NLRP3 inhibitory activity compounds and uses thereof

Abstract

The invention discloses application of a compound as shown in a formula I or a tautomer, a meso-mer, a raceme, an enantiomer, a diastereoisomer or a pharmaceutically acceptable salt of the compound in preparation of an NLRP3 inhibitor. The compound provided by the invention has excellent NLRP3 inhibitory activity, so that the compound can be used as a therapeutic drug for NLRP3-related diseases. The invention also provides a novel synthesis method of the isopentenyl modified flavonoid compound, so that the isopentenyl modified flavonoid compound can be synthesized at high yield. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically refers to flavonoid compounds, drug combinations thereof and applications thereof in inhibiting NLRP3 activation. Background Art

[0003] The inflammatory response is induced by a variety of inflammasomes. Inflammasome nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) is a key regulatory protein. NLRP3 is an inflammasome sensor protein. Its activation leads to the formation of oligomeric protein complexes including apoptosis-associated microparticle protein (ASC) and caspase-1. This oligomeric complex is called "inflammasome". The host can induce nuclear factor κB activation by recognizing pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), triggering the transcriptional expression of NLRP3, pro-caspase-1 and pro-IL-1β. The activated NLRP3 pyrin-like domain (PYD) binds to the PYD domain of ASC and assembles with the ASC helix to form ASC spots. Subsequently, the caspase recruitment domain (CARD) of ASC and the CARD of pro-caspase-1 interact and bind. The oligomeric form of procaspase-1 produces caspase-1 through its own proteolytic cleavage, thereby causing inflammation. Activated NLRP3 may lead to the occurrence of a variety of diseases, such as gout, rheumatoid arthritis, Alzheimer's disease and multiple sclerosis, but there are currently no NLRP3 inhibitors on the market and multiple clinical trials have been terminated due to safety reasons.

[0004] Isopentenyl is an important active group of flavonoids. In addition, the biological activity and bioavailability of drug-like natural products can be effectively improved by modifying their structures with isopentenyl. However, in the synthesis of flavonoids or the modification of natural products, isopentenyl substitution has many by-products, low substrate universality, or involves metal catalysis, which makes it difficult to recover the catalyst and has low cost performance. Base-catalyzed reaction is one of the commonly used isopentenyl substitution methods. Different systems are used to directly isopentenylate the ortho-hydroxyl position of the benzene ring, but for polyhydroxy substrates, there are problems of low selectivity and low yield. Summary of the invention

[0005] The present invention aims to provide the use of various flavonoid compounds in inhibiting the activation of NLRP3, and their use in preparing drugs for preventing or treating NLRP3-related diseases. The present invention further provides methods for preparing these compounds.

[0006] In a first aspect of the present invention, there is provided a use of a compound of formula I or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt in the preparation of an NLRP3 inhibitor.

[0007]

[0008] Wherein, X1 is selected from O or S;

[0009] X2 is selected from O, S or NR 11 ; R 11 Selected from the following group: H, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl;

[0010] R1, R2, R3, R4, R5 and R6 are independently selected from the following groups: H, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-10 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl, halogen, amino, nitro; or, R2 and R3 or R3 and R4 together with the carbon atoms to which they are attached form a 3-6 membered ring structure;

[0011] n is an integer from 1 to 5.

[0012] In a preferred embodiment, the "optionally substituted" refers to being optionally substituted by one or more substituents selected from the following: cyano, halogen, hydroxyl, amino, nitro, carboxyl, ester, sulfonyl, oxo, optionally substituted C 1-3 Alkyl, optionally substituted C 1-3 Alkoxy, optionally substituted C 5-10 Aryl, optionally substituted sulfonyl.

[0013] In a specific embodiment, X1 is selected from O;

[0014] X2 is selected from O or S;

[0015] R1 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, halogen, amino, nitro;

[0016] R2, R3 and R4 are independently selected from the following groups: H, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-10 alkenyl; or, R2 and R3 or R3 and R4 together with the carbon atoms to which they are attached form a 5- or 6-membered cyclic structure containing 1-3 heteroatoms selected from O, N or S or containing alkenyl;

[0017] R5 is selected from the group consisting of H, hydroxyl;

[0018] R6 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy;

[0019] n is an integer from 1 to 3.

[0020] In a specific embodiment, X1 is selected from O; X2 is selected from O;

[0021] R1 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy;

[0022] R2, R3 and R4 are independently selected from the following groups: H, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-10 alkenyl;

[0023] R5 is selected from the group consisting of H, hydroxyl;

[0024] R6 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy;

[0025] n is an integer from 1 to 3.

[0026] In a specific embodiment, the compound represented by formula I is a compound selected from the following group:

[0027]

[0028]

[0029] In a specific embodiment, the compound is selected from the group consisting of:

[0030]

[0031] In a specific embodiment, the compound is selected from the group consisting of:

[0032]

[0033] In a specific embodiment, the NLRP3 inhibitor is a drug for treating NLRP3-related diseases.

[0034] In a specific embodiment, the NLRP3-related disease is gout, enteritis, hepatitis, arthritis, type II diabetes, and neurodegenerative disease.

[0035] In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0036] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound of formula I described in the first aspect or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt and an optional excipient.

[0037] In a preferred embodiment, the pharmaceutical composition is used as a NLRP3 inhibitor.

[0038] In a preferred embodiment, the NLRP3 inhibitor is a drug for treating NLRP3-related diseases.

[0039] In a preferred embodiment, the NLRP3-related disease is gout, enteritis, hepatitis, arthritis, type II diabetes, and neurodegenerative disease.

[0040] In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0041] In a third aspect, the present invention provides a method for synthesizing an isopentenyl-modified flavonoid compound, the method comprising reacting the flavonoid compound with an isopentenyl halide and an inorganic base in an organic solvent to obtain the isopentenyl-modified flavonoid compound.

[0042] In a preferred embodiment, the isopentenyl halide is isopentenyl fluoride, isopentenyl chloride, isopentenyl bromide or isopentenyl iodide; preferably, the isopentenyl halide is isopentenyl bromide.

[0043] In a preferred embodiment, the inorganic base is potassium carbonate, sodium acetate, potassium tert-butoxide, potassium hydroxide; preferably, the inorganic base is potassium hydroxide.

[0044] In a preferred embodiment, the organic solvent is ethyl acetate, tetrahydrofuran, acetonitrile, isopropyl ether; preferably, the organic solvent is ethyl acetate.

[0045] In a preferred embodiment, the synthesis method is carried out at a temperature ranging from room temperature to the reflux temperature of the solvent; preferably at 70°C.

[0046] In a fourth aspect, the present invention provides a method for treating NLRP3-related diseases, comprising the step of administering a therapeutically effective amount of the compound of the first aspect or its tautomer, mesomer, racemate, enantiomer, diastereomer, a mixture thereof or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect to a subject in need thereof.

[0047] In a specific embodiment, the NLRP3-related diseases are gout, enteritis, hepatitis, arthritis, type II diabetes, neurodegenerative diseases, etc.

[0048] The neurodegenerative diseases include but are not limited to Alzheimer's disease and Parkinson's disease.

[0049] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The inhibitory activity of compound T1 on NLRP3 was shown.

[0051] Figure 2 The inhibitory activity of compound T1S on NLRP3 was shown.

[0052] Figure 3 The inhibitory activity of compound T2 on NLRP3 was shown.

[0053] Figure 4 The inhibitory activity of compound T3 on NLRP3 was shown.

[0054] Figure 5 The inhibitory activity of compound T3S on NLRP3 was shown.

[0055] Figure 6 The inhibitory activity of compound T4 on NLRP3 was shown.

[0056] Figure 7 The inhibitory activity of compound T4S on NLRP3 was shown.

[0057] Figure 8 The inhibitory activity of compound T5 on NLRP3 was shown.

[0058] Fig. 9 The inhibitory activity of compounds T6 and T6S against NLRP3 was shown. DETAILED DESCRIPTION

[0059] After extensive and in-depth research, the inventors unexpectedly found that a variety of flavonoid compounds have NLRP3 inhibitory activity, so that the compounds can be used to prepare NLRP3 inhibitors, and then used to prepare drugs for treating NLRP3-related diseases. On this basis, the present invention was completed.

[0060] Definition of terms

[0061] The terms used herein have the meanings conventionally understood by those skilled in the art. For the purpose of clearly understanding the present invention only, the terms used herein are defined as follows. However, it should be understood by those skilled in the art that the present invention is not limited by the specific definitions.

[0062] In this document, "a", "an", "a kind" or "a category" are meant to include the plural forms of the objects they modify, that is, "a", "an", "a kind" or "a category" means at least one / kind / category or one / kind / category or more than one / kind / category.

[0063] In this article, the form "C 1-n " means that the group has 1-n carbon atoms, for example, "C 1-10 ” means that the group has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms; similarly, “C2-C6” means that the group has 2, 3, 4, 5 or 6 carbon atoms.

[0064] The term "alkyl" as used herein has the same meaning as that generally understood by those of ordinary skill in the art, and refers to various saturated, straight-chain, side-chain hydrocarbon groups. For example, the alkyl described herein refers to a lower alkyl group of 1-10 carbon atoms; preferably, it refers to a lower alkyl group of 1-8 carbon atoms; more preferably, it refers to a lower alkyl group of 1-6 carbon atoms. In a specific embodiment, the alkyl described herein includes, but is not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and the like.

[0065] The term "alkenyl" as used herein has the same meaning as that generally understood by those of ordinary skill in the art, and refers to a hydrocarbon group with one or more double bonds, straight chain, side chain or ring. For example, the alkenyl described herein refers to an alkenyl group of 2-10 carbon atoms. In a specific embodiment, the alkenyl described herein is an isopentenyl group.

[0066] The term "cycloalkyl" as used herein refers to a cyclic alkyl group commonly understood by those of ordinary skill in the art. "C3-C10 cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclodecyl, etc.

[0067] The term "C 1-6 The term "alkoxy" has the same meaning as that generally understood by those skilled in the art, and refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy and butoxy.

[0068] The term "halogen" as used herein refers to F, Cl, Br or I.

[0069] Based on the teachings of the present invention and common knowledge in the art, those skilled in the art will appreciate that the compounds of the present invention and the various substituents defined above may be further substituted, as long as the desired combination of substituents is a stable or chemically feasible combination of substituents.

[0070] The term "substituted" as used herein means that one or more hydrogen atoms on a specific group are replaced by a specific substituent. The specific substituent may be a substituent described above, or a specific substituent appearing in each embodiment. Therefore, in the present invention, the substituents in Formula I may each independently be a corresponding group in a specific compound in the embodiment; that is, the present invention includes a combination of the substituents in the above Formula I, and also includes a combination of some of the substituents shown in Formula I with other specific substituents appearing in the embodiments.

[0071] Unless otherwise specified, a substituted group may have a specific substituent at any substitutable position of the group, and the substituent may be the same or different at each position. A cyclic substituent, such as a heterocycloalkyl, may be attached to another ring, such as a cycloalkyl, to form a spirobicyclic ring system, for example, the two rings have a common carbon atom. The substituents are, for example (but not limited to): C 1-8 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclic group, aryl, heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, C 1-8 Alkoxy, nitro, cyano, mercapto, amino, etc. In a specific embodiment, the hydrogen atoms on the alkyl and aryl groups are replaced by amino, halogen or other groups to become the groups defined above.

[0072] For the sake of convenience and in accordance with common understanding, the term "arbitrary substitution" or "optionally substituted" only applies to sites that can be substituted by substituents, and does not include those substitutions that are chemically unfeasible.

[0073] Compounds of the present invention and uses thereof

[0074] In this document, the terms "compound of the present invention", "flavonoid compound of the present invention" and the like have the same meaning, and all refer to the compound of formula I or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt having excellent NLRP3 inhibitory activity.

[0075]

[0076] wherein X1, X2, R1, R2, R3, R4, R5, R6 and n are as defined above, respectively.

[0077] In a specific embodiment, the compound of the present invention is a compound selected from the group consisting of:

[0078]

[0079]

[0080] In a preferred embodiment, the compound of the present invention is a compound selected from the group consisting of:

[0081]

[0082]

[0083] More preferably, the following compounds are:

[0084]

[0085] The compounds of the present invention have excellent NLRP3 inhibitory activity, and thus can be used as NLRP3 inhibitors, and further used to treat NLRP3-related diseases. In a specific embodiment, the NLRP3-related diseases are: gout, enteritis, hepatitis, arthritis, type II diabetes, neurodegenerative diseases. In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0086] Pharmaceutical compositions and treatment methods of the present invention

[0087] The compounds of the present invention have excellent NLRP3 inhibitory activity and can be used as NLRP3 inhibitors, and further as drugs for treating the above-mentioned NLRP3-related diseases. In view of this, on the basis of the above-mentioned compounds, the present invention also provides a pharmaceutical composition, which comprises the compound represented by the above-mentioned formula I or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt and optional pharmaceutically acceptable excipients.

[0088] In a specific embodiment, the pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.

[0089] "Pharmaceutically acceptable excipients or carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0090] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0091] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0092] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0093] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0094] Besides these inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.

[0095] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0096] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0097] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0098] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. When using a pharmaceutical composition, a safe and effective amount of the compounds of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage. The compounds and pharmaceutical compositions of the present invention can be administered via the oral, nasal, skin, lung or gastrointestinal tract. Oral administration, either once or in divided doses, is most preferred. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Usually, the dose is started with a small dose and gradually increased until the most suitable dose is found. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0099] Based on the teachings of the present invention, those skilled in the art can use the compounds or pharmaceutical compositions of the present invention to treat NLRP3-related diseases. In a specific embodiment, the method comprises administering a therapeutically effective amount of a compound of Formula I or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt to a patient in need thereof. The NLRP3-related diseases include, but are not limited to, gout, enteritis, hepatitis, arthritis, type II diabetes, and neurodegenerative diseases; the neurodegenerative diseases include, but are not limited to, Alzheimer's disease or Parkinson's disease.

[0100] Synthesis method of the present invention

[0101] In the process of studying the compounds of the present invention, the inventors unexpectedly discovered a method for synthesizing an isopentenyl-modified flavonoid compound. Compared with the methods in the prior art, the method can obtain an isopentenyl-modified flavonoid compound in high yield. In a specific embodiment, the method comprises reacting a flavonoid compound with an isopentenyl halide and an inorganic base in an organic solvent to obtain an isopentenyl-modified flavonoid compound.

[0102] In a preferred embodiment, the isopentenyl halide can be isopentenyl fluoride, isopentenyl chloride, isopentenyl bromide or isopentenyl iodide; preferably isopentenyl bromide; the inorganic base is potassium carbonate, sodium acetate, potassium tert-butoxide, potassium hydroxide; preferably potassium hydroxide; the organic solvent is ethyl acetate, tetrahydrofuran, acetonitrile, isopropyl ether; preferably ethyl acetate.

[0103] The inventors further found that as the reaction temperature increases, the yield of the isopentenyl-modified flavonoids prepared by the method of the present invention also increases accordingly. Therefore, the synthesis method of the present invention can be carried out at a temperature range from room temperature to the reflux temperature of the solvent. In a preferred embodiment, the synthesis method of the present invention is carried out at 70°C.

[0104] Advantages of the present invention:

[0105] 1. The compounds of the present invention have excellent NLRP3 inhibitory activity;

[0106] 2. The compounds of the present invention lay a new material foundation for the development of therapeutic drugs for NLRP3-related diseases; and

[0107] 3. The present invention provides a new method for synthesizing isopentenyl-modified flavonoid compounds, which can synthesize isopentenyl-modified flavonoid compounds in high yield.

[0108] Example 1. Preparation of flavonoid compounds T1 and T1S

[0109]

[0110] Take a dry and clean reaction bottle, dissolve S1 (168 mg, 1.0 mmol) in 2-3 mL of dry ethyl acetate, add S2 (239 mg, 1.6 mmol), stir at room temperature for 10 min, then add KOH (84 mg, 1.5 mmol), react at room temperature overnight, add 5 mL of water after the reaction is completed by TLC monitoring, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (6 mL) in turn, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S3 (59.7%) by column chromatography (ethyl acetate / petroleum ether=1:8).

[0111] Take a dry and clean reaction bottle, dissolve the above synthesized compound S3 (1.0 eq) in 5-10 mL DCM, add DIEA (3.0 eq), stir at room temperature for 10 min, add dropwise to dissolve in MOMBr (2.2 eq), react at room temperature for 1-24 h, add 10 mL of water to quench the reaction after TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S4 (60-90%) by column chromatography (ethyl acetate / petroleum ether = 1:15).

[0112] Take a dry and clean reaction bottle, dissolve the above synthesized compound S4 (1.0 eq) in 5-10 mL of ethanol, add S5 (1.0 eq), stir at room temperature for 10 min, then add KOH (7.0 eq), react at room temperature for 1-24 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine solution (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S6 (60-90%) by column chromatography (ethyl acetate / petroleum ether = 1:5).

[0113] Take a dry and clean reaction bottle, dissolve the above synthesized compound S6 (1.0 eq) in 5-10 mL of methanol, add 5.4% NaOH (10.0 eq), stir at room temperature for 10 min, then add 30% hydrogen peroxide (6.0 eq), react at room temperature for 1-24 h, add 10 mL of water to quench the reaction after the reaction is completed by TLC, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine solution (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S7 (50-80%) by column chromatography (ethyl acetate / petroleum ether = 1:3).

[0114] Take a dry and clean reaction bottle, dissolve the above synthesized compound S7 (1.0 eq) in 5-10 mL of methanol, add 3 M hydrochloric acid (60 μL), react at reflux temperature for 1-24 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T1S (50-70%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0115] Take a dry and clean reaction bottle, dissolve the above synthesized compound T1S (1.0 eq) in 5-10 mL of anhydrous toluene, add DDQ (1.1 eq), react at reflux temperature for 4 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T1 (50-90%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0116]

[0117] Example 2. Preparation of flavonoid compound T2

[0118]

[0119] Take a dry and clean reaction bottle, dissolve S8 (295 mg, 1.5 mmol) and aluminum chloride (1.2 g, 9.0 mmol) in 4 mL DCM, and react at reflux temperature for 4 h. After TLC detection, add 10 mL of water to quench the reaction, extract with dichloromethane three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and directly put into the next step.

[0120] Take a dry and clean reaction bottle, dissolve the above synthesized compound S9 (354 mg, 1.5 mmol) and DIPEA (3.0 eq) in 6 mL DCM, add MOMBr (2.2 eq) and stir at room temperature for 3 hours. After TLC detection, add 10 mL of water to quench the reaction, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S10 by column chromatography (ethyl acetate / petroleum ether=1:5) (two-step yield 60%).

[0121] Take a dry and clean reaction bottle, take the above synthesized S10 (256 mg, 1.0 mmol) and dissolve it in 2 mL of ethanol, add S4 (1.0 eq), stir at room temperature for 10 min, then add KOH (7.0 eq), react at room temperature overnight, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S11 (70-90%) by column chromatography (ethyl acetate / petroleum ether = 1:4).

[0122] Take a dry and clean reaction bottle, take the above synthesized S11 (281 mg, 0.5 mmol) and dissolve it in 3 mL of ethanol, add sodium acetate (6.0 eq), and react at reflux temperature for 24 h. After the reaction is completed by TLC detection, add 10 mL of water, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S12 (50-70%) by column chromatography (ethyl acetate / petroleum ether = 1:4).

[0123] Take a dry and clean reaction bottle, take the above synthesized S12 (140 mg, 0.25 mmol) and dissolve it in 3 mL of methanol, add 3 M hydrochloric acid (60 μL), react at reflux temperature for 4 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases and wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure and obtain compound T2 (40-60%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0124]

[0125] Example 3. Preparation of flavonoid compounds T3 and T3S

[0126]

[0127] Take a dry and clean reaction bottle, dissolve compound S13 (304 mg, 2.0 mmol) in 6 mL DCM, add DIEA (517 mg, 4.0 mmol), stir at room temperature for 10 min, add dropwise to dissolve in MOMBr (300 mg, 2.4 mmol), react at room temperature for 3 h, add 10 mL of water to quench the reaction after the reaction is completed by TLC, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S14 (83%) by column chromatography (ethyl acetate / petroleum ether = 1:8).

[0128] Take a dry and clean reaction bottle, dissolve the above synthesized compound S14 (1.0 eq) in 5-10 mL of ethanol, add S4 (1.0 eq), stir at room temperature for 10 min, then add KOH (7.0 eq), react at room temperature for 1-24 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine solution (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S15 (60-90%) by column chromatography (ethyl acetate / petroleum ether = 1:5).

[0129] Take a dry and clean reaction bottle, dissolve the above synthesized compound S15 (1.0 eq) in 5-10 mL of methanol, add 5.4% NaOH (10.0 eq), stir at room temperature for 10 min, then add 30% hydrogen peroxide (6.0 eq), react at room temperature for 1-24 h, add 10 mL of water to quench the reaction after the reaction is completed by TLC, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine solution (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound S16 (50-80%) by column chromatography (ethyl acetate / petroleum ether = 1:3).

[0130] Take a dry and clean reaction bottle, dissolve the above synthesized compound S16 (1.0 eq) in 5-10 mL of methanol, add 3 M hydrochloric acid (60 μL), react at reflux temperature for 1-24 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T3S (50-70%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0131] Take a dry and clean reaction bottle, dissolve the above synthesized compound T3S (1.0 eq) in 5-10 mL of anhydrous toluene, add DDQ (1.1 eq), react at reflux temperature for 4 h, add 10 mL of water after the reaction is completed by TLC detection, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T3 (50-90%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0132]

[0133] Example 4. Preparation of flavonoid compound T4

[0134]

[0135] Take a dry and clean reaction bottle, dissolve T4S (286 mg, 1.0 mmol) in 2-3 mL of dry ethyl acetate, add 1-bromo-3-methyl-2-butene (236 mg, 1.6 mmol) and then add KOH (84 mg, 1.5 mmol), react at room temperature overnight, add 5 mL of water after the reaction is completed by TLC monitoring, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (6 mL) in turn, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T4 (10-30%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0136]

[0137] Example 5. Preparation of flavonoid compound T5

[0138]

[0139] Take a dry and clean reaction bottle, dissolve T4S (286 mg, 1.0 mmol) in 2-3 mL of dry ethyl acetate, add geranyl bromide (347 mg, 1.6 mmol) and then KOH (84 mg, 1.5 mmol), react at room temperature overnight, add 5 mL of water after the reaction is completed by TLC monitoring, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (6 mL) in turn, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T5 (5-20%) by column chromatography (ethyl acetate / petroleum ether = 1:1).

[0140]

[0141] Example 6. Preparation of flavonoid compound T6

[0142]

[0143] Take a dry and clean reaction bottle, dissolve T6S (302 mg, 1.0 mmol) in 2-3 mL of dry ethyl acetate, add 1-bromo-3-methyl-2-butene (236 mg, 1.6 mmol) and then add KOH (84 mg, 1.5 mmol), react at room temperature overnight, add 5 mL of water after the reaction is completed by TLC monitoring, extract with ethyl acetate three times (10 mL*3), combine the organic phases, wash with saturated brine (6 mL) in turn, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound T6 (5-20%) by column chromatography (ethyl acetate / petroleum ether=1:1).

[0144]

[0145] Example 7. Inhibitory activity of flavonoids on NLRP3 inflammasome

[0146] 1. Reagents and Materials

[0147] Dimethyl sulfoxide (Aladdin, 67-68-5), RPMI 1640 medium (VM-2101BM, Vistech), Dulbecco's modified Eagle's medium (DMEM, Vistech, VM-1101BM), Opti-MEM medium (Gibco, 31985062), fetal bovine serum (FBS, Vistech, SE100-011), penicillin-streptomycin double antibody (PS, Gibco, 15140-122), macrophage stimulating factor (M-CSF, Bio-techne, 416-ML), lipopolysaccharide (LPS, MedChemExpress, HY-D1056), nigericin (MedChemExpress, HY-127019), Mouse IL-1beta / IL-1F2 Quantikine HS ELISA Kit (R&D Systems, MHSLB00), Mouse IL-1beta / IL-1F2 DuoSet ELISA kit (R&D Systems, DY401-05), ethanol (Shanghai Test, 10009159), cell lysate (Beyotime, P0013B), chloroform (Shanghai Test, 10006818), methanol (Shanghai Test, 10014128), NLRP3 antibody (Cell Signaling Technologies, 15101), Caspase-1 antibody (Cell Signaling Technologies, 24232), IL-1β antibody (Cell Signaling Technologies, 12426), GAPDH antibody (Cell Signaling Technologies, 2118), HRP-labeled affinity purified goat anti-rabbit IgG (H+L) secondary antibody (Bioker, BK-R050), enhanced chemiluminescence substrate (ECL, NCM Biotechnology). Biotech, P2300), dissecting scissors, cell scraper, 12-well cell culture plate (Corning, 3737), 24-well cell culture plate (Corning, 3738), disposable bacterial culture dish (Bbi-life sciences, F611004).

[0148] 2. Experimental instruments: ELISA reader (Tecan Group Ltd., Swiss), water bath (Shanghai Boxun Medical Biological Instrument Co., Ltd., DK-8D), high-speed centrifuge (Eppendorf AG, Centrifuge 5420), protein electrophoresis instrument (Bio-Rad Laboratories Co., Ltd., Tetra Blotting Module), exposure instrument (Ebiotrade, e-Blot).

[0149] 3. Experimental animals: Male ICR mice (6-12 weeks old) were purchased from the Experimental Animal Center of Hangzhou Normal University and kept in a constant temperature and humidity environment. The feeding and experimental use were in accordance with the "Guidelines for the Care and Use of Laboratory Animals".

[0150] 4. Experimental Methods

[0151] (1) Isolation and differentiation of BMDM cells

[0152] Removal of mouse leg bones: The mice were killed by dislocation, and the legs and backs of the mice were disinfected with sufficient 75% ethanol. The hind limbs were then removed along the greater trochanter at the root of the thigh with scissors, and the muscle tissue was removed and placed in cold PBS. (This step was completed in the clean bench, and the subsequent steps were transferred to the cell room)

[0153] BMDM cell extraction and induction: rinse the leg bones with PBS, remove excess muscle, and rinse about 2-3 times. Cut the ends of the femur and tibia with scissors, use a 1mL syringe to absorb cold induction culture medium to blow the bone marrow out of the femur and tibia, and repeat the washing 3 times until no obvious red color can be seen in the leg bones. Use a pipette to repeatedly blow the culture medium containing bone marrow cells to disperse the cell clumps, transfer to a 15mL centrifuge tube, and centrifuge at 1500rpm for 5min. Discard the supernatant, add about 1-2mL of red blood cell lysis buffer to resuspend, use a pipette to blow the cells into single cells, let stand at room temperature for 5min, and then add 10mL of culture medium to terminate the reaction. Filter with a 0.45μm cell filter membrane and centrifuge at 1500rpm / min for 5min. Discard the supernatant, resuspend the cells, and count the cells. Resuspend the cells with 1640 culture medium (10% FBS, 2% PS) containing 20ng / mL M-CSF factor, and transfer the cell suspension to a no-treated disposable bacterial culture dish for culture.

[0154] (2) Activation of the classical NLRP3 inflammasome

[0155] Use 4-6 days BMDM cells that have been well differentiated, discard the culture supernatant, add an appropriate amount of PBS to wash the cells. Add 10mL DMEM culture medium and scrape the cells with a cell scraper, resuspend the cells and transfer them to a centrifuge tube, centrifuge at 1500rpm for 10min. Remove the supernatant, resuspend the BMDM cells with DMEM culture medium and divide them into 12-well plates, so that the number of cells in each well is about 5×10 5 / mL. The next day, 500μL Opti-MEM medium (containing 500ng / mL LPS) was added to each well of BMDM cells for pretreatment for 4-5 hours. The compound was weighed on an analytical balance and placed in a sterile centrifuge tube, and then dimethyl sulfoxide (DMSO) was added to dissolve it and the compound was prepared into the required concentration. The drug was added for treatment for 30 minutes, and 10μM Nigericin was added for stimulation for 60 minutes, and the cell supernatant (SN) and cell lysate (Input) were collected. According to the obtained cell supernatant (SN) and cell lysate (Input), Western Blot detection and ELISA detection were performed.

[0156] (3) Protein extraction and sample preparation

[0157] Extraction of protein from cell culture supernatant: Place the sample in a centrifuge and centrifuge at 13,000 rpm for 5 minutes to remove dead cells and impurities; Transfer the supernatant to a new EP tube, add 500 μL methanol and 125 μL chloroform, vortex evenly, and centrifuge at 13,000 rpm for 5 minutes; Remove the upper liquid, and the middle layer is the protein solution. Add 500 μL methanol, vortex evenly, and centrifuge at 13,000 rpm for 5 minutes; The protein precipitates at the bottom of the EP tube, remove the supernatant, let it stand for 10 minutes to evaporate the methanol, add 60 μL 1×SDS loading buffer, fully dissolve, and denature at 100°C for 10 minutes.

[0158] Extraction of cell proteins: Add 120 μL of cell lysis buffer to the cells after removing the cell supernatant. After the cells are fully lysed, add 5×SDS loading buffer and denature at 100℃ for 10 minutes.

[0159] (4) Western Blot

[0160] Select 4-12% protein gel for WB experiment, the voltage line condition of protein electrophoresis is 70-80V, 20-30min, and the separation gel running condition is 130-160V, 60-90min. Soak the PVDF membrane in methanol for 5-10min in advance, soak the filter paper and sponge in the transfer solution in advance, and soak the protein gel in the transfer solution for 5-10min after the electrophoresis is completed. Clamp with a sandwich structure of sponge-filter paper-gel-membrane-filter paper-sponge for transfer, and the transfer condition is 250mA, 90min. Block with 5% skim milk for 1h, dilute the primary antibody with 5% BSA according to the dilution ratio of the antibody, and incubate overnight at 4°C. The next day, wash three times with 1xTBST, 5min apart. Dilute the secondary antibody with 1xTBST according to the dilution ratio of the antibody, incubate for 1h, and wash three times with 1xTBST, 5min apart. After washing the membrane, expose with ECL supersensitive exposure solution.

[0161] (5) ELISA test

[0162] The cell supernatant obtained from "Activation of the classical NLRP3 inflammasome" in step 2 of the experimental method was used for ELISA experiments according to the instructions of the Mouse IL-1beta / IL-1F2 Quantikine HS ELISA Kit (R&D Systems, MHSLB00) and Mouse IL-1beta / IL-1F2 DuoSet ELISA (R&D Systems, DY401-05) kits.

[0163] The above experimental results are detailed in the table below.

[0164] 5. Experimental Results

[0165] The biological activities of the compounds of the present invention were determined by the above experiments. All compounds had different degrees of inhibitory effects on NLRP3. The results are shown in the following table. IC values ​​provided by compounds designated as "A" for NLRP3 inhibitory activity 50 Value is IC 50 ≤20 μM; compounds with activity designated as “B” provided IC 50 The value is 20μM<IC 50 ≤50 μM; compounds with activity designated as “C” provided IC 50 The value is 50μM<IC 50 ≤100 μM; compounds with activity designated as “D” provided IC 50 Value is IC 50 >100μM.

[0166]

[0167] The experimental results showed that the above flavonoid compounds can inhibit the activation of NLRP3 inflammasome.

[0168] Example 8. Screening of isopentenyl substitution conditions on benzene ring

[0169] Based on the importance of isopentenyl in flavonoid natural products and the difficulty in synthesis, the present invention screened the conditions for the substitution of isopentenyl on the benzene ring, and the conditions and results are as follows:

[0170]

[0171]

[0172]

[0173] The optimal conditions are potassium hydroxide as base, ethyl acetate as solvent, and 70 degrees Celsius.

[0174] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description should be regarded as illustrative rather than restrictive.

[0175] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of the compound represented by formula I or its tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt in the preparation of NLRP3 inhibitors, Wherein, X1 is selected from O or S; X2 is selected from O, S or NR 11 ; R 11 Selected from the following group: H, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl; R1, R2, R3, R4, R5 and R6 are independently selected from the following groups: H, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-10 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl, halogen, amino, nitro; or, R2 and R3 or R3 and R4 together with the carbon atoms to which they are attached form a 3-6 membered ring structure; n is an integer from 1 to 5.

2. The use according to claim 1, characterized in that Wherein, X1 is selected from O; X2 is selected from O or S; R1 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, halogen, amino, nitro; R2, R3 and R4 are independently selected from the following groups: H, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-10 alkenyl; or, R2 and R3 or R3 and R4 together with the carbon atoms to which they are attached form a 5- or 6-membered cyclic structure containing 1-3 heteroatoms selected from O, N or S or containing alkenyl; R5 is selected from the group consisting of H, hydroxyl; R6 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy; n is an integer from 1 to 3.

3. The use according to claim 2, characterized in that Wherein, X1 is selected from O; X2 is selected from O; R1 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy; R2, R3 and R4 are independently selected from the following groups: H, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-10 alkenyl; R5 is selected from the group consisting of H, hydroxyl; R6 is selected from the group consisting of H, hydroxy, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy; n is an integer from 1 to 3.

4. The use according to claim 3, characterized in that At most one of R2, R3 and R4 is H.

5. The use according to claim 1, characterized in that The compound represented by formula I is a compound selected from the following group:

6. The use according to claim 5, characterized in that The compound represented by formula I is a compound selected from the following group:

7. The use according to claim 6, characterized in that The compound represented by formula I is a compound selected from the following group:

8. The use according to any one of claims 1 to 7, characterized in that The NLRP3 inhibitor is a drug used to treat NLRP3-related diseases.

9. The use according to claim 8, characterized in that The NLRP3-related diseases are: gout, enteritis, hepatitis, arthritis, type II diabetes, and neurodegenerative diseases.

10. A method for synthesizing an isopentenyl-modified flavonoid compound, characterized in that: The method comprises reacting the flavonoid compound with isopentenyl halide and inorganic base in an organic solvent to obtain the isopentenyl-modified flavonoid compound.

Images