Preparation and use of an NLRP3 inflammasome inhibitor

Abstract

The present invention relates to the production of a polyketide compound with NLRP3 inflammasome inhibitory activity using the Antarctic fungus Pseudogymnoascus sp. HDN17-895 (Deposit Number: CCTCC M20211640), and also relates to the use of such a compound in preventing or treating NLRP3 inflammasome-related diseases. The structural formula is: #imgabs0#. The fermentation product containing such a compound can be obtained by fermenting and culturing the fungus Pseudogymnoascus sp. HDN17-895, and then separated and purified by methods such as normal phase silica gel column chromatography, Sephadex LH20 gel column chromatography, medium pressure MPLC, and semi-preparative HPLC. The present invention aims to provide a novel polyketide compound with NLRP3 inflammasome inhibitory activity from a secondary metabolite derived from the fungus, as well as a preparation method and use thereof.

Description

Technical Field

[0001] The present invention belongs to the field of natural products and medicinal chemistry, and specifically relates to the use of the fungus Pseudogymnoascus sp. HDN17-895 (CCTCC NO: M 20211640, preservation date: December 17, 2021, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province) to produce a polyketide compound with NLRP3 inflammasome inhibitory activity; the present invention also relates to the use of such compounds in preventing or treating NLRP3 inflammasome-related diseases. Background Art

[0002] The NLRP3 inflammasome is a multiprotein complex composed of the intracellular innate immune receptor NLRP3, the adaptor protein ASC, and the protease pro-caspase-1. This complex assembly can induce the maturation and secretion of proinflammatory cytokines such as IL-1β and IL-18, thereby promoting inflammatory responses. NLRP3 inflammasome activation is closely associated with the pathogenesis of numerous major human diseases. Mutations in NLRP3 itself can lead to a class of autoinflammatory diseases, including familial cold autoinflammatory syndrome (FCAS), Mueller-Weiss syndrome (MWS), and chronic infantile neurocutaneous arthritis (CINCA). Furthermore, the NLRP3 inflammasome can be activated by various abnormal metabolites, including hyperglycemia, saturated fatty acids, cholesterol crystals, uric acid crystals, and β-amyloid protein. Therefore, the NLRP3 inflammasome plays a crucial role in the pathogenesis of diseases such as type 2 diabetes, atherosclerosis, gout, neurodegenerative diseases, and multiple sclerosis. Therefore, the NLRP3 inflammasome is an important candidate target for intervention in these diseases.

[0003] Despite the involvement of the NLRP3 inflammasome in the pathogenesis of numerous major human diseases, there are currently no clinical drugs targeting the NLRP3 inflammasome. While several small molecules have been reported to inhibit NLRP3 inflammasome activation, these small molecules either target upstream signaling pathways of NLRP3 or inhibit other inflammatory signaling pathways or the activation of other inflammasomes, and are not able to specifically inhibit NLRP3 inflammasome activation. Therefore, the search for small molecule drugs that can target NLRP3 and specifically inhibit NLRP3 inflammasome activation is of great significance.

[0004] The inventors isolated three polyketides from the fermentation products of the Antarctic fungus Pseudogymnoascus sp. HDN17-895. The findings indicate that these polyketides exhibit significant NLRP3 inflammasome inhibitory activity, suggesting their potential for further development as novel NLRP3 inflammasome inhibitors. Summary of the Invention

[0005] The present invention aims to provide a novel compound with novel structure and NLRP3 inflammasome inhibitory activity. Its structural formula is

[0006]

[0007] in

[0008] R x and R y Together they represent the following structure: wherein the carbon bearing R4 is connected to the O atom shown in formula I; or wherein the carbon bearing R6 is connected to the O atom shown in formula I;

[0009] R1-R5 are independently selected from the following substituents: hydrogen, hydroxyl, COOR', C 1-10 Alkoxy, C 2-10 Alkenyloxy and C 2-10 one or more of alkynyloxy, cyano, amino, halogen, nitro, chloroalkyl, mercapto, alkylamino, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, cycloalkyl, and cycloalkylalkyl;

[0010] wherein R' is selected from hydrogen, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 alkynyl, chloroalkyl, mercapto, alkylamino, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, cycloalkyl, and cycloalkylalkyl;

[0011] In this specification, the term "alkyl" refers to a straight or branched hydrocarbon group, preferably having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Representative groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, etc.

[0012] The term "alkenyl" refers to a straight or branched aliphatic hydrocarbon group having at least one carbon-carbon double bond, preferably having 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms. Representative examples include ethenyl, propenyl, allyl, butenyl, pentadienyl, and the like.

[0013] The term "alkynyl" refers to a straight or branched aliphatic hydrocarbon group having at least one carbon-carbon triple bond, preferably having 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms. Representative examples include ethynyl, propynyl, butynyl, etc.

[0014] The compounds of formula I of the present invention can be obtained by fermentation culture of microorganisms to obtain fermentation products containing the compounds, and then the crude fermentation extracts are separated and purified by Sephadex LH20 gel column chromatography, medium pressure MPLC and semi-preparative HPLC.

[0015] The following examples of the present invention illustrate the use of Pseudogymnoascus sp. HDN17-895 to prepare the compound of formula I of the present invention.

[0016] In one embodiment, diseases that can be prevented or treated by inhibiting inflammasomes include, but are not limited to, familial cold autoinflammatory syndrome (FCAS), Muir-Wei syndrome (MWS), chronic infantile neurocutaneous arthritis (CINCA), type II diabetes, atherosclerosis, gout, neurodegenerative diseases, multiple sclerosis, Alzheimer's disease, etc.

[0017] In another aspect, the present invention provides use of the compound of formula I or its sub-formula or pharmaceutically acceptable salt or stereoisomer as described above in the preparation of an NLRP3 inhibitor.

[0018] The following terms, phrases and / or symbols used in this application have the meanings described below unless the context indicates otherwise.

[0019] As used herein, the terms "optional," "optionally," or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted with" means that the group or atom in question is substituted with or without the substituent mentioned. When it means "substituted," it means that the given atom or group is substituted with 1, 2, or 3 (preferably 1) substituents independently selected from a given group (e.g., halogen and / or hydroxy). It should be understood that the structures and / or substitutions described herein are subject to the rules of valence, and that combinations of substituents and / or variables are permissible only if such combinations result in chemically correct and stable compounds.

[0020] The term "pharmaceutically acceptable salt" as used herein refers to a non-toxic, biologically tolerable or other biologically suitable salt of a compound of formula (I) formed with a free acid or base that is suitable for administration to an individual. The salts include acid addition salts and base addition salts. Acids commonly used to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as tartaric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, oxalic acid, and the like. Base addition salts include salts derived from inorganic bases such as ammonium or alkali metal or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases that can be used to prepare the salts of the present invention include, but are not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like. Organic salts of the present invention include acetate, propionate, butyrate, tartrate, maleate, hydroxymaleate, fumarate, citrate, lactate, mucate, gluconate, benzoate, succinate, oxalate, phenylacetate, methanesulfonate, p-toluenesulfonate, benzenesulfonate, p-aminosalicylate, aspartate, glutamate, edetate, stearate, palmitate, oleate, laurate, pantothenate, tannate, ascorbate, valerate or alkylammonium salts. For a general description of pharmaceutically acceptable salts, see, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, eds., Wiley-VCH and VHCA, Zurich, 2002.

[0021] It will be appreciated by those skilled in the art that some compounds of formula (I) may comprise one or more chiral centers, and therefore have two or more stereoisomers. Racemic mixtures of these isomers, single isomers, and a mixture enriched in enantiomers, as well as diastereomers when there are two chiral centers, and mixtures partially enriched in specific diastereomers are within the scope of the present invention. It will also be appreciated by those skilled in the art that the present invention includes all single stereoisomers (e.g., enantiomers), racemic mixtures, or partially resolved mixtures of compounds of formula (I), and, where appropriate, includes its single tautomers.

[0022] The term "pharmaceutically acceptable excipient" as used herein refers to one or more compatible solid or liquid fillers or gel substances that are pharmacologically inactive, compatible with the other ingredients in the composition, and should be acceptable for administration to warm-blooded animals such as humans, for example, used as a carrier or medium for the compound of the present invention in the administration form, examples of which include but are not limited to cellulose and its derivatives (such as sodium carboxymethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as magnesium stearate), calcium sulfate, vegetable oils, polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tweens), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, etc. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention, but the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental materials and reagents used in the following examples were obtained from commercial sources, prepared according to methods in the prior art, or prepared according to methods similar to those disclosed herein. The known compounds in the examples were commercially available, prepared by methods in the prior art, or identified by comparison with data and / or properties of corresponding compounds in the prior art.

[0025] Example 1 Fermentation production and separation and purification of compound 1-3

[0026] The fungus Pseudogymnoascus sp. HDN17-895 was first cultured on a PDA solid slant medium (200 g / L potato, 20 g / L glucose, 20 g / L agar) in an incubator at 15°C for 5 days, then inoculated again into a rice medium (80 g rice, 120 mL seawater) and the fungus was fermented at room temperature (15-20°C) for 30 days. The fermentation product was extracted three times with methanol, concentrated under reduced pressure to remove methanol, and then extracted three times with ethyl acetate and concentrated under reduced pressure to obtain a crude extract. The crude extract was initially separated by C-18 ODS column chromatography using a methanol / water (30%-100%) mobile phase for gradient elution; finally, it was separated and purified by reverse phase semi-preparative high performance liquid chromatography (YMC-Pack ODS-A, 10×250 mm, 5 μm, 3 mL / min) to obtain the following compound 1 (acetonitrile / water, 40%, t R =23min), 2(acetonitrile / water, 43%, t R =31min), 3 (methanol / water, 50%, t R =10min).

[0027] Compound 1 is a red powder with the molecular formula C 30 H 30 O 13 , HR-ESI-MS m / z: 597.1603[MH] - , calculated value 597.1614; 1 H and 13 C NMR is shown in Tables 1-1 and 1-2.

[0028] Compound 2 is a red powder with the molecular formula C 30 H 30 O 12 , HR-ESI-MS m / z: 581.1659[MH] - , calculated value 581.1664; 1 H and 13 C NMR is shown in Tables 1-1 and 1-2.

[0029] Compound 3 is a red powder with the molecular formula C 30 H 30 O 13 , HR-ESI-MS m / z: 597.1617[MH] - , calculated value 597.1603; 1 H and 13 C NMR is shown in Tables 1-1 and 1-2.

[0030] Table 1-1 Compounds measured in DMSO-d6 1 H NMR (500 MHz) data

[0031]

[0032] Table 1-2 Compounds measured in DMSO-d6 13 C (125 MHz) NMR data.

[0033]

[0034]

[0035] Example 2 In vitro biological experiment

[0036] 1. Experimental samples and methods

[0037] The test sample is the pure compound isolated and purified in Example 1. An appropriate amount of sample is accurately weighed and prepared into a solution of the desired concentration with DMSO for activity measurement.

[0038] J774A.1 mouse mononuclear macrophage cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin / amphotericin (37°C, 5% CO2). J774A.1 cells were plated at 1.5×10 5 Cells were seeded at a density of 100 μL in a 96-well plate containing 100 μL of growth medium. Cells were pretreated with 1 μg / mL of E. coli for 4.5 h. Test compounds were then added to each well and incubated for 30 minutes, followed by the addition of 5'-ATP (final concentration 5 mM) and a 30-minute incubation. After 30 minutes, the cells were centrifuged at 900 rpm for 3 minutes, and the supernatant was collected for further analysis of IL-1β levels using a mouse IL-1β ELISA kit (Duoset Elisa, R&D Systems).

[0039] 2. Experimental Results

[0040] Compounds 1-3 inhibited LPS / ATP-induced IL-1β release in a dose-dependent manner, IC 50 The values ​​were 1.13μM, 6.96μM, 5.92μM ( Figure 2 ).

[0041] 3. Conclusion

[0042] The above compounds have good NLRP3 inhibitory activity and can be used as new NLRP3 inhibitors for preventing or treating NLRP3 inflammasome-related diseases.

[0043] Example 3 In vivo biological experiment

[0044] 1. Experimental samples and methods

[0045] Male C57BL / 6 mice (5-6 weeks) were randomly divided into three groups (n=5 per group): a blank group, a model group, and a treatment group. The blank group received intraperitoneal administration of the vehicle; the model group received intraperitoneal injection of the blank vehicle, followed by intraperitoneal injection of LPS (35 mg / kg) 1 hour later; and the treatment group received intraperitoneal injection of compound 1 (50 mg / kg), followed by intraperitoneal injection of LPS (35 mg / kg) 1 hour later. Blood was collected from the eyeballs 2.5 hours later, allowed to stand for 1 hour, and then centrifuged. The supernatant was collected and serum levels of IL-1β, IL-6, and TNF-α were determined by ELISA.

[0046] 2. Experimental Results

[0047] In the LPS-induced acute peritonitis experiment in mice, the representative compound 1 can significantly inhibit the release of IL-1β in LPS-induced acute peritonitis in mice, without affecting the release of inflammatory factors IL-6 and TNF-α, indicating that the compound can specifically inhibit the NLRP3 inflammasome and has a certain selectivity ( Figure 3 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is the chemical structure of compound 1-3.

[0049] Figure 2 These are the results of in vitro NLRP3 inflammasome inhibitory assays of compounds 1-3.

[0050] Figure 3 The effect of compound 1 (50 mg / kg) on ​​LPS-induced IL-1β, IL-6 and TNF-α production in C57BL / 6 mice.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound shown in the following structure or a pharmaceutically acceptable salt thereof 2. The method for preparing the compound according to claim 1, characterized in that The following steps are involved: The fungus Pseudogymnoascus sp. HDN17-895, with a deposit number of CCTCC NO: M20211640, is first cultured on a PDA solid slant medium in an incubator at 15°C for 5 days, then inoculated into a rice culture medium and the fungus is statically cultured at room temperature for 30 days; the fermentation product is extracted three times with methanol, concentrated under reduced pressure to remove methanol, and then extracted three times with ethyl acetate and concentrated under reduced pressure to obtain a crude extract; the crude extract is initially separated by C-18 ODS column chromatography using methanol / water as the mobile phase; and finally, the compounds 1, 2, and 3 according to claim 1 are separated and purified by reverse-phase semi-preparative high-performance liquid chromatography.

3. Use of the compound of claim 1 in the preparation of a medicament for preventing or treating diseases associated with abnormal activation of NLRP3 inflammasome, characterized in that The disease associated with abnormal activation of NLRP3 inflammasome is selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, traumatic brain injury, inflammatory bowel disease, pneumonia, rheumatoid arthritis, rheumatoid arthritis, gouty arthritis, osteoarthritis, non-alcoholic hepatitis, acute nephritis, chronic nephritis, peritonitis, gout, non-alcoholic fatty liver disease, type II diabetes, heart failure, atherosclerosis, acute myocardial infarction, liver fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cryopyrin-associated periodic syndrome and systemic lupus erythematosus.

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