An NLRP3 inhibitor, its preparation method and application

Abstract

The present invention relates to an NLRP3 inhibitor, a pharmaceutical composition and their applications. Specifically, it provides a compound represented by Formula 1, a pharmaceutical composition and their applications in the preparation of a medicament for preventing or treating diseases associated with abnormal activation of NLRP3 inflammasome.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and relates to an NLRP3 inhibitor, a pharmaceutical composition, and their use in the preparation of a medicament for preventing or treating diseases associated with abnormal activation of the NLRP3 inflammasome. Background Art

[0002] The inflammasome is a multimeric protein complex that triggers an inflammatory response in response to exogenous pathogens or endogenous danger signals. NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) is an inflammasome that has been widely and deeply studied at present. It mainly consists of three parts: an N-terminal pyridine-rich domain (PYD) that plays an important role in inflammasome assembly, a central ATPase trimer domain (NACHT) that regulates protein oligomerization, and a 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, etc. Activation of the NLRP3 inflammasome can produce a large amount of interleukin-1β (IL-1β). IL-1β is an important pro-inflammatory cytokine released during the inflammatory response. NLRP3 forms an inflammasome complex by recruiting apoptosis-associated speck-like protein (ASC) and caspase-1. After ASC oligomerizes and induces self-cleavage and activation of caspase-1, it cleaves the precursors of IL-1β and I-18 to produce the corresponding mature cytokines I-1β and I-18, inducing programmed death of inflammation-related cells, that is, pyroptosis.

[0004] Normal activation of the NLRP3 inflammasome is beneficial for the host to resist pathogenic microorganism infections and maintain the stability of the internal environment of the body. However, over-activation of the NLRP3 inflammasome is closely related to the pathogenesis of human inflammatory, autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, and tumors, etc. Therefore, inhibiting the abnormal activation of the NLRP3 inflammasome is a promising disease treatment strategy.

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

[0006] Chinese Patent Application CN113784957A discloses a class of NLRP3 inflammasome inhibitors. Among them, the compound of Example EX 004 has high NLRP3 inhibitory activity and can effectively inhibit the secretion of IL-1β.

[0007]

[0008] EX004

[0009] However, there is currently no NLRP3 inhibitor 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, its preparation method, and its application in the preparation of drugs for preventing or treating diseases related to abnormal activation of the NLRP3 inflammasome.

[0011] The technical solution of the present invention is as follows:

[0012] The present invention provides an NLRP3 inhibitor, specifically Compound 1 described below, its pharmaceutically acceptable salts or stereoisomers.

[0013] 1

[0015] The present invention also provides a pharmaceutical composition, comprising any one of the above compounds, its pharmaceutically acceptable salts or stereoisomers, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquid preparations, granules, injections, or various sustained-release or controlled-release preparations, etc. The pharmaceutical composition can be administered orally or parenterally (such as intravenously, subcutaneously, or locally, etc.). The dosage can be appropriately adjusted according to the age, gender, and disease type of the patient, and the general daily dosage is about 1 - 200 mg.

[0016] 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 related diseases caused by abnormal activation of NLRP3 inflammasome. The diseases include central nervous system diseases, autoimmune diseases, cardiovascular diseases or metabolic diseases, etc. The diseases further include immune diseases, inflammatory diseases, autoimmune diseases, autoinflammatory fever syndromes, chronic liver diseases, viral hepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis, alcoholic liver diseases, inflammatory arthritis-related disorders, gout, chondrocalcinosis, osteoarthritis, rheumatoid arthritis, chronic arthropathy, acute arthropathy, kidney-related diseases, hyperoxaluria, lupus nephritis, type I and type II diabetes, nephropathy, retinopathy, hypertensive nephropathy, hemodialysis-related inflammation, neuroinflammation-related diseases, multiple sclerosis, brain infections, acute injuries, neurodegenerative diseases, Alzheimer's disease, stroke, intracerebral hemorrhage, hypertension, atherosclerosis, peripheral arterial diseases, acute heart failure, inflammatory skin diseases, acne, wound healing and scar formation, asthma, sarcoidosis, age-related macular degeneration, colon cancer, lung cancer, myeloproliferative neoplasms, leukemia, myelodysplastic syndromes or myelofibrosis. Detailed implementation manners

[0017] Example 1 Synthesis of Compound 1

[0018] 1

[0020] Synthesis route:

[0021]

[0022] Step 1: Synthesis of Compound 1-2

[0023] Add 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) to acetonitrile (300 mL), then add potassium carbonate (38.04 g, 275.22 mmol, 2 eq) at 25 °C, and stir the reaction solution at 80 °C for 18 hours. TLC plate monitoring shows that the reactants are basically consumed completely. Cool the reaction mixture to room temperature, filter, wash with ethyl acetate, and concentrate the filtrate under reduced pressure to obtain a residue. Purify the crude product by slurrying with petroleum ether (100 mL) to obtain compound 1-2 (30 g), which is a yellow solid. (ESI) m / z = 307.9 [M+H] + .

[0024] Step 2: Synthesis of Compound 1-3

[0025] Compound 1-2 (700 mg, 1.96 mmol, 1 eq) was dissolved in absolute 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, 6 eq) were added. The reaction mixture was stirred at 40 °C for 2 h. LCMS showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was diluted with water (200 mL) and extracted with ethyl acetate (300 mL × 3). The combined organic layers were 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), which was a white oil. (ESI) m / z = 278.1 [M+H] + 。

[0026] Step 3: Synthesis of Compound 1-4

[0027] 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. Then sodium nitrite (9.43 g, 136.62 mmol, 2 eq) was dissolved in water (50 mL) and slowly added dropwise to the reaction mixture over 10 min, and the mixture was stirred for 1 h. Then, at 0 °C, stannous dichloride dihydrate (46.24 g, 204.93 mmol, 3 eq) diluted with water (50 mL) was added dropwise, and the reaction mixture was stirred at 0 °C for 2 h. LCMS showed that the reaction was complete. The mixture was concentrated under reduced pressure to obtain Compound 1-4 (20 g, crude product), which was a yellow oil. (ESI) m / z = 293.0 [M+H] + 。

[0028] Step 4: Synthesis of Compound 1-5

[0029] Compound 1-4 (20 g, 68.22 mmol, 1 eq) was dissolved in dichloromethane (200 mL), and di-tert-butyl dicarbonate (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. The reaction mixture was stirred at 20 °C for 3 hours. LCMS showed that the reaction was complete. The mixture was concentrated under reduced pressure to obtain a residue. The crude product was purified by column chromatography (silica gel, 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+H-t-Bu] + 。

[0030] Step 5: Synthesis of Compound 1-6

[0031] 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(pinacolato)diboron (7.75 g, 30.51 mmol, 1.2 eq) and potassium acetate (4.99 g, 50.86 mmol, 2 eq) were added. After purging with nitrogen three times, the reaction mixture was stirred at 100 °C for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by column chromatography (column: Welch Ultimate XB-NH 2 250*50*10um; mobile phase: [hexane-ethanol]; gradient: 10%-50% B for 12 minutes) to obtain Compound 1-6 (2.2 g, 4.84 mmol, yield 19.04%, purity 96.9%) as a yellow oil. (ESI) m / z = 441.1 [M+H] + 。 1 H NMR (400 MHz, 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).

[0032] Step 6: Synthesis of Compound 1-7

[0033] Compound 1-6 (1.83 g, 4.15 mmol, 2 eq) and compound (R)-6-chloro-5-methyl-N-(1-methylpiperidin-3-yl)pyridazin-3-amine (500 mg, 2.08 mmol, 1 eq) were added to a reaction flask, followed by the addition of (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). Finally, dioxane (20 mL) and water (4 mL) were added. After purging with nitrogen three times, the reaction mixture was stirred at 90 °C for 12 h. LCMS indicated the completion of the reaction. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by high performance liquid chromatography (0.1% formic acid) and lyophilized to obtain Compound 1-7 (300 mg, 561.07 μmol, yield 27.01%, purity 97.504%), which was a brown oil. (ESI) m / z = 519.3 [M+H] + 。

[0034] Step 7: Synthesis of Compound 1-8

[0035] 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 mixture was stirred at 25 °C for 1 h. LCMS indicated the completion of the reaction. The reaction mixture was concentrated under reduced pressure to obtain Compound 1-8 (240 mg, crude product), which was a yellow oil. (ESI) m / z = 419.2 [M+H] + 。

[0036] Step 8: Synthesis of Compound 1-9

[0037] 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. The reaction mixture was stirred at 60 °C for 1 hour. LCMS showed that the reaction was complete, and the residue was obtained by concentration under reduced pressure. The crude product was purified by reverse phase (0.1% formic acid) and lyophilized to give 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] + 。

[0038] Step 9: Synthesis of Compound 1

[0039] 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 mixture was stirred at 40 °C for 12 hours. LCMS showed that the reaction was complete. The reaction mixture was added to saturated sodium bicarbonate solution at 0 °C until pH = 7, and then filtered and concentrated under reduced pressure to obtain the 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, which was lyophilized to give 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 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).

[0040] Test Example 1 Determination of the NLRP3 Inhibitory Activity of the Compounds of the Present Invention

[0041] 1.1 Test Materials

[0042]

[0043] 1.2 Test Instruments

[0044]

[0045] 1.3 Test Methods

[0046] a) Culture BMDM in a 6-well plate containing RPMI 1640 medium with M-CSF in an incubator at 37°C and 5% CO 2 2.

[0047] b) Wash the BMDM cells with PBS and re-seed the BMDM cells into a 96-well plate and culture in an incubator at 37°C and 5% CO 2 for 24 h.

[0048] c) Add LPS to the BMDM cells and incubate at 37°C and 5% CO 2 for 3 hours.

[0049] d) Add 5 μL of the compound (final 0.1% DMSO) to the cells and incubate the cells at 37°C and 5% CO 2 .

[0050] e) Add 2 uL of Nigericin to stimulate the cells and incubate at 37°C and 5% CO 2 .

[0051] f) Centrifuge the 96-well plate and transfer the supernatant to a new 96-well plate.

[0052] g) Detect IL-1β according to the detection method of the IL-1β Elisa kit.

[0053] h) Read the OD value at 450 nm on an Envision microplate reader.

[0054] i) Fit a curve based on the OD value and concentration and calculate the IC 50 value. The IC 50 is the compound concentration corresponding to an inhibition rate of 50%.

[0055] 1.4 Test Results

[0056] Table 1 IC 50 Values of Compounds Inhibiting IL-1β Expression

[0057]

[0058] * Compound of Example EX004 of CN113784957A

[0059] Conclusion: Compared with MCC950 and EX004, Compound 1 of the present invention can significantly improve the inhibitory activity against NLRP3 and effectively reduce the secretion of IL-1β.

Claims

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

3. Use of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, 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 selected from central nervous system diseases, autoimmune diseases, cardiovascular diseases and metabolic diseases.

5. The use according to claim 3, wherein the disease is selected from autoinflammatory febrile syndrome, viral hepatitis, nonalcoholic fatty hepatitis, alcoholic fatty hepatitis, gout, chondrocalcinosis, rheumatoid arthritis, hyperoxaluria, lupus nephritis, type I and type II diabetes, retinopathy, hypertensive nephropathy, hemodialysis-related inflammation, multiple sclerosis, brain infection, Alzheimer's disease, stroke, hypertension, acute heart failure, acne, wound healing and scar formation, asthma, sarcoidosis, colon cancer, lung cancer, myeloproliferative tumors and leukemia.

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