Preparation method of thiazole amide derivative and application of thiazole amide derivative in treatment of inflammation

Abstract

The invention discloses a preparation method of a thiazole amide derivative and application of the thiazole amide derivative in treatment of inflammation. Comprising the following steps: mixing 3-acetyl-2, 5-dichlorothiophene, absolute ethyl alcohol, iodine and copper oxide for reaction; and adding thiourea and continuously stirring, adding dichloromethane and pyridine after the reaction is completed, mixing, and adding a dichloromethane solution containing 5-nitro-2-furoyl chloride while stirring to obtain the thiazole amide derivative. The invention discovers that the thiazole amide derivative, especially the compound 21, has good anti-inflammatory activity for the first time. The compound 21 has remarkable inhibitory activity on gene transcription and release of inflammatory factors by effectively inhibiting NLRP3 inflammasome activation and GSDMD-mediated pyroptosis, so that inflammatory injury is relieved.

Description

Technical Field

[0001] The invention relates to a preparation method of a thiazole amide derivative and application of the thiazole amide derivative in treating inflammation, and belongs to the field of thiazole amide derivatives. Background Art

[0002] Intracellular pattern recognition receptors (PRRs), including the NLR family, CARD8, pyrin, and AIM2, are essential for maintaining cellular homeostasis. Upon recognition of pathogenic microorganisms or detection of cellular damage, these PRRs are activated and assembled into multiprotein complexes called inflammasomes. Typical inflammasomes are usually composed of sensors (such as NLRP3, NLRC4, or AIM2), adapters (ASC), and effectors (Caspase-1). Upon stimulation, sensor proteins form oligomers and bind to ASC, which then recruits Caspase-1 to assemble the inflammasome complex.

[0003] The NLRP3 complex has been recognized as one of the most thoroughly studied and well-defined inflammasomes. The assembly of the NLRP3 inflammasome is tightly regulated and usually requires two distinct signals. The first signal involves the recognition of exogenous or endogenous threats by transmembrane PRRs such as TLRs and the activation of the NF-κB pathway. The second signal, triggered by various PAMPs and DAMPs such as ATP, nigericin, and SiO2, initiates the assembly and activation of the NLRP3 inflammasome. This process activates the effector protein Caspase-1, which cleaves inflammatory factors such as IL-1β pro- and IL-18 pro- into their functional forms. Caspase-1 also cleaves and activates GSDMD by cleaving it at the active site ASP275 (ASP276 in mice) of the pyroptosis executioner GSDMD. The cleaved N-terminal fragment of GSDMD (GSDMD-NT) undergoes conformational changes, oligomerizes, and selectively binds negatively charged phospholipids, which promotes the formation of transmembrane pores by punching holes from the inside of the membrane. The extensive pores disrupt the structural integrity of the cell membrane, allowing the flow of non-selective ions and water, as well as the selective secretion of cytokines. Ultimately, when the cell membrane ruptures, it leads to the release of a large amount of intracellular materials, including inflammatory cytokines, HMGB1 and LDH, thus triggering severe inflammation.

[0004] Abnormal pyroptosis mediated by inflammasome activation triggers a series of inflammatory responses, including dysregulated ion flux, organelle dysfunction, cell rupture, and sustained cytokine release. Numerous studies have shown that sustained activation of the NLRP3 inflammasome-associated pyroptosis pathway is closely related to a variety of diseases, including Alzheimer's disease, sepsis, atherosclerosis, inflammatory bowel disease (IBD), gout, type 2 diabetes, and cancer. Therefore, NLRP3 inflammasome and pyroptosis effector protein GSDMD have become attractive targets for drug development to treat these inflammatory diseases.

[0005] Recent advances have identified and validated a number of small molecule inhibitors that effectively modulate the NLRP3 inflammasome pathway. These NLRP3 inhibitors exhibit diverse chemical structures, including sulfonylureas (e.g., MCC950, N14, ZYIL-1, IZD-174, and GDC-2394), sulfonamides (e.g., D-6 and 15Z), amides (e.g., NP3-562), and Michael acceptors (e.g., CY-09, tranilast, and compound M-49). In addition, several natural products, including oridonin, coumarin, and berberine, have also shown potential therapeutic effects on the NLRP3 inflammasome. Figure 1 Representative NLRP3 inhibitors are summarized. In addition, peptides targeting amino acids critical for NLRP3-ASC interaction have shown effectiveness in disrupting NLRP3 inflammasome assembly and activation. Currently, several NLRP3 inhibitors with therapeutic potential have entered clinical trials, including IZD-334 from Infrazome (clinical phase I, NCT04086602, for CAPS), OLT1177 from Olatec Therapeutics LLC (clinical phase II / III, NCT05658575, for acute gout attacks), ZYIL1 from Zydus (clinical phase II, NCT05186051, for CAPS; clinical phase I, NCT04972188), RG-6418 from Roche (clinical phase I, NCT05924243, for Parkinson's disease), DFV890 (also known as IFM-2427) from Novartis (clinical phase II, NCT04382053, for pneumonia; clinical phase II, NCT04972188), and NF-6418 from Roche (clinical phase I, NCT05924244, for Parkinson's disease). T04886258, for knee osteoarthritis) and NodThera Ltd.'s NT-0796 (clinical phase II, NCT06129409, for cardiovascular disease risk). Despite these advances, no candidate drug targeting the NLRP3 inflammasome has been approved for clinical use, highlighting the continued need for novel compounds in this field. Therefore, the discovery of more new compounds that inhibit the NLRP3 inflammasome remains of great significance. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing a thiazole amide derivative and its application in treating inflammation.

[0007] Technical solution: To solve the above technical problems, the present invention provides a method for preparing thiazole amide derivatives, comprising the following steps: mixing 3-acetyl-2,5-dichlorothiophene, anhydrous ethanol, iodine and copper oxide for reaction; adding thiourea and continuing to stir the reaction, adding dichloromethane and pyridine after the reaction is completed, and adding a dichloromethane solution containing 5-nitro-2-furoyl chloride under stirring to obtain the thiazole amide derivative; the structural formula of the thiazole amide derivative is:

[0008]

[0009] The present invention also provides the use of thiazole amide derivatives or pharmaceutically acceptable salts, hydrates, solvates, and pharmaceutically acceptable carriers thereof in the preparation of drugs for treating and / or improving inflammation-related diseases. The thiazole amide derivatives include compounds having a general structural formula as shown in Formula I:

[0010]

[0011] Among them, R is C1~C 30 Aryl, heteroaryl, alkyl, C1~C 30 Alkyl aryl, C1~C 30 Alkyl heteroaryl, C1~C 12 Heterocyclic, C1~C 30 Substituted aryl or C1~C 30 Substituted heteroaryl; the C1~C 30 Substituted aryl or C1~C 30 The substituted heteroaryl group is substituted by one or more of the following identical or different substituents: C1-C 30 alkyl, alkoxy, alkylamino, amide, sulfonamide, nitro, halogen, benzyl, nitrile, amino, carboxyl, carboxyl, carbonyl, trifluoromethyl;

[0012] R' is C1~C 30 Alkyl, aryl, heteroaryl, C1~C 30 Alkyl aryl, C1~C 30 Alkyl heteroaryl, C1~C 12 Heterocyclic, C1~C 30 Substituted aryl or C1~C 30 Substituted heteroaryl; the C1~C 30 Substituted aryl or C1~C 30 The substituted heteroaryl group is substituted by one or more of the following identical or different substituents: C1-C 30 The alkyl, alkoxy, alkylamino, amide, sulfonamide, nitro, halogen, benzyl, nitrile, amino, carboxyl, carboxyl, carbonyl, trifluoromethyl.

[0013] Wherein, the thiazole amide derivatives include

[0014]

[0015] Wherein, the inflammation-related diseases include sepsis, inflammatory bowel disease, diabetes, gout, rheumatoid arthritis, rheumatic arthritis, non-alcoholic fatty liver disease, atherosclerosis or neurodegenerative diseases.

[0016] Wherein, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease or multiple sclerosis.

[0017] The present invention also provides the use of the compound represented by Formula I or its pharmaceutically acceptable salt, hydrate, solvate, and pharmaceutically acceptable carrier in the preparation of NLRP3 inflammasome or GSDMD inhibitor.

[0018] The present invention also provides a pharmaceutical composition for treating and / or improving inflammation-related diseases, which contains the thiazole amide derivative or a pharmaceutically acceptable salt, hydrate, solvate, or a pharmaceutically acceptable carrier thereof.

[0019] Wherein, the inflammation-related diseases include sepsis, inflammatory bowel disease, diabetes, gout, rheumatoid arthritis, rheumatic arthritis, non-alcoholic fatty liver disease, atherosclerosis or neurodegenerative diseases.

[0020] Wherein, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease or multiple sclerosis.

[0021] Wherein, the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

[0022] The present invention also provides an NLRP3 inflammasome or GSDMD inhibitor, which contains the thiazole amide derivative or a pharmaceutically acceptable salt, hydrate, solvate, or pharmaceutically acceptable carrier thereof.

[0023] Among the gasdermin family, GSDMD is the most extensively studied and best characterized pyroptotic effector protein. GSDMD consists of GSDMD-NT, which is known to form membrane pores, and GSDMD-CT, which has an autoinhibitory effect. A flexible linker region connects these two domains and serves as a cleavage site for inflammatory caspases. Cleavage of this site activates GSDMD-NT, thereby initiating pyroptosis. Currently, only a limited number of GSDMD-targeted inhibitors have been identified, most of which are covalent inhibitors, such as disulfiram (DSF), necrosulphonamide (NSA), dimethyl fumarate (DMF), BAY 11-7082, LDC7559, and itaconate. These inhibitors mainly act by forming covalent bonds with cysteine ​​residues of GSDMD, thereby inhibiting pyroptosis. The development of non-covalent GSDMD inhibitors is necessary to improve drug safety and precisely regulate inflammatory responses.

[0024] The present invention discovered a new thiazole amide derivative through laboratory screening, which significantly inhibits NLRP3 inflammasome activation-mediated pyroptosis.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] 1. The present invention first discovered that thiazole amide derivatives (Table 1), especially compound 21, have good anti-inflammatory activity. Compound 21 effectively inhibits NLRP3 inflammasome activation and GSDMD-mediated cell pyroptosis, and has significant inhibitory activity on gene transcription and release of inflammatory factors, thereby alleviating inflammatory damage.

[0027] 2. The present invention proposes a new synthetic route for compound 21, which has a simple preparation method, cheap and readily available raw materials, a safe synthetic process, and is easy for large-scale production.

[0028] 3. NLRP3 inflammasome activation and GSDMD-mediated pyroptosis are involved in the occurrence and development of a variety of diseases, including sepsis, multiple organ damage, inflammatory bowel disease, autoimmune diseases, metabolic diseases, neurodegenerative diseases, etc. Compound 21 has good anti-inflammatory activity and no obvious toxicity to THP-1 cells. It can be used to prepare anti-inflammatory drugs and can be used for the development of multiple disease indications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the chemical structure of a representative NLRP3 inhibitor;

[0030] Figure 2 is the hydrogen spectrum (400 MHz) of compound m-21 in deuterated chloroform (CDCl3);

[0031] Figure 3is the hydrogen spectrum (400 MHz) of compound 21 in deuterated dimethyl sulfoxide (DMSO-d6);

[0032] Figure 4 is the compound m-21 in chloroform (CDCl3) 13 C NMR spectroscopy (101 MHz);

[0033] Figure 5 is the reaction mixture of compound 21 in deuterated dimethyl sulfoxide (DMSO-d6) 13 C NMR spectroscopy (101 MHz);

[0034] Figure 6 is the HPLC chromatogram of compound 21;

[0035] Figure 7 is the mass spectrum of compound 21;

[0036] Figure 8 Compound 21 inhibits GSDMD-induced pyroptosis in THP-Ms cells: (A) Chemical structure of compound 21; (B) Dose-response curve of compound 21 in pyroptosis assay; (C) After THP-Ms were stimulated with LPS, compound 21 was applied for 6 h, followed by stimulation with nigericin, and the formation of pyroptotic membrane pores was evaluated by PI uptake; (D) Bright field and fluorescence microscopy images of THP-Ms, scale bar, 100 μm;

[0037] Fig. 9 Compound 21 inhibits multiple inflammasome-induced pyroptosis: (A) Effect of compound 21 on BMDMs pyroptosis assessed by CCK-8 assay; (B) IL-1β quantification of GSDMD-mediated inflammation by ELISA; (C) Pyroptosis assessed by LDH release; (D) LPS-stimulated THP-Ms treated with compound 21 before exposure to nigericin; (EF) Effect of compound 21 on AIM2 and NLRC4 inflammasomes; (G) THP-Ms pretreated with compound 21 and then stimulated with LPS and nigericin; Values ​​are mean ± SEM (n = 3);

[0038] Fig.10Inhibition of GSDMD cleavage and GSDMD-NT oligomerization by compound 21: (A) Western blot analysis of NLRP3 antibody and GSDMD-related proteins in THP-Ms; (B) Western blot analysis of active GSDMD-NT, Caspase-1 and IL-1β in the supernatant of THP-Ms; (C) Western blot analysis of THP-Ms incubated with compound 21 before LPS stimulation; (D) Western blot analysis of GSDMD-NT oligomerization; (E-F) qPCR analysis of NLRP3, IL-1β and IL-6 gene levels, with GAPDH as an internal reference. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0040] 1. Chemical and biological reagents: PMA (Abcam, ab120297), CCK-8 (Topscience), Poly(dA:dT) / Lyovec (Invivogen), Salmonella typhimurium flagellin (Invivogen, FLA-ST), penicillin-streptomycin (Beyotime, C0222), LPS (Sigma, L2880), LDH detection kit (Nanjing Jiancheng, A020-2-2), nigericin (Invivogon, tlrl-nig), BCA cytotoxicity detection kit (Beyotime), Lipo 3000 reagent (Invitrogen). Thiazole amide derivatives (Table 1) were purchased from TopScience Limited Liability Company.

[0041] 2. Cell culture conditions: Human THP-1 cells, HEK293T cells, and L-929 cells were derived from the Shanghai Institute of Biochemistry and Cell Biology. Human THP-1 cells were maintained in RPMI 1640 medium enriched with 10% (v / v) FBS. HEK293T and L929 cells were grown in DMEM and minimum essential medium (MEM, Procell) containing 10% (v / v) FBS, respectively. Bone marrow-derived macrophages (BMDM) were collected from the tibia and femur of C57BL / 6 mice, and the bone marrow was flushed with cold PBS and DMEM medium. The cells were differentiated in DMEM medium enriched with 20% L-929 mouse fibroblast supernatant and 15% FBS for 7 days. The medium was changed after 3 days and incubated at 37°C in a humidified 5% CO2 environment.

[0042] 3. Cell treatment: Human THP-1 cells were cultured at 5×10 5Cells were plated on 96-well plates at a concentration of 1×10 cells / mL and induced to differentiate into macrophages (THP-Ms) overnight with 100 ng / mL phorbol 12-myristic acid 13-acetate (PMA). 6 Cells / mL were plated overnight in 96-well plates at a density of 1 μg / mL. The next morning, the medium was discarded and the cells were subsequently maintained in fresh medium containing 10% FBS for 24 hours. THP-Ms were stimulated with 1 μg / mL LPS for 3 hours and then replaced with fresh medium containing compounds 1-compound 27 (5 μM and 20 μM) and incubated for 6 hours. Then, they were stimulated with 10 μM nigericin, 1 μg / mL poly(dA:dT) / Lyovec, or 500 ng / mL Salmonella flagellin for 1 hour, and Salmonella typhimurium was treated for 4 hours to induce inflammation and pyroptosis. After treatment with compound 21, human THP-1 cells were exposed to LPS to assess NLRP3 inflammasome activation. The effects of these compounds on inflammasome-induced pyroptosis were then tested by CCK-8.

[0043] Example 1 Preliminary structure-activity relationship study on the anti-pyroptotic activity of thiazole amide derivative analogs

[0044] The present invention found that thiazole amide derivatives have anti-pyroptosis activity through laboratory activity screening. Therefore, preliminary anti-pyroptosis activity determination and structure-activity relationship study were carried out on thiazole amide derivatives with various substituents. NLRP3 inflammasome activation induces Caspase-1 cleavage and activation, which then mediates GSDMD cleavage and triggers cell pyroptosis. In order to evaluate the effect of thiazole amide derivatives on GSDMD-mediated pyroptosis, PMA-differentiated THP-macrophages (THP-Ms) were used. THP-Ms were stimulated with LPS for 3 hours and then stimulated with nigericin for 1 hour to establish a cell pyroptosis model. Among them, the covalent GSDMD inhibitor disulfiram was used as a positive control.

[0045] The effects of two substituents, namely an aromatic or heteroaromatic ring attached to the thiazole ring (denoted as R) and a substituent attached to the carbonyl group (denoted as R'), on the anti-pyroptotic activity of the thiazole amide core were subsequently evaluated. As shown in Table 1, most thiazole amide derivatives showed significant inhibitory activity against GSDMD-mediated pyroptosis at high concentrations (20 μM). Among them, compounds with different R groups exhibited different activities. Specifically, derivatives with electron-donating aromatic groups, such as p-tolyl (compounds 3 and 4) and p-methoxyphenyl (compounds 7 and 8), were found to promote pyroptosis after NLRP3 inflammasome activation. In contrast, compounds whose R groups were substituted with moderate electron-withdrawing aromatic groups, such as p-fluorophenyl and p-bromophenyl (compounds 6, 9, and 10), exhibited moderate anti-pyroptotic activity.

[0046] The effect of the R' substituent attached to the carbonyl group on the activity of the thiazole core was further investigated. When R was 2,4-dichlorophenyl, compounds with electron-withdrawing aromatic rings (compound 12) or heteroaromatic rings (compound 13) showed considerable anti-pyroptotic activity, while compound 11 substituted with a pyridyl group showed relatively weak activity. When R was substituted with 2,5-dichlorothiophen-3-yl, thiazole amide compounds with various phenyl R' substituents (compounds 15-19) showed weak to moderate anti-pyroptotic activity. Compounds with a phenylsulfonylmethyl group (compounds 20 and 22) promoted cell death at high concentrations, which was due to cytotoxicity. Among them, the o-nitrofuranyl R' substituent (compound 21) showed the strongest anti-pyroptotic activity among the tested compounds, reaching 46.72±2.86% and 62.59±1.29% inhibition rates at 5μM and 20μM, respectively, exceeding the positive control disulfiram.

[0047] The CCK-8 assay was used to evaluate the cytotoxicity of compounds with a thiazole amide skeleton. THP-1 cells were cultured in 96-well plates at 8 × 10 3 The cells were plated at a density of 100 ng / mL and differentiated into THP-Ms using 100 ng / mL PMA overnight. THP-Ms were incubated with different concentrations of thiazole amide analogs (ranging from 3.125 μM to 100 μM) for 24 hours. Subsequently, 10.0 μL CCK-8 reagent was added and incubated for 2 hours. The absorbance at 450 nm was recorded using a PerkinElmer Victor Nivo Alpha 5 microplate reader. The results are shown in Table 1.

[0048] Table 1 Anti-pyroptotic activity of thiazole amide derivatives

[0049]

[0050]

[0051]

[0052] in, a b THP-Ms pretreated with LPS were treated with compounds (5 or 20 μM) for 6 h and then stimulated with nigericin (10 μM) for 1 h; b The cytotoxicity of the listed compounds was evaluated by CCK-8 assay.

[0053] In addition, the anti-pyroptotic effects of other thiazole analogs were also evaluated. As shown in Table 2, compound 23 containing a thiourea group showed a significant anti-pyroptotic effect, achieving an inhibition rate of 64.95±1.48% at 20 μM. In contrast, compound 26 containing an α,β-unsaturated carbonyl group promoted cell pyroptosis. The cytotoxicity of these thiazole amide derivatives was evaluated in THP-Ms cells. As shown in Table 1, compounds with an α,β-unsaturated ester group (compound 10) and compounds with a benzenesulfonylmethyl substituent (compounds 20 and 22) showed higher toxicity, but their effective concentrations were lower than the toxic IC 50 The IC values ​​of most thiazole amide derivatives on THP-Ms cell viability are about 10 times lower than that of the 50 The values ​​were over 100 μM, indicating no obvious toxicity to THP-Ms cells.After the initial antipyroptotic activity screening, compound 21 was identified for further pharmacological evaluation.

[0054] Table 2 Anti-pyroptotic activity of other series of compounds

[0055]

[0056] a THP-Ms pretreated with LPS were treated with compounds (5 μM or 20 μM) for 6 h and then stimulated with nigericin (10 μM) for 1 h. b The cytotoxicity of the compounds in the list was evaluated by CCK-8 assay, in which disulfiram was used as a positive control. The data are mean ± SEM (n = 3).

[0057] Example 3 Synthesis of Compound 21

[0058] Synthesis route:

[0059]

[0060] 1. Synthesis of 4-(2,5-dichlorothiophene-3-yl)thiazole-2-amine (m-21)

[0061] 3-Acetyl-2,5-dichlorothiophene (500 mg, 2.56 mmol), 77 mL of anhydrous ethanol, iodine (I2, 715.7 mg, 2.82 mmol) and copper (II) oxide (CuO, 224.3 mg, 2.82 mmol) were added to a dry reaction flask in sequence and stirred at 80 ° C. The reaction process was monitored by thin layer chromatography (TLC) to ensure that 3-acetyl-2,5-dichlorothiophene was completely consumed. Then, thiourea (194.9 mg, 2.56 mmol) was introduced into the reaction flask and continued to stir. After one hour, TLC detection was performed at 254 nm, confirming that the intermediate α-halogen compound had completely reacted. Subsequently, excess iodine was consumed with sodium thiosulfate solution. The mixture was extracted with ethyl acetate, and the organic layer was separated and evaporated under reduced pressure. The remaining residue was purified by column chromatography to obtain 156.6 mg of compound m-21 (white solid, yield: 24%). The H and C NMR spectra of compound m-21 are as follows Figure 2 and Figure 4 shown. 1 H NMR (Bruker DRX-40, 400MHz, CDCl3) δ7.25 (s, 1H), 7.05 (s, 1H), 4.92 (s, 2H). 13 C NMR (Bruker DRX-40, 101MHz, CDCl3) δ 166.43, 143.66, 132.45, 127.59, 126.08, 121.55, 106.93.

[0062] 2. Synthesis of N-(4-(2,5-dichlorothiophene-3-yl)thiazol-2-yl)-5-nitrofuran-2-carboxamide (Compound 21)

[0063] Compound m-21 (100 mg, 0.4 mmol), 4 mL of dichloromethane and 48 μL of pyridine (0.6 mmol) were mixed and stirred in a reaction vessel. Subsequently, a dichloromethane solution containing 105.3 mg of 5-nitro-2-furoyl chloride (0.6 mmol) was added dropwise to the above mixture. After 4 hours, the reaction was detected by TLC to confirm completion. The mixture was then filtered and the residue was purified by recrystallization in methanol to obtain 131.1 mg of orange-yellow solid compound 21 (yield: 84%). LC-MS mass spectra were collected using a TOF mass spectrometer in ESI positive ion mode. The hydrogen nuclear magnetic resonance spectrum, carbon spectrum, HPLC chromatogram, and mass spectrum of compound 21 are as follows: Figure 3 , Figures 5 to 7 shown. 1H NMR (Bruker DRX-40, 400MHz, DMSO-d6) δ13.33(s,1H),7.91(s,1H),7.88-7.76(m,2H),7.51-7.43(m,1H). 13 C NMR(Bruker DRX-40,101MHz,DMSO-d6)δ157.13,154.70,152.47,145.85,141.80,132.32,127.54,125.34,121.24,118.06,113.28,113.12.LC-MS(ESI)calcd for C 12 H6Cl2N3O4S2[M+H] + 389.9and 391.9.HPLC(30-100%acetonitrile),t R =6.436min;purity>99%.

[0064] Example 4 Compound 21 inhibits GSDMD-induced pyroptosis

[0065] Screening for small molecule inhibitors of GSDMD-mediated pyroptosis led to the identification of compound 21 with an IC 50 About 11μM ( Figure 8 A, 8B). To establish the pyroptosis model, THP-Ms were first stimulated with LPS and then with nigericin.

[0066] After NLRP3 inflammasome activation, THP-Ms were treated and stained with propidium iodide (PI, 1ug / mL). Subsequently, the cells were incubated in the dark for half an hour, and the PI solution was carefully discarded. The cells were gently rinsed three times with PBS to remove residual PI. Finally, a microscope (Nikon ECLIPSE Ti2) was used to observe and capture the occurrence of cell pyroptosis ( Figure 8 D). Pyroptotic cells showed obvious swelling, formed pyroptotic bodies, and organelles were redistributed to one side of the cell, eventually leading to cell membrane rupture. After treatment with compound 21, THP-Ms were stained with PI (1 ug / mL) in the dark for 30 minutes. Fluorescence intensity was quantified at 535nm / 615nm using a multifunctional microplate reader (PerkinElmer, Victor Nivo Alpha 5). The highest fluorescence signal was obtained by adding 1% Triton X-100. The relative PI uptake was determined using the following equation: Relative PI uptake = (OD 样本 -OD 背景 ) / (OD 最大 -OD 背景). Experimental data showed that compound 21 reduced the uptake of propidium iodide in a concentration-dependent manner, indicating that it can inhibit the formation of membrane pores ( Figure 8 C, 8D).

[0067] The anti-pyroptosis activity of compound 21 was further explored in primary bone marrow-derived macrophages (BMDM). BMDM were pretreated with LPS and then exposed to different concentrations of compound 21 for 6 h before nigericin stimulation. The results showed that compound 21 exhibited significant activity in inhibiting pyroptosis in both human and mouse cells ( Fig. 9 A).

[0068] After THP-1 cell differentiation, THP-Ms were incubated in RPMI 1640 medium containing 1% FBS. The activity of LDH was evaluated using an LDH detection kit (Nanjing Jiancheng). The absorbance value at 440 nm was recorded using a microplate reader (PerkinElmer, Victor Nivo Alpha5). After LPS-pretreated THP-Ms were stimulated with nigericin, the formation of pyroptotic membrane pores was caused, which ultimately led to cell lysis and the excretion of intracellular substances such as LDH and inflammatory cytokines. The supernatant of THP-Ms culture was collected, and the IL-1β level was determined using an ELISA kit (Boster, EK0392) according to the manufacturer's guidelines. The absorbance value at 450 nm was recorded using a microplate reader (PerkinElmer, Victor Nivo Alpha 5). The results show ( Fig. 9 B, 9C), compound 21 reduced the release of IL-1β and LDH in a concentration-dependent manner.

[0069] In addition to the effect on NLRP3, the present invention also tested the effect of compound 21 on pyroptosis caused by activation of other inflammasomes. AIM2 and NLRC4 inflammasomes respond to cytoplasmic double-stranded DNA and pathogenic bacterial infection, respectively. Compound 21 significantly inhibited pyroptosis triggered by activation of NLRP3 and AIM2 inflammasomes. However, compound 21 had less effect on pyroptosis induced by NLRC4 inflammasomes and exhibited anti-pyroptosis activity at high concentrations (30 μM) ( Fig. 9 D-9G). These findings suggest that compound 21 inhibits pyroptosis and subsequent inflammatory cytokine release induced by multiple inflammasome activation.

[0070] Example 5 Compound 21 inhibits GSDMD cleavage and upstream NLRP3 signaling pathway

[0071] The present invention studies how compound 21 inhibits pyroptosis. NLRP3 inflammasome activation triggers the cleavage of Caspase-1, which subsequently promotes the processing and release of IL-1β and pyroptosis execution protein GSDMD. In the pyroptosis model, Western blot analysis detected significant GSDMD-NT, mature IL-1β, active caspase-1p20, and caspase-1 cleavage fragments in the cell supernatant. Specifically, cells were treated and cultured in a medium without FBS to prevent other proteins in the serum from interfering with the detection. After cell treatment, the supernatant was collected and stored at -80°C. The supernatant was concentrated to 500 μL using an ultrafiltration tube (Millipore, 3kDa) at 4000 rpm. Methanol and chloroform were introduced into the concentrated 500 μL cell supernatant in a ratio of 4:1. The mixture was vortexed for 30 seconds to form a milky suspension and centrifuged at 13000 rpm for 5 minutes. The solution was divided into three layers: an upper layer (methanol-water mixture), a middle layer (protein), and a lower layer (chloroform). The top and bottom layers were carefully aspirated, leaving the middle protein layer. Methanol (1 mL) was added to each sample and centrifuged at 13,000 rpm, 4°C for 5 minutes. The methanol was removed and air-dried. 50 μL of loading buffer was added to each sample tube, dissolved on ice for 2 hours, boiled for 10 minutes, and the processed protein samples were stored at -20°C.

[0072] After THP-Ms stimulation or transfection of HEK-293T cells with GSDMD-NT plasmid, cells were lysed using Western and IP lysis buffer. The corresponding volume of loading buffer was added to the protein solution, boiled in water for 10 min to denature the protein, and then stored at -20 °C for immunoblot analysis.

[0073] The results of the study showed that ( Fig.10 A, 10B), compound 21 effectively inhibited the cleavage and activation of caspase-1 and GSDMD in a concentration-dependent manner. In addition, the levels of mature IL-1β and caspase-1p20 in the cell supernatant were also significantly reduced.

[0074] In addition, it was observed that compound 21 inhibited the expression of NLRP3 protein. Therefore, the effect of compound 21 on the upstream NF-κB pathway was explored next. THP-Ms were pretreated with different doses of compound 21 for 6 h and then exposed to LPS for 3 h. Compound 21 showed concentration-dependent inhibition of NF-κB activation and phosphorylation and downregulated the expression of NLRP3 and pro-IL-1β ( Fig.10 C).

[0075] qT-PCR was used to analyze the effect of compound 21 on target gene transcription. Total RNA was isolated from THPMs using TRIzol (Takara, 9108) and converted into cDNA using the reverse transcription PrimeScriptTM RT Master Mix kit. cDNA was subjected to quantitative PCR analysis using the LightCycler 480Ⅱ system with Taq Pro Universal SYBR qPCR Master Mix and aRoche. Gene expression levels were measured using the ΔΔCT method, with GAPDH as an internal control. The threshold cycle (CT) of each gene was normalized to GAPDH. Gene expression was calculated as 2 -ΔCT Among them, the target gene primer sequences are shown in Table 4.

[0076] Table 4

[0077]

[0078] The results show that ( Fig.10 E-10G), compound 21 significantly reduced the gene transcription levels of IL-6, NLRP3 and IL-1β.

[0079] GSDMD is cleaved into active GSDMD-NT using inflammatory caspases, which then transfer to the membrane, oligomerize, form extensive pores, and lead to cell death and rupture. To further investigate the effect of compound 21 on GSD MD-NT oligomerization during pyroptosis, human eGFP GSDMD-NT-plasmid custom-synthesized from GenScript Biotech was transfected into HEK-293T cells using Lipofectamine 3000. Specifically, HEK-293T cells were transfected with 5×10^ 6 The cells were plated at a density of 10 cells / well in a 6-well culture dish and left overnight. The human eGFP-GSDMD-NT plasmid (name: pcDNA3.1(+)-N-eGFP, cloning site is BamHI / XhoI, length is 966bp) contains a 3×Flag epitope tag at the N-terminus, and it was inserted into the pcDNA3.1 / Zeo(+) vector to obtain the recombinant eGFP GSDMD NT plasmid; the recombinant eGFP GSDMD-NT plasmid was transfected into HEK-293T cells at a dose of 2μg / well using Lipofectamine 3000 for 1 day. Subsequently, HEK-293T cells were treated with compound 21 for 6 hours. Pyroptosis was observed under a microscope, and then the intracellular proteins were extracted for immunoblot analysis. Fig.10 As shown in D, compound 21 inhibited GSDMD-NT oligomerization at high concentrations, thereby inhibiting pore formation and pyroptosis to a certain extent. Among them, the gene sequence of the recombinant eGFP GSDMD NT-plasmid is shown in SEQ ID NO.9.

[0080] In summary, the present invention has identified a series of thiazole amide compounds with anti-pyroptosis activity through laboratory activity screening. SAR analysis showed that compound 21 is the most effective inhibitor of inflammasome-induced pyroptosis, with IC 50 About 11 μM. The experimental results showed that compound 21 inhibited the formation of pyroptotic membrane pores in a concentration-dependent manner, as shown by the reduction of PI uptake. In addition, compound 21 significantly inhibited the release of IL-1β and LDH, which are hallmark indicators of pyroptosis. In addition, compound 21 showed inhibitory activity on pyroptosis mediated by several typical inflammasomes (NLRP3, NLRC4 and AIM2), indicating that its effect is not limited to the NLRP3 inflammasome activation stage. Mechanistically, compound 21 showed partial inhibition of the upstream NF-κB pathway and reduced the expression of inflammation-related proteins. In addition, compound 21 can effectively inhibit the cleavage and activation of GSDMD and the oligomerization of GSDMD-NT, thereby effectively inhibiting pyroptosis.

Claims

1. A method for preparing a thiazole amide derivative, characterized in that: The following steps are involved: 3-acetyl-2,5-dichlorothiophene, anhydrous ethanol, iodine and copper oxide are mixed for reaction; thiourea is added and stirred continuously; after the reaction is completed, dichloromethane and pyridine are added and mixed; a dichloromethane solution containing 5-nitro-2-furoyl chloride is added under stirring to obtain the thiazole amide derivative; the structural formula of the thiazole amide derivative is:

2. Use of a thiazole amide derivative or a pharmaceutically acceptable salt, hydrate, solvate, or pharmaceutically acceptable carrier thereof in the preparation of a drug for treating and / or improving inflammation-related diseases, characterized in that: The thiazole amide derivatives include compounds with a general structural formula as shown in Formula I: Among them, R is C1~C 30 Aryl, heteroaryl, alkyl, C1~C 30 Alkyl aryl, C1~C 30 Alkyl heteroaryl, C1~C 12 Heterocyclic, C1~C 30 Substituted aryl or C1~C 30 Substituted heteroaryl; the C1~C 30 Substituted aryl or C1~C 30 The substituted heteroaryl group is substituted by one or more of the following identical or different substituents: C1-C 30 alkyl, alkoxy, alkylamino, amide, sulfonamide, nitro, halogen, benzyl, nitrile, amino, carboxyl, carboxyl, carbonyl, trifluoromethyl; R' is C1~C 30 Alkyl, aryl, heteroaryl, C1~C 30 Alkyl aryl, C1~C 30 Alkyl heteroaryl, C1~C 12 Heterocyclic, C1~C 30 Substituted aryl or C1~C 30 Substituted heteroaryl; the C1~C 30 Substituted aryl or C1~C 30 The substituted heteroaryl group is substituted by one or more of the following identical or different substituents: C1-C 30 The alkyl, alkoxy, alkylamino, amide, sulfonamide, nitro, halogen, benzyl, nitrile, amino, carboxyl, carboxyl, carbonyl, trifluoromethyl.

3. The application according to claim 2, characterized in that: The thiazole amide derivatives include 4. The application according to claim 2, characterized in that: The inflammation-related diseases include sepsis, inflammatory bowel disease, diabetes, gout, rheumatoid arthritis, rheumatic arthritis, non-alcoholic fatty liver disease, atherosclerosis or neurodegenerative diseases.

5. The use according to claim 4, characterized in that: The neurodegenerative disease includes Alzheimer's disease, Parkinson's disease or multiple sclerosis.

6. Use of the compound of formula I according to claim 2 or 3 or its pharmaceutically acceptable salt, hydrate, solvate, or pharmaceutically acceptable carrier in the preparation of an NLRP3 inflammasome or GSDMD inhibitor.

7. A pharmaceutical composition for treating and / or improving inflammation-related diseases, characterized in that: It contains the thiazole amide derivative according to claim 2 or 3 or a pharmaceutically acceptable salt, hydrate, solvate thereof, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that: The inflammation-related diseases include sepsis, inflammatory bowel disease, diabetes, gout, rheumatoid arthritis, rheumatic arthritis, non-alcoholic fatty liver disease, atherosclerosis or neurodegenerative diseases; the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease or multiple sclerosis.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that The pharmaceutical composition is in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

10. An NLRP3 inflammasome or GSDMD inhibitor, characterized in that It contains the thiazole amide derivative according to claim 2 or 3 or a pharmaceutically acceptable salt, hydrate, solvate thereof, and a pharmaceutically acceptable carrier.

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