CN118146202A

CN118146202A - Isoalantolactone derivative and preparation method and application thereof

Info

Publication number: CN118146202A
Application number: CN202410174166.9A
Authority: CN
Inventors: 叶能; 赵旻
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2024-02-07
Filing date: 2024-02-07
Publication date: 2024-06-07

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an isoalantolactone derivative, a preparation method and application thereof. The invention provides a novel compound IALN, the structural formula of which is shown in a formula I. The compound can be used as an NLRP3 inhibitor, and can realize anti-inflammatory effect by directly interacting with NLRP3, has remarkable treatment effect on acute inflammatory diseases, namely ulcerative colitis, and provides a new way for widening the screening and research of anti-inflammatory drugs.

Description

Isoalantolactone derivative and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an isoalantolactone derivative, a preparation method and application thereof.

Background

NLRP3 inflammatory corpuscles are a vital part of the innate immune system. It has important functions of helping organism sense danger and quickly respond, and maintaining physiological balance of organism. But its abnormal activation may lead to various inflammation-related diseases such as cold-imidazoline-related periodic syndrome, inflammatory bowel disease, atherosclerosis, type ii diabetes, neurodegenerative diseases, etc. The current medicines for treating NLRP3 inflammation small-scale related diseases are mainly IL-1 beta receptor antagonists, but besides NLRP3, IL-1 beta can be produced by other ways, so that adverse reactions such as excessive immunosuppression and the like can be caused by using the IL-1 beta receptor antagonists.

A number of small molecule inhibitors targeting NLRP3 and exhibiting good therapeutic potential for their related inflammatory diseases have been reported, a few have entered clinical studies, but whether these inhibitors can ultimately be approved by drug administration for clinical treatment of NLRP3 inflammatory small-body related diseases remains to be further investigated. Searching for inhibitors which are better in activity and higher in safety and can be applied to clinic is a target of wide scientific researchers at present.

Among these reported small molecule inhibitors, the discovery of natural products such as oridonin, wogonin, erianin, parthenolide, cardamomin and the like provides a new idea for the development of NLRP3 inhibitors. Natural products have been an important source of new drug development candidates. Currently 60% of the world's developed drugs are derived from natural product active ingredients and analogues thereof. Inula racemosa hook. F. is a perennial herb belonging to Inula genus of Compositae family, and its dried root has effects of invigorating spleen, regulating stomach, regulating qi, and resolving stagnation. Isoalantolactone is one of the main components of dry roots of inula racemosa, and has been reported to have good anti-inflammatory effects by several documents, and its structural formula is as follows:

In order to meet the requirements of clinical applications, it is necessary to further enhance the anti-inflammatory effect of isoalantolactone. Therefore, the detection of a novel NLRP3 inflammatory corpuscle regulatory factor has important significance by taking isoalantolactone as a probe. However, during derivatization of isoalantolactone, derivatives with different substituents were found to exhibit a large difference in anti-inflammatory activity. Therefore, how to design the molecular structure and obtain isoalantolactone derivatives having excellent anti-inflammatory activity is an important subject in the art.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an isoalantolactone derivative, and a preparation method and application thereof.

A compound of formula I, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterate thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a co-crystal thereof:

The invention also provides a preparation method of the compound, or a stereoisomer, or a solvate, or a metabolite, or a deuterated product, or a prodrug, or a pharmaceutically acceptable salt or a eutectic crystal thereof, which comprises the following steps:

Step 1, oxidizing isoalantolactone to obtain an intermediate A;

And 2, reacting the intermediate A with a raw material B to obtain a compound shown in a formula I.

Preferably, in step 1, the oxidizing agent is selected from the group consisting of SeO ₂ and TBHP; the solvent is selected from DCM; the reaction temperature is 0-30 ℃; the reaction time is 6-12h.

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 ℃; the reaction time is 12-18h.

The invention also provides application of the compound, or a stereoisomer, or a solvate, or a metabolite, or a deuterated product, or a prodrug, or a pharmaceutically acceptable salt or a eutectic crystal thereof in preparing anti-inflammatory drugs.

Preferably, the medicament is for the treatment of acute inflammatory diseases.

Preferably, the medicament is for the treatment of ulcerative colitis.

8. Use of a compound according to any one of claims 1-3, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterate thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a co-crystal thereof, in the preparation of an NLRP3 inhibitor.

Preferably, the NLRP3 inhibitor is used to inhibit NLRP3 inflammatory body activation;

and/or, the NLRP3 inhibitor is used for inhibiting cell apoptosis caused by activation of NLRP3 inflammatory bodies;

And/or, the NLRP3 inhibitor is used to inhibit NLRP3 inflammatory small body assembly;

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

The invention also provides an anti-inflammatory drug or NLRP3 inhibitor, which is prepared by taking the compound, or a stereoisomer, or a solvate, or a metabolite, or a deuterated product, or a prodrug, or a pharmaceutically acceptable salt or a eutectic crystal thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

The invention optimizes the structure of isoalantolactone, provides a novel tricyclic sesquiterpene lactone-6-nicotinic acid ester compound IALN which can be used as an NLRP3 inhibitor, realizes anti-inflammatory effect by directly interacting with NLRP3, has obvious treatment effect on acute inflammatory diseases, namely ulcerative colitis, and provides a novel approach for widening screening and research and development of anti-inflammatory drugs.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a ¹ H spectrum of the product IALN prepared in example 1.

FIG. 2 is a ¹³ C spectrum of the product IALN prepared in example 1.

FIG. 3 is a high resolution mass spectrum (HRESIMS) spectrum of the product IALN prepared in example 1.

Fig. 4 is IALN inhibiting NLRP3 inflammatory body activation in a variety of macrophages. (A-C) LPS-induced THP-1 (A), BMDM (B), PBMC (C) cells were treated with IALN for 40min and then stimulated with 10 μ M NIGERICIN for 40min. The supernatant was analyzed for IL-1β release by ELISA. (D-F) IALN was incubated with THP-1 (D), BMDM (E), PBMC (F) cells for 24h, and cell viability was measured by CCK-8 method. (G, H) expression of NLRP 3-related proteins in THP-1 and BMDM cell supernatants (Sup.) and cell extracts (Lys.) was analyzed by immunoblotting. (I, J) LPS-induced THP-1 (I) and BMDM (J) cells were treated with IALN or MCC950 for 40min and then stimulated with 10 μ M NIGERICIN for 40min. The supernatant was analyzed for IL-18 release by ELISA. Statistical differences were calculated using one-way anova, compared to LPS alone groups: # # P <0.0001, compared to nigericin stimulation group: * P <0.0001, P <0.001, P <0.01, P <0.05, ns represent no significant difference.

FIG. 5 is IALN inhibiting apoptosis caused by NLRP3 activation in macrophages. (A, B) THP-1 cells (A) and BMDMs (B) were treated with LPS for 3h, treated with MCC950 or IALN at different concentrations for 40min, and stimulated with nigericin for 40min. GSDMD-NT in cells was analyzed by immunoblotting. (C, D) THP-1 cells (C) and BMDMs (D) were treated with LPS for 3h, and then treated with MCC950 or IALN at different concentrations for 40min and then stimulated with nigericin for 40min. After the treatment, the cells were stained with propidium iodide (5. Mu.g/mL) for 20 minutes at room temperature and the images were collected using a Nikon ECLIPSE TS R inverted microscope. (E, F) THP-1 cells (E) and BMDMs (F) were treated with LPS for 3h, treated with MCC950 or IALN at different concentrations for 40min and stimulated with nigericin for 40min. Cell supernatants were collected and assayed for LDH content. Statistical differences were calculated using one-way anova, compared to LPS alone groups: # # P <0.0001, # P <0.001, compared to nigericin stimulation group: * P <0.0001, P <0.001, P <0.01, P <0.05.

Fig. 6 is a IALN assembly process to inhibit NLRP3 inflammatory bodies. (A) THP-1 cells and NLRP3 KO THP-1 cells were treated with LPS for 3h, and then treated with IALN at different concentrations for 40min and then stimulated with nigericin for 40min. Effect of immunoblot analysis IALN on ASC oligomerization in cells. (B) The effect of IALN on ASC spots was observed by immunofluorescence microscopy. (C, D) THP-1 cells were treated with LPS for 3h, then IALN (1. Mu.M) was added for 40min, and then nigericin was added for stimulation for 40min. The interaction of NLRP3-NEK7 (C) and NLRP3-ASC (D) was analyzed by co-immunoprecipitation.

FIG. 7 is a diagram showing IALN covalent binding to cysteine 279 of the NACHT domain of NLRP3 protein. (A, B) DARTS (A) and CETSA (B) assays were performed using THP-1 cell lysates. (C, D) DARTS (C) and CETSA (D) assays were performed using purified NLRP 3. DELTA. LRR protein. (E, F) DARTS analysis was performed using purified NLRP3 NACHT protein (E) and PYD protein (F). (G) THP-1 cells were treated with LPS for 3h, then incubated with IALN at various concentrations for 40min, and cells were washed three times with PBS or without washing, and stimulated with nigericin for 40min. ELISA detects secretion of IL-1. Beta. In cell supernatants. Shotgun mass spectra of (H) IALN and NACHT proteins. (I) HEK-293T cells were transfected with HA-NLRP3 and HA-NLRP 3C 279A plasmids for 24h and DARTS analysis was performed with IALN. (J) HEK-293T cells were co-transfected with HA-NLRP3 and FLAG-NEK7 plasmid or HA-NLRP 3C 279A and FLAG-NEK7 plasmid and co-incubated for 24h with IALN (1. Mu.M) for immunoprecipitation analysis. Molecular docking simulation of (K) IALN and NACHT proteins. Statistical differences were calculated using one-way anova, compared to LPS alone groups: # # P <0.0001, compared to nigericin stimulation group: * P <0.0001, P <0.01, ns represents no significant difference.

Figure 8 is IALN effective in inhibiting DSS-induced ulcerative colitis. Percent change in body weight of mice. (B) disease Activity index. (C) colon length. (D) colon tissue HE staining. (E-F) production of IL-1. Beta. -E) and TNF-alpha. -F in DSS-induced colon tissues of mice was examined by ELISA. (G) Caspase-1 expression was examined by western blotting in colon tissue. Statistical differences were calculated using one-way anova, compared to normal group: # # # p <0.0001, compared to DSS group: * P <0.0001, P <0.001, P <0.01, P <0.05.

Detailed Description

In the following examples, reagents and materials not specifically described are commercially available.

Example 1 Compound IALN and method for preparing same

This example provides compound IALN having the structural formula:

The preparation method comprises the following steps:

Into a 500mL eggplant-shaped bottle were charged 416mg (3.75 mmol) of selenium dioxide and 1.35g (15 mmol) of t-butyl hydroperoxide, and 40mL of methylene chloride was added to activate at 0℃for 30min. Then 5.8g (25 mmol) of isoalantolactone is dissolved in methylene dichloride, and the mixture of activated selenium dioxide and tert-butyl hydroperoxide is added dropwise at 0 ℃, and the mixture is transferred to normal temperature for reaction for 18h. TLC monitoring, adding saturated Na ₂S₂O₃ solution after the reaction is completed to quench the reaction, placing the reaction solution in a separating funnel, standing and layering, collecting the lower reaction solution, and neutralizing the residual Na ₂S₂O₃ in the reaction solution by using saturated NaHCO ₃ solution. The solvent in the reaction solution was then spin-dried. The Flash column is used for separation and purification to obtain the off-white solid product A with the yield of about 66 percent.

Then 100mg (0.4 mmol) of intermediate A,230mg (1.2 mmol) of raw material B and 20mg (0.16 mmol) of DMAP were taken and put into a 25mL eggplant-shaped bottle, 10mL of methylene chloride was used as a solvent, and 198mg (0.96 mmol) of DDC was then added thereto, and the reaction was stirred at room temperature overnight. TLC monitoring, spin-drying the solvent after the reaction is finished, and separating and purifying by a Flash fast column to obtain a white solid product. The yield was 48%.

The nuclear magnetic resonance hydrogen spectrum and carbon spectrum data of the product are shown in fig. 1 and 2, and the specific data are as follows:

(3aR,4aR,6R,8aR,9aR)-8a-methyl-3,5-dimethylene-2-oxododecahydronapht ho[2,3-b]furan-6-yl 4,6-dichloronicotinate(49).White solid,48％yield.C₂₁H₂₁Cl₂NO₄.HRESIMS:m/z 444.0743[M+Na]⁺(calcd.for C₂₁H₂₁Cl₂NO₄Na,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(td,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.

Mass spectrum data of the product are shown in fig. 3, and specific results are as follows:

Sample No.	Formula(M)	Ion Formula	Measured m/z	Calc m/z	Diff(ppm)
						IALN	C₂₁H₂₁Cl₂NO₄	C₂₁H₂₁Cl₂NO₄Na	444.0743	444.0745	-0.45

EXAMPLE 2 Activity Studies of Compound IALN

1. Experimental method

Compound IALN in this example was prepared according to the procedure of example 1.

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

THP-1 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Differentiated THP-1 cells were obtained by co-culture with 100ng/mL PMA for 24 h. BMDMs from C57BL/6 mice femur and tibia, cultured in DMEM medium supplemented with 10% fetal calf serum, and 30% L929 culture supernatant was added. PBMCs were isolated from venous blood contributed by healthy volunteers and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Induction of activation of NLRP3 inflammatory bodies: PMA induced differentiation THP-1 cells, BMDMs and PBMCs were seeded at a density of 2X 10 ⁵/mL in appropriate well plates. The following day, the medium was changed to Opti-MEM with 1. Mu.g/mL LPS to stimulate cells for 3 hours, followed by treatment of cells with IALN at different concentrations for 40 minutes and finally stimulation of cells with 10. Mu. M NIGERICIN for 40 minutes.

(2) Detection of cytokines and LDH secretion

After the cells have been stimulated, the cell supernatants are collected and tested for IL-1. Beta. Or IL-18 concentration using ELISA kits or by Cytotox according to manufacturer's instructionsThe non-radioactive cytotoxicity detection kit detects release of LDH.

(3) Cell viability

Viability of PMA-differentiated THP-1 cells, BMDMs and PBMCs was assessed by using the CCK-8 assay. Briefly, cells (2X 10 ⁵/mL) were seeded into 96-well plates and incubated with IALN for 24 hours the next day. Then, 10. Mu.L of CCK-8 reagent was added to each well, and absorbance was measured at 450nm using a microplate reader after incubation for a certain period of time.

(4) PI staining

After the cells were stimulated, the cell supernatant was removed, the cells were gently washed three times with pre-chilled PBS, 200. Mu.L of 5. Mu.g/mL Propidium Iodide (PI) was added to each well, and incubated at room temperature for 10min. After staining, cells were gently washed three times with pre-chilled PBS and then photographed under a microscope.

(5) Western immunoblotting

Cells were lysed in RIPA lysis buffer, then loading buffer was added and mixed well and denatured by heating at 100 ℃. Proteins were separated by SDS-PAGE and then transferred onto PVDF membrane. Blocking with 5% skim milk, incubating overnight with primary antibody at 4deg.C, incubating at room temperature the next day with appropriate secondary antibody, and developing with developer in an imager.

(6) Immunofluorescence

After the cells were stimulated, the supernatant was discarded, the cells were washed with pre-chilled PBS, fixed with ice-cold methanol at-20℃for 20min, PBST rinsed for 5min, and blocked with 0.5% bovine serum albumin (w/v, PBST dilution) for 1h. Followed by incubation with antibody overnight. The nuclei were then stained with 4',6' -diamino-2-phenylindole (DAPI) by a PBST rinse, followed by 1:500 secondary antibody treatment with 0.5% bovine serum albumin for 1h. Then the mixture was photographed under a fluorescence microscope.

(7) ASC oligomerization

Cells were lysed with 300. Mu.L of NP-40 lysis buffer, which was lysed on a shaker at 4℃for 30min, centrifuged at 6000 Xg for 15min at 4 ℃. After removal of the supernatant, insoluble cell debris was incubated in 2mM DSS at 37℃for 30min, centrifuged at 4℃and 6000 Xg for 15min, and the pellet was resuspended in 30. Mu.L of 1 Xloading buffer and heated for immunoblotting.

(8) Co-immunoprecipitation

For endogenous IP detection, the supernatant was removed after the cells were stimulated, THP-1 cells were washed three times with PBS, and then cells were lysed with NP-40 for 30 minutes. Cell lysates were incubated with primary antibodies overnight at 4 ℃, protein a/G magnetic beads were added to the cell lysates and incubated with rotation at 4 ℃ for 4 hours. The beads were washed several times and immunoblotted after heating in a1×loading buffer.

For exogenous IP detection, HEK-293T cells were seeded overnight in 6-well plates, followed by plasmid transfection using polyethylenimine and co-incubation with IALN (1. Mu.M) added. After 24 hours, cells were collected and lysed with NP-40 lysis buffer. Primary antibody was added and incubated overnight at 4 ℃, protein a/G magnetic beads were added to the cell lysate and incubated at 4 ℃ with rotation for 4 hours. The beads were washed several times and heated in a1×loading buffer before immunoblotting detection.

(9) Drug Affinity Reaction Target Stability (DARTS)

PMA-differentiated THP-1 cells were plated in 10cm dishes at a density of 5X 10 ⁵/mL. The following day, the medium was removed and replaced with Opti-MEM containing LPS for 3 hours. Or HEK-293T cells were seeded in 6-well plates at a density of 5X 10 ⁵/mL and transfected with plasmid for 24 hours.

Cell supernatants were removed, cells were washed with PBS, and then lysed with NP-40 for 30 min. The lysate was centrifuged at 17000g for 15min at 4 ℃. After centrifugation, 1. Mu. L IALN or DMSO and 99. Mu.L of supernatant or purified protein were incubated at room temperature. After 50 minutes, pronase was added to the above mixture at room temperature, and the mixture was incubated at room temperature. After 30 minutes the reaction was stopped by adding the protease inhibitor cocktail. And adding a loading buffer for heating, and performing immunoblotting detection.

(10) Thermal displacement analysis

PMA-differentiated THP-1 cells were plated at a density of 5X 10 ⁵/mL in whole medium in a 10cm dish. The following day, the medium was removed and replaced with Opti-MEM containing LPS for 3 hours. Cells were then washed with PBS, scraped with a cell scraper, lysed by three rounds of freezing and thawing with liquid nitrogen and room temperature water, and centrifuged at 17000g for 15min at 4 ℃. Cell lysates or purified NLRP3 Δlrr proteins were split into two groups in PCR tubes. One group was mixed with 1 μ L IALN and the other group was mixed with DMSO as a negative control. The two groups of proteins were heated to 45 to 63 or 78 ℃ under the same conditions using a thermal cycler. Each sample was heated at a single temperature for 2 minutes. And adding a loading buffer for heating, and performing immunoblotting detection.

(11) Mass spectrometry identification of protein modification sites

Recombinant NACHT protein was incubated with IALN for 12 hours, and the mixture was SDS-PAGE and stained with Coomassie blue. The band corresponding to NACHT was excised and digested with trypsin in the gel. The tryptic peptides were separated on a C ₁₈ column and analyzed using a Obitrap Fusion Lumos mass spectrometer. Mass spectral data were identified and quantitatively analyzed by software pFind.2.1.

(12) Molecular docking

By using2018-1 Software molecular covalent docking IALN with NLRP3 NACHT protein mode of action. The appropriate crystal structure (PDB ID:7 ALV) was selected in the RCSB PDB database (PDB, http:// www.rcsb.org /), and protein preparation, including amino acid deletion residue supplementation and protein optimization, was performed in docking software. Ligand preparation was performed on IALN. The covalent docking module was used to select amino acid residue (position 279) as the active site and this was used as the docking box center, and the reaction type was Michael addition.

(13) Animal experiment

8 Week old male C57BL/6 mice (20-22 g), available from Beijing Fukang Biotechnology Co., ltd. All mice were housed in a university of Sichuan laboratory animal center-specific pathogen-free animal facility and animal experiments were performed according to animal experimental protocols approved by the ethical committee for animal experiments in the national emphasis laboratory for animal experiments for biological treatment of Sichuan university.

Mice were fed with 3.25% (w/v) DSS in drinking water, and were free to drink, inducing experimental colitis. Mice were randomly divided into 4 experimental groups (n=6). Untreated groups, mice drink normal water; model group, mice drink DSS water and change into normal drinking water after 6 days; IALN groups, mice were challenged with DSS water, 6 days later with normal drinking water and daily intraperitoneal injections of IALN (15 mg/kg) or (30 mg/kg); IAL group, mice were given DSS water, 6 days later changed to normal drinking water and daily intraperitoneal injection of IAL (30 mg/kg). From the day of modeling, mice were observed daily for feeding, activity, hair, animal body weight was weighed, mice fecal trait and fecal occult blood, bleeding status were observed, colitis severity was assessed, and quantification was performed using DAI (disease activity index) scores based on three parameters, body weight, rectal bleeding and fecal consistency. Mice were sacrificed after the end of the experiment and the colon of the mice was collected for determining the colon length. A portion of the colon was frozen directly for subsequent experimental analysis and a portion of the mouse colon was fixed with 4% paraformaldehyde for H & E staining.

2. Experimental results

1. IALN inhibit NLRP3 inflammatory platelet activation in macrophages

To preliminary investigate whether IALN inhibited NLRP3 activation in vitro, the effect of IALN on the secretion of inflammatory factor IL-1 beta after NLRP3 activation was tested using PMA-induced differentiated THP-1 cells, BMDMs and PBMCs. Cells were first primed with LPS for 3h, then treated with IALN for 40min, and finally stimulated with NLRP3 inflammatory ponema stimulator nigericin for 40 min. IALN inhibited the release of IL-1β in THP-1 cells, BMDMs and PBMCs in a dose-dependent manner, with half-maximal inhibitory concentrations (IC ₅₀) of 293.4nM, 738.8nM and 44.97nM, respectively (FIGS. 4A-C).

To ensure that IALN inhibition of IL-1β was not caused by cell killing, the cytotoxic effects of IALN in three cells, THP-1, BMDMs and PBMCs were subsequently examined using the CCK-8 method. The results indicate that the concentration of effect used in this study by IALN did not cause significant cytotoxicity, indicating that the inhibition of IL-1β by IALN in this study was not caused by cytotoxicity (FIGS. 4D-F).

During NLRP3 inflammatory body activation pro-caspase-1 is cleaved to mature caspase-1, and then caspase-1 cleaves inactive pro-IL-1β to active mature form IL-1β and releases it extracellular. To further confirm IALN's inhibition of NLRP3 inflammatory minibodies, the effects on NLRP3 inflammatory minibody-related components, IL-1β, caspase-1, pro-IL-1β, pro-caspase-1, NLRP3 and ASC expression were examined by western blot. The results showed that IALN dose-dependently inhibited the expression of IL-1β, caspase-1 in THP-1 cells and BMDMs supernatant, and had no effect on the expression of intracellular NLRP3 inflammatory corpuscle components (FIGS. 4G and H).

In addition to IL-1. Beta. And caspase-1, IL-18 is also considered one of the major markers of NLRP3 inflammatory body activation. Secretion of IL-18 was also examined in THP-1 cells and BMDMs supernatant. The results showed IALN dose-dependently inhibited secretion of IL-18 in cell supernatants (FIGS. 4I and J).

The above results demonstrate that IALN can inhibit maturation and secretion of IL-1β, IL-18 and caspase-1 by NLRP3 inflammatory body activation in macrophages.

2. IALN inhibit apoptosis caused by activation of NLRP3 inflammatory corpuscle in macrophage

Activation of NLRP3 inflammatory bodies results in activation of caspase-1, which cleaves the GSDMD N-end (GSDMD-NT) that binds to phosphoinositide and cardiolipin, disrupting cell membranes resulting in pore formation, and thus causing cell scorch and LDH release. The effect of IALN on cleavage of GSDMD after activation of NLRP3 inflammatory corpuscles was examined by western blot. The results showed that IALN dose-dependently inhibited production of GSDMD-NT in THP-1 cells and BMDMs, suggesting that IALN could inhibit GSDMD-induced apoptosis (FIGS. 5A and B).

PI is a red fluorescent dye that, due to its impermeability to living cell membranes, can penetrate through pyro-apoptotic cells to stain DNA and RNA and exhibit red fluorescence, whereas living cells are not stained. Experimental results found that IALN inhibited the production of red fluorescence in THP-1 cells and BMDMs after NLRP3 activation in a dose dependent manner (FIGS. 5C and D).

After activation of NLRP3 inflammatory corpuscles, cell scorch is initiated, holes are formed on the surface of cell membranes, and LDH is released. The effect of IALN on cellular LDH release after NLRP3 activation was examined by LDH detection kit, and the results showed that IALN dose-dependent inhibition of LDH release in THP-1 cells and BMDMs (fig. 5E and F).

These results demonstrate that IALN can inhibit apoptosis caused by NLRP3 activation.

3. IALN inhibit NLRP3 inflammatory corpuscle Assembly Process

Activation of NLRP3 inflammatory bodies results in ASC formation of ASC spots by self-oligomerization of their PYD domains, an upstream key event in caspase-1 activation. In order to get a thorough understanding of IALN on the regulation of NLRP3 inflammatory corpuscles, after chemical crosslinking of cell lysates with disuccinimide suberate, ASC oligomerization in cells was detected by western blot; the results showed that LPS and nigericin induced NLRP3 activation promoted the formation of ASC multimers, and that addition IALN dose-dependently inhibited the oligomerization of ASC in THP-1 cells, whereas no ASC oligomerization event was detected in THP-1 cells of NLRP3 KO (FIG. 6A). The formation of ASC spots was further assessed by immunofluorescence. Consistent with the western blot results, the dose-dependence inhibited ASC-spot formation after IALN treatment, whereas no ASC-spot formation was observed in THP-1 cells of NLRP3 KO (fig. 6B). These results indicate that IALN inhibited ASC oligomerization during NLRP3 inflammatory body activation and suggested that IALN may act upstream of ASC aggregation by acting on NLRP3 protein.

Under normal physiological conditions, NLRP3 protein forms a double-ring cage structure with NACHT domain and LRR domain through LRR-LRR interaction, wraps PYD domain therein, and is in self-inhibition state, thereby avoiding abnormal activation. When stimulated by PAMPs or DAMPs, NLRP3 protein binds to NEK7, opening the self-inhibiting structure, through which PYD domain homotypic recruitment ASC adaptor proteins are further assembled into inflammatory bodies. Therefore, the effect of IALN on the interaction of NLRP3 with NEK7 or ASC upstream of ASC oligomerization was examined. Co-immunoprecipitation results showed that LPS and nigericin co-stimulation promoted interaction between NLRP3-NEK7 and NLRP3-ASC, while IALN significantly inhibited interaction between NLRP3 and NEK7 (fig. 6C) and between NLRP3 and ASC (fig. 6D) after the interaction.

These results demonstrate that IALN inhibits the assembly process of NLRP3 inflammatory bodies, revealing that IALN may target NLRP3 directly.

4. IALN can be covalently bound to cysteine 279 of the NACHT domain of NLRP3 protein

Whereas small molecules can increase protein stability by forming ligand-protein complexes, two experiments, DARTS and thermal displacement, were employed to further verify IALN and NLRP3 endogenous interactions. DARTS experiment results show that NLRP3 protein is degraded after protease is added into THP-1 cell lysate; after IALN, the ability of NLRP3 protein to resist proteolytic hydrolysis increases with increasing concentration of action (FIG. 7A). The thermal displacement experimental result shows that NLRP3 protein is degraded along with the temperature rise; after IALN addition, the stability of the NLRP3 protein was increased, increasing its sensitivity to temperature (FIG. 7B). NLRP3 protein contains three domains, PYD domain, NACHT domain and LRR domain. The primary function of the PYD domain is to promote NLRP3 inflammatory body assembly through PYD/PYD interactions between ASC and NLRP 3. ATP hydrolysis of the NACHT domain regulates NLRP3 self-oligomerization and assembly of the inflammation partner complex required for caspase-1 and IL-1. Beta. Activation. However, truncated NLRP3 proteins lacking the LRR domain are reported to remain fully activated by some typical NLRP3 inflammatory small triggers (e.g., nigericin, ATP, siO2, etc.). To further illustrate the direct interaction of IALN with NLRP3, DARTS experiments and thermal shift experiments were performed using purified NLRP3 Δlrr protein. Consistent with previous experimental results, the addition of IALN significantly increased the protease hydrolysis resistance (fig. 7C) and high temperature degradation resistance (fig. 7D) of the NLRP3 Δlrr protein. To determine which domain of the NLRP3 Δlrr protein IALN acts on, the binding site of IALN in the NLRP3 Δlrr protein was next further examined using the purified PYD domain and the NACHT domain. In DARTS experiments, PYD and NACHT domains were examined for different protease concentrations and different compound concentrations, respectively. The results show that IALN enhances the resistance of NACHT proteins (fig. 7E) to proteolytic hydrolysis, but not PYD proteins (fig. 7F), under whatever conditions. IALN were shown to function by binding to the NACHT domain of NLRP 3.

The alpha, beta-unsaturated carbonyl part contained in IALN structure is used as the receptor of Michael addition reaction, can be covalently bound with various amino acids of protein, and is a group which is critical to the biological activity of the compound. Withanolides Tubocapsanolide A have been reported as irreversible covalent inhibitors of NLRP3 inflammatory bodies, in which the α, β -unsaturated carbonyl moiety in the structure forms a covalent bond with Cys514 of the NLRP3NACHT domain, blocking the interaction of NLRP3 with NEK7, ASC. In light of this, it is believed that IALN might be covalently bound to the NLRP3NACHT domain. First, to confirm the nature of the action of IALN, the reversibility of its inhibition of NLRP3 inflammatory body activation was examined. In THP-1 cells, LPS was used for priming for 3h, IALN was added for 40min, then cells were washed 3 times with PBS or not, and nigericin was added for stimulation for 40min, and cell supernatants were collected for ELISA analysis. The results indicate that IALN, whether washed or not, inhibited IL-1β production following NLRP3 inflammatory body activation in a concentration-dependent manner (fig. 7G); the effect of IALN was irreversible, suggesting that IALN might be covalently bound to the NLRP3NACHT domain. To further determine IALN covalent binding to the NLRP3NACHT domain and to find its site of action, a shotgun proteomic analysis was performed after co-incubation of IALN with NACHT protein, which showed IALN binding to cysteine 279 in the NLRP3NACHT domain (FIG. 7H).

To verify that IALN functions by binding to cysteine 279 in the NLRP3 NACHT domain, a plasmid was constructed that mutated the cysteine 279 in the NLRP3 protein to alanine. DARTS experiments were performed using 293T cell lysates and IALN that overexpressed the HA-NLRP3 or HA-NLRP 3C 279A protein. The results showed that IALN lost the protective effect on the HA-NLRP 3C 279A protein (fig. 7I). In addition, exogenous co-immunoprecipitation results showed that IALN inhibited the interaction of HA-NLRP3 and FLAG-NEK7 in 293T cells, but did not affect the interaction of HA-NLRP 3C 279A and FLAG-NEK7 (FIG. 7J).

Next, the binding of IALN to its site of action was simulated using molecular docking, which indicated that the α, β -unsaturated carbonyl group of IALN forms a covalent bond with cysteine 279 (fig. 7K).

From the above results, it was concluded that IALN was covalently bound to cysteine 279 in the NLRP3 NACHT domain through an alpha, beta-unsaturated carbonyl group.

5. IALN treatment can alleviate mouse DSS-induced ulcerative colitis

In vitro cell experiment results prove that IALN can effectively inhibit NLRP3 inflammatory body activation, and the IALN can protect the immune system in the body from damage caused by excessive activation of inflammation. The effect of IALN in DSS-induced mouse ulcerative colitis model was further explored.

DSS-induced colitis is a classical mouse model of human inflammatory bowel disease, whose pathogenesis is associated with the over-activation of NLRP3 inflammatory bodies. Firstly, mice are fed with drinking water containing 3.25% DSS for 6 days to induce colonitis of the mice, then 15mg/kg or 30mg/kg of positive control isoalantolactone of IALN or 30mg/kg is injected intraperitoneally every day, and the treatment condition of the mice is evaluated by detecting the weight change and disease activity index of the mice every day; the colon and ceca of the mice were removed on day 11, the colon length was measured, and subsequent analyses were performed. IALN significantly alleviated the assessment of the index of weight loss and disease activity due to colitis in mice (figures 8A and B); the pathological features of shortening the colon of the mice were significantly improved (fig. 8C). The results of histological analysis of the colon of the mice showed that colonic epithelial cell structure damage, goblet cell loss and inflammatory cell infiltration caused by the colonitis of the mice were improved upon the administration IALN (fig. 8D). ELISA experiments showed that IALN reduced IL-1. Beta. And TNF-. Alpha.levels in colon tissue of mice (FIGS. 8E and F). Western blot results indicate that IALN significantly inhibited caspase-1 maturation in mouse colon tissue (FIG. 8G). The positive control isoalantolactone also shows a certain improvement effect; but less effective than IALN at the same dose.

The above results demonstrate a good therapeutic effect of IALN in DSS-induced ulcerative colitis in mice.

Overall, this example found IALN in the study that targets NLRP3 to inhibit activation of NLRP3 inflammatory bodies, and found by studying IALN as a mechanism of NLRP3 inhibitory activity: IALN can inhibit the assembly process of NLRP3 inflammatory corpuscles, and further, IALN is directly targeted to NLRP3 protein and plays an anti-inflammatory role by covalently binding to cysteine 279 of NACHT domain. In addition, in a DSS-induced colitis model, the expression of the NLRP 3-related inflammatory factor caspase-1 is high, and the expression of the caspase-1 is reduced after IALN treatment, which shows that IALN can be used as a potential candidate medicament for NLRP3 inflammatory small body abnormal activation related diseases.

According to the embodiment, the invention provides a novel compound IALN which can be used as an NLRP3 inhibitor, and can be directly and covalently combined with cysteine 279 in an NLRP3 NACHT structural domain to inhibit NLRP3 inflammatory body activation, so that the anti-inflammatory effect is realized, the anti-inflammatory compound has remarkable treatment effect on ulcerative colitis induced by DSS, and a novel reference is provided for widening screening research and development of anti-inflammatory drugs.

Claims

1. A compound of formula I, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterate thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a co-crystal thereof:

2. the method for preparing a compound of claim 1, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterate thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a co-crystal thereof, comprising the steps of:

Step 1, oxidizing isoalantolactone to obtain an intermediate A;

3. The method of manufacturing according to claim 2, wherein: in step 1, the oxidizing agent is selected from SeO ₂ and TBHP; the solvent is selected from DCM; the reaction temperature is 0-30 ℃; the reaction time is 6-12h.

4. The method of manufacturing according to claim 2, wherein: 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 ℃; the reaction time is 12-18h.

5. Use of a compound according to any one of claims 1-3, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterate thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a co-crystal thereof, in the manufacture of an anti-inflammatory medicament.

6. Use according to claim 5, characterized in that: the medicament is used for treating acute inflammatory diseases.

7. Use according to claim 6, characterized in that: the medicament is used for treating ulcerative colitis.

9. Use according to claim 7, characterized in that: the NLRP3 inhibitor is used for inhibiting NLRP3 inflammatory small body activation;

10. An anti-inflammatory agent or an NLRP3 inhibitor, characterized in that: a compound according to any one of claims 1 to 3, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterated product thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a co-crystal thereof as an active ingredient, and adding pharmaceutically acceptable excipients.