Chalcone derivatives and uses thereof

Abstract

The application discloses a chalcone derivative with a structure shown in formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof and application thereof as an active ingredient in preparation of an NLRP3 inflammasome inhibitor and / or a STATs pathway inhibitor. The compound can selectively inhibit the activation of the NLRP3 inflammasome and can inhibit the STATs pathway, thereby treating or improving diseases related to the NLRP3 inflammasome and / or the STATs pathway, such as acute peritonitis and colitis, and can be used in preparation of a therapeutic drug for diseases related to the NLRP3 inflammasome and / or the STATs pathway.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a class of chalcone derivatives and their applications. Background Technology

[0002] 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 procaspase-1 (Pro-caspase-1). It is a multi-protein complex located in the cytoplasm. Upon activation of the NLRP3 inflammasome, 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), ultimately leading to an inflammatory response and pyroptosis. Extensive evidence suggests a close association between the NLRP3 inflammasome and many human diseases, such as Alzheimer's disease, gout, multiple sclerosis, type II diabetes, and inflammatory bowel disease. To date, several NLRP3 inflammasome inhibitors have been discovered, but none are currently available for clinical use.

[0003] Furthermore, the JAK / STAT pathway is also a key pro-inflammatory signaling pathway that mediates the biological effects of various cytokines in inflammatory diseases. Interfering with the JAK and STAT families offers novel approaches for some patients unresponsive to current therapies. For example, tofacitinib, a non-specific inhibitor of JAK, affects multiple pro-inflammatory cytokine-dependent and STAT-mediated pathways, potentially aiding in the treatment of patients who have failed or are intolerant of conventional or biologic therapies, such as those with rheumatoid arthritis, ulcerative colitis, psoriasis, and other inflammatory diseases. Researching the impact of STAT proteins on uncontrolled inflammatory pathology has become an active area of ​​study.

[0004] Therefore, the discovery of novel dual inhibitors of the NLRP3 inflammasome / STAT pathway is of great significance for the treatment of inflammation-related diseases. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a class of chalcone derivatives that can inhibit the activation of the NLRP3 inflammasome and suppress the STAT pathway, thereby treating or improving diseases related to the NLRP3 inflammasome and / or the STAT pathway, such as acute peritonitis and colitis.

[0006] This invention includes the following technical solutions:

[0007] Chalcone derivatives having the structure shown in formula (I), or pharmaceutically acceptable salts thereof, or stereoisomers thereof,

[0008]

[0009]

[0010] Among them, R1 and R2 are independently selected from: H, -C(=O)OR3, -C(=O)N(R4)2, and -N(R5)-C(=O)R6;

[0011] R3 is selected from: H, C1-C6 alkyl groups;

[0012] Each R4 is independently selected from: H, one or more R7-substituted C1-C6 alkyl groups, and one or more R7-substituted C3-C6 cycloalkyl groups;

[0013] R5 is selected from: H, C1-C6 alkyl groups;

[0014] R6 is selected from: one or more R7-substituted C1-C6 alkyl groups, or one or more R7-substituted C3-C6 cycloalkyl groups;

[0015] Each R7 is independently selected from: H, -C(=O)OR3, C6-C 10 Aryl, C6-C 10 Arylmethyl, -C(=O)N(R8)2;

[0016] Each R8 is independently selected from: H, C1-C6 alkyl, C6-C 10 Aryl or borate-substituted C1-C8 alkyl groups;

[0017] Alternatively, R1, R2, and the carbon atom attached to them can form a 5-10 membered heterocyclic group.

[0018] In some embodiments, R1 and R2 are independently selected from: H, -C(=O)OR3, -C(=O)N(R4)2, -N(R5)-C(=O)R6;

[0019] R3 is selected from: H, C1-C3 alkyl groups;

[0020] Each R4 is independently selected from: H, one or more R7-substituted C1-C3 alkyl groups;

[0021] R5 is selected from: H, C1-C3 alkyl groups;

[0022] R6 is selected from one or more R7-substituted cyclopropyl groups;

[0023] Each R7 is independently selected from: H, -C(=O)OR3, phenyl, benzyl, -C(=O)N(R8)2;

[0024] Each R8 is independently selected from: H, C1-C3 alkyl, phenyl, borate-substituted C3-C6 alkyl;

[0025] Alternatively, R1, R2, and the carbon atom attached to them can form an 8-10 membered heterocyclic group.

[0026] In some embodiments, R1 and R2 are independently selected from: H, -C(=O)OR3, -C(=O)N(R4)2, -N(R5)-C(=O)R6;

[0027] R3 is selected from: methyl, ethyl;

[0028] Each R4 is independently selected from: H, or one or more R7-substituted methyl groups;

[0029] R5 is selected from: H;

[0030] R6 is selected from one or more R7-substituted cyclopropyl groups;

[0031] Each R7 group is independently selected from: H, -C(=O)OR3, carboxyl, benzyl, -C(=O)N(R8)2;

[0032] Each R8 is independently selected from: H, methyl, ethyl, or isopentyl groups substituted with borate;

[0033] Alternatively, R1, R2, and the carbon atom attached to them can form an 8-membered heterocyclic group.

[0034] In some embodiments, one of R4 is H and the other is not H.

[0035] In some embodiments, when one R7 is a carboxyl group, the number of R7s is 2, and the other R7 is selected from: -C(=O)OR3, benzyl, -C(=O)N(R8)2.

[0036] In some embodiments, R2 is H and R1 is not H.

[0037] This invention also provides applications of the chalcone derivatives, or their pharmaceutically acceptable salts, or their stereoisomers, including the following technical solutions:

[0038] The use of the chalcone derivatives, or their pharmaceutically acceptable salts, or their stereoisomers, in the preparation of NLRP3 inflammasome inhibitors and / or STAT pathway inhibitors.

[0039] The use of the chalcone derivatives, or their pharmaceutically acceptable salts, or their stereoisomers, in the preparation of medicaments for the prevention and / or treatment of diseases associated with the NLRP3 inflammasome and / or the STAT pathway.

[0040] In some embodiments, the diseases associated with the NLRP3 inflammasome and / or STAT pathway are inflammatory diseases, Alzheimer's disease, gout, multiple sclerosis, and type II diabetes.

[0041] In some embodiments, the inflammatory disease is inflammatory bowel disease, rheumatoid arthritis, or psoriasis.

[0042] In some embodiments, the inflammatory disease is peritonitis and colitis.

[0043] In some embodiments, the peritonitis is acute peritonitis.

[0044] The present invention also provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases, comprising the following technical solutions:

[0045] A pharmaceutical composition for the prevention and treatment of inflammatory diseases, prepared from an active ingredient and pharmaceutically acceptable excipients, said active ingredient including the chalcone derivatives or their pharmaceutically acceptable salts or stereoisomers.

[0046] The chalcone derivatives or pharmaceutically acceptable salts thereof provided by this invention can selectively inhibit the activation of the NLRP3 inflammasome and inhibit the STAT pathway, thereby treating or improving diseases associated with the NLRP3 inflammasome and / or the STAT pathway, such as acute peritonitis and colitis, and can be used to prepare therapeutic drugs for diseases associated with the NLRP3 inflammasome and / or the STAT pathway. Attached Figure Description

[0047] Figure 1 The figure shows the results of compound 10v inhibiting the activation of NLRP3 inflammasome in vitro; where A is the result of Western blot analysis, B is the result of IL-1β secretion in BMDMs cell model activated by NLRP3 inflammasome, C is the result of IL-1β secretion in BMDMs cell model activated by NLRC4 inflammasome, and D is the result of IL-1β secretion in BMDMs cell model activated by AIM2 inflammasome.

[0048] Figure 2 The figure shows the results of compound 10v inhibiting NLRP3 inflammasome assembly and STAT protein expression.

[0049] Figure 3 The figure shows the results of compound 10v improving sodium dextran sulfate (DSS)-induced colitis.

[0050] Figure 4 The results of pathological sections of mouse colon tissue. Detailed Implementation

[0051] In the compounds described in this invention, when any variable (e.g., R) 4 If a component (e.g., a substituent) appears more than once in any component, the definition of each occurrence is independent of the definition of each subsequent occurrence. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. A line drawn from a substituent into the ring system indicates that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It should be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is itself substituted by more than one group, it should be understood that these groups can be on the same carbon atom or on different carbon atoms, as long as the structure is stable.

[0052] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0053] The term "heterocyclic group" as used in this invention refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent (including monocyclic, spirocyclic, fused, bridged rings, etc.), wherein one or more ring atoms are selected from heteroatoms of N, O or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0055] The raw materials used in the following examples may be commercially available, or prepared by methods known in the art, or prepared according to the methods described herein.

[0056] The synthetic route of the compounds of this invention is as follows:

[0057]

[0058] Synthetic reaction conditions for chalcone derivatives: (a) DMAP, DIPEA, dichloromethane, room temperature; (b) sodium hydroxide, methanol, room temperature; (c) 1M hydrochloric acid aqueous solution, methanol, 60°C; (d) concentrated sulfuric acid, ethanol, 60°C; (e) various amino acid methyl esters, DIPEA, HATU, dichloromethane, from 0°C to room temperature; (f) lithium hydroxide, water, methanol, room temperature; (g) various aminoacetamides, EDCI-hydrochloric acid, HOBt, DIPEA, dichloromethane, from 0°C to room temperature; (h) trifluoroacetic acid, dichloromethane, room temperature; (i) isobutylboronic acid, 1M hydrochloric acid aqueous solution, n-hexane / methanol.

[0059] The following are specific examples.

[0060] Example 1: Synthesis of (E)-4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzoic acid (5a):

[0061]

[0062] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0063] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0064] (3) The product (4a) obtained in step (2) was mixed with MeOH, and then 1M HCl (2.0 equiv.) was added dropwise. The mixture was heated under reflux in an oil bath at 60°C for 1 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by evaporation under reduced pressure, dried with anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography (DCM:MeOH = 10:1) to obtain a yellow solid (5a), with a yield of 58%. 1HNMR (400MHz, DMSO-d6) δ 8.20–8.13 (m, 2H), 8.08–8.02 (m, 2H), 7.69 (d, J = 15.5 Hz, 1H), 7.61 (d, J = 15.5 Hz, 1H), 7.48 (s, 2H), 2.17 (s, 6H). HRMS (ESI) calculated values: C 18 H 16 O4[M+H] + Experimental value: 297.1082; Value: 297.1122.

[0065] Example 2(E) Synthesis of ethyl 4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzoate (5b):

[0066]

[0067] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0068] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0069] (3) The carboxylic acid (4a) obtained in step (2) was mixed with ethanol, and concentrated sulfuric acid (3.0 equiv) was added. The mixture was heated under reflux in an oil bath at 60°C for 4 h, and the reaction progress was monitored by TLC. After the reaction was complete, the solvent was evaporated under reduced pressure, and the mixture was extracted with EA and water. The organic phase was retained, dried with anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography to obtain a white solid (5b) (PE:EA = 3:1), with a yield of 98%. 1HNMR (400MHz, Methanol-d4) δ 8.12 (d, J = 8.0Hz, 2H), 8.08 (d, J = 8.0Hz, 2H), 7.68 (d, J = 15.7Hz, 1H), 7.50 (d, J = 15.6, 3.0Hz, 1H), 7.34 (s, 2H), 4.37 (q, J = 7.2Hz, 2H), 2.21 (s, 6H), 1.38 (t, J = 7.1Hz, 3H); HRMS (ESI) calculated values: C 20 H 20 O4[M+H] + Experimental value: 325.1395; 325.1432.

[0070] Example 3: Synthesis of (E)-3-(4-hydroxy-3,5-dimethylphenyl)-1-(3'H-spiro[azacyclobutane-3,1'-isobenzofuran]-5'-yl)prop-2-en-1-one (7):

[0071]

[0072] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0073] (2) 5'-acetyl-3'H-spiro[azacyclobutane-3,1'-isobenzofuran]-1-carboxylic acid tert-butyl ester (6) (1.0 equiv.) was mixed with the product (2) from step (1) in MeOH, and then a saturated solution of NaOH (2.0 equiv.) was added. The mixture was stirred and reacted at room temperature for 24 h. The reaction progress was monitored by TLC. Subsequently, the MeOH in the solution was removed by vacuum evaporation, and the obtained product (7a) (DCM:MeOH = 10:1) was purified by thin-layer chromatography.

[0074] (3) The product (7a) obtained in step (2) was dissolved in DCM, and trifluoroacetic acid (10 equiv) was added. The mixture was stirred and reacted at room temperature for 4 h. The mixture was then rotary evaporated, extracted with EA and water, and the organic layers were combined, dried, and purified by thin-layer chromatography to obtain a pale yellow solid (7) with a yield of 63%. 1HNMR (400MHz, CD3OD) δ 8.13 (d, J = 8.0Hz, 1H), 7.98 (s, 1H), 7.83 (d, J = 7.9Hz, 1H), 7.69 (d, J = 15.5Hz, 1H), 7.52 (d, J = 15.5Hz, 1H), 7.35 (s, 2H), 5.21 (s, 2H), 4.50 (d, J = 11.8Hz, 2H), 4.44 (d, J = 11.9Hz, 2H), 2.22 (s, 6H); HRMS (ESI) calculated values: C 21 H 21 NO3[M+H] + Experimental value: 336.1555; 336.1593.

[0075] Example 4: Synthesis of methyl (4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)phenyl)carbamoyl)cyclopropane-1-carboxylic acid (5d):

[0076]

[0077] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0078] (2) P-acetylanisole (3c) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4c).

[0079] (3) 1,1-Cyclopropyldicarboxylic acid monomethyl ester (1.0 equiv.) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, the compound (4c) (1.2 equiv.) obtained in step (2) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography to obtain a pale yellow solid (4d).

[0080] (4) The product (4d) obtained in step (3) was mixed with MeOH, and then 1M HCl (2.0 equiv.) was added dropwise. The mixture was heated under reflux in an oil bath at 60°C for 1 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by evaporation under reduced pressure, dried with anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography to obtain a deep yellow solid (5d) with a yield of 85%. 1 HNMR (400MHz, DMSO-d6) δ 10.63 (s, 1H), 8.07 (dd, J = 8.8, 2.0Hz, 2H), 7.73 (dd, J = 8.8, 2.0Hz, 2H), 7.67 (d, J = 15.5Hz, 1H), 7.54 (d, J = 15.4Hz, 1H), 7.43 (s, 2H), 3.64 (s, 3H), 2.16 (s, 6H), 1.44–1.39 (m, 2H), 1.39–1.33 (m, 2H); HRMS (ESI) calculated values: C 23 H 23 NO5[M+H] + Experimental value: 394.1610; 394.1647.

[0081] Example 5: Synthesis of (4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzoyl)glycine methyl ester (5e):

[0082]

[0083] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0084] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation. The pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0085] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, glycine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography to obtain a pale yellow solid (4e).

[0086] (4) The product (4e) obtained in step (3) was mixed with MeOH, and then 1M HCl (2.0 equiv.) was added dropwise. The mixture was refluxed in an oil bath at 60°C for 1 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by evaporation under reduced pressure, dried with anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography to obtain a yellow solid (5e) with a yield of 65%. 1 HNMR (400MHz, CD3OD) δ 9.17 (t, J = 5.9Hz, 1H), 8.24–8.18 (m, 2H), 8.04–7.98 (m, 2H), 7.73 (d, J = 15.4Hz, 1H), 7.65 (d, J = 15.4Hz, 1H), 7.51 (s, 2H), 4.06 (d, J = 5.7Hz, 2H), 3.67 (s, 3H), 2.21 (s, 6H); HRMS (ESI) calculated values: C 21 H21 NO5[M+H] + Experimental value: 368.1453; 368.1493.

[0087] Example 6: Synthesis of (E)-(4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzoyl)glycine (5f):

[0088]

[0089] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0090] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0091] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, glycine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography (DCM) to obtain a pale yellow solid (4e).

[0092] (4) The methyl ester (4e) synthesized in step (3) was mixed with MeOH and stirred. LiOH aqueous solution (2.0 equiv.) was added, and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the pH was adjusted to acidic with 1M HCl, and a large amount of solid precipitated. The yellow solid (4f) was collected. The collected 4f was mixed with methanol, and 1M HCl was added to make the solution acidic. The mixture was heated under reflux at 60 °C for 1 h. The solvent was removed by vacuum evaporation. The product was extracted with EA and water. The organic layers were combined, dried over anhydrous sodium sulfate, and the yellow solid (5f) was obtained by thin-layer chromatography (DCM:MeOH = 10:1), with a yield of 52%. 1 HNMR (400MHz, DMSO-d6) δ 9.00 (t, J = 5.9Hz, 1H), 8.16 (d, J = 8.2Hz, 2H), 7.97 (d, J = 8.2Hz, 2H), 7.69 (d, J = 15.5Hz, 1H), 7.60 (d, J = 15.4Hz, 1H), 7.46 (s, 2H), 3.91 (d, J = 4.9Hz, 2H), 2.16 (s, 6H); HRMS (ESI) calculated values: C 20 H 19 NO5[M+H] + Experimental value: 354.1297; Value: 354.1334.

[0093] Example 7: Synthesis of (4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzoyl)phenylalanine methyl ester (5g):

[0094]

[0095] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0096] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0097] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, L-phenylalanine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography (DCM) to obtain a pale yellow solid (4 g).

[0098] (4) The product (4g) obtained in step (3) was mixed with MeOH, and then 1M HCl (2.0 equiv.) was added dropwise. The mixture was heated under reflux in an oil bath at 60°C for 1 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by evaporation under reduced pressure, dried with anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography to obtain a yellow solid (5g), with a yield of 86%. 1 HNMR (400MHz, DMSO-d6) δ 9.04 (d, J = 7.8Hz, 1H), 8.94 (s, 1H), 8.14 (d, J = 8.0Hz, 2H), 7.89 (d, J = 7.9Hz, 2H), 7.68 (J = 15.4Hz, 1H), 7.60 (J = 15.4Hz, 1H), 7.47 (s, 2H), 7.31–7.20 (m, 5H), 7.19–7.13 (m, 1H), 4.70–4.60 (m, 1H), 3.61 (s, 3H), 2.17 (s, 6H); HRMS (ESI) calculated values: C 28 H 27 NO5[M+H] + Experimental value: 458.1923; Value: 458.1959.

[0099] Example 8: Synthesis of (E)-(4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzoyl)phenylalanine (5h):

[0100]

[0101] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0102] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0103] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, L-phenylalanine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography (DCM) to obtain a pale yellow solid (4 g).

[0104] (4) The methyl ester (4g) synthesized in step (3) was mixed with MeOH and stirred. LiOH aqueous solution (2.0 equiv.) was added, and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the pH was adjusted to acidic with 1M HCl, and a large amount of solid precipitated. The yellow solid was collected (4 h). The collected solid was mixed with methanol, and 1M HCl was added to make the solution acidic. The mixture was heated under reflux at 60 °C for 1 h. The solvent was removed by vacuum evaporation. The product was extracted with EA and water. The organic layers were combined, dried over anhydrous sodium sulfate, and the yellow solid was obtained by thin-layer chromatography (DCM:MeOH = 10:1) (5 h), with a yield of 75%. 1HNMR (400MHz, DMSO-d6) δ 8.99 (s, 1H), 8.92 (d, J = 8.2Hz, 1H), 8.18–8.11 (m, 2H), 7.93–7.86 (m, 2H), 7.69 (d, J = 15.4Hz, 1H), 7.60 (d, J = 15.4Hz, 1H), 7.48 (s, 2H), 7.33–7.22 (m, 4H), 7.20–7.11 (m, 1H), 4.66–4.56 (m, 1H), 3.23–3.14 (m, 1H), 3.10–3.00 (m, 1H), 2.18 (s, 6H); HRMS (ESI) calculated values: C 27 H 25 NO5[M+H] + Experimental value: 444.1766; Value: 444.1805.

[0105] Example 9: Synthesis of 4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)-N-(2-methylamino)-2-oxoethylbenzamide (5i):

[0106]

[0107] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0108] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0109] (3) The carboxylic acid (4a) from step (2) was mixed with HOBt and DCM and stirred in an ice-water bath for 10 min. Then EDCI was added and stirred in an ice-water bath for 30 min. 2-Amino-N-methylacetamide (1.2 equiv.) was added and stirred for 10 min. Finally, DIPEA was added and stirred in an ice-water bath for 1 h. The reaction was then brought to room temperature for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation and redissolved with EA. The solution was washed three times with 3% citric acid aqueous solution and saturated NaHCO3. The organic phase was dried with anhydrous sodium sulfate and filtered. The residue was separated and purified by thin-layer chromatography (DCM:MeOH = 10:1) to obtain a pale yellow solid (4i).

[0110] (4) Dissolve the product obtained in step (3) with MeOH, adjust the pH to acidic with 1M HCl, stir the reaction at 60°C for 1 h, a large amount of solid precipitates, filter to obtain yellow solid (5i), yield 63%. 1 HNMR (400MHz, DMSO-d6) δ 8.97-8.86 (m, 2H), 8.20-8.13 (m, 2H), 8.03-7.96 (m, 2H), 7.83 (d, J = 4.6Hz, 1H), 7.71 (d, J = 15.5Hz, 1H), 7.60 (d, J = 15.5Hz, 1H), 7.48 (s, 2H), 3.81 (d, J = 5.9Hz, 2H), 2.56 (d, J = 4.5Hz, 3H), 2.17 (s, 6H); HRMS (ESI) calculated values: C 21 H 22 N₂O₄[M+Na] + Experimental value: 389.1477; 389.1471.

[0111] Example 10: Synthesis of (E)-N-(2-ethylamino)-2-oxoethyl)-4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzamide (5j):

[0112]

[0113] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0114] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0115] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, glycine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography (DCM) to obtain a pale yellow solid (4e).

[0116] (4) The methyl ester (4e) synthesized in step (3) was mixed with MeOH and stirred. LiOH aqueous solution (2.0 equiv.) was added and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the pH was adjusted to neutral with 1M HCl, and the mixture was extracted three times with EA. The organic layers were combined and the solvent was evaporated to obtain a pale yellow solid (4f).

[0117] (5) The pale yellow solid (4f) was collected and mixed with HOBt and DCM. The mixture was stirred in an ice-water bath at 0°C for 10 min, then EDCI was added and stirred in an ice-water bath for another 30 min. Ethylamine hydrochloride was then added and stirred for another 10 min. Finally, DIPEA was added and the mixture was stirred in an ice-water bath for 1 h. The mixture was then allowed to return to room temperature and reacted for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by evaporation under reduced pressure. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3. The organic layer was retained, dried with anhydrous sodium sulfate, and filtered. The residue was purified by thin-layer chromatography (DCM:MeOH = 10:1) to obtain the pale yellow solid (4j).

[0118] (6) Dissolve the product (4j) obtained in step (5) with MeOH, adjust the pH to acidic with 1M HCl, stir the reaction at 60℃ for 1h, a large amount of solid precipitates, filter to obtain a dark yellow solid (5j), yield 26%. 1HNMR (400MHz, DMSO-d6) δ 8.97 (s, 1H), 8.91 (t, J = 6.0Hz, 1H), 8.22–8.15 (m, 2H), 8.04–7.98 (m, 2H), 7.95 (t, J = 5.6Hz, 1H), 7.73 (d, J = 15.5Hz, 1H), 7.62 (d, J = 15.4Hz, 1H), 7.50 (s, 2H), 3.83 (d, J = 5.9Hz, 2H), 3.15–3.02 (m, 2H), 2.18 (s, 6H), 1.00 (t, J = 7.2Hz, 3H); HRMS (ESI) calculated values: C 22 H 24 N₂O₄[M+Na] + Experimental value: 403.1667; Value: 403.1624.

[0119] Example 11: Synthesis of 2-dimethylamino-2-oxoethyl-4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzamide (5l):

[0120]

[0121] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0122] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0123] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, glycine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography (DCM) to obtain a pale yellow solid (4e).

[0124] (4) The methyl ester (4e) synthesized in step (3) was mixed with MeOH and stirred. LiOH aqueous solution (2.0 equiv.) was added and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the pH was adjusted to neutral with 1M HCl, and the mixture was extracted with EA and water. The organic layers were combined and the solvent was removed by rotary evaporation to obtain a pale yellow solid (4f).

[0125] (5) The carboxylic acid (4f) from step (4) was mixed evenly with HOBt and DCM, and stirred in an ice-water bath at 0°C for 10 min. Then EDCI was added, and stirring was continued in an ice-water bath for 30 min. Then N,N-dimethyl hydrochloride (1.2 equiv.) was added, and stirring was continued for 10 min. Finally, DIPEA was added, and stirring was carried out in an ice-water bath for 1 h. The reaction was then brought back to room temperature for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by thin-layer chromatography (DCM:MeOH = 10:1) to obtain a pale yellow solid (4l).

[0126] (5) Dissolve the product (4l) obtained in step (4) with MeOH, adjust the pH to acidic with 1M HCl, stir the reaction at 60°C for 1 h, a large amount of solid precipitates, and filter to obtain a dark yellow solid (5l) with a yield of 23%. 1 HNMR (400MHz, DMSO-d6) δ 8.93 (s, 1H), 8.72 (t, J = 5.6Hz, 1H), 8.16 (d, J = 8.1Hz, 2H), 7.98 (d, J = 8.2Hz, 2H), 7.70 (d, J = 15.5Hz, 1H), 7.60 (d, J = 15.4Hz, 1H), 7.48 (s, 2H), 4.09 (d, J = 5.6Hz, 2H), 2.99 (s, 3H), 2.82 (s, 3H), 2.17 (s, 6H); HRMS (ESI) calculated values: C 22H 24 N₂O₄[M+Na] + Experimental value: 403.1634; Value: 403.1628.

[0127] Example 12: Synthesis of (E)-N-(2-(diethylamino)-2-oxoethyl)-4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzamide (5m):

[0128]

[0129] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the remaining product (2) (PE:EA = 6:1) by column chromatography.

[0130] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0131] (3) The carboxylic acid (4a) from step (2) was mixed evenly with HOBt and DCM, and stirred in an ice-water bath at 0°C for 10 min. Then EDCI was added, and stirring was continued in an ice-water bath for 30 min. Then 2-amino-N,N-diethylacetamide hydrochloride (1.2 equiv.) was added, and stirring was continued for 10 min. Finally, DIPEA was added, and stirring was carried out in an ice-water bath for 1 h. The reaction was then brought to room temperature for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation, and the product was redissolved with EA. The product was washed three times with 3% citric acid aqueous solution and saturated NaHCO3, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by thin-layer chromatography (DCM:MeOH = 10:1) to obtain a pale yellow solid (4m).

[0132] (5) Dissolve the product (4m) obtained in step (4) with MeOH, adjust the pH to acidic with 1M HCl, stir the reaction at 60℃ for 1h, a large amount of solid precipitates, filter to obtain a dark yellow solid (5m), yield 76%. 1 HNMR (400MHz, DMSO-d6) δ 8.93 (s, 1H), 8.72 (t, J = 5.6Hz, 1H), 8.16 (d, J = 8.1Hz, 2H), 7.98 (d, J = 8.2Hz, 2H), 7.70 (d, J = 15.5Hz, 1H), 7.60 (d, J = 15.4Hz, 1H), 7.48 (s, 2H), 4.09 (d, J = 5.6Hz, 2H), 2.99 (s, 3H), 2.82 (s, 3H), 2.17 (s, 6H); HRMS (ESI) calculated values: C 22 H 24 N₂O₄[M+Na] + Experimental value: 403.1634; Value: 403.1628.

[0133] Example 13: Synthesis of (E)-4-(3-(4-hydroxy-3,5-dimethylphenyl)acryloyl)-N-(2-oxo-2-phenylamino)ethylbenzamide (5k)

[0134]

[0135] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0136] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0137] (3) The carboxylic acid (4a) from step (2) was mixed with HATU and DCM and stirred at room temperature for 30 min. Then, glycine methyl ester hydrochloride (1.2 equiv.) was added and reacted at room temperature for 10 min. DIPEA (3.0 equiv.) was added and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3 aqueous solution, respectively. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by column chromatography (DCM) to obtain a pale yellow solid (4e).

[0138] (4) The methyl ester (4e) synthesized in step (3) was mixed with MeOH and stirred. LiOH aqueous solution (2.0 equiv.) was added and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the pH was adjusted to neutral with 1M HCl, and the mixture was extracted with EA and water. The organic layers were combined and the solvent was removed by rotary evaporation to obtain a pale yellow solid (4f).

[0139] (5) The pale yellow solid (4f) was collected and mixed with HOBt and DCM. The mixture was stirred in an ice-water bath at 0°C for 10 min. Then, EDCI was added, and the mixture was stirred in an ice-water bath for 30 min. Aniline (1.2 equiv.) was added, and the mixture was stirred for another 10 min. Finally, DIPEA was added, and the mixture was stirred in an ice-water bath for 1 h. The reaction was then allowed to proceed at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by evaporation under reduced pressure. The mixture was redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography to obtain the pale yellow solid (4k).

[0140] (6) The product (4k) obtained in step (5) was mixed with MeOH and stirred. The pH was adjusted to acidic with 1M HCl and reacted at 60°C for 1 h. A large amount of solid was precipitated. The solid (5k) was obtained by filtration and purified by thin-layer chromatography with a yield of 38%. 1HNMR (400MHz, DMSO-d6) δ 10.10 (s, 1H), 9.06 (t, J = 5.9Hz, 1H), 9.01 (s, 1H), 8.22–8.17 (m, 2H), 8.05–8.01 (m, 2H), 7.73 (d, J = 15.4Hz, 1H), 7.63 (d, J = 15.4Hz, 1H), 7.60–7.56 (m, 2H), 7.50 (s, 2H), 7.32–7.25 (m, 2H), 7.06–6.99 (m, 1H), 4.07 (d, J = 5.8Hz, 2H), 2.18 (s, 6H); HRMS (ESI) calculated values: C 26 H 24 N₂O₄[M+H] + Experimental value: 429.1770; Value: 429.1809.

[0141] Example 14: Synthesis of (R)-1-(S)-2-(4-hydroxy-3,5-dimethylphenyl)acryloyl)benzamide)-3-phenylpropionamido)-3-methylbutyl acid (10 v):

[0142]

[0143] (1) Mix 3,5-dimethyl-p-hydroxybenzaldehyde (1) (1.0 equiv.) with DCM, add DIPEA (2.0 equiv.) and DAMP (0.03 equiv.) to the mixture, stir until homogeneous, then add chloromethyl ethyl ether (2.0 equiv.) dropwise under ice-water bath conditions, gradually restore to room temperature for 3 h, and monitor the reaction progress by TLC. Evaporate the solvent from the reaction solution under reduced pressure, and separate and purify the residue to obtain product (2) (PE:EA = 6:1) by column chromatography.

[0144] (2) P-acetylbenzoic acid (3a) (1.0 equiv.) was mixed evenly with MeOH, and then a saturated NaOH solution (2.0 equiv.) was added. After stirring for 10 min, the product (2) obtained in step (1) was added dropwise to the solution. The reaction was carried out at room temperature for 24 h, and the reaction progress was monitored by TLC. Then, the MeOH in the solution was removed by vacuum evaporation, and the pH of the remaining solution was adjusted to acidic with 1M hydrochloric acid until a large amount of pale yellow solid precipitated. The solution was filtered, and the filter residue was washed three times with water to obtain a pale yellow solid (4a).

[0145] (3) The product 4a (1.0 equiv.) obtained in step (2) was mixed with HOBt and DCM and stirred in an ice-water bath at 0°C for 10 min. Then EDCI was added and stirred in an ice-water bath for 30 min. Then (alphaS)-alpha-amino-N-[(1R)-1-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methylbridged-1,3,2-benzodioxoborane-2-yl]-3-methylbutyl]phenylpropionamide hydrochloride (8, 1.2 equiv.) was added and stirred for 10 min. Finally, DIPEA was added and stirred in an ice-water bath for 1 h. The mixture was then allowed to return to room temperature and reacted for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum evaporation. The mixture was then redissolved with EA and extracted three times with 3% citric acid aqueous solution and saturated NaHCO3. The organic layer was retained, dried with anhydrous sodium sulfate, filtered, and the residue was separated and purified by thin-layer chromatography (DCM:MeOH = 10:1) to obtain a pale yellow solid (9v).

[0146] (6) The borate ester (9 v) synthesized in step (5) was dissolved in MeOH, and isobutylboronic acid (2.0 equiv.) and n-hexane (2.0 equiv.) were added. The solution was adjusted to acidity with 1M HCl, and stirred overnight at room temperature. The reaction was detected by TLC. After the reaction was complete, the MeOH phase was collected, the solvent was removed by evaporation under reduced pressure, dissolved in DCM, extracted twice with water, dried over anhydrous sodium sulfate, filtered, and the residue was separated and extracted by column chromatography to obtain a yellow solid (10 v) (DCM:MeOH = 20:1), with a yield of 68%. 1 HNMR(400MHz,CD3OD)δ8.08(d,J=8.1Hz,2H),7.89(d,J=7.9Hz,2H),7.68(d,J=15.7,3.1Hz,1H), 7.51(d,J=15.5,3.1Hz,1H),7.34(s,2H),7.29(d,J=4.4Hz,4H),7.25-7.20(m,1H),4.96(t,J=8.1 Hz, 1H), 3.21(dd, J=7.9, 2.6Hz, 2H), 2.64(t, J=7.7Hz, 1H), 2.22(s, 6H), 1.36-1.23(m, 2H), 1.12(t, J=7.5Hz, 2H), 0.84-0.78(m, 6H); HRMS(ESI) calculated value: C32H37BN2O6[M+Na]+579.2642; experimental value: 579.2639.

[0147] Example 15: In vitro study of the inhibitory effect of chalcone derivatives on the NLRP3 inflammasome.

[0148] J774A.1 cells were divided into 96-well plates, approximately 5 × 10⁶ cells per well. 5 Cells were seeded overnight, and the supernatant was discarded. 100 μL of DMEM medium containing bacterial lipopolysaccharide (LPS, 1 μg / ml) and 10% serum was added to each well. Then, different concentrations of chalcone derivatives (20 μM, 10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM) were added for 1 hour, followed by treatment with nigericin (10 μM) for 1 hour. The cell supernatant was then collected, and the IL-1β content was measured using a Mouse IL-1β ELISA kit to calculate the inhibitory effect of the compound of this invention on the NLRP3 inflammasome.

[0149] The results are shown in Table 1: The chalcone derivatives of the present invention have good inhibitory activity against the NLRP3 inflammasome.

[0150] Table 1: Inhibitory activity of chalcone derivatives against NLRP3 / IL-1β in J774A.1 cells

[0151]

[0152]

[0153]

[0154] Example 16: Compound 10V specifically inhibits the activation of the NLRP3 inflammasome in vitro.

[0155] 1. NLRP3 inflammasome activation and IL-1β detection: J774A.1 cells or mouse bone marrow-derived macrophages (BMDMs) were aliquoted into 96-well plates, with 5 × 10⁶ cells per well. 5 Cells were seeded overnight, and the supernatant was discarded. 100 μL of DMEM medium containing 10% serum and bacterial lipopolysaccharide (LPS) (1 μg / ml) was added to each well. Then, different concentrations of compounds (200 nM, 100 nM, 50 nM) were added for 1 h, followed by treatment with Nigericin (10 μM) for 1 h. The cell supernatant was then collected, and the IL-1β content was determined using the Mouse IL-1β ELISA kit.

[0156] 2. Western blot analysis of proteins: J774A.1 cell samples treated in step 1 were lysed in RIPA lysis buffer containing protease inhibitors at 4°C for 30 min. Proteins in the lysis buffer or supernatant were separated using a 12% SDS-polyacrylamide gel, transferred to a PVDF membrane, and subjected to Western blot analysis with anti-mouse IL-1β antibody, anti-ASC antibody, anti-casepase-1 antibody, anti-NLRP3 antibody, and anti-β-actin antibody.

[0157] 3. NLRC4 and AIM2 inflammasome activation and IL-1β detection: For NLRC4 or AIM2 inflammasome activation, BMDMs cells were stimulated with 1 μg / mL LPS for 5 h, then treated with different concentrations (50 nM, 100 nM, 200 nM) of compounds for 1 h. Cells were then infected with bacterial flagellin (FLA-ST Ultrapure) (2.5 μg / mL) for 4 h, or transfected with poly(dA:dT) (0.25 μg / mL) for 4 h. Cell supernatants were then collected, and IL-1β levels were measured using the Mouse IL-1β ELISA kit.

[0158] The results are as follows Figure 1 As shown, from Figure 1 The results showed that in the J774A.1 and BMDMs cell models with NLRP3 inflammasome activation, compound 10v could inhibit IL-1β secretion in a concentration-dependent manner. Figure 1 Figures A and B in the diagram). Western blotting experiments showed that compound 10V inhibited caspase-1p20 maturation and IL-1β secretion in a dose-dependent manner, but did not affect pro-IL-1β, pro-caspase-1, NLRP3, or ASC in cell lysates. Figure 1 (Figure A in the diagram). Meanwhile, compound 10v did not inhibit the activation of the NLRC4 inflammasome ( Figure 1 Figure C in the diagram shows a slight inhibitory effect on the AIM2 inflammasome. Figure 1 (See Figure D in the diagram). The above results indicate that 10v can selectively inhibit NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion.

[0159] Example 17: Mechanism of compound 10v in inhibiting NLRP3 inflammasome activation

[0160] NLRP3 inflammasome assembly detection: J774A.1 cells in 6-well plates were treated with LPS (1 μg / mL) and incubated in a CO2 incubator for 5 h. Then, 10 μM of the appropriate concentration of the compound was added and incubated for 1 h. Finally, Nigericin (10 μM) was added and incubated for 1 h. Cells treated in the above steps were lysed with IP lysis buffer containing proteasome and PMSF on ice for 10 min. Cells were then gently scraped off with a cell scraper and transferred to EP tubes for further lysis for 30 min. After lysis, the cells were centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected, the precipitate was discarded, and 20 μL of agar beads were added to each tube of supernatant. The mixture was vortexed thoroughly before adding the beads and incubated overnight at 4 °C. The supernatant was then removed, and anti-NLRP3 antibody was added. After mixing well, the cells were incubated at 4 °C for 6-8 h. Then, add 20 μL of agar beads after shaking and incubation at 4°C for 6-8 hours. Discard the supernatant, retaining the bottom beads. Wash the beads 5 times with pre-cooled D-PBS, add 2X Loading Buffer, mix gently, and boil in a 100°C metal bath for 10 minutes. Separate the proteins in the lysis buffer or supernatant using a 10% SDS-polyacrylamide gel, transfer to a PVDF membrane, and perform Western blotting analysis with anti-Ubiquitin antibody.

[0161] like Figure 2 As shown in Figure A, compared to the model group, the drug-treated group showed ubiquitinated NLRP3 protein, indicating that 10V promotes the accumulation of NLRP3 ubiquitination. Figure 2 As shown in Figure B, NLRP3 and ASC interact significantly in LPS and Nigericin-treated J774A.1 cells, while compound 10v reduces this interaction. This suggests that compound 10v may inhibit IL-1β release by promoting NLRP3 ubiquitination, inhibiting the binding of NLRP3 to ASC, and inhibiting the assembly of the NLRP3 inflammasome.

[0162] Example 18: Compound 10v inhibits the expression of STAT1 and STAT5.

[0163] J774A.1 cells in large culture dishes were treated with compound 10V for 2 h, the supernatant was removed, and LPS (1 μg / mL) was added and incubated for 6 h. The J774A.1 cell samples treated in the above steps were lysed in RIPA lysis buffer containing protease inhibitors at 4°C for 30 min. Proteins in the lysis buffer or supernatant were separated using a 10% SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and analyzed by Western blotting with anti-rabbit STAT1 antibody, anti-STAT3 antibody, anti-STAT5 antibody, anti-pSTAT1 antibody, anti-pSTAT3 antibody, and anti-pSTAT5 antibody.

[0164] The results are as follows Figure 2 As shown in the CE diagram, compound 10v does not affect the expression and phosphorylation of STAT3, but it can reduce the expression and phosphorylation of STAT1 and STAT5 in a dose-dependent manner. This indicates that compound 10v can also inhibit immune-related STAT pathways.

[0165] Example 19: Compound 10v improves sodium dextran sulfate (DSS)-induced colitis

[0166] 1. Six- to eight-week-old female C57BL / 6 mice were divided into five groups of six each. The specific grouping and treatment were as follows:

[0167] Group 1: Distilled water was given in the diet from day 1 to day 10, along with a gavage medium administered daily.

[0168] Group 2: Distilled water was given in the diet on days 1-3. From days 4-10 onwards, 2.5% DSS distilled water was given in the diet daily, along with a gavage medium daily.

[0169] Group 3: Sulfasalazine (10 mg / kg) was administered by gavage every three days. Starting from the fourth day, 2.5% DSS distilled water was given to the diet daily.

[0170] Group 4: 10v (0.2mg / kg) of compound was injected intraperitoneally every three days. Starting from the fourth day, 2.5% DSS distilled water was given to the diet daily.

[0171] Group 5: 10v (0.6mg / kg) of compound was injected intraperitoneally every three days. Starting from the fourth day, 2.5% DSS distilled water was given to the diet daily.

[0172] 2. Starting from the day before DSS administration, the degree of fecal bleeding and body weight of each group of mice were monitored daily. On day 11, the length of the mouse colon was measured and the IL-1β content in the colon was determined.

[0173] The results are as follows Figure 3 As shown, from Figure 3 The results showed that administration of 2.5% DSS to the diet increased the severity of fecal hemorrhage in mice, shortened the length of the colon, and significantly increased IL-1β levels in the colon. Intraperitoneal injection of compound 10v could dose-dependently improve fecal hemorrhage, shorten the length of the colon, and reduce the level of IL-1β in the colon.

[0174] 3. Pathological section results of mouse colon tissue as follows: Figure 4As shown, the colonic mucosal structure in the DSS group was significantly damaged, with goblet cells completely disappearing, crypt structures completely destroyed, and the colonic surface essentially exhibiting ulceration and severe inflammatory infiltration. In contrast, the colons of mice treated with compound 10v showed better therapeutic effects, with intact mucosa, well-preserved goblet cells and crypt structures, almost no ulceration, and extremely low inflammatory infiltration. Therefore, compound 10v can significantly improve DSS-induced colitis.

[0175] Activity assays showed that the chalcone compounds of the present invention exhibited inhibitory activity against the NLRP3 inflammasome in J774A.1 cells. Simultaneously, the representative compound 10v selectively inhibited NLRP3 inflammasome activation and could suppress immune-related STAT pathways, improving DSS-induced colitis. Therefore, the chalcone compounds of the present invention have potential use in treating diseases related to the NLRP3 inflammasome and / or STAT pathways.

[0176] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0177] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A chalcone derivative or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The chalcone derivatives are selected from the following compounds: 。 2. The use of the chalcone derivatives of claim 1, or their pharmaceutically acceptable salts, or their stereoisomers, in the preparation of NLRP3 inflammasome inhibitors and / or STAT pathway inhibitors.

3. The use of the chalcone derivative of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for the prevention and / or treatment of diseases associated with the NLRP3 inflammasome and / or the STAT pathway, wherein the inflammatory disease is colitis.

4. A pharmaceutical composition, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the chalcone derivatives of claim 1 or their pharmaceutically acceptable salts or their stereoisomers.

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