NLRP3 inhibitor as well as preparation method and application thereof

Abstract

The invention relates to an NLRP3 inhibitor, a pharmaceutical composition and application of the NLRP3 inhibitor. Specifically, the invention provides a compound as shown in a formula 1, a pharmaceutical composition and application of the compound and the pharmaceutical composition in preparation of drugs for preventing or treating diseases related to abnormal activation of NLRP3 inflammasome. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to an NLRP3 inhibitor, a pharmaceutical composition and an application thereof in the preparation of a drug for preventing or treating diseases associated with abnormal activation of NLRP3 inflammasomes. Background Art

[0002] Inflammasomes are multimeric protein complexes that trigger inflammatory responses in response to exogenous pathogens or endogenous danger signals. NOD-like receptor pyrin domain-related protein 3 (NLRP3) is an inflammasome that has been extensively and deeply studied at this stage. It is mainly composed of three parts: the N-terminal pyridinium-rich domain (PYD) that plays an important role in inflammasome assembly, the central ATPase trimer domain (NACHT) that regulates protein oligomerization, and the C-terminal leucine-rich repeat domain (LRR) that serves as a signal sensor.

[0003] NLRP3 is expressed by a variety of cells, including neutrophils, macrophages, microglia, lymphocytes, epithelial cells, osteoblasts, neurons, and dendritic cells. Activation of the NLRP3 inflammasome can produce a large amount of interleukin-1β (IL-1β). IL-1β is an important proinflammatory cytokine released during the inflammatory response. NLRP3 forms an inflammasome complex by recruiting apoptosis-associated speck-like protein (ASC) and caspase-1. After oligomerization, ASC induces Caspase-1 to self-cleave and activate, which will cleave the precursors of IL1β and I-18 to produce the corresponding mature cytokines I-1β and I-18, inducing programmed cell death related to inflammation, namely cell pyroptosis.

[0004] Normal activation of NLRP3 inflammasomes helps the host resist infection by pathogenic microorganisms and maintain the stability of the body's internal environment. However, excessive activation of NLRP3 inflammasomes is closely related to the pathogenesis of human inflammatory diseases, autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, and tumors. Therefore, inhibiting abnormal activation of NLRP3 inflammasomes is a promising disease treatment strategy.

[0005] MCC950 is the earliest discovered NLRP3 inflammasome inhibitor. Studies have shown that MCC950 has therapeutic effects on central nervous system diseases, autoimmune diseases, cardiovascular diseases and metabolic diseases. Many clinical studies on MCC950 have also been carried out.

[0006] Chinese invention patent application CN113784957A discloses a class of NLRP3 inflammasome inhibitors, among which Example EX 004 compound has high NLRP3 inhibitory activity and can effectively inhibit the secretion of IL-1β.

[0007]

[0008] EX004

[0009] However, there are currently no NLRP3 inhibitors on the market, and the development of NLRP3 inhibitors is of great significance. Summary of the invention

[0010] The present invention provides a novel NLRP3 inhibitor and a preparation method thereof, and application of the inhibitor in the preparation of a drug for preventing or treating diseases associated with abnormal activation of NLRP3 inflammasomes.

[0011] The technical solution of the present invention is as follows: The present invention provides an NLRP3 inhibitor, specifically the compound 1 described below, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0012] 1

[0014] The present invention also provides a pharmaceutical composition, comprising any one of the above compounds, a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquids, granules, injections, or various sustained-release preparations. The pharmaceutical composition can be administered orally or parenterally (such as intravenously, subcutaneously, or topically). The dosage can be appropriately adjusted according to the patient's age, gender, and disease type, and the general daily dosage is about 1-200 mg.

[0015] The present invention also provides the use of the above-mentioned compound, its pharmaceutically acceptable salt or stereoisomer or pharmaceutical composition in the preparation of a drug for preventing or treating diseases related to abnormal activation of NLRP3 inflammasomes. The diseases are central nervous system diseases, autoimmune diseases, cardiovascular diseases or metabolic diseases, etc. The disease is further an immune disease, an inflammatory disease, an autoimmune disease, an autoinflammatory febrile syndrome, a chronic liver disease, a viral hepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis, alcoholic liver disease, inflammatory arthritis-related disorders, gout, chondrocalcinosis, osteoarthritis, rheumatoid arthritis, chronic joint disease, acute joint disease, kidney-related disease, hyperoxaluria, lupus nephritis, type I and type II diabetes, kidney disease, retinopathy, hypertensive kidney disease, hemodialysis-related inflammation, neuroinflammation-related disease, multiple sclerosis, brain infection, acute injury, neurodegenerative disease, Alzheimer's disease, stroke, cerebral hemorrhage, hypertension, atherosclerosis, peripheral arterial disease, acute heart failure, inflammatory skin disease, acne, wound healing and scar formation, asthma, sarcoidosis, age-related macular degeneration, colon cancer, lung cancer, myeloproliferative tumors, leukemia, myelodysplastic syndrome or myelofibrosis. DETAILED DESCRIPTION

[0016] Example 1 Synthesis of Compound 1 1

[0018] Synthesis route:

[0019] Step 1: Synthesis of compound 1-2 Compound 2-bromo-5-nitrophenol (30 g, 137.61 mmol, 1 eq) and benzyl bromide (24.71 g, 144.49 mmol, 17.16 mL, 1.05 eq) were added to acetonitrile (300 mL), and potassium carbonate (38.04 g, 275.22 mmol, 2 eq) was added at 25°C. The reaction solution was stirred at 80°C for 18 hours. TLC spot plate showed that the reactants were almost completely consumed. The reaction mixture was cooled to room temperature, filtered, washed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain a residue. The crude product was purified by slurrying with petroleum ether (100 mL) to obtain compound 1-2 (30 g) as a yellow solid. (ESI) m / z = 307.9[M+H] + .

[0020] Step 2: Synthesis of Compound 1-3 Compound 1-2 (700 mg, 1.96 mmol, 1 eq) was dissolved in anhydrous ethanol (200 mL) and water (50 mL), and then iron (16.31 g, 292.09 mmol, 6 eq) and ammonium chloride (15.62 g, 292.09 mmol, 6eq) were added, and the reaction solution was stirred at 40°C for 2 hours. LCMS showed that the reaction was complete, and the reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was diluted with water (200 mL), extracted with ethyl acetate (300 mL * 3), and the combined organic layer was washed with brine (300 mL * 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 1-3 (19 g, crude product) as a white oil. (ESI) m / z = 278.1 [M+H] + .

[0021] Step 3: Synthesis of Compound 1-4 Compound 1-3 (19 g, 68.31 mmol, 1 eq) was added to 6 M hydrochloric acid (2.46 g, 68.31 mmol, 20 mL, 1 eq) at -10°C. Sodium nitrite (9.43 g, 136.62 mmol, 2 eq) was then dissolved in water (50 mL) and slowly added to the reaction solution over 10 minutes and stirred for 1 hour. Tin dichloride dihydrate (46.24 g, 204.93 mmol, 3 eq) was then diluted with water (50 mL) at 0°C and added dropwise, and the reaction solution was stirred at 0°C for 2 hours. LCMS showed that the reaction was complete, and the mixture was concentrated under reduced pressure to obtain compound 1-4 (20 g, crude product) as a yellow oil. (ESI) m / z = 293.0[M+H] + .

[0022] Step 4: Synthesis of Compound 1-5 Compound 1-4 (20 g, 68.22 mmol, 1 eq) was dissolved in dichloromethane (200 mL), di-tert-butyl carbonate (14.89 g, 68.22 mmol, 15.67 mL, 1 eq) and triethylamine (13.81 g, 136.44 mmol, 18.99 mL, 2 eq) were added, and the reaction solution was stirred at 20 °C for 3 hours. LCMS showed that the reaction was complete, and the mixture was concentrated under reduced pressure to obtain a residue. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 0~25%) to obtain compound 1-5 (10 g, 24.17 mmol, yield 35.43%, purity 95.054%) as a yellow oil. (ESI) m / z = 337.1 [M+Ht-Bu]+ .

[0023] Step 5: Synthesis of Compound 1-6 Compound 1-5 (10 g, 25.43 mmol, 1 eq) was dissolved in dioxane (100 mL), and then [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium (1.86 g, 2.54 mmol, 0.1 eq), bis(triphenylphosphino)ferrocene (7.75 g, 30.51 mmol, 1.2 eq) and potassium acetate (4.99 g, 50.86 mmol, 2 eq) were added. After replacing with nitrogen three times, the reaction solution was stirred at 100°C for 12 hours. LCMS showed that the reaction was complete, and the reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by column chromatography (chromatographic column: Welch Ultimate XB-NH2 250*50*10um; mobile phase: [hexane-ethanol]; gradient: 10%-50% B, lasting 12 minutes) to obtain compound 1-6 (2.2 g, 4.84mmol, yield 19.04%, purity 96.9%) as a yellow oil. (ESI) m / z = 441.1 [M+H] + . 1 H NMR (400MHz, CHLOROFORM-d) δ 7.66 - 7.59 (m, 3H), 7.42 - 7.34 (m, 2H), 7.30 (d, J =7.2 Hz, 1H), 6.51 - 6.24 (m, 3H), 5.97 - 5.68 (m, 1H), 5.09 (s, 2H), 1.47 (brs, 9H), 1.36 (s, 12H).

[0024] Step 6: Synthesis of Compound 1-7 Compound 1-6 (1.83 g, 4.15 mmol, 2 eq) and compound (R)-6-chloro-5-methyl-N-(1-methylpiperidin-3-yl)pyridazine-3-amine (500 mg, 2.08 mmol, 1 eq) were added to a reaction flask, followed by (2-dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (175.81 mg, 207.70 μmol, 0.1 eq) and cesium carbonate (2.03 g, 6.23 mmol, 3 eq), and finally dioxane (20 mL) and water (4 mL). After replacing the atmosphere with nitrogen three times, the reaction solution was stirred at 90°C for 12 hours. LCMS showed that the reaction was complete, and the reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by HPLC (0.1% formic acid) and lyophilized to obtain compound 1-7 (300 mg, 561.07 μmol, yield 27.01%, purity 97.504%) as a brown oil. (ESI) m / z = 519.3 [M+H] + .

[0025] Step 7: Synthesis of Compound 1-8 Compound 1-7 (300 mg, 578.43 μmol, 1 eq) was dissolved in dichloromethane (10 mL), and then trifluoroacetic acid (3.07 g, 26.93 mmol, 2.00 mL, 46.55 eq) was added at 0°C. The reaction solution was stirred at 25°C for 1 hour. LCMS showed that the reaction was complete, and the mixture was concentrated under reduced pressure to obtain compound 1-8 (240 mg, crude product) as a yellow oil. (ESI) m / z = 419.2 [M+H] + .

[0026] Step 8: Synthesis of Compound 1-9 Compound 1-8 (240 mg, 573.43 μmol, 1 eq) was dissolved in acetonitrile (3 mL), and then ethyl acetoacetate (89.55 mg, 688.12 μmol, 87.11 μL, 1.2 eq) was added, and the reaction solution was stirred at 60°C for 1 hour. LCMS showed that the reaction was complete, and the residue was concentrated under reduced pressure. The crude product was purified by reverse phase (0.1% formic acid) and lyophilized to obtain compound 1-9 (220 mg, 449.45 μmol, yield 78.38%, purity 99.451%) as a yellow solid. (ESI) m / z = 485.2 [M+H] + .

[0027] Step 9: Synthesis of Compound 1 Compound 1-9 (100 mg, 206.36 μmol, 1 eq) was dissolved in hexafluoroisopropanol (2 mL), and then methanesulfonic acid (99.16 mg, 1.03 mmol, 73.73 μL, 5 eq) was added at 0°C. The reaction solution was stirred at 40°C for 12 hours. LCMS showed that the reaction was complete. Saturated sodium bicarbonate solution was added to the reaction mixture at 0°C until pH = 7, and then filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by high performance liquid chromatography, column: Waters Xbridge 150*25mm* 5um, mobile phase: [water (ammonium bicarbonate)-acetonitrile], gradient: 1%-25% B, 10 min to obtain the pure product, and lyophilized to obtain compound 1 (6 mg, 15.15 μmol, yield 7.34%, purity 99.602%) as a white solid. (ESI) m / z = 395.1 [M+H] + . 1 HNMR (400 MHz, CHLOROFORM-d) δ 12.44 - 11.54 (m, 1H), 7.62 (d, J = 2.2 Hz,1H), 7.56 - 7.49 (m, 1H), 7.47 - 7.42 (m, 1H), 6.66 - 6.62 (m, 1H), 5.34 -5.14 (m, 1H), 4.26 - 4.07 (m, 1H), 3.53 - 3.39 (m, 2H), 2.64 - 2.49 (m, 2H), 2.45 (s, 3H), 2.34 - 2.24 (m, 4H), 2.21 (s, 3H), 1.87 - 1.61 (m, 5H).

[0028] Test Example 1 Determination of the inhibitory activity of the compounds of the present invention on NLRP3 1.1 Test materials

[0029] 1.2 Test equipment

[0030] 1.3 Test methods a) BMDM were cultured in 6-well plates in RPMI 1640 medium containing M-CSF at 37°C in a 5% CO2 incubator.

[0031] b) Wash the BMDM cells with PBS, re-seed the BMDM cells into a 96-well plate, and culture them in a 37°C, 5% CO2 incubator for 24 h.

[0032] c) LPS was added to BMDM cells and incubated at 37°C and 5% CO2 for 3 h.

[0033] d) Add 5 μL of compound (final 0.1% DMSO) to cells and incubate cells at 37°C, 5% CO2.

[0034] e) Stimulate the cells by adding 2 uL of Nigericin and incubate at 37°C, 5% CO2.

[0035] f) Centrifuge the 96-well plate and transfer the supernatant to a new 96-well plate.

[0036] g) Detect IL-1β using the IL-1β Elisa kit.

[0037] h) Read the OD value at 450 nm on an Envision microplate reader.

[0038] i) Fit the curve based on OD value and concentration and calculate IC 50 Value, IC 50 is the concentration of compound corresponding to 50% inhibition rate.

[0039] 1.4 Test results Table 1 IC of compounds inhibiting IL-1β expression 50 value

[0040] * CN113784957A Example EX004 Compound Conclusion: Compared with MCC950 and EX004, compound 1 of the present invention can significantly enhance the inhibitory activity against NLRP3 and effectively reduce the secretion of IL-1β.

Claims

1. A compound represented by Formula 1, a pharmaceutically acceptable salt or a stereoisomer thereof, 1。 2. A pharmaceutical composition comprising the compound according to claim 1, a pharmaceutically acceptable salt or stereoisomer thereof and a pharmaceutically acceptable carrier.

3. Use of the compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, or the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing or treating diseases associated with abnormal activation of NLRP3 inflammasome.

4. The use according to claim 3, wherein the disease is a central nervous system disease, an autoimmune disease, a cardiovascular disease or a metabolic disease.

5. The use according to claim 3, wherein the disease is an inflammatory disease, autoinflammatory fever syndrome, chronic liver disease, viral hepatitis, nonalcoholic fatty hepatitis, alcoholic fatty hepatitis, alcoholic liver disease, inflammatory arthritis-related disorders, gout, chondrocalcinosis, osteoarthritis, rheumatoid arthritis, chronic joint disease, acute joint disease, kidney-related disease, hyperoxaluria, lupus nephritis, type I and type II diabetes, kidney disease, retinopathy, hypertensive nephropathy, hemodialysis-related inflammation, neuroinflammation-related diseases, multiple sclerosis, brain infection, acute injury, neurodegenerative disease, Alzheimer's disease, stroke, cerebral hemorrhage, hypertension, atherosclerosis, peripheral arterial disease, acute heart failure, inflammatory skin disease, acne, wound healing and scar formation, asthma, sarcoidosis, age-related macular degeneration, colon cancer, lung cancer, myeloproliferative tumors, leukemia, myelodysplastic syndrome or myelofibrosis.

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