Compound for inhibiting IL-1beta as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of anti-inflammatory pharmacy, and relates to a compound for inhibiting IL-1beta as well as a preparation method and application thereof, and the structural formula of the compound for inhibiting IL-1beta is shown as a formula I or a formula II. The compound for inhibiting the IL-1beta or the pharmaceutically acceptable salt thereof has the activity of inhibiting the IL-1beta, does not generate cytotoxicity, and can be used for preparing medicines for treating diseases caused by high expression of the IL-1beta; the invention develops a method for rapidly constructing a series of compounds for inhibiting IL-1beta from the raw materials which are simple and easy to obtain, and the anti-inflammatory potential of the Disprocochleroids A-C derivatives can be further explored. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-inflammatory medicine, and in particular relates to a compound for inhibiting IL-1β, a preparation method and an application thereof. Background Art

[0002] Ganoderma lucidum fungus is widely used as a precious traditional Chinese medicine. In traditional Chinese medicine, it can enhance human resistance, prevent and fight cancer, protect the liver and detoxify, reduce the three highs, and improve the cardiovascular system. It has a good conditioning effect on long-term insomnia, neurasthenia, gastrointestinal diseases, diabetes, chronic pneumonia, bronchitis, asthma, kidney deficiency, etc. in the middle-aged and elderly. The active ingredients in Ganoderma lucidum have significant medicinal properties. So far, more than 680 kinds of Ganoderma lucidum terpenoid natural products have been isolated from this fungal family. (±)-Dispirocochlearoids AC are three non-polyketide terpenoid natural products isolated from Ganoderma lucidum by Cheng Yongxian's research group at Shenzhen University in 2020. They belong to the dimeric terpenoid class. In terms of biological activity, (-)-Dispirocochlearoid B and its analogues have the effects of inhibiting COX-2 and anti-inflammatory both in vitro and in vivo. In vitro results showed that (-)-Dispirocochlearoid B has good anti-inflammatory activity, IC 50 =386nM; selective for COX-2 over COX-1 (IC 50 =29.621μM); in vitro cytotoxicity tests showed no cytotoxicity. Preliminary in vitro mechanism studies showed that the mRNA level and protein expression of proinflammatory factors IL-6 and IL-1β can be inhibited in the lipopolysaccharide (LPS)-induced macrophage polarization model. In vivo studies confirmed that (-)-Dispirocochlearoid B can significantly inhibit inflammation in mice with LPS-induced acute lung injury (ALI) animal model. (-)-Dispirocochlearoid B can inhibit the expression of COX-2 in the lung tissue of ALI mice and reduce lung damage. This type of natural product contains a unique structure of a 6 / 6 / 5 / 6 / 6 / 6 six-ring common skeleton with two consecutive spirocyclic chiral centers, which has a very novel natural product skeleton. In addition, we believe that Dispirocochlearoids have potential anti-inflammatory research value, and their anti-inflammatory activity has a lot of room for improvement.

[0003] Inflammation is the body's defensive response to pathogen invasion, physical damage, etc. Moderate inflammation is conducive to the removal of pathogens and repair of damaged tissues, while excessive, uncontrolled inflammation will further aggravate physical damage and even lead to death. The inflammatory process is inseparable from the participation of various inflammatory mediators such as cytokines. IL-1β is a typical pro-inflammatory cytokine that can induce the release of inflammatory cytokines such as IL-6 and TNF-α and stimulate T cell activation, which can stimulate local and systemic inflammatory responses.

[0004] Interleukin 1β (IL-1β) is also called leukocyte pyogen, leukocyte endogenous mediator, mononuclear cytokine, lymphocyte activation factor, etc. Human IL-1β is a cytokine protein encoded by the IL-1β gene. The expression (synthesis, processing and secretion) of L-1β is regulated by dual signals. The initial signal induces cells to express pro-IL-1β without biological activity, and the activation signal activates the inflammasome. Subsequently, mature Caspase-1 cuts pro-IL-1β into biologically active IL-1β, which is finally secreted outside the cell through different pathways to exert its effect.

[0005] IL-1β was first cloned in the 1980s and was soon recognized as a key factor in regulating the inflammatory process. IL-1β is a typical proinflammatory cytokine that is essential for the host's defense response to infection and injury. It is expressed in various tissues and cells, especially in macrophages in lymphoid organs, including the thymus, spleen, lymph nodes, and bone marrow. IL-1β is also expressed in tissue macrophages in non-lymphoid organs such as the lungs, digestive tract, and liver.

[0006] IL-1β is considered a typical multifunctional cytokine that affects almost all cell types, either acting alone or in combination with other cytokines. IL-1β is essential for cellular defense and tissue repair of almost all tissues and is associated with pain, inflammation, and autoimmunity. IL-1β is also involved in neuroprotection, tissue remodeling, and repair. Due to the central role of IL-1β in the inflammatory process, human IL-1β has been associated with a variety of inflammatory diseases, such as rheumatoid arthritis, acute liver injury, acute lung injury, gout, periodic fever, and neuroinflammation. Recently, hIL-1β has also been linked to the pathogenesis of atherosclerosis and the progression of cancer.

[0007] While approaches based on anti-hIL-1β antibodies have demonstrated substantial benefits for patients, oral IL-1β inhibitors could target a wider range of diseases. In particular, increasing evidence suggests the contribution of inflammasomes to neuroinflammation, which cannot be well studied with antibody tools due to the limited passage of anti-hIL-1β antibodies through the blood-brain barrier. Therefore, the development of IL-1β small molecule compounds has broad research prospects for the treatment of related inflammatory diseases. Summary of the invention

[0008] In view of the deficiencies of the above-mentioned prior art, the present invention starts from the natural product Dispirocochlearoids, simplifies the structure of this type of novel natural product, conducts innovative research on synthetic methods and performs structural diversity transformation to obtain a series of Dispirocochlearoids derivatives of natural products that inhibit IL-1β, which have good anti-inflammatory effects and no toxic side effects. The specific technical scheme is as follows:

[0009] The first object of the present invention is to provide a compound for inhibiting IL-1β, the structural formula of which is shown in Formula I or Formula II:

[0010]

[0011] Wherein, ring A is phenyl or other five-membered or six-membered heterocyclic ring;

[0012] R 1 and R 2 Each independently, R1 is independently selected from H, 3-CH3, 4-CH3, 5-CH3, 6-CH3, 5-OMOM, 4-OMe, 4-F, 4-Cl, 4-Br, 4-I, 4-OMOM, R 2 H; or R 1 and R 2 for

[0013] R 3 independently selected from OMOM, OMe, OH, H, R 4 independently selected from OMe and OEt;

[0014] R 5 and R 6 for

[0015] R 7 is 4-methylpentyl-3-enyl, methyl, ethyl, propyl, isopropyl, isobutyl, cyclopentyl, cyclohexyl, phenyl or trifluoromethyl.

[0016] Furthermore, the compound for inhibiting IL-1β is one of the following structures:

[0017]

[0018]

[0019] The above-mentioned compound for inhibiting IL-1β is a compound of formula I, wherein the A ring is a phenyl ring or other five-membered or six-membered heterocyclic ring; 1 and R2 Independently, R 1 independently selected from H, 3-CH3, 4-CH3, 5-CH3, 6-CH3, 5-OMOM, 4-OMe, 4-F, 4-Cl, 4-Br, 4-I, 4-OMOM, R 2 H; R 3 and R 4 Independently, R 3 independently selected from OMOM, OMe, OH, H, R 4 independently selected from OMe, OEt; R 5 and R 6 for R 7 The preferred structure is 4-methylpentyl-3-enyl, methyl, ethyl, propyl, isopropyl, isobutyl, cyclopentyl, cyclohexyl, phenyl or trifluoromethyl.

[0020] Furthermore, the compound for inhibiting IL-1β has the following structure:

[0021]

[0022] The above-mentioned compound for inhibiting IL-1β is a compound of formula I, wherein the A ring is a phenyl ring or other five-membered or six-membered heterocyclic ring; 1 and R 2 for R 3 and R 4 Independently, R 3 independently selected from OMOM, OMe, OH, H, R 4 independently selected from OMe, OEt; R 5 and R 6 for R 7 The preferred structure is 4-methylpentyl-3-enyl, methyl, ethyl, propyl, isopropyl, isobutyl, cyclopentyl, cyclohexyl, phenyl or trifluoromethyl. Wherein, ring A is phenyl; R 3 For OMOM, R 4 OMe; R 5 and R 6 for R 7 is 4-methylpentyl-3-enyl. Further, the compound for inhibiting IL-1β is one of the following structures:

[0023]

[0024]

[0025] The above-mentioned compound for inhibiting IL-1β is of formula II, wherein ring A is phenyl or other five-membered or six-membered heterocyclic ring; 1 and R 2 Independently, R 1 independently selected from H, 3-CH3, 4-CH3, 5-CH3, 6-CH3, 5-OMOM, 4-OMe, 4-F, 4-Cl, 4-Br, 4-I, 4-OMOM, R 2 H; R 5 and R 6 for R 7 The preferred structure is 4-methylpentyl-3-enyl, methyl, ethyl, propyl, isopropyl, isobutyl, cyclopentyl, cyclohexyl, phenyl or trifluoromethyl.

[0026] The second object of the present invention is to provide a method for preparing a compound for inhibiting IL-1β, wherein the compound is as shown in formula I, starting from the compound of formula S1, S1 reacts with dimethyl methyl phosphate under strong alkaline conditions to obtain the corresponding phosphate S2, S2 undergoes Horner-Wadsworth-Emmons reaction to obtain the corresponding (Z)-unsaturated γ-keto acid ester S4; then starting from S4 and S5, an intermediate Z1 is obtained by Aldol condensation, and then cyclization is performed to obtain the compound shown in formula I, and the reaction equation is as follows:

[0027]

[0028] The present invention starts from the simple and readily available raw material S1. 3 When it is a hydroxyl group, the hydroxyl group is protected with bromomethyl methyl ether, and S1 reacts with dimethyl methyl phosphate under strong alkaline conditions to obtain the corresponding phosphate S2, and S2 undergoes a Horner-Wadsworth-Emmons reaction to obtain the corresponding (Z)-unsaturated γ-keto acid ester S4. Then, starting from S4 and S5, a series of compounds for inhibiting IL-1β as shown in Formula I can be successfully constructed through Aldol condensation / cyclization reactions, i.e., derivatives of Dispirocochlearoids AC, and the anti-inflammatory potential of these Dispirocochlearoids AC derivatives can be further explored.

[0029] Furthermore, the preparation method of the compound for inhibiting IL-1β, the compound is as shown in Formula II, which is obtained by deprotecting the compound shown in Formula I to obtain the intermediate state Z2, and then cyclizing to obtain the compound shown in Formula II with a tetracyclic skeleton derivative product having two consecutive spiro rings. The reaction equation is as follows:

[0030]

[0031] R 3 Independently selected from OMOM, OMe, OH.

[0032] The present invention can further react the compound shown in formula I to quickly construct a series of compounds for inhibiting IL-1β as shown in formula II, i.e., derivatives of Dispirocochlearoids AC. A novel synthetic method for quickly constructing a tetracyclic skeleton derivative with two consecutive spiro rings in two steps by connecting Aldol condensation / cyclization / deprotection / cyclization cascade reactions is developed, and the anti-inflammatory potential of these Dispirocochlearoids AC derivatives can be further explored.

[0033] Furthermore, the method for preparing the compound as shown in Formula I comprises the following steps:

[0034] Under nitrogen protection, S1 (1.0 equiv) and dimethyl methylphosphonate (DMMP) (1.0-2.0 equiv) were dissolved in anhydrous tetrahydrofuran, and LDA (2 mol / L, 1.1-4.0 equiv) was slowly added at -40 to 0°C; the mixture was stirred at this temperature for 1.5 to 3 hours; the reaction was quenched with saturated NH4Cl, and extracted with ethyl acetate three times; the organic phases were combined, and the combined organic layers were washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure; the crude product was purified by column chromatography to obtain intermediate S2;

[0035] The intermediate S2 (1.0 equiv) and α-ketoester S3 (1.0-2.0 equiv) were dissolved in tetrahydrofuran and stirred at -30 to 10°C; then sodium tert-butoxide (1.0-3.0 equiv) was added at the same temperature; after the reaction was completed, the reaction was quenched with saturated NH4Cl, and extracted with ethyl acetate three times; the organic phases were combined, and the combined organic layers were washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure; the crude product was purified by column chromatography to obtain the intermediate S4;

[0036] Under nitrogen protection, S5 (1.0-3.0 equiv) was dissolved in tetrahydrofuran at -80 to -50°C, and then added dropwise to a tetrahydrofuran solution of lithium diisopropylamide (LDA) (2 mol / L, 1.1-3.3 equiv), and then S4 (1.0 equiv) was added dropwise. The temperature was slowly raised to room temperature. After the reaction of S4 was completed, a saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (EA) three times. The EA layers were combined, washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the organic phase was concentrated and subjected to silica gel column chromatography to obtain the compound of formula I.

[0037] Furthermore, the method for preparing the compound as shown in Formula II comprises the following steps:

[0038] The compound represented by formula I (1.0 equiv) is dissolved in toluene, and after catalytic reaction with p-toluenesulfonic acid (0.1-1.0 equiv) at 50-70° C., the compound represented by formula II is directly subjected to silica gel column chromatography.

[0039] The third object of the present invention is to provide a pharmaceutical composition comprising the compound for inhibiting IL-1β (compound of formula I or formula II) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0040] The pharmaceutically acceptable salt of the present invention is a salt obtained by reacting the compound for inhibiting IL-1β (compound of formula I or formula II) with an inorganic base or an organic base compound. The salt obtained by reacting the compound for inhibiting IL-1β (compound of formula I or formula II) with an inorganic base compound includes but is not limited to: aluminum salt, ammonium salt, calcium salt, copper salt, iron salt, ferrous salt, lithium salt, magnesium salt, manganic salt, manganous salt, potassium salt, sodium salt, zinc salt, etc., preferably ammonium salt, calcium salt, magnesium salt, potassium salt and sodium salt. The compound for inhibiting IL-1β (compound of formula I or formula II) reacts with an organic base compound to obtain a pharmaceutically acceptable salt of an organic non-toxic base, including but not limited to salts of primary amines, secondary amines and tertiary amines, and substituted amines include naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, guanidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0041] "Pharmaceutically acceptable carrier" refers 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 active ingredient of the present invention (the compound for inhibiting IL-1β or its pharmaceutically acceptable salt) and with each other without significantly reducing the efficacy of the active ingredient.

[0042] Furthermore, the pharmaceutically acceptable carrier is 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, wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0043] Furthermore, the pharmaceutical composition is administered orally in the form of capsules, tablets, granules, solutions, powders, granules or syrups, or is administered non-orally in the form of injections.

[0044] The compounds and pharmaceutical compositions of the present invention may be present in suitable solid or liquid carriers and suitable sterile devices for injection or infusion. The above preparations may be prepared by conventional pharmaceutical methods.

[0045] The fourth object of the present invention is to provide the use of the compound or its pharmaceutically acceptable salt or pharmaceutical composition, which uses include preparing IL-1β inhibitors; using as IL-1β inhibitors; and preparing drugs for treating related diseases caused by high expression of IL-1β.

[0046] Furthermore, the disease is rheumatoid arthritis, acute liver injury, acute lung injury, gout, periodic fever, neuroinflammation, etc.

[0047] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0048] 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.

[0049] The beneficial effects of the present invention are:

[0050] The compound for inhibiting IL-1β of the present invention or a pharmaceutically acceptable salt thereof has the activity of inhibiting IL-1β, can better inhibit IL-1β than celecoxib, shows good anti-inflammatory activity and does not produce cytotoxicity, can be used as an IL-1β inhibitor, or used to prepare a drug for treating diseases caused by high expression of IL-1β, wherein the diseases include rheumatoid arthritis, acute liver injury, acute lung injury, gout, periodic fever and neuroinflammation.

[0051] The present invention develops a method for rapidly constructing a series of compounds (compounds of formula I and formula II) for inhibiting IL-1β, namely, derivatives of Dispirocochlearoids AC, based on simple and readily available raw materials, and further exploring the anti-inflammatory potential of these Dispirocochlearoids AC derivatives. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0053] In the following examples, NMR was measured using an Avance NEO 600 instrument, and NMR calibration was: δH

[0054] 7.26ppm (CDCl3); reagents were mainly provided by Shanghai Chemical Reagent Company; TLC thin layer chromatography silica gel plates were produced by Shandong Yantai Huiyou Silica Gel Development Co., Ltd., model HSGF254; the normal phase column chromatography silica gel used for compound purification was produced by Shandong Qingdao Ocean Chemical Plant Branch, model 200-300 mesh.

[0055] Embodiment 1:

[0056] Preparation of compound 442-1 for inhibiting IL-1β:

[0057]

[0058] At 0°C, NaH (2.2 g, 52.8 mmol) was slowly added to a solution of stirred compound A1 (5.0 g, 33 mmol) in anhydrous THF (50 mL). The mixture was stirred at 0°C for 10 minutes. MOMBr (6.2 g, 49.5 mmol) was slowly added. The mixture was stirred at 0°C for 1 hour. The reaction was quenched with NH4Cl at 0°C and extracted 3 times with ethyl acetate. The organic phases were combined, the combined organic layers were washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate=10 / 1) to obtain intermediate A2.

[0059] Under nitrogen protection, intermediate A2 (2.0 g, 10.2 mmol) and dimethyl methylphosphonate (DMMP) (2.3 g, 18.4 mmol) were dissolved in anhydrous THF (50 mL), and LDA (2 mol / L, 18.4 mL 36.7 mmol) was slowly added at -30 ° C. The mixture was stirred at -30 ° C for 2 hours. The reaction was quenched with NH4Cl at -30 ° C and extracted with ethyl acetate 3 times. The organic phases were combined, and the combined organic layers were washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate) to obtain intermediate A3.

[0060] At 0°C, intermediate A3 (1.0 g, 3.5 mmol) and α-ketoester A4 (0.9 g, 5.2 mmol) were dissolved in THF (40 mL) and stirred. Then t-BuNa (0.7 g, 6.9 mmol) was added at the same temperature. After the reaction was completed, NH4Cl was quenched at 0°C and extracted with ethyl acetate 3 times. The organic phases were combined, and the combined organic layers were washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 15 / 1) to obtain intermediate A5.

[0061] Under nitrogen protection, methyl cyclohexanecarboxylate (0.9 g, 6.0 mmol) was dissolved in tetrahydrofuran at -78 °C and added dropwise to a tetrahydrofuran solution of LDA (2 mol / L, 3.3 mL 6.6 mmol), then a tetrahydrofuran solution of A5 (1.0 g, 3.0 mmol) was added dropwise, stirred at -78 °C for 30 min, and slowly warmed to room temperature. After the reaction of A5 was completed, a saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate 3 times. The organic phases were combined, and the combined organic layer was washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 15 / 1) to obtain 442-1. 1 HNMR(600MHz, CDCl3)δ7.98(t,J=1.4Hz,1H),7.57(dd,J=7.9,1.7Hz,1H),7.28-7.25(m,1H),7.07(dd,J=8.4,1 .2Hz,1H),7.00(ddd,J=8.2,7.3,1.2Hz,1H),5.22(d,J=6.6Hz,1H),5.08(d,J=6.6Hz,1H),5.06(ddt,J=6.9,5.4 ,1.5Hz,1H),3.64(s,3H),3.49(s,3H),2.33-2.19(m,4H),2.16-2.10(m,1H),2.05-1.99(m,1H),1.66-1.60(m, 5H),1.52(s,4H),1.46(td,J=13.2,4.0Hz,1H),1.28(d,J=3.6Hz,1H),1.21-1.14(m,1H),1.13-1.02(m,2H)ppm.

[0062] The following compounds were synthesized using the same preparation method as in Example 1, as shown in Table 1:

[0063] Table 1 Compounds synthesized by the same preparation method as Example 1

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Example 2

[0074] Preparation of compound 366 for inhibiting IL-1β:

[0075]

[0076] 442-1 (1000 mg, 2.26 mmol) was dissolved in 40 mL of toluene, and water and p-toluenesulfonic acid (43 mg, 0.23 mmol) were added to catalyze the reaction at 60°C. After the reaction of 442-1 was completed, 366 was obtained by direct silica gel column chromatography (petroleum ether / ethyl acetate = 15 / 1). 1 H NMR (600MHz, CDCl3) δ7.37 (ddd, J=8.5, 6.6, 2.3Hz, 1H), 7.13 (q, J=7.3Hz, 2H), 7.08(d,J=8.1Hz,1H),6.92(s,1H),5.02(t,J=7.2Hz,1H),2.39(q,J=7.6Hz,2H) ,2.27(q,J=8.4,7.9Hz,2H),1.84(q,J=16.9,14.5Hz,2H),1.77-1.61(m,8H),1. 57(s,4H),1.26(td,J=13.2,4.5Hz,1H),1.10(tdd,J=13.0,9.0,3.6Hz,1H)ppm.

[0077] The following compounds were synthesized using the same preparation method as in Example 2, as shown in Table 2:

[0078] Table 2 Compounds synthesized by the same preparation method as Example 2

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] IL-1β anti-inflammatory activity test of compounds:

[0085] 1. Sources

[0086] Male C57BL / 6J mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and kept in an SPF animal room with free access to water and food. All animal experiments were carried out in accordance with the relevant regulations of the national laboratory animal administration department and approved by the laboratory animal ethics committee of the *.

[0087] 2. Experimental Procedure

[0088] Isolation and culture of bone marrow-derived macrophages

[0089] C57BL / 6J mice around 6-8 weeks old were killed by dislocating the cervical vertebrae and then placed in 75% alcohol for 5 minutes for disinfection. The femur was separated, the muscle tissue was stripped off, and it was placed in sterile PBS and transferred to a biosafety cabinet. Cut the two ends of the femur, use a 10mL syringe to absorb PBS to blow out the bone marrow, and blow and wash repeatedly until the leg bone turns white. Use a pipette to repeatedly blow and beat until a single cell suspension is formed, transfer it to a 50mL centrifuge tube, and centrifuge at 500g for 5 minutes. Discard the supernatant, add 1mL of red blood cell lysis buffer to resuspend the cells, and let it stand for 5 minutes. Add 5mL of complete culture medium (HG DMEM+10% FBS) to stop the lysis, mix gently with a pipette, and centrifuge at 500g for 5 minutes. Discard the supernatant, add complete culture medium to resuspend, pass through a 40μm cell strainer, and transfer to a 50mL centrifuge tube. Cell count, 1×10 6 Cells / mL were inoculated into a 10cm dish. On the second day of culture, 10mL of complete culture medium was added. On the fifth day, the medium was changed halfway. Treatment can be performed on the seventh day. Macrophage colony stimulating factor (M-CSF, final concentration of 20ng / mL) should be added throughout the culture process for induction.

[0090] LPS induces BMDM differentiation

[0091] After BMDM cells matured, 5×10 5Cells / mL were inoculated into 12-well plates (1 mL / well) and placed in an incubator (37°C, 5% CO2) for overnight culture. After the cells adhered to the wall, drugs were treated at concentrations of 5, 10, and 20 μM. After 2 hours, LPS (10 ng / mL) was used for treatment. After 4 hours, protein samples were collected to measure the expression of IL-1β protein. After BMDM cells matured, 5×10 5 Cells / mL were inoculated in a 96-well plate (100 μL / well), placed in an incubator (37°C, 5% CO2) for overnight culture, and treated with synthetic natural product Dispirocochlearoids derivatives after the cells adhered to the wall, with an action concentration of 3 and 30 μM. After 2 hours, they were treated with LPS (10 ng / mL), and 4 hours later, adenosine triphosphatase (ATP) (5 mM) was treated. After 45 minutes, the cell supernatant was collected and the IL-1β content in the supernatant was determined. After the cell experiment was completed, the supernatant was collected, centrifuged at 4000 rpm / 4°C for 5 minutes, the cell precipitate was removed, and the supernatant was collected for detection. The detection was performed according to the determination method in the kit instructions. (MOUSE IL1 BETA KIT#62MIL1BPEG) The specific operation steps are as follows:

[0092] ① Thaw all reagents and samples on ice;

[0093] ② Preparation of standard products;

[0094] ③ Take equal volumes of IL-1βEμ antibody and IL-1βd2 antibody and mix them evenly to form the antibody working solution;

[0095] ④In a 384-well plate, add 2 μL of antibody working solution and 8 μL of standard / sample to each well;

[0096] ⑤Incubate at 4℃ overnight;

[0097] ⑥ Use Envision to read the fluorescence value at 620 / 655nm, use the four-parameter logistic curve to create a standard curve, and calculate the sample concentration.

[0098] The experimental results are shown in Table 3:

[0099] Table 3 Inhibition of pro-inflammatory factor IL-1β by compounds

[0100]

[0101]

[0102] Note: 0<*<40%, 40%≤**<80%, 80%≤***<100%

[0103] It can be seen from the inhibition of the proinflammatory factor IL-1β by the compounds in Table 3 that the compounds for inhibiting IL-1β of the present invention, i.e., the natural product Dispirocochlearoids derivatives, can effectively inhibit the proinflammatory factor IL-1β, and the activity of some compounds is better than celecoxib, indicating that the compounds prepared by the present invention have good anti-inflammatory activity and no cytotoxicity, showing the possibility of being used to treat related diseases caused by high expression of IL-1β, including rheumatoid arthritis, acute liver injury, acute lung injury, gout, periodic fever and neuroinflammation.

[0104] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound for inhibiting IL-1β, characterized in that The structural formula is shown in Formula I or Formula II: Wherein, ring A is phenyl or other five-membered or six-membered heterocyclic ring; R 1 and R 2 Independently, R 1 independently selected from H, 3-CH3, 4-CH3, 5-CH3, 6-CH3, 5-OMOM, 4-OMe, 4-F, 4-Cl, 4-Br, 4-I, 4-OMOM, R 2 H; or R 1 and R 2 for R 3 independently selected from OMOM, OMe, OH, H, R 4 independently selected from OMe and OEt; R 5 and R 6 for R 7 is 4-methylpentyl-3-enyl, methyl, ethyl, propyl, isopropyl, isobutyl, cyclopentyl, cyclohexyl, phenyl or trifluoromethyl.

2. The compound for inhibiting IL-1β according to claim 1, characterized in that One of the following structures:

3. A method for preparing a compound for inhibiting IL-1β as claimed in claim 1, characterized in that: The compound is as shown in formula I. Starting from compound S1, S1 reacts with dimethyl methyl phosphate under strong alkaline conditions to obtain the corresponding phosphate S2, and S2 undergoes Horner-Wadsworth-Emmons reaction to obtain the corresponding (Z)-unsaturated γ-keto acid ester S4; then starting from S4 and S5, an intermediate Z1 is obtained through Aldol condensation, and then cyclization is performed to obtain the compound shown in formula I. The reaction equation is as follows:

4. A method for preparing a compound for inhibiting IL-1β as claimed in claim 1, characterized in that: The compound is shown in Formula II, which is obtained by deprotecting the compound shown in Formula I to obtain the intermediate state Z2, and then cyclizing to obtain the compound shown in Formula II. The reaction equation is as follows: R 3 Independently selected from OMOM, OMe, OH.

5. The method for preparing a compound for inhibiting IL-1β according to claim 3, characterized in that: The steps include: Under nitrogen protection, S1 and dimethyl methylphosphonate DMMP are dissolved in anhydrous tetrahydrofuran, and lithium diisopropylamide LDA is slowly added at -40 to 0°C, and the mixture is stirred for 1.5 to 3 hours; the reaction is quenched with saturated NH4Cl, and extracted with ethyl acetate three times; the organic phases are combined, and the combined organic layers are washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent is evaporated under reduced pressure; the crude product is purified by column chromatography to obtain intermediate S2; The intermediate S2 and α-ketoester S3 were dissolved in tetrahydrofuran and stirred at -30 to 10°C; then sodium tert-butoxide was added at the same temperature; after the reaction was completed, the reaction was quenched with saturated NH4Cl, and extracted with ethyl acetate three times; the organic phases were combined, and the combined organic layer was washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure; the crude product was purified by column chromatography to obtain the intermediate S4; Under nitrogen protection, S5 was dissolved in tetrahydrofuran at -80 to -50°C, added dropwise to a tetrahydrofuran solution of lithium diisopropylamide LDA, and then S4 was added dropwise. The temperature was slowly raised to room temperature. After the reaction of S4 was completed, a saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with ethyl acetate EA for 3 times. The ethyl acetate EA layers were combined, washed with saturated sodium chloride, dried over anhydrous Na2SO4, and the organic phase was concentrated and subjected to silica gel column chromatography to obtain the compound of formula I.

6. The method for preparing a compound for inhibiting IL-1β according to claim 4, characterized in that: The steps include: The compound represented by formula I is dissolved in toluene, and the reaction is catalyzed by p-toluenesulfonic acid at 50-70°C, and then directly subjected to silica gel column chromatography to obtain the compound represented by formula II.

7. A pharmaceutical composition, characterized in that The invention comprises the compound for inhibiting IL-1β as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutically acceptable carrier is one or more of cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavoring agents, stabilizers, antioxidants, preservatives, or pyrogen-free water.

9. A use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7 or 8, characterized in that: The purposes described are: (1) For the preparation of IL-1β inhibitors; (2) Used as an IL-1β inhibitor; (3) Used for preparing drugs for treating diseases related to high expression of IL-1β.

10. The use according to claim 9, characterized in that: The diseases are rheumatoid arthritis, acute liver injury, acute lung injury, gout, periodic fever, and neuroinflammation.

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