (methanol / water as mobile phase, 1.0 mL / min). -1 (Detected at 254 nm). Nuclear magnetic resonance spectrum ( 1 H NMR 400MHz, 13 C NMR (100 MHz) was performed using a Bruker ARX-400 instrument, with DMSO-d6 as the solvent (TMS as the internal standard). Chemical shifts are expressed as δ (ppm), and peak shapes are expressed as s, brs, d, dd, t, q, and m.

[0068] I. Preparation of Compounds 1-35

[0069] The synthetic route of compound 1-18 (A1-18) is shown below:

[0070] .

[0071] Reagents and conditions: In reaction (i): a) SOCl2, DMF, DCM, 40℃; b) TEA, DCM, 0℃-room temperature; In reaction (ii): NaOH, THF, H2O, 50℃; In reaction (iii): Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane, H2O, 105℃.

[0072] Example 1: Preparation of (E)-2-[(3-phenylprop-2-enoyl)amino]benzoic acid (compound 1, A1)

[0073] (1) Preparation of intermediate: (E)-2-cinnamamidobenzoate methyl ester (AMA1)

[0074] Cinnamic acid (2.00 g, 13.5 mmol) was dissolved in dichloromethane (20 mL), and thionyl chloride (5.9 mL, 48.6 mmol) was added. Two drops of DMF were added as a catalyst, and the mixture was heated and stirred at 40 °C. After the reaction was complete, the solvent was removed under reduced pressure. The resulting acyl chloride intermediate was dissolved in dichloromethane and added dropwise under ice bath conditions to a mixed solution of methyl 4-aminobenzoate (2.04 g, 13.5 mmol) and triethylamine (2.2 mL, 16.2 mmol) in dichloromethane (20 mL). After the addition was complete, the mixture was stirred at room temperature for 8 h. After the reaction was monitored by thin-layer chromatography, the reaction solution was poured into water and extracted three times with ethyl acetate. The organic phases were combined, dried over sodium sulfate, and concentrated. The crude product was recrystallized from methanol / water to give 3.22 g of a white solid, with a yield of 85%, which was used directly in the next reaction without purification.

[0075] (2) Preparation of the target compound: (E)-2-cinnamamidobenzoic acid, i.e. (E)-2-[(3-phenylprop-2-enoyl)amino]benzoic acid

[0076] Methyl 2-cinnamamidobenzoate (AMA1) (1.00 g, 3.53 mmol) was dissolved in 10 mL of THF, and 5 mL of 4 M sodium hydroxide solution was added. The reaction was stirred at 50 °C for 4 h, and the reaction was monitored by TLC until completion. After cooling to room temperature, the pH was adjusted to 2-3 with dilute hydrochloric acid, and a large amount of white solid precipitated. The product was filtered, washed with water, and dried to obtain 0.84 g of white solid, with a yield of 88%. mp: 196.3-197.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.63(s, 1H), 11.36 (s, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.02 (dd, J = 7.9, 1.3 Hz,1H), 7.75 - 7.70 (m, 2H), 7.67 - 7.62 (m, 1H), 7.61 (d, J = 6.3 Hz, 1H), 7.46- 7.40 (m, 3H), 7.18 (t, J = 7.5 Hz, 1H), 6.88 (d, J = 15.7 Hz, 1H). 13 C NMR(101 MHz, DMSO-d6) δ 169.48, 141.34, 140.86, 134.01, 131.16, 130.03, 128.92,128.19, 122.89, 122.43, 120.44, 116.87. ESI-HRMS calcd. for C 16 H 13 NO3Na [M+Na] + 290.0793, found 290.0798. HPLC purity: 99.54%.

[0077] Example 2: Preparation of (E)-2-(3-(3-methoxyphenyl)acrylamido)benzoic acid (compound 2, A2)

[0078] (1) Preparation of intermediate: (E)-2-(3-(3-methoxyphenyl)acrylamido)methyl benzoate (AMA2)

[0079] Starting with 3-methoxycinnamic acid, the product was synthesized according to the method of AMA1, yielding a white solid in 78% yield. It was used directly in the next reaction without purification.

[0080] (2) Preparation of the target compound: (E)-2-(3-(3-methoxyphenyl)acrylamido)benzoic acid

[0081] Starting with intermediate AMA2, the mixture was synthesized following the method described in A1, yielding a white solid in 90% yield. mp: 172.4–178.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.67 (dd, J = 8.4, 0.8 Hz,1H), 8.06 (dd, J = 7.9, 1.6 Hz, 1H), 7.67 - 7.62 (m, 2H), 7.39 - 7.29 (m,3H), 7.23 - 7.18 (m, 1H), 7.01 (d, J = 7.9 Hz, 1H), 6.94 (d, J = 15.7 Hz,1H), 3.84 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 169.50, 163.74, 159.64,141.16, 140.84, 135.86, 133.74, 131.11, 129.87, 122.81, 122.76, 120.68,120.34, 117.21, 115.95, 112.95, 55.19. ESI-HRMS calcd. for C 17 H 15 NO4Na [M+Na] + 320.0899, found 320.0903. HPLC purity: 97.71%.

[0082] Example 3: Preparation of (E)-2-(3-(4-methoxyphenyl)acrylamido)benzoic acid (compound 3, A3)

[0083] (1) Preparation of intermediate: (E)-2-(3-(4-methoxyphenyl)acrylamido)methyl benzoate (AMA3)

[0084] Starting with 4-methoxycinnamic acid, it was synthesized according to the method of AMA1 to obtain a white solid with a yield of 73%, which was used directly in the next reaction without purification.

[0085] (2) Preparation of the target compound: (E)-2-(3-(3-methoxyphenyl)acrylamido)benzoic acid

[0086] Starting with intermediate AMA3, the mixture was synthesized following the method described in A1, yielding a white solid in 78% yield. mp: 185.6–188.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 8.68 (d, J = 7.8 Hz, 1H), 8.06 (dd, J = 7.9, 1.5 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.63 (ddd, J =11.6, 4.6, 2.2 Hz, 2H), 7.22 - 7.17 (m, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.74(d, J = 15.6 Hz, 1H), 3.83 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 169.59,164.03, 160.80, 141.06, 141.02, 133.76, 131.11, 129.82, 127.01, 122.54,120.18, 119.83, 116.96, 114.34, 55.25. ESI-HRMS calcd. for C 17 H 15 NO4Na [M+Na] + 320.0899, found 320.0901. HPLC purity: 98.86%.

[0087] Example 4: Preparation of (E)-2-(3-(3-hydroxyphenyl)acrylamido)benzoic acid (compound 4, A4)

[0088] (1) Preparation of intermediate: (E)-2-(3-(3-hydroxyphenyl)acrylamido)methyl benzoate (AMA4)

[0089] Starting with 3-hydroxycinnamic acid, the mixture was synthesized according to the method described in AMA1, yielding a white solid in 69% yield. mp: 240.7-243.3℃. 1H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 9.63 (s, 1H), 8.41(d, J = 8.3 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.57(d, J = 15.6 Hz, 1H), 7.26 (dt, J = 10.6, 7.8 Hz, 2H), 7.19 - 7.10 (m, 2H), 6.86 (t, J = 11.4 Hz, 2H), 3.91 (s, 3H).

[0090] (2) Preparation of the target compound: (E)-2-(3-(3-hydroxyphenyl)acrylamido)benzoic acid

[0091] Starting with intermediate AMA4, the mixture was synthesized following the method described in A1, yielding a white solid in 85% yield. mp: 223.5–225.9 °C. 1 H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 10.10 (s, 1H), 8.66 (d, J= 8.3 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.53 (dd, J = 15.4, 10.5 Hz, 2H),7.25 (t, J = 7.8 Hz, 1H), 7.18 - 7.07 (m, 3H), 6.87 (d, J = 7.8 Hz, 1H), 6.72(d, J = 15.7 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 170.30, 163.55, 157.89,140.89, 140.78, 135.80, 132.26, 131.29, 129.94, 122.79, 122.31, 120.55,119.51, 118.98, 117.1, 114.39. ESI-HRMS calcd. for C 16 H 13 NO4Na [M+Na] + 306.0742, found 306.0740. HPLC purity: 99.42%.

[0092] Example 5: Preparation of (E)-2-(3-(4-hydroxyphenyl)acrylamido)benzoic acid (compound 5, A5)

[0093] (1) Preparation of intermediate: (E)-2-(3-(4-hydroxyphenyl)acrylamido)methyl benzoate (AMA5)

[0094] Starting with 4-hydroxycinnamic acid, the product was synthesized according to the method of AMA1, yielding a white solid in 63% yield. It was used directly in the next reaction without purification.

[0095] (2) Preparation of the target compound: (E)-2-(3-(4-hydroxyphenyl)acrylamido)benzoic acid

[0096] Starting with intermediate AMA5, the mixture was synthesized following the method described in A1, yielding a white solid in 87% yield. mp: 221.0–222.5 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 10.03 (s, 1H), 8.68 (d, J= 8.1 Hz, 1H), 8.05 (dd, J = 7.9, 1.4 Hz, 1H), 7.67 - 7.62 (m, 1H), 7.60 (dd,J = 12.0, 3.2 Hz, 3H), 7.21 - 7.15 (m, 1H), 6.86 (d, J = 8.5 Hz, 2H), 6.66(d, J = 15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.58, 164.25, 159.51,141.64, 141.18, 134.03, 131.17, 130.11, 125.47, 122.62, 120.27, 118.64,116.54, 115.80. ESI-HRMS calcd. for C 16 H 13 NO4Na [M+Na] + 306.0742, found306.0746. HPLC purity: 99.92%.

[0097] Example 6: Preparation of (E)-2-(3-(3-methylphenyl)acrylamido)benzoic acid (compound 6, A6)

[0098] (1) Preparation of intermediate: (E)-2-(3-(3-methylphenyl)acrylamido)methyl benzoate (AMA6)

[0099] Starting with 3-methylcinnamic acid, the mixture was synthesized according to the method of AMA1 to obtain a white solid with a yield of 71%. It was used directly in the next reaction without purification.

[0100] (2) Preparation of the target compound: (E)-2-(3-(3-methylphenyl)acrylamido)benzoic acid

[0101] Starting with intermediate AMA6, the mixture was synthesized according to the method described in A1, yielding a white solid in 90% yield. mp: 135.7-137.7℃. 1 H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.67 - 8.63 (m, 1H), 8.06 (dd, J = 7.8, 1.6 Hz, 1H), 7.57 (d, J = 15.7 Hz, 1H), 7.51 (ddd, J = 9.0,6.1, 1.8 Hz, 2H), 7.49 - 7.44 (m, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.21 (d, J =7.5 Hz, 1H), 7.12 (td, J = 7.9, 1.1 Hz, 1H), 6.78 (d, J = 15.7 Hz, 1H), 2.33(s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 170.07, 163.56, 140.89, 140.68, 138.13,134.44, 132.35, 131.22, 130.59, 128.78, 128.37, 125.38, 122.75, 122.29,120.20, 119.50. ESI-HRMS calcd. for C 17 H 15 NO3Na [M+Na] + 304.0950, found304.0952. HPLC purity: 99.63%.

[0102] Example 7: Preparation of (E)-2-(3-(4-methylphenyl)acrylamido)benzoic acid (compound 7, A7)

[0103] (1) Preparation of intermediate: (E)-2-(3-(4-methylphenyl)acrylamido)methyl benzoate (AMA7)

[0104] Starting with 4-methylcinnamic acid, the mixture was synthesized according to the method of AMA1 to obtain a white solid with a yield of 65%, which was used directly in the next reaction without purification.

[0105] (2) Preparation of the target compound: (E)-2-(3-(4-methylphenyl)acrylamido)benzoic acid

[0106] Starting with intermediate AMA7, the mixture was synthesized according to the method described in A1, yielding a white solid in 89% yield. mp: 201.1–203.6 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 11.35 (s, 1H), 8.62 (dd, J= 8.4, 0.8 Hz, 1H), 8.01 (dd, J = 7.9, 1.6 Hz, 1H), 7.63 (d, J = 2.3 Hz, 1H), 7.61 (d, J = 3.3 Hz, 2H), 7.60 - 7.56 (m, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.19- 7.15 (m, 1H), 6.81 (d, J = 15.7 Hz, 1H), 2.33 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 169.50, 163.91, 141.33, 140.94, 139.93, 133.97, 131.68, 131.15,129.53, 128.18, 122.79, 121.31, 120.36, 116.83, 21.01. ESI-HRMS calcd. forC 17 H 15 NO3Na [M+Na] + 304.0950, found 304.0952. HPLC purity: 96.37%.

[0107] Example 8: Preparation of (E)-2-(3-(3-(trifluoromethyl)phenyl)acrylamido)benzoic acid (compound 8, A8)

[0108] (1) Preparation of intermediate: (E)-2-(3-(3-(trifluoromethyl)phenyl)acrylamido)methyl benzoate (AMA8)

[0109] Starting with 3-(trifluoromethyl)cinnamic acid, it was synthesized according to the method of AMA1 to obtain a white solid with a yield of 78%, which was used directly in the next reaction without purification.

[0110] (2) Preparation of the target compound: (E)-2-(3-(3-(trifluoromethyl)phenyl)acrylamido)benzoic acid

[0111] Starting with intermediate AMA8, the mixture was synthesized according to the method in A1, yielding a white solid in 82% yield. 1 H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.65 (d, J = 8.3 Hz, 1H), 8.10 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.73 - 7.67 (m, 2H), 7.64- 7.56 (m, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 15.7 Hz, 1H). 13 C NMR(101 MHz, DMSO-d6) δ 169.57, 163.46, 140.78, 139.59, 135.71, 133.75, 132.01,131.20, 129.91, 129.66, 126.15, 125.43, 124.71, 124.64, 122.93, 120.38,117.46. ESI-HRMS calcd. for C 17 H 12 F3NONa [M+Na] + 358.0667, found 358.0670. HPLCpurity: 98.35%.

[0112] Example 9: Preparation of (E)-2-(3-(4-(trifluoromethyl)phenyl)acrylamido)benzoic acid (compound 9, A9)

[0113] (1) Preparation of intermediate: (E)-2-(3-(4-(trifluoromethyl)phenyl)acrylamido)methyl benzoate (AMA9)

[0114] Starting with 4-(trifluoromethyl)cinnamic acid, it was synthesized according to the method of AMA1 to obtain a white solid with a yield of 73%, which was used directly in the next reaction without purification.

[0115] (2) Preparation of the target compound: (E)-2-(3-(4-(trifluoromethyl)phenyl)acrylamido)benzoic acid

[0116] Starting with intermediate AMA9, the mixture was synthesized according to the method described in A1, yielding a white solid in 87% yield. mp: 218.7–220.8 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.05 (dd, J = 7.9, 1.5 Hz, 1H), 7.97 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 8.2Hz, 2H), 7.72 (d, J = 15.7 Hz, 1H), 7.66 - 7.61 (m, 1H), 7.24 - 7.18 (m, 1H), 7.07 (d, J = 15.7 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.53, 163.30,140.67, 139.48, 138.51, 133.81, 131.18, 128.79, 125.70, 125.35, 123.02,120.42, 117.42. ESI-HRMS calcd. for C 17 H 12 F3NO3Na [M+Na] + 358.0667, found358.0664. HPLC purity: 98.11%.

[0117] Example 10: Preparation of (E)-2-(3-(3-fluorophenyl)acrylamido)benzoic acid (compound 10, A10)

[0118] (1) Preparation of intermediate: (E)-2-(3-(3-fluorophenyl)acrylamido)methyl benzoate (AMA10)

[0119] Starting with 3-fluorocinnamic acid, the mixture was synthesized according to the method of AMA1 to obtain a white solid with a yield of 72%, which was used directly in the next reaction without purification.

[0120] (2) Preparation of the target compound: (E)-2-(3-(3-fluorophenyl)acrylamido)benzoic acid

[0121] Starting with intermediate AMA10, the mixture was synthesized according to the method described in A1, yielding a white solid in 90% yield. mp: 189.8–191.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 10.36 (s, 1H), 7.81- 7.76 (m, 4H), 7.72 - 7.69 (m, 2H), 7.28 - 7.24 (m, 2H), 2.09 (s, 3H). 13 CNMR (101 MHz, DMSO-d6) δ 169.46, 163.54, 161.28, 140.76, 140.02, 137.01,133.97, 131.17, 130.88, 124.69, 124.05, 117.00, 116.79, 116.57, 114.45,114.23. ESI-HRMS calcd. for C 16 H 12 FNO3Na [M+Na] + 308.0699, found 308.0701. HPLC purity: 99.44%.

[0122] Example 11: Preparation of (E)-2-(3-(4-fluorophenyl)acrylamido)benzoic acid (compound 11, A11)

[0123] (1) Preparation of intermediate: (E)-2-(3-(4-fluorophenyl)acrylamido)methyl benzoate (AMA11)

[0124] Starting with 4-fluorocinnamic acid, the mixture was synthesized according to the method of AMA1 to obtain a white solid with a yield of 69%, which was used directly in the next reaction without purification.

[0125] (2) Preparation of the target compound: (E)-2-(3-(4-fluorophenyl)acrylamido)benzoic acid

[0126] Starting with intermediate AMA11, the mixture was synthesized following the method described in A1, yielding a white solid in 93% yield. mp: 184.9–185.7 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.66 (d, J = 8.3 Hz,1H), 8.06 (d, J = 7.7 Hz, 1H), 7.87 - 7.81 (m, 2H), 7.65 (dd, J = 15.6, 9.6Hz, 2H), 7.29 (t, J = 8.6 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H), 6.88 (d, J =15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.56, 164.27, 163.71, 161.81,140.87, 140.11, 133.85, 131.17, 130.51, 130.43, 122.83, 122.36, 120.32,117.14, 115.99, 115.77. ESI-HRMS calcd. for C 16 H 12 FNO3Na [M+Na] + 308.0699, found 308.0704. HPLC purity: 99.44%.

[0127] Example 12: Preparation of (E)-2-(3-(3-bromophenyl)acrylamido)benzoic acid (compound 12, A12)

[0128] (1) Preparation of intermediate: (E)-2-(3-(3-bromophenyl)acrylamido)methyl benzoate (AMA12)

[0129] Starting with 3-bromocinnamic acid, it was synthesized according to the method of AMA1 to obtain a white solid in 64% yield, which was used directly in the next reaction without purification.

[0130] (2) Preparation of the target compound: (E)-2-(3-(3-bromophenyl)acrylamido)benzoic acid

[0131] Starting with intermediate AMA12, the mixture was synthesized according to the method described in A1, yielding a white solid in 85% yield. mp: 184.7–185.7 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 8.63 (d, J = 8.3 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.95 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.55(d, J = 4.2 Hz, 1H), 7.55 (s, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.8Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 15.7 Hz, 1H). 13 C NMR (101MHz, DMSO-d6) δ 169.98, 163.23, 140.74, 138.96, 137.11, 132.38, 132.33,131.22, 130.90, 130.42, 127.08, 124.69, 122.45, 122.32, 120.25, 119.60. ESI-HRMS calcd. for C 16 H 12 BrNO3Na [M+Na] + 367.9898, found 367.9902. HPLC purity:99.15%.

[0132] Example 13: Preparation of (E)-2-(3-(4-bromophenyl)acrylamido)benzoic acid (compound 13, A13)

[0133] (1) Preparation of intermediate: (E)-2-(3-(4-bromophenyl)acrylamido)methyl benzoate (AMA13)

[0134] Starting with 4-bromocinnamic acid, the mixture was synthesized according to the method described in AMA1, yielding a white solid in 65% yield. mp: 149.8–151.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.42 (d, J = 8.4 Hz,1H), 7.94 (dd, J = 7.9, 1.6 Hz, 1H), 7.70 - 7.58 (m, 6H), 7.24 - 7.18 (m,1H), 6.97 (d, J = 15.7 Hz, 1H), 3.88 (s, 3H). ESI-MS(m / z): 384.00 [M+Na] + .

[0135] (2) Preparation of the target compound: (E)-2-(3-(4-bromophenyl)acrylamido)benzoic acid

[0136] Starting with intermediate AMA13, the mixture was synthesized according to the method described in A1, yielding a white solid in 86% yield. mp: 218.2–220.1 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.64 (s, 1H), 11.35 (s, 1H), 8.63- 8.58 (m, 1H), 8.01 (dd, J = 7.9, 1.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.64 - 7.61 (m, 3H), 7.59 (d, J = 8.2 Hz, 1H), 7.21 - 7.16 (m, 1H), 6.94 (d,J = 15.7 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.42, 163.58, 140.74, 140.06,133.98, 133.75, 131.86, 131.15, 130.16, 123.31, 123.28, 122.97, 120.45,116.96. ESI-HRMS calcd. for C 16 H 12 BrNO3Na [M+Na] + 367.9898, found 367.9898.HPLC purity: 98.47%.

[0137] Example 14: Preparation of (E)-2-(3-(2-chlorophenyl)acrylamido)benzoic acid (compound 14, A14)

[0138] (1) Preparation of intermediate: (E)-2-(3-(2-chlorophenyl)acrylamido)methyl benzoate (AMA14)

[0139] Starting with 2-chlorocinnamic acid, the mixture was synthesized according to the method of AMA1 to obtain a white solid with a yield of 68%, which was used directly in the next reaction without purification.

[0140] (2) Preparation of the target compound: (E)-2-(3-(2-chlorophenyl)acrylamido)benzoic acid

[0141] Starting with intermediate AMA14, the mixture was synthesized according to the method described in A1, yielding a white solid in 90% yield. mp: 197.3–199.2 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.69 (s, 1H), 11.48 (s, 1H), 8.61 (d, J = 8.2 Hz, 1H), 8.03 (dd, J = 8.0, 1.4 Hz, 1H), 8.00 (dd, J = 7.3, 2.2Hz, 1H), 7.94 (d, J = 15.7 Hz, 1H), 7.68 - 7.61 (m, 1H), 7.56 (dd, J = 7.4,1.7 Hz, 1H), 7.50 - 7.44 (m, 1H), 7.43 (dd, J = 4.1, 2.1 Hz, 1H), 7.24 - 7.17(m, 1H), 6.97 (d, J = 15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.47,163.30, 140.59, 136.04, 133.98, 133.62, 132.13, 131.46, 131.15, 129.96,128.20, 127.74, 125.51, 123.11, 120.48, 117.17. ESI-HRMS calcd. forC 16 H 12 ClNO3Na [M+Na] + 324.0403, found 324.0407. HPLC purity: 99.50%.

[0142] Example 15: Preparation of (E)-2-(3-(3-chlorophenyl)acrylamido)benzoic acid (compound 15, A15)

[0143] (1) Preparation of intermediate: (E)-2-(3-(3-chlorophenyl)acrylamido)methyl benzoate (AMA15)

[0144] Starting with 3-chlorocinnamic acid, the mixture was synthesized according to the method of AMA1 to obtain a white solid with a yield of 73%, which was used directly in the next reaction without purification.

[0145] (2) Preparation of the target compound: (E)-2-(3-(3-chlorophenyl)acrylamido)benzoic acid

[0146] Starting with intermediate AMA15, the mixture was synthesized according to the method described in A1, yielding a white solid in 83% yield. mp: 185.1–187.5 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 1H), 11.40 (s, 1H), 8.65- 8.60 (m, 1H), 8.02 (dd, J = 7.9, 1.5 Hz, 1H), 7.87 (s, 1H), 7.68 (tt, J =5.3, 2.8 Hz, 1H), 7.62 (dd, J = 6.6, 1.9 Hz, 1H), 7.59 (d, J = 2.6 Hz, 1H),7.47 - 7.42 (m, 2H), 7.20 - 7.15 (m, 1H), 7.01 (d, J = 15.6 Hz, 1H). 13 C NMR(101 MHz, DMSO-d6) δ 169.47, 163.52, 140.78, 139.80, 136.76, 133.96, 133.77,131.18, 130.67, 129.60, 127.64, 126.98, 124.18, 122.98, 120.44, 117.01. ESI-HRMS calcd. for C 16 H 12 ClNO3Na [M+Na] + 324.0403, found 324.0407. HPLC purity:98.68%.

[0147] Example 16: Preparation of (E)-2-(3-(4-chlorophenyl)acrylamido)benzoic acid (compound 16, A16)

[0148] (1) Preparation of intermediate: (E)-2-(3-(4-chlorophenyl)acrylamido)methyl benzoate (AMA16)

[0149] Starting with 4-chlorocinnamic acid, the mixture was synthesized according to the method of AMA1, yielding a white solid in 72% yield. It was used directly in the next reaction without purification.

[0150] (2) Preparation of the target compound: (E)-2-(3-(4-chlorophenyl)acrylamido)benzoic acid

[0151] Starting with intermediate AMA16, the mixture was synthesized according to the method described in A1, yielding a white solid in 84% yield. mp: 179.2–180.1 °C. 1 H NMR (400 MHz, DMSO-d6) δ 16.34 (s, 1H), 14.18 (s, 1H), 11.41(d, J = 8.3 Hz, 1H), 10.85 - 10.78 (m, 1H), 10.56 (d, J = 8.4 Hz, 2H), 10.45- 10.38 (m, 2H), 10.27 (d, J = 8.3 Hz, 2H), 9.97 (t, J = 7.5 Hz, 1H), 9.71 (d, J = 15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.53, 163.57, 140.84,139.94, 134.49, 133.91, 133.42, 131.17, 129.90, 128.91, 123.27, 122.89,120.37, 117.04. ESI-HRMS calcd. for C 16 H 12 ClNO3Na [M+Na] + 324.0403, found324.0408. HPLC purity: 97.32%.

[0152] Example 17: Preparation of (E)-2-(3-([1,1'-biphenyl]-3-yl)acrylamido)benzoic acid (compound 17, A17)

[0153] AMA12 (1.00 g, 2.78 mmol), benzyl pinacol ester (0.57 g, 2.78 mmol), bis(triphenylphosphine)palladium dichloride (5.9 mg, 0.3 mol%), and potassium carbonate (1.92 g, 13.9 mmol) were placed in a reaction vessel, and a mixed solvent of 1,4-dioxane and water (2:1) was added. The reaction mixture was heated to 105 °C and stirred, and TLC was monitored until the starting material was completely converted. After the reaction was complete, the reaction solution was cooled to room temperature, quenched with water, and extracted with ethyl acetate. The aqueous phase was adjusted to pH 2-3 with dilute hydrochloric acid in an ice bath, and then back-extracted with a mixed solvent of ethyl acetate and tetrahydrofuran. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give a white solid in 54% yield. mp: 155.8-157.4 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.65 (s, 1H), 8.02 (d, J = 52.6 Hz, 2H), 7.69 (d, J = 7.8 Hz, 3H), 7.62 (d, J = 41.8 Hz, 3H), 7.47 (s, 3H), 7.44 - 7.32 (m, 2H), 7.12 - 6.83 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.40, 140.84, 140.10, 139.60, 135.30, 131.44, 131.31, 129.52,128.95, 128.08, 127.72, 126.86, 126.38, 123.80, 122.22, 121.99, 119.12. ESI-HRMS calcd. for C 22 H 17 NO3Na [M+Na] + 366.1106, found 366.1109. HPLC purity:98.60%.

[0154] Example 18: Preparation of (E)-2-(3-([1,1'-biphenyl]-4-yl)acrylamido)benzoic acid (compound 18, A18)

[0155] Starting with intermediate AMA13, the mixture was synthesized following the method described in A17, yielding a white solid in 63% yield. mp: 187.0–190.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 10.37 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H), 8.13 (s, 1H), 8.06 - 7.95 (m, 3H), 7.84 (d, J = 7.3 Hz,1H), 7.63 (s, 1H), 7.60 (d, J = 6.8 Hz, 2H), 7.57 (s, 1H), 7.54 (d, J = 7.0Hz, 1H), 7.47 (d, J = 7.2 Hz, 1H), 7.45 - 7.40 (m, 1H), 7.18 (t, J = 7.4 Hz,1H), 6.81 (d, J = 15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.36, 165.49,163.37, 140.92, 140.55, 139.53, 134.56, 133.56, 131.50, 130.96, 129.10,128.24, 127.49, 123.62, 122.70, 122.42, 121.86, 120.18, 119.19, 117.34. ESI-HRMS calcd. for C 22 H 17 NO3Na [M+Na] + 366.1106, found 366.1106. HPLC purity:95.40%.

[0156] The synthetic route of compound 19-30 (B1-12) is shown below:

[0157] .

[0158] Reagents and conditions: In reaction (i): various haloalkanes, K2CO3, DMF, 60℃; In reaction (ii): NaOH, THF, H2O, 50℃.

[0159] Example 19: Preparation of (E)-2-(3-(3-(benzyloxy)phenyl)acrylamido)benzoic acid (compound 19, B1)

[0160] (1) Preparation of intermediate: (E)-2-(3-(3-(benzyloxy)phenyl)acrylamido)methyl benzoate (BMB1)

[0161] Methyl (E)-2-(3-(3-hydroxyphenyl)acrylamido)benzoate (AMA4) (1.00 g, 3.36 mmol), benzyl chloride (0.42 mL, 3.69 mmol), and potassium carbonate (0.93 g, 6.72 mmol) were placed in a reaction vessel, and N,N-dimethylformamide (16.8 mL) was added. The reaction mixture was heated to 60 °C and stirred until the reactants were completely converted by TLC. After the reaction was complete, the reaction solution was cooled to room temperature, quenched with water, and extracted three times with ethyl acetate. The organic phases were combined, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate) to give 1.06 g of white solid, 85% yield. mp: 134.3–135.3 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.94 (dd, J = 8.0, 1.7 Hz,1H), 7.66 - 7.57 (m, 2H), 7.50 - 7.46 (m, 2H), 7.43 - 7.28 (m, 6H), 7.21 (td,J = 7.6, 1.2 Hz, 1H), 7.06 (dd, J = 7.6, 2.9 Hz, 1H), 6.97 (d, J = 15.6 Hz,1H), 5.16 (s, 2H), 3.87 (s, 3H). ESI-MS(m / z): 386.10 [MH] - .

[0162] (2) Preparation of the target compound: (E)-2-(3-(3-(benzyloxy)phenyl)acrylamido)benzoic acid

[0163] Starting with intermediate BMB1, the mixture was synthesized according to method A1, yielding a white solid in 92% yield. mp: 206.3–209.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.72 (s, 1H), 11.49 (s, 1H), 8.68 (d, J= 8.3 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.68 - 7.61 (m, 2H), 7.52 (d, J =7.3 Hz, 2H), 7.48 - 7.41 (m, 3H), 7.40 - 7.35 (m, 2H), 7.33 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 6.95 (d, J = 15.6Hz, 1H), 5.20 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.77, 158.73, 141.24,135.89, 133.87, 131.16, 129.98, 128.45, 127.89, 127.84, 122.84, 121.09,120.38, 116.79, 113.83, 69.34. ESI-HRMS calcd. for C 23 H 19 NO4Na [M+Na] + 396.1212, found 396.1214. HPLC purity: 98.75%.

[0164] Example 20: Preparation of (E)-2-(3-(3-(phenylethoxy)phenyl)acrylamido)benzoic acid (compound 20, B2)

[0165] (1) Preparation of intermediate: (E)-2-(3-(3-(phenylethoxy)phenyl)acrylamido)methyl benzoate (BMB2)

[0166] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with (2-chloroethyl)benzene to give a yellow solid in 62% yield, which was used directly in the next reaction without purification.

[0167] (2) Preparation of the target compound: (E)-2-(3-(3-(phenethoxy)phenyl)acrylamido)benzoic acid

[0168] Starting with intermediate BMB2, the mixture was synthesized according to method A1, yielding a white solid in 83% yield. mp: 159.0–161.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.61 (s, 1H), 11.30 (s, 1H), 8.60 (d, J= 7.8 Hz, 1H), 8.01 (dd, J = 7.9, 1.5 Hz, 1H), 7.66 - 7.61 (m, 1H), 7.59 (d,J = 15.6 Hz, 1H), 7.35 (d, J = 1.9 Hz, 2H), 7.33 (s, 3H), 7.32 - 7.26 (m,2H), 7.26 - 7.22 (m, 1H), 7.21 - 7.16 (m, 1H), 7.01 - 6.97 (m, 1H), 6.92 (d,J = 15.6 Hz, 1H), 4.26 (t, J = 6.9 Hz, 2H), 3.06 (t, J = 6.9 Hz, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 169.37, 163.80, 158.81, 141.29, 140.78, 138.31, 133.97,131.12, 129.95, 129.00, 128.32, 126.29, 122.90, 122.76, 120.94, 120.48,116.60, 113.35, 68.26, 34.96. ESI-HRMS calcd. for C 24 H 21 NO4Na [M+Na] + 410.1368, found 410.1367. HPLC purity: 95.89%.

[0169] Example 21: Preparation of (E)-2-[3-(3-(4-pyridinemethoxy)phenyl)acryloylamino]benzoic acid (compound 21, B3)

[0170] (1) Preparation of intermediate: (E)-2-(3-(3-((pyridin-4-yl)ethoxy)phenyl)acrylamido)methyl benzoate (BMB3)

[0171] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 4-(2-chloroethyl)pyridine to give a yellow solid in 54% yield. It was used directly in the next reaction without purification.

[0172] (2) Preparation of the target compound: (E)-2-(3-(3-((pyridin-4-yl)ethoxy)phenyl)acrylamido)benzoic acid

[0173] Starting with intermediate BMB3, the mixture was synthesized according to method A1 to obtain a white solid in 86% yield. mp: 200.2–202.8 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 8.61 (d, J = 16.7 Hz, 3H), 8.02 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 13.7 Hz, 2H), 7.53 - 7.39 (m, 3H), 7.39 - 7.27 (m, 2H), 7.21 - 7.05 (m, 2H), 6.91 (d, J = 15.3 Hz, 1H), 5.25 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.56, 163.74, 158.34, 149.69, 146.17,141.12, 140.86, 136.01, 133.88, 131.18, 130.09, 122.99, 122.87, 121.95,121.44, 120.37, 117.14, 116.75, 113.86, 67.61. ESI-HRMS calcd. for C 22 H 20 N₂O₄[M+H] + 375.1345, found 375.1344. HPLC purity: 99.07%.

[0174] Example 22: Preparation of (E)-2-(3-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)acrylamido)benzoic acid (compound 22, B4)

[0175] (1) Preparation of intermediate: (E)-2-(3-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)acrylamido)methyl benzoate (BMB4)

[0176] Starting with AMA4, the product was synthesized according to the method of BMB1. It was reacted with 1-chloro-3-methylbut-2-ene to give a yellow solid with a yield of 71%. It was used directly in the next reaction without purification.

[0177] (2) Preparation of the target compound: (E)-2-(3-(3-((3-methylbut-2-en-1-yl)oxy)phenyl)acrylamido)benzoic acid

[0178] Starting with intermediate BMB4, the mixture was synthesized according to method A1, yielding a white solid in 84% yield. mp: 144.7–147.2 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.63 (s, 1H), 11.36 (s, 1H), 8.63 (d, J= 8.3 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.65 - 7.56 (m, 2H), 7.31 (d, J =6.8 Hz, 2H), 7.26 (d, J = 7.3 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 6.97 (d, J =7.5 Hz, 1H), 6.90 (d, J = 15.6 Hz, 1H), 5.44 (s, 1H), 4.57 (d, J = 6.3 Hz,2H), 1.74 (s, 3H), 1.72 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 169.49, 163.80,158.84, 141.40, 140.89, 137.21, 135.81, 133.99, 131.16, 129.88, 122.86,122.67, 120.73, 120.42, 116.83, 116.69, 113.60, 64.41, 25.43, 18.04. ESI-HRMScalcd. for C 21 H 21 NO4Na [M+Na] + 374.1368, found 374.1367. HPLC purity: 98.47%.

[0179] Example 23: Preparation of (E)-2-(3-(3-(isopentoxy)phenyl)acrylamido)benzoic acid (compound 23, B5)

[0180] (1) Preparation of intermediate: (E)-2-(3-(3-(isopentoxy)phenyl)acrylamido)methyl benzoate (BMB5)

[0181] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 1-chloro-3-methylbutane to give a yellow solid in 58% yield, which was used directly in the next reaction without purification.

[0182] (2) Preparation of the target compound: (E)-2-(3-(3-(isopentoxy)phenyl)acrylamido)benzoic acid

[0183] Starting with intermediate BMB5, the mixture was synthesized according to method A1, yielding a white solid in 90% yield. mp: 140.7–142.5 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.88 (s, 1H), 8.61 (d, J = 8.2 Hz, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.54 (d, J = 15.7 Hz, 1H), 7.38 (t, J = 7.4 Hz,1H), 7.30 (t, J = 7.8 Hz, 1H), 7.26 - 7.19 (m, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 6.73 (d, J = 15.7 Hz, 1H), 4.03 (t, J = 6.4 Hz,2H), 1.78 (dt, J = 13.2, 6.6 Hz, 1H), 1.61 (q, J = 6.4 Hz, 2H), 0.93 (d, J =6.5 Hz, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 170.14, 163.32, 159.07, 139.91,136.07, 131.22, 130.90, 129.90, 123.71, 123.08, 121.80, 120.31, 118.89,116.19, 113.24, 65.96, 37.48, 24.58, 22.45. ESI-HRMS calcd. for C 21 H 23 NO4Na [M+Na] + 376.1525, found 376.1529. HPLC purity: 98.92%.

[0184] Example 24: Preparation of (E)-2-(3-(3-((4-methylbenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 24, B6)

[0185] (1) Preparation of intermediate: (E)-2-(3-(3-((4-methylbenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB6)

[0186] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 4-methylbenzyl chloride to give a yellow solid in 67% yield, which was used directly in the next reaction without purification.

[0187] (2) Preparation of the target compound: (E)-2-(3-(3-((4-methylbenzyl)oxy)phenyl)acrylamido)benzoic acid

[0188] Starting with intermediate BMB6, the mixture was synthesized according to method A1, yielding a white solid in 88% yield. mp: 185.7–187.1 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.67 (d, J = 8.3 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 12.0 Hz, 1H), 7.57 (d, J = 10.3 Hz,1H), 7.39 (d, J = 13.0 Hz, 2H), 7.36 - 7.31 (m, 2H), 7.28 (d, J = 7.4 Hz,1H), 7.20 (d, J = 7.7 Hz, 2H), 7.16 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 7.5 Hz,1H), 6.90 (d, J = 15.6 Hz, 1H), 5.10 (s, 2H), 2.31 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 169.94, 163.68, 158.76, 140.97, 137.12, 133.89, 133.25, 131.22, 129.94, 129.00, 127.92, 123.03, 122.63, 120.92, 120.02, 116.73, 113.83,69.24, 20.79. ESI-HRMS calcd. for C 24 H 21 NO4Na [M+Na] + 410.1368, found 410.1372. HPLC purity: 99.19%.

[0189] Example 25: Preparation of (E)-2-(3-(3-((4-fluorobenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 25, B7)

[0190] (1) Preparation of intermediate: (E)-2-(3-(3-((4-fluorobenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB7)

[0191] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 4-fluorobenzyl chloride to give a yellow solid in 73% yield, which was used directly in the next reaction without purification.

[0192] (2) Preparation of the target compound: (E)-2-(3-(3-((4-fluorobenzyl)oxy)phenyl)acrylamido)benzoic acid

[0193] Starting with intermediate BMB7, the mixture was synthesized according to method A1, yielding a white solid in 91% yield. mp: 182.3–185.0 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.66 (s, 1H), 11.40 (s, 1H), 8.64 (d, J= 8.3 Hz, 1H), 8.02 (d, J = 6.9 Hz, 1H), 7.64 - 7.58 (m, 2H), 7.53 (dd, J =8.2, 5.8 Hz, 2H), 7.42 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.29 (d, J = 7.5Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 7.19-7.15 (m, 1H), 7.05 (d, J = 7.1 Hz,1H), 6.92 (d, J = 15.6 Hz, 1H), 5.14 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ169.51, 163.79, 163.04, 160.61, 158.65, 141.27, 140.91, 135.92, 133.97,133.16, 131.18, 130.14, 130.06, 129.98, 122.84, 121.16, 120.41, 116.87,116.76, 115.38, 115.16, 113.87, 68.63. ESI-HRMS calcd. for C 23 H 18 FNO4Na [M+Na] + 414.1118, found 414.1115. HPLC purity: 99.16%.

[0194] Example 26: Preparation of (E)-2-(3-(3-((4-chlorobenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 26, B8)

[0195] (1) Preparation of intermediate: (E)-2-(3-(3-((4-chlorobenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB8)

[0196] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 4-chlorobenzyl chloride to give a yellow solid in 78% yield, which was used directly in the next reaction without purification.

[0197] (2) Preparation of the target compound: (E)-2-(3-(3-((4-chlorobenzyl)oxy)phenyl)acrylamido)benzoic acid

[0198] Starting with intermediate BMB8, the mixture was synthesized according to method A1, yielding a white solid in 90% yield. mp: 188.3–191.7 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H), 8.65 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 7.9, 1.5 Hz, 1H), 7.63 - 7.57 (m, 2H), 7.50 (s, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.41 (s, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 7.6 Hz, 1H), 7.18 - 7.14 (m, 1H), 7.06 - 7.01 (m, 1H), 6.90 (d, J = 15.6 Hz, 1H),5.15 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.65, 163.73, 158.55, 141.10,140.90, 135.99, 135.95, 133.67, 132.49, 131.19, 129.98, 129.59, 128.45,122.96, 122.76, 121.18, 120.24, 117.46, 116.72, 113.85, 68.47. ESI-HRMScalcd. for C 23 H 18 ClNO4Na [M+Na] +430.0822, found 430.0822. HPLC purity: 99.70%.

[0199] Example 27: Preparation of (E)-2-(3-(3-((4-bromobenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 27, B9)

[0200] (1) Preparation of intermediate: (E)-2-(3-(3-((4-bromobenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB9)

[0201] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 4-bromobenzyl chloride to give a yellow solid in 65% yield, which was used directly in the next reaction without purification.

[0202] (2) Preparation of the target compound: (E)-2-(3-(3-((4-bromobenzyl)oxy)phenyl)acrylamido)benzoic acid

[0203] Starting with intermediate BMB9, the mixture was synthesized according to method A1, yielding a white solid in 73% yield. mp: 198.7–200.2 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 11.38 (s, 1H), 8.62 (d, J= 8.0 Hz, 1H), 8.02 (dd, J = 7.9, 1.5 Hz, 1H), 7.63 (dd, J = 10.1, 1.8 Hz,2H), 7.59 (t, J = 4.9 Hz, 2H), 7.45 (s, 1H), 7.43 (s, 2H), 7.35 (t, J = 7.8Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.21 - 7.15 (m, 1H), 7.08 - 7.03 (m, 1H),6.92 (d, J = 15.6 Hz, 1H), 5.15 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.45,141.21, 140.83, 136.43, 133.95, 131.38, 131.15, 130.01, 129.93, 122.89,122.86, 121.22, 120.41, 116.76, 113.87, 68.49. ESI-HRMS calcd. forC 23 H 18BrNO4Na [M+Na] + 474.0317, found 474.0321. HPLC purity: 99.23%.

[0204] Example 28: Preparation of (E)-2-(3-(3-((3-fluorobenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 28, B10)

[0205] (1) Preparation of intermediate: (E)-2-(3-(3-((3-fluorobenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB10)

[0206] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 3-fluorobenzyl chloride to give a yellow solid in 78% yield, which was used directly in the next reaction without purification.

[0207] (2) Preparation of the target compound: (E)-2-(3-(3-((3-fluorobenzyl)oxy)phenyl)acrylamido)benzoic acid

[0208] Starting with intermediate BMB10, the mixture was synthesized according to method A1, yielding a white solid in 86% yield. mp: 169.7-171.1℃. 1 H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.64 (d, J = 8.2 Hz,1H), 8.03 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 14.8 Hz, 2H), 7.45 (d, J = 14.0Hz, 2H), 7.33 (d, J = 7.4 Hz, 2H), 7.30 (s, 2H), 7.17 (d, J = 7.2 Hz, 2H), 7.06 (d, J = 7.2 Hz, 1H), 6.91 (d, J = 15.6 Hz, 1H), 5.18 (s, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 169.64, 163.74, 158.53, 141.13, 140.90, 139.94, 135.96,133.72, 131.19, 130.42, 130.00, 123.64, 122.95, 121.24, 120.28, 117.38,116.73, 114.73, 114.44, 114.22, 113.87, 68.48. ESI-HRMS calcd. for C23 H 18 FNO4Na[M+Na] + 414.1118, found 414.1119. HPLC purity: 99.28%.

[0209] Example 29: Preparation of (E)-2-(3-(3-((3-chlorobenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 29, B11)

[0210] (1) Preparation of intermediate: (E)-2-(3-(3-((3-chlorobenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB11)

[0211] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 3-chlorobenzyl chloride to give a yellow solid in 56% yield, which was used directly in the next reaction without purification.

[0212] (2) Preparation of the target compound: (E)-2-(3-(3-((3-chlorobenzyl)oxy)phenyl)acrylamido)benzoic acid

[0213] Starting with intermediate BMB11, the mixture was synthesized according to method A1, yielding a white solid in 76% yield. mp: 175.0-175.7℃. 1 H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 1H), 11.38 (s, 1H), 8.64 (d, J = 6.3 Hz, 1H), 8.07 - 7.97 (m, 1H), 7.66 - 7.57 (m, 2H), 7.54 (s, 1H), 7.42 (d, J = 13.2 Hz, 4H), 7.33 (d, J = 13.0 Hz, 2H), 7.12 (d, J = 42.1 Hz, 2H), 6.92 (d, J = 15.2 Hz, 1H), 5.17 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ169.50, 163.77, 158.50, 141.24, 140.91, 139.53, 135.95, 133.98, 133.18,131.17, 130.35, 130.01, 127.80, 127.42, 126.28, 122.87, 121.26, 120.41,116.81, 116.68, 113.96, 68.41. ESI-HRMS calcd. for C23 H 18 ClNO4Na [M+Na] + 430.0822, found 430.0825. HPLC purity: 99.46%. The test results are as follows. Figures 1 to 4 As shown.

[0214] Example 30: Preparation of (E)-2-(3-(3-((3-bromobenzyl)oxy)phenyl)acrylamido)benzoic acid (compound 30, B12)

[0215] (1) Preparation of intermediate: (E)-2-(3-(3-((3-bromobenzyl)oxy)phenyl)acrylamido)methyl benzoate (BMB12)

[0216] Starting with AMA4, it was synthesized according to the method of BMB1. It reacted with 3-bromobenzyl chloride to give a yellow solid in 69% yield, which was used directly in the next reaction without purification.

[0217] (2) Preparation of the target compound: (E)-2-(3-(3-((3-bromobenzyl)oxy)phenyl)acrylamido)benzoic acid

[0218] Starting with intermediate BMB12, the mixture was synthesized according to method A1, yielding a white solid in 85% yield. mp: 164.1-165.8℃. 1 H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.64 (d, J = 7.0 Hz,1H), 8.11 - 7.98 (m, 1H), 7.67 (s, 1H), 7.60 (d, J = 12.6 Hz, 2H), 7.52 (d, J= 5.6 Hz, 1H), 7.47 (d, J = 5.6 Hz, 1H), 7.42 (s, 1H), 7.39 - 7.25 (m, 3H), 7.16 (s, 1H), 7.06 (s, 1H), 5.16 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.63,163.74, 158.50, 141.11, 140.88, 139.78, 135.96, 133.73, 131.19, 130.71,130.65, 130.31, 130.02, 126.69, 122.97, 122.80, 121.75, 121.24, 120.28,116.67, 113.93, 68.35. ESI-HRMS calcd. for C 23 H 18 BrNO4Na [M+Na] + 474.0317, found 474.0317. HPLC purity: 98.70%.

[0219] The synthetic route of compound 31-35 (C1-5) is shown below:

[0220] .

[0221] Reagents and conditions: In reaction (i): a) SOCl2, DMF, DCM, 40℃; b) TEA, DCM, 0℃ to room temperature; In reaction (ii): SnCl2, MeOH, 60℃; In reaction (iii): various halogenated hydrocarbons, HATU, DIPEA, DMF, room temperature; In reaction (iv): NaOH, THF, H2O, 50℃.

[0222] (1) Preparation of intermediate CMA1

[0223] (E)-2-(3-(3-nitrophenyl)acrylamido)methyl benzoate (CMA1)

[0224] 3-Nitrocinnamic acid (4.85 g, 25.1 mmol) was dissolved in dichloromethane (30 mL), and thionyl chloride (9.2 mL, 75.3 mmol) was added. Two drops of DMF were added as a catalyst, and the mixture was heated and stirred at 40 °C. After the reaction was complete, the solvent was removed under reduced pressure to obtain an acyl chloride intermediate. This intermediate was dissolved in dichloromethane (10 mL) and added dropwise under ice bath conditions to a dichloromethane mixture of methyl 2-aminobenzoate (3.79 g, 25.1 mmol) and triethylamine (4.2 mL, 30.1 mmol). After the addition was complete, the mixture was stirred at room temperature for 8 h. After monitoring the reaction by thin-layer chromatography, the reaction solution was poured into water, extracted with dichloromethane, and the organic layer was dried over sodium sulfate and concentrated. The crude product was recrystallized from methanol / tetrahydrofuran to give a white solid with a yield of 70%. 1H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.62 (s, 1H), 8.46 (d, J = 8.1 Hz, 1H), 8.26 (dd, J= 8.1, 1.6 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.98 (dd, J = 7.9, 1.5 Hz, 1H),7.78 (d, J = 11.6 Hz, 1H), 7.75 (d, J = 3.9 Hz, 1H), 7.70 - 7.65 (m, 1H),7.25 (d, J = 4.1 Hz, 1H), 7.24 - 7.18 (m, 1H), 3.93 (s, 3H).

[0225] (2) Preparation of intermediate CMB1

[0226] (E)-2-(3-(3-aminophenyl)acrylamido)methyl benzoate (CMB1)

[0227] Intermediate CMA1 (5.00 g, 15.33 mmol) and stannous chloride (14.51 g, 76.65 mmol) were placed in a round-bottom flask, and methanol (50 mL) was added. The mixture was stirred at 70 °C for 2 h. After the reaction was complete, 1 M sodium hydroxide solution was added to adjust the pH to 12. The mixture was then filtered, and the filter cake was washed with water. The filtrates were combined, evaporated to dryness under reduced pressure, and the crude product was recrystallized from methanol / tetrahydrofuran to give a white solid in 64% yield. 1 H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.32(d, J = 8.4 Hz, 1H), 7.94 (dd, J = 7.9, 1.4 Hz, 1H), 7.65 - 7.60 (m, 1H),7.20 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 7.7 Hz, 1H), 6.50 (s, 1H), 6.44 (d, J= 7.7 Hz, 2H), 4.99 (s, 2H), 3.88 (s, 3H).

[0228] Example 31: Preparation of (E)-2-(3-(3-benzamidophenyl)acrylamido)benzoic acid (compound 31, C1)

[0229] (1) Preparation of intermediate: (E)-2-(3-(3-benzamidophenyl)acrylamido)methyl benzoate (CMC1)

[0230] Intermediate CMB1 (2.00 g, 6.75 mmol) and benzoic acid (0.82 g, 6.75 mmol) were placed in a reaction flask, and HATU (7.70 g, 20.25 mmol) and DIPEA (3.5 mL, 20.25 mmol) were added. DMF (50 mL) was added to dissolve the precipitate, and the mixture was stirred at room temperature. After the reaction was complete, the reaction solution was poured into water, and a yellow solid precipitated. The solid was filtered, washed with water and methanol, and dried to obtain a yellow solid with a yield of 65%. This solid was used directly in the next reaction without further purification.

[0231] (2) Preparation of the target compound: (E)-2-(3-(3-benzamidophenyl)acrylamido)benzoic acid

[0232] Starting with intermediate CMC1, the mixture was synthesized according to method A1, yielding a white solid in 89% yield. mp: 221.6–223.4 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 10.37 (s, 1H), 8.60 (d, J= 8.4 Hz, 1H), 8.13 (s, 1H), 8.01 (dd, J = 11.0, 7.7 Hz, 3H), 7.84 (d, J =7.8 Hz, 1H), 7.62 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 9.8 Hz, 2H), 7.55 (s,1H), 7.53 (s, 1H), 7.46 (d, J = 6.5 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.81(d, J = 15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 169.55, 165.70, 163.58,141.13, 140.76, 139.74, 134.77, 133.78, 131.72, 131.17, 129.31, 128.45,127.70, 123.84, 122.92, 122.63, 122.07, 120.39, 119.40, 117.55. ESI-HRMScalcd. for C 23 H 18 N₂O₄Na [M+Na] +409.1164, found 409.1167. HPLC purity: 96.53%.

[0233] Example 32: Preparation of (E)-2-(3-(3-(3-chlorobenzamido)phenyl)acrylamido)benzoic acid (compound 32, C2)

[0234] (1) Preparation of intermediate: (E)-2-(3-(3-(3-chlorobenzamido)phenyl)acrylamido)methyl benzoate (CMC2)

[0235] Starting with intermediate CMB1 and 3-chlorobenzoic acid, the mixture was synthesized following the method described for CMC1, yielding a yellow solid in 72% yield. It was used directly in the next reaction without purification.

[0236] (2) Preparation of the target compound: (E)-2-(3-(3-(3-chlorobenzamido)phenyl)acrylamido)benzoic acid

[0237] Starting with intermediate CMC2, the mixture was synthesized according to method A1, yielding a white solid in 83% yield. mp: 266.5–267.4 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 10.49 (s, 1H), 8.63 (d, J= 8.2 Hz, 1H), 8.09 (s, 1H), 8.06 (d, J = 8.9 Hz, 2H), 7.95 (d, J = 7.7 Hz,1H), 7.88 - 7.82 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H),7.55 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 6.4 Hz, 2H),7.09 (t, J = 7.4 Hz, 1H), 6.72 (d, J = 15.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 170.18, 164.16, 163.25, 140.87, 140.25, 139.47, 136.69, 134.92, 133.26,131.92, 131.50, 131.24, 130.41, 129.32, 127.48, 126.54, 124.02, 123.27,122.22, 121.87, 121.13, 119.32, 119.14. ESI-HRMS calcd. for C 23 H 17 ClN₂O₄Na [M+Na] + 443.0775, found 443.0779. HPLC purity: 99.06%.

[0238] Example 33: Preparation of (E)-2-(3-(3-(4-chlorobenzamido)phenyl)acrylamido)benzoic acid (compound 33, C3)

[0239] (1) Preparation of intermediate: (E)-2-(3-(3-(4-chlorobenzamido)phenyl)acrylamido)methyl benzoate (CMC3)

[0240] Starting with intermediate CMB1 and 4-chlorobenzoic acid, the mixture was synthesized following the method described for CMC1, yielding a yellow solid in 59% yield. It was used directly in the next reaction without purification.

[0241] (2) Preparation of the target compound: (E)-2-(3-(3-(4-chlorobenzamido)phenyl)acrylamido)benzoic acid

[0242] Starting with intermediate CMC3, the mixture was synthesized according to method A1 to obtain a white solid in 84% yield. mp: 224.8–226.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 10.42 (s, 1H), 8.60 (d, J= 8.3 Hz, 1H), 8.10 (s, 1H), 8.02 (d, J = 8.4 Hz, 3H), 7.82 (d, J = 7.8 Hz,1H), 7.63 (d, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 7.6 Hz, 1H), 7.43(t, J = 7.7 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 6.80 (d, J = 15.6 Hz, 1H). 13 CNMR (101 MHz, DMSO-d6) δ 169.55, 164.55, 163.54, 141.04, 140.76, 139.52,136.55, 134.78, 133.75, 133.43, 131.16, 129.66, 129.32, 128.51, 123.97,122.88, 122.68, 122.09, 120.35, 119.46, 117.52. ESI-HRMS calcd. forC 23 H 17 ClN₂O₄Na [M+Na] + 443.0775, found 443.0770. HPLC purity: 98.32%.

[0243] Example 34: Preparation of (E)-2-(3-(3-(2-(3-chlorophenyl)acetamido)phenyl)acrylamido)benzoic acid (compound 34, C4)

[0244] (1) Preparation of intermediate: (E)-2-(3-(3-(2-(3-chlorophenyl)acetamido)phenyl)acrylamido)methyl benzoate (CMC4)

[0245] Starting with intermediate CMB1 and 2-(3-chlorophenyl)acetic acid, the mixture was synthesized following the method described for CMC1, yielding a yellow solid in 81% yield. It was used directly in the next reaction without purification.

[0246] (2) Preparation of the target compound: (E)-2-(3-(3-(2-(3-chlorophenyl)acetamido)phenyl)acrylamido)benzoic acid

[0247] Starting with intermediate CMC4, the mixture was synthesized according to method A1, yielding a white solid in 82% yield. mp: 146.4–147.0 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.58 (s, 1H), 10.43 (s, 1H), 8.61 (d, J= 8.2 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.93 (s, 1H), 7.63 (s, 1H), 7.53 (d,J = 15.6 Hz, 1H), 7.41 (d, J = 9.3 Hz, 2H), 7.35 (d, J = 4.9 Hz, 3H), 7.31(d, J = 6.7 Hz, 2H), 7.06 (t, J = 7.4 Hz, 1H), 6.64 (d, J = 15.7 Hz, 1H),3.70 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 170.19, 168.82, 163.14, 140.85,140.03, 139.68, 138.27, 135.02, 132.83, 131.40, 131.24, 130.11, 129.42,129.16, 128.00, 126.57, 123.40, 123.35, 122.20, 122.04, 120.58, 119.10,117.70, 42.66. 24 H 19 ClN₂O₄Na [M+Na] + 457.0931, found457.0929. HPLC purity: 95.33%.

[0248] Example 35: Preparation of (E)-2-(3-(3-(2-(4-chlorophenyl)acetamido)phenyl)acrylamido)benzoic acid (compound 35, C5)

[0249] (1) Preparation of intermediate: (E)-2-(3-(3-(2-(4-chlorophenyl)acetamido)phenyl)acrylamido)methyl benzoate (CMC5)

[0250] Starting with intermediate CMB1 and 2-(4-chlorophenyl)acetic acid, the mixture was synthesized following the method described for CMC1, yielding a yellow solid in 68% yield. It was used directly in the next reaction without purification.

[0251] (2) Preparation of the target compound: (E)-2-(3-(3-(2-(4-chlorophenyl)acetamido)phenyl)acrylamido)benzoic acid

[0252] Starting with intermediate CMC5, the mixture was synthesized according to method A1, yielding a white solid in 80% yield. mp: 199.1–202.3 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 10.33 (s, 1H), 8.58 (d, J= 8.2 Hz, 1H), 8.02 (dd, J = 7.9, 1.3 Hz, 1H), 7.93 (s, 1H), 7.62 (d, J = 7.7Hz, 1H), 7.60 - 7.53 (m, 2H), 7.38 (d, J = 2.8 Hz, 5H), 7.36 - 7.34 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 6.74 (d, J = 15.7 Hz, 1H), 3.68 (s, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 169.61, 168.98, 163.45, 140.81, 140.72, 134.86, 134.84,133.40, 131.3, 131.15, 131.09, 129.42, 128.22, 123.35, 122.77, 120.74,120.18, 118.24, 118.06, 42.42. ESI-HRMS calcd. for C 24 H 19 ClN₂O₄Na [M+Na] + 457.0931, found 457.0934. HPLC purity: 96.64%.

[0253] II. Bioactivity Test

[0254] Cell culture and animal feeding were performed before the experiment, as shown below:

[0255] Peritoneal macrophages (PMs) and THP-1 cells were collected from male mice and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin at 37°C in a 5% CO2 incubator. Bone marrow-derived macrophages (BMDMs) were obtained from the bone marrow of male mice and cultured in DMEM medium containing 10% FBS and 20% L929 cell supernatant. After 6 days, BMDMs were ready for subsequent experiments. HK2 and HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin at 37°C in a 5% CO2 incubator. Only primary PMs and BMDMs were used in the experiments. Kunming mice and SD rats were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All mice and rats were housed under 50% humidity, 12:12 h diurnal cycle conditions, and provided with standard feed and sterilized water for free consumption. Animals need to acclimatize to the environment for one week before the experiment.

[0256] 1. Activity screening experiment of target compound

[0257] The isolated primary mouse macrophages were cultured at 1.0 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of cells / mL in 96-well plates and cultured overnight. The next day, the medium was discarded, and medium containing LPS (100 ng / mL) was added and incubated for 3 h (the blank group was replaced with fresh serum-free medium). Subsequently, the test group was incubated with drug-containing medium at the set concentration for 1 h, while the model group and blank group were replaced with fresh serum-free medium. ATP (25 mM) solution was prepared fresh and adjusted to neutral with 1 M NaOH. After drug incubation, 11.14 μL of ATP (final concentration 2.5 mM) was added to each well via replenishment, and the blank group was incubated with an equal volume of serum-free medium for another 30 min. Cell supernatant was collected, centrifuged, and appropriately diluted according to the ELISA kit instructions before IL-1β content was measured.

[0258] a PMs cells were pretreated with LPS (100 ng / mL) for 3 h as stimulation. Subsequently, different concentrations of compounds were added for 1 h, followed by ATP (2.5 mM) for 30 min. Cell supernatants were collected and analyzed using ELISA. b The concentration of MCC950 was 0.1 μM. As shown in Table 1-3, "nd" indicates not detected, and "NA" indicates no activity. IC 50 The units are in nM. Most compounds showed significant inhibitory activity against NLRP3 inflammasome-mediated inflammatory responses, with the representative compound B11 showing the best inhibitory effect (IC50). 50 = 0.57 ± 0.08 μM).

[0259] Table 1. Inhibitory activity of compounds 1-19 on NLRP3 inflammasome-mediated IL-1β release.

[0260]

[0261] Table 2. Inhibitory activity of compounds 19-29 on NLRP3 inflammasome-mediated IL-1β release.

[0262]

[0263] Table 3. Inhibitory activity of compounds 30-35 on NLRP3 inflammasome-mediated IL-1β release.

[0264]

[0265] Based on the chemical structure of tranilast, this invention employs a drug design strategy combining ligand-directed and structure-directed approaches. While retaining its advantageous skeletal structure, precise structural modification and functional optimization were carried out to design and synthesize 35 novel 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds in three series (IA, IB, and IC). Preliminary cell activity screening results show that most compounds in this series can inhibit LPS / ATP-induced NLRP3 inflammasome activation to varying degrees and reduce IL-1β release levels, indicating that this class of compounds possesses overall NLRP3 inflammasome inhibitory activity.

[0266] Based on this, the present invention selected the representative compound B11, which exhibits the best activity, for further mechanistic studies, SPR binding experiments, metabolic stability evaluation, and in vivo pharmacodynamic studies. The results showed that this representative compound can directly bind to the NACHT domain of the NLRP3 protein and significantly inhibit ASC spot formation, caspase-1 activation, and the release of mature IL-1β. Given that the compounds in this series have highly similar core scaffold structures, electronic effect characteristics, and spatial conformational properties, they are expected to have similar binding modes and mechanisms of action with NLRP3 target proteins. Therefore, those skilled in the art can reasonably infer that other compounds in the same series also possess similar NLRP3 inflammasome regulatory activity and anti-inflammatory application potential.

[0267] 2. MTT cytotoxicity assay

[0268] To eliminate false positive results due to cytotoxicity in the in vitro activity screening of compounds, the cytotoxicity of the representative compound B11 and the existing drug MCC950 was evaluated in mouse primary macrophages (PMs). Mouse primary macrophages were extracted and processed at 10 × 10⁻⁶ cells. 4The cells were seeded at a density of [missing value] mL in 96-well plates and incubated overnight. The next day, the medium was aspirated and replaced with the appropriate concentration of drug-containing medium, and incubated for 1 h. The control group was replaced with serum-free fresh medium. For HK2 and HepG2 cells, the appropriate concentration of drug-containing medium was added via replenishment and incubated for 48 h. After incubation, 20 μL of MTT solution was added to each well via replenishment in the dark, and incubated for 4 h. The liquid containing MTT was carefully aspirated, and 100 μL of DMSO was added to each well. The plates were shaken for 10 min to fully dissolve the formazan produced by the reaction of live cells with MTT. The absorbance was then measured at wavelengths of 490 nm, 570 nm, and 630 nm. A Synergy H1 multi-functional microplate reader was used for absorbance testing. The formula for calculating the cytotoxicity inhibition rate of the test compound is as follows:

[0269] .

[0270] like Figure 5 As shown, PMs cells were pre-incubated with the test compound for 1 h. Cell viability was assessed using the MTT assay. Statistical analysis was performed using the Student's test, *p < 0.05. Within the critical test concentration range, compound B11 did not exhibit significant cytotoxicity; in contrast, the positive control drug MCC950 showed some degree of cytotoxicity at a concentration of 1000 nM. These results indicate that the representative compound B11 is safer than the positive control drug MCC950 in PMs cells, demonstrating the reliability of the activity data obtained from in vitro screening and the absence of false-positive bias due to cell death, thus providing effective assurance for subsequent biological evaluation.

[0271] 3. Surface Plasmon Resonance (SPR) Experiment

[0272] To further confirm that the in vitro anti-inflammatory activity of the compound originates from its direct targeting of the NLRP3 inflammasome, surface plasmon resonance (SPR) technology was used to conduct a molecular interaction study to confirm the target of the candidate compound B11. NLRP3 protein was used as the SPR ligand, diluted to 50 μg / mL with pH 5.0 sodium acetate buffer (BR-1003-51, Cytiva) before injection. The test compound was used as the analyte, diluted to different concentrations with PBS-P running buffer (calibration buffer) containing 5% DMSO. The ligands were manually conjugated onto a Series S SensorChip CM7 (Cytiva) chip at a conjugation level of approximately 11000 RU at a flow rate of 5 μL / min. After ligand immobilization, the analyte, standard curve, and calibration buffer were injected sequentially for analysis at a flow rate of 30 μL / min for 120 s. The recombinant human NLRP3 NACHT domain protein used in the experiment was purchased from MedChemExpress. Data were acquired using a Biacore T200 instrument, and the equilibrium dissociation constant (K0) was automatically calculated using the accompanying software. - D) and response unit (RU).

[0273] The results are as follows Figure 6 As shown, compound B11 exhibits rapid binding and dissociation characteristics with the NLRP3 NACHT domain, showing a clear dose-dependent relationship. Its binding affinity K0 - D is 405.7 nM. These results indicate that this class of compounds can directly bind to the key domains of the NLRP3 inflammasome and exert an inhibitory effect.

[0274] 4. Selective test of inflammasomes

[0275] Besides the most thoroughly studied NLRP3, the inflammasome family includes several other types, such as the NLRC4 and AIM2 inflammasomes. These inflammasomes share some structural similarities with NLRP3 and can all be activated by specific stimuli, thereby promoting IL-1β release and triggering an inflammatory response. Therefore, poly(dA:dT) and Flagellin were used as stimuli to induce the activation of AIM2 and NLRC4 inflammasomes to evaluate the selectivity of candidate compounds for different inflammasomes. Statistical analysis was performed using Student's t-test; *p < 0.05, ns indicates no significant difference. Results are as follows: Figure 7As shown, compound B11 significantly inhibited NLRP3 inflammasome-mediated IL-1β release, but had no significant effect on NLRC4 and AIM2 inflammasomes, indicating that the compound has good selective inhibitory activity against NLRP3 inflammasomes.

[0276] 5. ASC dot test

[0277] To investigate the mechanism of action of the compound in the activation stage of the NLRP3 inflammasome, its effect on ASC spot formation was examined using immunofluorescence assay. ASC oligomerization promotes caspase-1 activation, a key step in the assembly and activation of the NLRP3 inflammasome, and its occurrence can be observed as a typical spot signal through fluorescent staining. PMs cells were cultured at 2.0 × 10⁶ cells / year. 5 Cells / mL were seeded in 24-well plates with climbing smears and cultured overnight. The next day, the cells were stimulated with LPS (1 μg / mL) for 3 h, followed by incubation with different concentrations of compounds for 1 h, and then stimulated with ATP (final concentration 5 mM) for 30 min. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and incubated with anti-ASC primary antibody (1:1000, 4℃ overnight) and FITC-labeled secondary antibody. After DAPI staining, the cells were imaged under a fluorescence microscope.

[0278] The results are as follows Figure 8 A and Figure 8 As shown in Figure B, (A) Immunofluorescence staining of ASC spots (combined graph: green represents ASC, blue represents DAPI). (B) Statistical graph of the proportion of ASC spot-positive cells. Statistical analysis was performed using the Student's test, **p < 0.01; ##p < 0.01; ns indicates no significant difference. The representative compound B11 can inhibit the formation of ASC spots in a dose-dependent manner, indicating that the compound can inhibit the activation of inflammasomes by blocking the assembly process of NLRP3 inflammasomes.

[0279] 6. Western Blot Experiment

[0280] To further elucidate the molecular mechanism by which a series of compounds block NLRP3 inflammasome activation, Western blot was used to detect related pathway proteins. Bone marrow-derived macrophages (BMDMs) were cultured at 2.0 × 10⁻⁶ cells / mL. 5Cells / mL were seeded in 6-well plates and cultured for 7 days in DMEM medium containing 10% FBS and 20% L929 cell supernatant, followed by NLRP3 inflammasome activation protocol. After treatment, cell supernatant and intracellular proteins were collected separately: the supernatant was precipitated with methanol / chloroform and then lysed with RIPA containing a protease inhibitor; cells were washed with pre-cooled PBS, scraped, and lysed with RIPA in the same manner. The obtained proteins were quantified using the BCA method (562 nm), denatured with 5×SDS loading buffer, and then subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk powder, IL-1β, caspase-1, NLRP3, and ASC primary antibody (all 1:1000) were added and incubated overnight at 4°C. The next day, the corresponding secondary antibodies were incubated, and detection was performed using ECL chemiluminescence immunoassay. Analysis was conducted using a Tanon-5200 imaging system.

[0281] The results are as follows Figure 9 The image shows Western blot analysis of IL-1β and caspase-1 in the culture supernatant (Sup.) and ASC, NLRP3, pro-IL-1β, and pro-caspase-1 in the cell lysate (Lys.) of BMDMs. The representative compound B11 dose-dependently reduced the levels of mature IL-1β and caspase-1 proteins, while having no significant effect on the expression of ASC, NLRP3, pro-IL-1β, and pro-caspase-1, indicating that it primarily acts on the activation phase of the NLRP3 inflammasome without affecting the related signaling pathways in the initiation phase.

[0282] 7. Safety assessment experiment

[0283] Cytotoxicity assays were performed in the hepatocyte (HepG2) and kidney (HK2) cell lines to assess the safety of the candidate compounds, with colchicine used as a reference compound. As a marketed drug for the clinical treatment of gouty arthritis, colchicine has significant adverse reactions, requiring strict dosage control in clinical application. Cytotoxicity evaluation of colchicine and B11: HepG2 and HK2 cells were pre-incubated with the test compounds for 48 h. Cell viability was measured using the MTT assay. Statistical analysis was performed using Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001. Results are as follows: Figure 10 As shown, colchicine exhibited significant cytotoxicity against HepG2 and HK2 cells in the concentration range of 0.39-100 μM; in contrast, the representative compound B11 did not show significant cytotoxicity under the same conditions, indicating good in vitro safety.

[0284] 8. In vivo pharmacokinetic experiments

[0285] In vivo pharmacokinetic studies of the compounds were conducted using Sprague-Dawley rats (SD rats), consistent with in vivo pharmacodynamic experiments, to systematically evaluate their absorption, distribution, metabolism, and excretion characteristics in vivo. Two administration routes were investigated: gavage (10 mg / kg) and tail vein injection (10 mg / kg). The results are shown in Table 4. Compound B11, representing the compound, exhibited relatively ideal pharmacokinetic characteristics: its half-life (T0) was [not specified in the original text]. 1 / 2 The efficacy was 5.00 h and 6.85 h under oral and intravenous administration, respectively; the area under the curve (AUC0-∞) was 12.89 mg·h / L and 73.52 mg·h / L, respectively; and the maximum plasma concentration (Cmax) was 3.98 mg / L and 70.03 mg / L, respectively.

[0286] Furthermore, the representative compound B11 exhibits a low clearance rate (0.78 L·h⁻¹ for oral and intravenous administration, respectively). -1 ·kg -1 and 0.14 L·h -1 ·kg -1 The oral distribution volume was moderate (Vz = 5.55 L / kg), while the intravenous distribution volume was relatively low (Vz = 1.34 L / kg). This indicates that this class of compounds has a good pharmacokinetic basis and possesses potential for further development.

[0287] Table 4. Pharmacokinetic parameters of B11

[0288]

[0289] 9. Pharmacodynamic experiment of MSU-induced acute gouty arthritis in rats

[0290] To evaluate the in vivo anti-inflammatory therapeutic effects of the compounds, an MSU-induced acute gouty arthritis model in Gout rats was used, with colchicine, a first-line clinical drug, as a positive control. Representative compounds B11 and colchicine at various doses were administered prophylactically via gavage 1 hour before model establishment. Ankle joint diameters were measured at 0, 2, 4, 6, 12, and 24 hours post-modeling, and representative images of the animal's foot joints were taken at 0, 12, and 24 hours. Joint tissue samples were collected at 12 hours for HE staining analysis.

[0291] (A) Flowchart of the MSU-induced rat gouty arthritis model. (B) Representative images of foot appearance at 0, 12, and 24 h in each group. (C) Changes in ankle joint diameter measured at different time points after MSU injection. ***p < 0.001. (D) Serial sections of paraffin-embedded joint tissue stained with hematoxylin and eosin (H&E), showing representative sections from 5 samples (n = 5). Magnification: ×40 and ×400.

[0292] Results of the anti-inflammatory activity of B11 in vivo: Figure 11 As shown in A and B, no significant joint swelling was observed in the blank group, while varying degrees of joint swelling were observed in both the model group and the drug-treated group within 0-12 hours, indicating that MSU successfully induced an acute gouty arthritis model. Compared with the model group, all drug-treated groups significantly reduced ankle joint swelling, demonstrating good therapeutic effects; HE staining results further showed ( Figure 11 As shown in Figure C, compounds B11 and colchicine can significantly reduce inflammatory cell infiltration in joint tissues. These results indicate that the compounds can effectively alleviate MSU-induced acute gouty arthritis in rats and possess good in vivo anti-inflammatory activity.

[0293] In summary, the 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds provided by this invention exhibit significant NLRP3 inflammasome inhibitory activity, effectively inhibiting inflammasome activation and IL-1β release both in vivo and in vitro, thereby achieving precise regulation of the inflammatory response. These compounds can be used to prepare drugs for the treatment and / or prevention of diseases associated with abnormal NLRP3 inflammasome activation (such as gouty arthritis, rheumatoid arthritis, inflammatory bowel disease, and atherosclerosis), demonstrating good activity and safety profiles, and possessing significant research value and broad prospects for drug development and application.

[0294] The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds and their pharmaceutically acceptable salts provided by this invention, when formulated into any dosage form, possess pharmacological effects in preventing, alleviating, and / or treating NLRP3 inflammasome overactivation and its complications. Any pharmaceutical preparation containing 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds of general formulas IA, IB, and IC, or prepared solely as a component of 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds of general formulas IA and IB, as long as its packaging, instructions, or other promotional materials indicate or suggest its therapeutic effect on NLRP3 inflammasome overactivation and its complications, is also within the scope of protection of this invention.

Claims

1. A 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or a pharmaceutically acceptable salt, characterized in that, The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compounds are shown in general formula IA: ； Where R1 is located at any position in the benzene ring 2, 2', 3, 3', 4; R1 is independently selected from: hydrogen, alkoxy, hydroxyl, alkyl, haloalkyl, halogen, phenyl, -O-R2, -NH-C=O-R3; R2 is independently selected from: substituted or unsubstituted benzyl, substituted or unsubstituted phenethyl, substituted or unsubstituted ethylpyridyl, C 1-5 Saturated or unsaturated alkyl groups; R3 is independently selected from: substituted or unsubstituted phenyl, substituted or unsubstituted benzyl.

2. The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or pharmaceutically acceptable salt according to claim 1, characterized in that, R1 is independently selected from: methoxy, hydroxy, methyl, trifluoromethyl, fluorine, bromine, phenyl, and R1 is located at the meta or para position of the phenyl group, corresponding to the 3, 3', or 4 position of the benzene ring in formula IA; when R1 is a halogen chlorine, R1 is located at the meta, ortho, or para position of the phenyl group, corresponding to the 2, 2', 3, 3', or 4 position of the benzene ring in formula IA.

3. The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or pharmaceutically acceptable salt according to claim 1, characterized in that, When R1 is -O-R2, it is as shown in the general formula IB: ； In the formula, R2 is independently selected from: benzyl, phenethyl, 4-ethylpyridinyl, 2-methyl-2-pentenyl, 2-methylpentyl, 4-methylbenzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl.

4. The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or pharmaceutically acceptable salt according to claim 1, characterized in that, When R1 is -NH-C=O-R3, it is as shown in the general formula IC: ； In the formula, R3 is independently selected from: phenyl, 3-chlorophenyl, 4-chlorophenyl, 3-chlorobenzyl, 4-chlorobenzyl.

5. The 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or pharmaceutically acceptable salt according to claim 1, characterized in that, Selected from any of the following compounds: 。 6. A method for preparing the 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or a pharmaceutically acceptable salt as described in claim 1, characterized in that, When R1 is a non-aromatic group selected from hydrogen, alkoxy, hydroxyl, alkyl, haloalkyl, or halogen, the following steps are included: Compound Ia reacts with compound IIa to obtain IIIa, which is then hydrolyzed to obtain formula IA; #imgpt38#; where: a is the amidation reaction; b is the hydrolysis reaction.

7. A method for preparing the 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or a pharmaceutically acceptable salt as described in claim 1, characterized in that, When R1 is phenyl, the following steps are included: Compound Ib and compound IIb were simultaneously coupled and hydrolyzed under alkaline conditions to obtain formula IA; #imgpt39#; In the formula: a represents the Suzuki-Miyaura coupling reaction.

8. A method for preparing a 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or a pharmaceutically acceptable salt as described in claim 1 or 3, characterized in that, When R1 is -0-R2, the following steps are included: Compound Ic is reacted with a haloalkane to give compound IIc, which is then hydrolyzed to give IB. #imgpt40# In the formula: a represents the alkylation reaction; b represents the hydrolysis reaction.

9. A method for preparing a 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound or a pharmaceutically acceptable salt as described in claim 1 or 4, characterized in that, When R1 is -NH-C=O-R3, the following steps are included: Compound Id and compound IId are condensed to obtain compound IIId, which is then reduced to obtain compound IVd. Finally, compound V is condensed with various acids to obtain compound V. Compound V is hydrolyzed to obtain formula IC. #imgpt41#; In the formula: a represents condensation reaction; b represents reduction reaction; c represents amidation reaction; and d represents hydrolysis reaction.

10. A pharmaceutical composition, characterized in that, Its active ingredient is selected from the compounds of any one of claims 1-5 or pharmaceutically acceptable salts.

11. Use of any 2-[(3-phenylprop-2-enoyl)amino]benzoic acid compound of claims 1-5 and its pharmaceutically acceptable salt in the preparation of medicaments for the prevention, relief and / or treatment of diseases and complications associated with abnormal activation of the NLRP3 inflammasome; The diseases mentioned include: gouty arthritis, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, type 2 diabetes, and Alzheimer's disease; The complications mentioned include: joint erosion, intestinal mucosal barrier damage, kidney injury, cardiovascular events, and neurodegenerative diseases.

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