An isodomesticenol derivative, a preparation method and use thereof

Abstract

The application belongs to the technical field of medicines, and particularly relates to a kind of isoalantolactone derivatives, a preparation method and purposes thereof.The application provides a new compound IALN, and a structural formula is as shown in formula I.The compound can be used as NLRP3 inhibitor, realizes anti-inflammatory effect by directly interacting with NLRP3, has remarkable therapeutic effect on acute inflammatory disease-ulcerative colitis, and provides a new way for widening the screening and development of anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an isocarboxylic acid lactone derivative, its preparation method, and its uses. Background Technology

[0002] The NLRP3 inflammasome is a crucial part of the innate immune system. It plays a vital role in helping the body sense danger and respond rapidly, maintaining physiological homeostasis. However, its abnormal activation can lead to various inflammation-related diseases, such as cryopyridine-associated periodic syndrome, inflammatory bowel disease, atherosclerosis, type II diabetes, and neurodegenerative diseases. Currently, the main drugs used clinically to treat NLRP3 inflammasome-related diseases are IL-1β receptor antagonists. However, besides NLRP3, IL-1β can also be produced through other pathways; therefore, the use of IL-1β receptor antagonists may cause adverse reactions such as excessive immunosuppression.

[0003] Several small-molecule inhibitors targeting NLRP3 and showing promising therapeutic potential for related inflammatory diseases have been reported, with a few already in clinical trials. However, whether these inhibitors will ultimately receive regulatory approval for the clinical treatment of NLRP3 inflammasome-related diseases remains to be further investigated. Finding inhibitors with better activity, higher safety profiles, and clinical applicability is currently the goal of many researchers.

[0004] Among these reported small molecule inhibitors, the discovery of natural products such as oridonin, baicalin, romaine, fenestrate lactone, and cardamom has provided new insights for the development of NLRP3 inhibitors. Natural products have always been an important source of candidate compounds for new drug development. Currently, 60% of drugs developed worldwide are derived from the active ingredients of natural products and their analogues. *Inula racemosa* Hook.f. is a perennial herb belonging to the genus *Inula* of the Asteraceae family. Its dried root has the effects of strengthening the spleen and stomach, regulating qi, and relieving depression. Iso-indolactone is one of the main components of the dried root of *Inula racemosa* Hook.f., and several studies have reported its good anti-inflammatory effects. Its structural formula is as follows:

[0005]

[0006] To meet the needs of clinical applications, it is necessary to further enhance the anti-inflammatory effects of isothomolecin. Therefore, using isothomolecin as a probe to discover new NLRP3 inflammasome regulatory factors is of great significance. However, during the derivatization of isothomolecin, it was found that derivatives with different substituents exhibited significant differences in anti-inflammatory activity. Therefore, how to design molecular structures to obtain isothomolecin derivatives with excellent anti-inflammatory activity is an important issue in this field. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides an isocarboxylic acid lactone derivative, its preparation method, and its uses.

[0008] The compound represented by Formula I, or its stereoisomer, or its solvate, or its metabolite, or its deuterated form, or its prodrug, or its pharmaceutically acceptable salt, or its eutectic:

[0009]

[0010] The present invention also provides a method for preparing the above-mentioned compound, or its stereoisomer, or its solvate, or its metabolite, or its deuterated product, or its prodrug, or its pharmaceutically acceptable salt, or its cocrystal, comprising the following steps:

[0011]

[0012] Step 1: Oxidize isocarboxylide to obtain intermediate A;

[0013] Step 2: React the intermediate A with the raw material B to obtain the compound shown in Formula I.

[0014] Preferably, in step 1, the oxidant is selected from SeO2 and TBHP; the solvent is selected from DCM; the reaction temperature is 0-30℃; and the reaction time is 6-12h.

[0015] Preferably, in step 2, the reaction is carried out under the action of a condensing agent selected from DCC; the reaction is carried out under the action of a catalyst selected from DMAP; the solvent is selected from DCM; the reaction temperature is 20-30℃; and the reaction time is 12-18h.

[0016] The present invention also provides the use of the above-mentioned compound, or its stereoisomer, or its solvate, or its metabolite, or its deuterated form, or its prodrug, or its pharmaceutically acceptable salt, or its cocrystal, in the preparation of anti-inflammatory drugs.

[0017] Preferably, the drug is used to treat acute inflammatory diseases.

[0018] Preferably, the drug is used to treat ulcerative colitis.

[0019] The use of the compound, or its stereoisomer, or its solvate, or its metabolite, or its deuterated form, or its prodrug, or its pharmaceutically acceptable salt, or its cocrystal, in the preparation of an NLRP3 inhibitor.

[0020] Preferably, the NLRP3 inhibitor is used to inhibit NLRP3 inflammasome activation;

[0021] And / or, the NLRP3 inhibitor is used to inhibit pyroptosis induced by NLRP3 inflammasome activation;

[0022] And / or, the NLRP3 inhibitor is used to inhibit NLRP3 inflammasome assembly;

[0023] And / or, the NLRP3 inhibitor is used to covalently bind to cysteine ​​279 of the NACHT domain of the NLRP3 protein.

[0024] The present invention also provides an anti-inflammatory drug or NLRP3 inhibitor, which is prepared by adding pharmaceutically acceptable excipients to the above-mentioned compound, or its stereoisomer, or its solvate, or its metabolite, or its deuterated product, or its prodrug, or its pharmaceutically acceptable salt, or its cocrystal as the active ingredient.

[0025] This invention optimizes the structure of isothomyl lactone and provides a novel tricyclic sesquiterpene lactone-6-nicotinic acid ester compound, IALN. This compound can act as an NLRP3 inhibitor, achieving an anti-inflammatory effect through direct interaction with NLRP3. It has a significant therapeutic effect on acute inflammatory diseases, such as ulcerative colitis, and provides a new approach for broadening the screening and development of anti-inflammatory drugs.

[0026] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0027] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0028] Figure 1 The product IALN prepared in Example 1 1 H spectrum.

[0029] Figure 2 The product IALN prepared in Example 1 13 C-spectrum.

[0030] Figure 3 The image shows the high-resolution mass spectrometry (HRESIMS) spectrum of the product IALN prepared in Example 1.

[0031] Figure 4IALN inhibits NLRP3 inflammasome activation in various macrophages. (AC) LPS-induced THP-1 (A), BMDM (B), and PBMC (C) cells were treated with IALN for 40 min, followed by stimulation with 10 μM nigericin for 40 min. IL-1β release from the supernatant was analyzed by ELISA. (DF) THP-1 (D), BMDM (E), and PBMC (F) cells were incubated with IALN for 24 h, and cell viability was detected by CCK-8 assay. (G, H) The expression of NLRP3-related proteins in the supernatant (Sup.) and cell extract (Lys.) of THP-1 and BMDM cells was analyzed by Western blotting. (I, J) LPS-induced THP-1 (I) and BMDM (J) cells were treated with IALN or MCC950 for 40 min, followed by stimulation with 10 μM nigericin for 40 min. IL-1β release from the supernatant was analyzed by ELISA. Statistical differences were calculated using one-way ANOVA. Compared with the LPS-only group: ####P<0.0001; compared with the nigericin stimulation group: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns represents no significant difference.

[0032] Figure 5 IALN inhibited pyroptosis induced by NLRP3 activation in macrophages. (A, B) THP-1 cells (A) and BMDMs (B) were treated with LPS for 3 h, followed by treatment with MCC950 or different concentrations of IALN for 40 min, and then stimulation with nigericin for 40 min. GSDMD-NT in the cells was analyzed by Western blotting. (C, D) THP-1 cells (C) and BMDMs (D) were treated with LPS for 3 h, followed by treatment with MCC950 or different concentrations of IALN for 40 min, and then stimulation with nigericin for 40 min. After treatment, the cells were stained with propidium iodide (5 μg / mL) at room temperature for 20 min, and the images were acquired using a Nikon Eclipse Ts2R inverted microscope. (E, F) THP-1 cells (E) and BMDMs (F) were treated with LPS for 3 h, followed by treatment with MCC950 or different concentrations of IALN for 40 min, and then stimulation with nigericin for 40 min. The cell supernatant was collected for LDH content detection. Statistical differences were calculated using one-way ANOVA. Compared with the LPS-only group: ####P<0.0001, ###P<0.001; compared with the nigericin stimulation group: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.

[0033] Figure 6IALN inhibits the assembly of the NLRP3 inflammasome. (A) THP-1 cells and NLRP3 KOTHP-1 cells were treated with LPS for 3 h, then treated with different concentrations of IALN for 40 min, followed by stimulation with nigericin for 40 min. Immunoblot analysis was used to examine the effect of IALN on ASC oligomerization in the cells. (B) Immunofluorescence microscopy was used to observe the effect of IALN on ASC spots. (C, D) THP-1 cells were treated with LPS for 3 h, then treated with IALN (1 μM) for 40 min, followed by stimulation with nigericin for 40 min. Immunoprecipitation was used to analyze the interaction between NLRP3-NEK7 (C) and NLRP3-ASC (D).

[0034] Figure 7 IALN covalently binds to cysteine ​​279 of the NACHT domain of the NLRP3 protein. (A, B) DARTS (A) and CETSA (B) analyses were performed using THP-1 cell lysates. (C, D) DARTS (C) and CETSA (D) analyses were performed using purified NLRP3ΔLRR protein. (E, F) DARTS analyses were performed using purified NLRP3 NACHT protein (E) and PYD protein (F). (G) THP-1 cells were treated with LPS for 3 h, then treated with different concentrations of IALN for 40 min. Cells were washed three times with PBS or not washed, and then stimulated with nigericin for 40 min. IL-1β secretion in the cell supernatant was detected by ELISA. (H) Shotgun mass spectra of IALN and NACHT protein. (I) HEK-293T cells were transfected with HA-NLRP3 and HA-NLRP3 C279A plasmids for 24 h, and then analyzed with IALN using DARTS. (J) HEK-293T cells were co-transfected with HA-NLRP3 and FLAG-NEK7 plasmids or HA-NLRP3 C279A and FLAG-NEK7 plasmids, and incubated with IALN (1 μM) for 24 h before immunoprecipitation analysis. (K) Molecular docking simulation of IALN and NACHT protein. Statistical differences were calculated using one-way ANOVA. Compared with the LPS-only group: ####P<0.0001; compared with the nigericin-stimulated group: ****P<0.0001, **P<0.01, ns represents no significant difference.

[0035] Figure 8IALN effectively inhibits DSS-induced ulcerative colitis. (A) Percentage change in mouse body weight. (B) Disease activity index. (C) Colon length. (D) HE staining of colon tissue. (EF) ELISA examination of IL-1β (E) and TNF-α (F) production in DSS-induced mouse colon tissue. (G) Western blot examination of caspase-1 expression in colon tissue. Statistical differences were calculated using one-way ANOVA. Compared with the normal group: ####p<0.0001; compared with the DSS group: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Detailed Implementation

[0036] In the following examples, reagents and materials not specifically described are all commercially available products.

[0037] Example 1: Compound IALN and its preparation method

[0038] This embodiment provides the compound IALN, with the following structural formula:

[0039]

[0040] Its preparation method is as follows:

[0041]

[0042] 416 mg (3.75 mmol) of selenium dioxide and 1.35 g (15 mmol) of tert-butyl hydroperoxide were added to a 500 mL round-bottom flask, followed by activation with 40 mL of dichloromethane at 0 °C for 30 min. Then, 5.8 g (25 mmol) of isotretinoin was dissolved in dichloromethane and added dropwise to the activated selenium dioxide and tert-butyl hydroperoxide mixture at 0 °C. The mixture was then transferred to room temperature and reacted for 18 h. TLC monitoring was performed. After the reaction was complete, saturated Na₂S₂O₃ solution was added to quench the reaction. The reaction mixture was then placed in a separatory funnel and allowed to separate into layers. The lower layer was collected, and the residual Na₂S₂O₃ was neutralized with saturated NaHCO₃ solution. The solvent in the reaction mixture was then evaporated to dryness. The product A, a white solid, was obtained by separation and purification using a Flash column chromatography with a yield of approximately 66%.

[0043] Then, 100 mg (0.4 mmol) of intermediate A, 230 mg (1.2 mmol) of starting material B, and 20 mg (0.16 mmol) of DMAP were added to a 25 mL round-bottom flask. 10 mL of dichloromethane was used as the solvent, followed by the addition of 198 mg (0.96 mmol) of DDC. The reaction was stirred overnight at room temperature. TLC monitoring was performed. After the reaction was complete, the solvent was evaporated to dryness, and the product was purified by Flash column chromatography to obtain a white solid. The yield was 48%.

[0044] The proton and carbon NMR spectra of the product are as follows: Figure 1 and Figure 2 As shown, specifically:

[0045] (3aR,4aR,6R,8aR,9aR)-8a-methyl-3,5-dimethylene-2-oxododecahydronaphtho[2,3-b]furan-6-yl 4,6-dichloronicotinate(49).White solid,48%yield.C 21 H 21 Cl2NO4.HRESIMS:m / z 444.0743[M+Na] + (calcd.for C 21 H 21 Cl2NO4Na,444.0745).1H NMR (400MHz, CDCl3) δ8.87 (s, 1H), 7.48 (s, 1H), 6.16 (d, J = 0.5Hz, 1H), 5.66 (t, J = 2.5Hz, 1H),5.60(s,1H),5.27(s,1H),4.82(d,J=1.1Hz,1H),4.53(td,J=4.7,1.1Hz,1H),3.01( m,1H),2.30-2.25(m,2H),1.99-1.89(m,2H),1.75(ddd,J=14.0,7.0,2.6Hz,1H),1.67(t d,J=13.0,5.2Hz,1H),1.58-1.48(m,2H),1.39(dt,J=13.6,12.5Hz,1H),0.89(s,3H).13C NMR (100MHz, CDCl3) δ170.38,161.91,154.53,152.41,145.65,144.54,141.77,125.96,125 .07,120.45,113.72,77.62,76.48,41.62,40.90,40.19,36.45,33.81,26.98,26.81,17.00.

[0046] Mass spectrometry data of the product, such as Figure 3 As shown, the specific results are as follows:

[0047] IALN <![CDATA[C 21 H 21 Cl2NO4]]> <![CDATA[C 21 H 21 Cl2NO4Na]]> 444.0743 444.0745 -0.45

[0048] Example 2: Activity study of compound IALN

[0049] I. Experimental Methods

[0050] The compound IALN in this embodiment was prepared according to the method in Example 1.

[0051] (1) Cell culture and stimulation of THP-1, BMDMs and PBMCs

[0052] THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Differentiated THP-1 cells were obtained by co-culturing with 100 ng / mL PMA for 24 h. BMDMs were obtained from the femurs and tibias of C57BL / 6 mice and cultured in DMEM medium supplemented with 10% fetal bovine serum and 30% L929 culture supernatant. PBMCs were isolated from venous blood donated by healthy volunteers and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum.

[0053] Induction of NLRP3 inflammasome activation: PMA-induced differentiated THP-1 cells, BMDMs, and PBMCs activated at 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 μg / mL in appropriate wells. The next day, the culture medium was replaced with Opti-MEM containing 1 μg / mL LPS to stimulate the cells for 3 hours, followed by treatment with different concentrations of IALN for 40 minutes, and finally stimulation with 10 μM nigericin for 40 minutes.

[0054] (2) Detection of cellular inflammatory factors and LDH secretion

[0055] After cell stimulation, the cell supernatant was collected, and the concentrations of IL-1β and IL-18 were detected using an ELISA kit or via CytoTox, according to the manufacturer's instructions. A non-radioactive cytotoxicity assay kit was used to detect the release of LDH.

[0056] (3) Cell viability

[0057] The viability of PMA-differentiated THP-1 cells, BMDMs, and PBMCs was assessed using a CCK-8 assay. In short, cells (2 × 10⁻⁶) were used to differentiate into different types of cells. 5 The reagent ( / mL) was inoculated into 96-well plates and incubated with IALN for 24 hours the next day. Then, 10 μL of CCK-8 reagent was added to each well, and after incubation for a certain period of time, the absorbance was measured at 450 nm using a microplate reader.

[0058] (4) PI staining

[0059] After cell stimulation, the cell supernatant was removed, and the cells were gently washed three times with pre-chilled PBS. 200 μL of 5 μg / mL propidium iodide (PI) was added to each well, and the cells were incubated at room temperature for 10 min. After staining, the cells were gently washed three times with pre-chilled PBS and then photographed under a microscope.

[0060] (5) Western blot

[0061] Cells were lysed in RIPA lysis buffer, then mixed with loading buffer and denatured at 100°C. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Cells were blocked with 5% skim milk, incubated overnight with primary antibody at 4°C, and then incubated at room temperature the next day with appropriate secondary antibody. Finally, the cells were developed in an imaging system using developing solution.

[0062] (6) Immunofluorescence

[0063] After cell stimulation, the supernatant was discarded, and the cells were washed with pre-chilled PBS, fixed with methanol at -20°C for 20 min, washed with PBST for 5 min, and blocked with 0.5% bovine serum albumin (w / v, diluted with PBST) for 1 h. They were then incubated with antibody overnight. Afterward, they were washed with PBST, treated with secondary antibody diluted 1:500 with 0.5% bovine serum albumin for 1 h, and the nuclei were stained with 4',6'-diamino-2-phenylindole (DAPI). Finally, they were photographed under a fluorescence microscope.

[0064] (7) ASC oligomerization

[0065] Cells were lysed using 300 μL of NP-40 lysis buffer. The lysis buffer was incubated on a shaker at 4°C for 30 min, followed by centrifugation at 6000×g for 15 min at 4°C. After removing the supernatant, insoluble cell debris was incubated in 2 mM DSS at 37°C for 30 min, then centrifuged at 6000×g for 15 min at 4°C. The pellet was resuspended in 30 μL of 1× loading buffer, heated, and then subjected to Western blotting for detection.

[0066] (8) Immunoprecipitation

[0067] For endogenous IP assays, after cell stimulation, the supernatant was removed, and THP-1 cells were washed three times with PBS, followed by lysis with NP-40 for 30 minutes. The cell lysates were incubated overnight at 4°C with primary antibody. Protein A / G magnetic beads were added to the cell lysates, and the mixture was incubated at 4°C with rotation for 4 hours. The magnetic beads were washed several times, heated in 1× loading buffer, and then subjected to Western blotting for detection.

[0068] For exogenous IP assays, HEK-293T cells were seeded overnight in 6-well plates, then transfected with polyethyleneimine plasmids and co-incubated with IALN (1 μM). After 24 hours, cells were collected and lysed with NP-40 lysis buffer. Primary antibody was added and incubated overnight at 4°C. Protein A / G magnetic beads were added to the cell lysates and incubated at 4°C with rotation for 4 hours. The magnetic beads were washed several times, added to 1× loading buffer, heated, and then subjected to Western blotting for detection.

[0069] (9) Drug affinity response target stability (DARTS)

[0070] THP-1 cells differentiated from PMA were used at a rate of 5 × 10⁻⁶. 5 Seed the cells at a density of 1 mL in 10 cm culture dishes. The next day, remove the culture medium and replace it with Opti-MEM containing LPS, incubating for 3 hours. Alternatively, seed HEK-293T cells at a density of 5 × 10⁶ cells / mL. 5 The cells were seeded at a density of 1 / mL in 6-well plates and transfected with plasmids for 24 hours.

[0071] Remove cell supernatant, wash cells with PBS, and then lyse with NP-40 for 30 minutes. Centrifuge the lysate at 17000g for 15 minutes at 4°C. After centrifugation, incubate 1 μL of IALN or DMSO and 99 μL of supernatant or purified protein at room temperature. After 50 minutes, add streptomycin to the mixture and incubate at room temperature. After 30 minutes, terminate the reaction by adding a mixture of protease inhibitors. After adding loading buffer and heating, perform Western blotting.

[0072] (10) Thermal displacement analysis

[0073] THP-1 cells differentiated from PMA were used at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 10cm culture dishes with complete culture medium. The next day, the medium was removed and replaced with Opti-MEM containing LPS, and incubated for 3 hours. Cells were then washed with PBS, scraped off with a cell scraper, and lysed using liquid nitrogen and room temperature water in three rounds of freezing and thawing. Cells were then centrifuged at 17000g for 15 minutes at 4°C. Cell lysates or purified NLRP3ΔLRR protein were divided into two groups in PCR tubes. One group was mixed with 1 μL IALN, and the other with DMSO as a negative control. Both groups of proteins were heated to 45–63 or 78°C under identical conditions using a thermal cycler. Each sample was heated at a single temperature for 2 minutes. After adding loading buffer and heating, immunoblotting was performed.

[0074] (11) Mass spectrometry identification of protein modification sites

[0075] Recombinant NACHT protein was incubated with IALN for 12 hours, and the mixture was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. Bands corresponding to NACHT were excised and digested with trypsin in a gel. Trypsin peptides were analyzed at C1... 18 Separation was performed on the column and analyzed using an Obitrap Fusion Lumos mass spectrometer. Mass spectrometry data were identified and quantified using pFind 3.2.1 software.

[0076] (12) Molecular docking

[0077] use The 2018-1 software was used to perform molecular covalent docking between IALN and NLRP3 NACHT proteins. An appropriate crystal structure (PDB ID: 7ALV) was selected from the RCSB PDB database (PDB, http: / / www.rcsb.org / ). Protein preparation was performed in the docking software, including supplementing missing amino acid residues and optimizing the protein. Ligand preparation for IALN was also performed. Using the covalent docking module, amino acid residue (position 279) was selected as the active site and the center of the docking box. Michael addition was selected as the reaction type.

[0078] (13) Animal experiments

[0079] Eight-week-old male C57BL / 6 mice (20–22 g) were provided by Beijing Huafukang Biotechnology Co., Ltd. All mice were housed in a specific pathogen-free animal facility at the Experimental Animal Center of Sichuan University, and animal experiments were conducted in accordance with the animal experimentation procedures approved by the Animal Experimentation Ethics Committee of the State Key Laboratory of Biotherapy, Sichuan University.

[0080] Mice were fed drinking water prepared with 3.25% (w / v) DSS and allowed free access to induce experimental colitis. Mice were randomly divided into four experimental groups (n=6). The untreated group drank normal water; the model group drank DSS water, which was replaced with normal drinking water after 6 days; the IALN group drank DSS water, which was replaced with normal drinking water after 6 days, and the mice received daily intraperitoneal injections of IALN (15 mg / kg or 30 mg / kg); the IAL group drank DSS water, which was replaced with normal drinking water after 6 days, and the mice received daily intraperitoneal injections of IAL (30 mg / kg). From the start of the modeling process, daily observations were made of the mice's food intake, activity, and fur condition. Animal weight was measured, and fecal characteristics, fecal occult blood, and bleeding were observed to assess the severity of colitis. The severity was quantified using the DAI (Disease Activity Index) score, which is based on three parameters: body weight, rectal bleeding, and fecal consistency. Mice were sacrificed at the end of the experiment, and their colons were collected for colon length determination. A portion of the colon was directly frozen for subsequent experimental analysis, while another portion of the mouse colon was fixed with 4% paraformaldehyde for H&E staining.

[0081] II. Experimental Results

[0082] 1. IALN inhibits NLRP3 inflammasome activation in macrophages.

[0083] To preliminarily investigate whether IALN has an inhibitory effect on NLRP3 activation in vitro, the effect of IALN on the secretion of the inflammatory factor IL-1β after NLRP3 activation was tested using PMA-induced differentiated THP-1 cells, BMDMs, and PBMCs. First, cells were induced with LPS for 3 h, then treated with IALN for 40 min, and finally stimulated with the NLRP3 inflammasome stimulator nigericin for 40 min. IALN dose-dependently inhibited the release of IL-1β from THP-1 cells, BMDMs, and PBMCs, with a half-maximal inhibitory concentration (IC50) of [value missing]. 50 The values ​​were 293.4 nM, 738.8 nM, and 44.97 nM, respectively. Figure 4 AC).

[0084] To ensure that the inhibitory effect of IALN on IL-1β was not mediated by cell killing, the cytotoxic effects of IALN in THP-1, BMDMs, and PBMCs were assessed using the CCK-8 assay. The results showed that the concentration of IALN used in this study did not induce significant cytotoxicity, indicating that the inhibitory effect of IALN on IL-1β in this study was not cytotoxic. Figure 4 DF).

[0085] During NLRP3 inflammasome activation, pro-caspase-1 is cleaved into mature caspase-1, which then cleaves inactive pro-IL-1β into the active mature form of IL-1β, releasing it extracellularly. To further confirm the inhibitory effect of IALN on the NLRP3 inflammasome, its effects on the expression of NLRP3 inflammasome-related components—IL-1β, caspase-1, pro-IL-1β, pro-caspase-1, NLRP3, and ASC—were examined by Western blot. The results showed that IALN dose-dependently inhibited the expression of IL-1β and caspase-1 in the supernatant of THP-1 cells and BMDMs, without affecting the intracellular expression of NLRP3 inflammasome components. Figure 4 G and H).

[0086] Besides IL-1β and caspase-1, IL-18 is also considered one of the main markers of NLRP3 inflammasome activation. The secretion of IL-18 in the supernatant of THP-1 cells and BMDMs was also detected. The results showed that IALN dose-dependently inhibited the secretion of IL-18 in the cell supernatant. Figure 4 I and J).

[0087] The above results indicate that IALN can inhibit the maturation and secretion of IL-1β, IL-18 and caspase-1 induced by NLRP3 inflammasome activation in macrophages.

[0088] 2. IALN inhibits pyroptosis induced by NLRP3 inflammasome activation in macrophages.

[0089] NLRP3 inflammasome activation leads to caspase-1 activation, which cleaves the N-terminus of GSDMD (GSDMD-NT). This N-terminus binds to phosphatidylinositol and cardiolipin, disrupting the cell membrane and causing pore formation, subsequently inducing pyroptosis and LDH release. Western blot was used to examine the effect of IALN on GSDMD cleavage following NLRP3 inflammasome activation. The results showed that IALN dose-dependently inhibited GSDMD-NT production in THP-1 cells and BMDMs, suggesting that IALN can inhibit GSDMD-induced pyroptosis. Figure 5 (A and B).

[0090] PI is a red fluorescent dye that, due to its impermeability to living cell membranes, can penetrate pyroptosis cells to stain DNA and RNA, displaying red fluorescence, while living cells remain unstained. Experimental results showed that IALN inhibited the production of red fluorescence in THP-1 cells and BMDMs after NLRP3 activation in a dose-dependent manner. Figure 5 (C and D).

[0091] NLRP3 inflammasome activation triggers pyroptosis, forming pores on the cell membrane surface and releasing LDH. The effect of IALN on LDH release after NLRP3 activation was investigated using an LDH detection kit. The results showed that IALN dose-dependently inhibited LDH release in THP-1 cells and BMDMs. Figure 5 E and F).

[0092] These results indicate that IALN can inhibit pyroptosis induced by NLRP3 activation.

[0093] 3. IALN inhibits the assembly process of NLRP3 inflammasomes.

[0094] Activation of the NLRP3 inflammasome leads to the self-oligomerization of ASCs through their PYD domains, forming ASC spots, a key upstream event of caspase-1 activation. To further understand the regulation of the NLRP3 inflammasome by IALN, cell lysates were chemically cross-linked with bis(succinimide) octanoate, and ASC oligomerization in cells was detected by Western blot. The results showed that LPS and nigericin-induced NLRP3 activation promoted the formation of ASC polymers, while the addition of IALN dose-dependently inhibited ASC oligomerization in THP-1 cells, and no ASC oligomerization event was detected in NLRP3-KO THP-1 cells. Figure 6 A). The formation of ASC spots was further evaluated by immunofluorescence. Consistent with Western blot results, IALN treatment dose-dependently inhibited the formation of ASC spots, while no ASC spot formation was observed in NLRP3 KO THP-1 cells. Figure 6 B). These results indicate that IALN inhibits ASC oligomerization during NLRP3 inflammasome activation and suggest that IALN may function upstream of ASC aggregation by acting on the NLRP3 protein.

[0095] Under normal physiological conditions, NLRP3 protein, through LRR-LRR interactions, forms a double-ring cage-like structure between the NACHT and LRR domains, encapsulating the PYD domain within it, thus maintaining a self-inhibited state and preventing abnormal activation. When stimulated by PAMPs or DAMPs, NLRP3 protein binds to NEK7, opening the self-inhibitory structure and isomorphically recruiting ASC adaptor proteins through its PYD domain for further assembly into inflammasomes. Therefore, the effect of IALN on the upstream of ASC oligomerization: the interaction between NLRP3 and NEK7 or ASC. Immunoprecipitation results showed that co-stimulation with LPS and nigericin promoted the interaction between NLRP3-NEK7 and NLRP3-ASC, while IALN treatment significantly inhibited the interaction between NLRP3 and NEK7. Figure 6 C) and NLRP3 and ASC ( Figure 6 The interaction between D).

[0096] These results indicate that IALN inhibits the assembly process of the NLRP3 inflammasome, revealing that IALN may directly target NLRP3.

[0097] 4. IALN can covalently bind to cysteine ​​residue 279 of the NACHT domain of the NLRP3 protein.

[0098] Given that small molecules can increase protein stability by forming ligand-protein complexes, two experiments, DARTS and thermal shift, were used to further verify the endogenous interaction between IALN and NLRP3. The DARTS results showed that NLRP3 protein was degraded after the addition of protease to THP-1 cell lysate; while after IALN treatment, the resistance of NLRP3 protein to protease hydrolysis increased with increasing concentration. Figure 7 A). Thermal displacement experiments showed that NLRP3 protein degraded with increasing temperature; the addition of IALN increased the stability of NLRP3 protein and improved its temperature sensitivity. Figure 7B). The NLRP3 protein contains three domains: the PYD domain, the NACHT domain, and the LRR domain. The main function of the PYD domain is to promote NLRP3 inflammasome assembly through the PYD / PYD interaction between ASC and NLRP3. ATP hydrolysis of the NACHT domain regulates the assembly of the inflammasome complex required for NLRP3 self-oligomerization and caspase-1 and IL-1β activation. However, it has been reported that truncated NLRP3 proteins lacking the LRR domain are still fully activated by some typical NLRP3 inflammasome triggers (such as nigericin, ATP, SiO2, etc.). To further illustrate the direct interaction between IALN and NLRP3, purified NLRP3ΔLRR protein was used for DARTS and thermal displacement experiments. Consistent with previous results, the addition of IALN significantly increased the anti-protease hydrolysis ability of the NLRP3ΔLRR protein. Figure 7 C) and resistance to high-temperature degradation ( Figure 7 D). To determine which domain of the NLRP3ΔLRR protein IALN acts on, the binding site of IALN in the NLRP3ΔLRR protein was further investigated using purified PYD and NACHT domains. In DARTS experiments, the PYD and NACHT domains were examined at different protease concentrations and different compound concentrations. The results showed that, regardless of the conditions, IALN enhanced the binding of the NACHT protein (…). Figure 7 E) instead of PYD protein ( Figure 7 F) Resistance to protease hydrolysis. This indicates that IALN functions by binding to the NACHT domain of NLRP3.

[0099] The α,β-unsaturated carbonyl group in the IALN structure acts as an acceptor for the Michael addition reaction, covalently binding to various amino acids in proteins, making it a crucial group for the biological activity of compounds. Tubocapsanolide A, a solanine lactone, has been reported as an irreversible covalent inhibitor of the NLRP3 inflammasome. Its α,β-unsaturated carbonyl group forms a covalent bond with the Cys514 of the NLRP3 NACHT domain, blocking the interaction between NLRP3 and NEK7 and ASC. Inspired by this, we believe that IALN may covalently bind to the NLRP3 NACHT domain. First, to confirm the nature of IALN's action, we investigated the reversibility of its inhibition of NLRP3 inflammasome activation. In THP-1 cells, after LPS induction for 3 hours, IALN was added and treated for 40 minutes. Cells were then washed three times with PBS or not washed, followed by stimulation with nigericin for 40 minutes. The cell supernatant was collected for ELISA analysis. The results showed that regardless of washing, IALN could inhibit the production of IL-1β after NLRP3 inflammasome activation in a concentration-dependent manner. Figure 7 G); This indicates that the effect of IALN is irreversible, suggesting that IALN may covalently bind to the NLRP3NACHT domain. To further confirm the covalent binding of IALN to the NLRP3NACHT domain and identify its binding site, IALN and NACHT protein were co-incubated and then subjected to shotgun proteomics analysis. The results showed that IALN binds to cysteine ​​residue 279 in the NLRP3NACHT domain. Figure 7 H).

[0100] To verify that IALN functions by binding to cysteine ​​at position 279 of the NLRP3 NACHT domain, a plasmid was constructed with cysteine ​​at position 279 of the NLRP3 protein mutated to alanine. DARTS experiments were performed using lysates of 293T cells overexpressing HA-NLRP3 or HA-NLRP3C279A protein and IALN. The results showed that IALN lost its protective effect against HA-NLRP3C279A protein. Figure 7 I). Furthermore, exogenous immunoprecipitation results showed that IALN inhibited the interaction between HA-NLRP3 and FLAG-NEK7 in 293T cells, but did not affect the interaction between HA-NLRP3 C279A and FLAG-NEK7 (I). Figure 7 J).

[0101] Next, molecular docking was used to simulate the binding of IALN to its interaction site. The results showed that the α,β-unsaturated carbonyl group of IALN formed a covalent bond with cysteine ​​residue at position 279. Figure 7 K).

[0102] Based on the above results, it can be concluded that IALN is covalently bound to cysteine ​​at position 279 in the NLRP3 NACHT domain via an α,β-unsaturated carbonyl group.

[0103] 5. IALN treatment can alleviate DSS-induced ulcerative colitis in mice.

[0104] In vitro cell experiments have demonstrated that IALN effectively inhibits NLRP3 inflammasome activation, suggesting that IALN can protect the body's immune system from damage caused by excessive inflammatory activation. Further investigation was conducted into the effects of IALN in a DSS-induced mouse model of ulcerative colitis.

[0105] DSS-induced colitis is a classic mouse model of human inflammatory bowel disease, and its pathogenesis is related to the overactivation of the NLRP3 inflammasome. Mice were first fed drinking water containing 3.25% DSS for 6 days to induce colitis. Then, they were intraperitoneally injected daily with 15 mg / kg or 30 mg / kg of IALN or 30 mg / kg of the positive control isotretinoin. The treatment effect was assessed by daily monitoring of weight changes and disease activity index. On day 11, the colon and cecum of the mice were removed, colon length was measured, and subsequent analysis was performed. IALN significantly alleviated the weight loss and disease activity index in mice induced by colitis. Figure 8 A and B); significantly improved the pathological feature of colon shortening in mice ( Figure 8 C). Histological analysis of mouse colons showed that the structural damage to colonic epithelial cells, loss of goblet cells, and inflammatory cell infiltration caused by colitis in mice were improved after administration of IALN. Figure 8 D). ELISA results showed that IALN reduced the levels of IL-1β and TNF-α in mouse colon tissue. Figure 8 E and F). Western blot results showed that IALN significantly inhibited the maturation of caspase-1 in mouse colon tissue (E and F). Figure 8 G). The positive control, isothomyl lactone, also showed some improvement; however, the effect was weaker compared with the same dose of IALN.

[0106] The above results demonstrate the good therapeutic effect of IALN in DSS-induced ulcerative colitis in mice.

[0107] In summary, this study found that IALN targets NLRP3, thereby inhibiting the activation of the NLRP3 inflammasome. Further investigation into the mechanism by which IALN exerts its NLRP3 inhibitory activity revealed that IALN can inhibit the assembly process of the NLRP3 inflammasome. Furthermore, it was found that IALN directly targets the NLRP3 protein and exerts its anti-inflammatory effect by covalently binding to cysteine ​​residue 279 of its NACHT domain. Additionally, in a DSS-induced colitis model, the NLRP3-related inflammatory cytokine caspase-1 was highly expressed; caspase-1 expression decreased after IALN treatment, indicating that IALN could be a potential drug candidate for diseases related to abnormal NLRP3 inflammasome activation.

[0108] As can be seen from the above embodiments, the present invention provides a novel compound IALN, which can be used as an NLRP3 inhibitor. It inhibits the activation of the NLRP3 inflammasome by directly covalently binding to cysteine ​​at position 279 in the NACHT domain of NLRP3, thereby achieving an anti-inflammatory effect. It has a significant therapeutic effect on DSS-induced ulcerative colitis and provides a new reference for expanding the screening and development of anti-inflammatory drugs.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof: Formula I.

2. A method for preparing the compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: Step 1: Oxidize isocarboxylide to obtain intermediate A; Step 2: React the intermediate A with the raw material B to obtain the compound shown in Formula I.

3. The preparation method according to claim 2, characterized in that: In step 1, the oxidant is selected from SeO2 and TBHP; the solvent is selected from DCM; the reaction temperature is 0-30 ºC; and the reaction time is 6-12 h.

4. The preparation method according to claim 2, characterized in that: In step 2, the reaction is carried out under the action of a condensing agent selected from DCC; the reaction is carried out under the action of a catalyst selected from DMAP; the solvent is selected from DCM; the reaction temperature is 20-30 ºC; and the reaction time is 12-18 h.

5. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug.

6. The use according to claim 5, characterized in that: The drug is used to treat acute inflammatory diseases.

7. The use according to claim 5, characterized in that: The drug is used to treat ulcerative colitis.

8. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an NLRP3 inhibitor.

9. The use according to claim 8, characterized in that: The NLRP3 inhibitor is used to inhibit NLRP3 inflammasome activation.

10. The use according to claim 8, characterized in that: The NLRP3 inhibitor is used to inhibit pyroptosis caused by NLRP3 inflammasome activation.

11. The use according to claim 8, characterized in that: The NLRP3 inhibitor is used to inhibit NLRP3 inflammasome assembly.

12. The use according to claim 8, characterized in that: The NLRP3 inhibitor is used to covalently bind to cysteine ​​at position 279 of the NACHT domain of the NLRP3 protein.

13. An anti-inflammatory drug or NLRP3 inhibitor, characterized in that: It is prepared by adding pharmaceutically acceptable excipients to the compound of claim 1 or its pharmaceutically acceptable salt as the active ingredient.

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