Amide phenol derivatives and uses thereof

Abstract

The present invention belongs to the field of chemical medicine and provides a class of amide phenol derivatives and uses thereof. The amide phenol derivatives provided by the present invention can inhibit the activity of NOD-like receptor protein 3 inflammasomes, provide a new option for the development of NLRP3 small molecule inhibitors, and have good application prospects.

Description

Technical Field

[0001] The invention relates to amide phenol derivatives and uses thereof, belonging to the field of chemical medicine. Background Art

[0002] Innate immunity is the body's first line of defense for identifying pathogens and endogenous molecules, and is an essential defense mechanism for the host. Innate immunity recognizes exogenous pathogen-associated molecular patterns (PAMPs) and endogenous danger-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs). PRRs are mainly divided into five categories, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), A1M2-like receptors (ALRs), RIG-like receptors (RLRs) and NOD-like receptors (NLRs). NLR S Located inside the cell, in addition to participating in immune defense, it also plays an important role in biological processes such as inflammatory response and early embryonic development. According to the difference in the N-terminal domain, NLRs can be divided into five categories: NLRA, NLRB, NLRC, NLRP, and NLRX. The NLRP subfamily is the largest subfamily of NLRs, with a total of 14 members (NLRP1-14), of which NLRP1, NLRP3, NLRP6 and other members can participate in the formation of inflammasomes.

[0003] Inflammasome is a multi-protein complex composed of sensors, adaptor proteins and effector proteins, which is associated with immune response, cell pyroptosis, and many inflammatory diseases. NLRP3 inflammasome can be activated by a variety of stimuli, including pathogen-associated molecular patterns such as viruses and bacteria, and danger-associated molecular patterns such as uric acid, ATP, and β-amyloid protein. NLRP3 inflammasome is associated with the pathogenesis of many diseases, such as atherosclerosis, gout, Alzheimer's disease, etc. Studies have shown that NLRP3 inflammasome plays a key role in the pathological regulation of diseases such as acute myocardial infarction, systemic lupus erythematosus, and inflammatory bowel disease. In addition, studies have shown that NLRP3 inflammasome is also closely related to Corona Virus Disease 2019 (COVID-19).

[0004] There are already some drugs that block IL-1β for the treatment of NLRP3-related diseases, but because the activated NLRP3 inflammasome not only produces IL-1β, it may also produce cytokines such as IL-18, which may also lead to NLRP3-related diseases. In addition, the production of IL-1β can be mediated by other inflammasomes or non-inflammatory pathways, and these inhibitors against IL-1β may lead to unexpected immunosuppressive reactions. Therefore, drug inhibitors that only target the NLRP3 inflammasome may be a better choice for the treatment of NLRP3-related diseases. Summary of the invention

[0005] The present invention develops a new type of amide phenol derivatives, which have the activity of inhibiting NOD-like receptor protein 3 (NLRP3) inflammasome and can be used to prepare NLRP3 small molecule inhibitors.

[0006] The present invention first provides a compound represented by formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008]

[0009] n=0~1;m=0~3;

[0010] X is selected from N or C-R4;

[0011] R1, R2, R3, and R4 are independently selected from H, halogen, hydroxyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 oxycarbonyl, substituted or unsubstituted 6-10 membered aryl, or R1 is connected to the nitrogen on the amide in formula I to form a 5-10 membered alkyl ring;

[0012] In R1, R2, R3, and R4, the substituent of the substituted 6- to 10-membered aryl group is selected from amino and C1-C8 alkyl;

[0013] R5 is selected from H, D, substituted or unsubstituted C1-C8 acyl;

[0014] In R5, the substituent of the substituted C1-C8 acyl group is selected from a 6-10-membered aryl group and a 5-10-membered heteroaryl group; in R5, the number of heteroatoms of the 5-10-membered heteroaryl group is 1-3, and the heteroatoms are N, S, and O;

[0015] R6 is selected from H, C1-C8 alkyl;

[0016] Y is selected from a bond, O, N-R8;

[0017] R8 is selected from H, C1-C8 alkyl;

[0018] R7 is selected from substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, In R7, the number of heteroatoms of the 5- to 10-membered heteroaryl group is 1 to 3, and the heteroatoms are N, S, and O; in R7, W and Z are independently selected from O, NH, and CH; o=0 to 1; p=1 to 2;

[0019] In R7, the substituent of the substituted 6-10 membered aryl group or substituted 5-10 membered heteroaryl group is selected from substituted or unsubstituted C1-C8 alkyl group, substituted or unsubstituted C1-C8 alkoxy group, 6-10 membered aryl group, hydroxyl group, halogen group, nitro group,

[0020] In R7, the substituents of the substituted C1-C8 alkoxy and substituted C1-C8 alkyl are selected from halogen and 6-10 membered aryl;

[0021] R9 is selected from C1-C8 alkyl.

[0022] Among them, in the above compounds, m=0-2.

[0023] Among them, in the above compounds, R1, R2, R3, and R4 are independently selected from H, halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 oxycarbonyl, substituted or unsubstituted 6-10 membered aryl, or R1 is connected to the nitrogen on the amide in formula I to form a 5-6 membered alkyl ring; among R1, R2, R3, and R4, the substituent of the substituted 6-10 membered aryl is selected from amino, C1-C6 alkyl.

[0024] Preferably, in the above compounds, R1, R2, R3, and R4 are independently selected from H, halogen, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 oxycarbonyl, substituted or unsubstituted 6-membered aryl, or R1 is connected to the nitrogen on the amide in formula I to form a 5-6-membered alkyl ring; among R1, R2, R3, and R4, the substituent of the substituted 6-membered aryl is selected from amino, C1-C4 alkyl.

[0025] More preferably, in the above compounds, R1, R2, R3, and R4 are independently selected from H, chlorine, fluorine, hydroxyl, methyl, methoxy, methoxycarbonyl, and amino-substituted phenyl, or R1 is connected to the nitrogen on the amide in formula I to form a 5-6 membered alkyl ring.

[0026] Most preferably, in the above compounds, R1 is selected from H, fluorine, methyl, 3- or 4-amino substituted phenyl, or R1 is connected to the nitrogen on the amide in formula I to form a 5-6 membered alkyl ring, R2 is selected from H, chlorine, methyl, methoxy, hydroxyl, R3 is selected from H, chlorine, methyl, and R4 is selected from H, methyl, methoxycarbonyl.

[0027] Among them, in the above compounds, R5 is selected from H, D, substituted or unsubstituted C1~C6 acyl; in R5, the substituent of the substituted C1~C6 acyl is selected from 6-10 membered aryl and 5-10 membered heteroaryl; in R5, the number of heteroatoms of the 5-10 membered heteroaryl is 1, and the heteroatom is N, S, or O.

[0028] Preferably, in the above compounds, R5 is selected from H, D, substituted or unsubstituted C1~C4 acyl; in R5, the substituent of the substituted C1~C4 acyl is selected from 6-10 membered aryl and 5-10 membered heteroaryl; in R5, the number of heteroatoms of the 5-10 membered heteroaryl is 1, and the heteroatom is N.

[0029] More preferably, in the above compounds, R5 is selected from H, D, substituted or unsubstituted C1-C4 acyl; in R5, the substituent of the substituted C1-C4 acyl is selected from phenyl and indolyl.

[0030] Most preferably, in the above compounds, R5 is selected from H, D,

[0031] Among them, in the above compounds, R6 is selected from H, C1~C8 alkyl.

[0032] Preferably, in the above compounds, R6 is selected from H, C1~C6 alkyl.

[0033] More preferably, in the above compounds, R6 is selected from H, C1-C4 alkyl.

[0034] Most preferably, in the above compounds, R6 is selected from H and methyl.

[0035] Among them, in the above compounds, R8 is selected from H, C1~C8 alkyl.

[0036] Preferably, in the above compounds, R8 is selected from H, C1-C6 alkyl.

[0037] More preferably, in the above compounds, R8 is selected from H, C1-C4 alkyl.

[0038] Most preferably, in the above compounds, R8 is selected from H and ethyl.

[0039] Among them, in the above compounds, the substituents of the substituted aryl and substituted heteroaryl in R7 are selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, 6-10 membered aryl, hydroxyl, halogen, nitro, In R7, the substituents of the substituted C1-C6 alkoxy and substituted C1-C6 alkyl are selected from halogen and 6-10 membered aryl; and R9 is selected from C1-C6 alkyl.

[0040] Preferably, in the above compounds, in R7, the substituent of the substituted aryl or substituted heteroaryl is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, 6-membered aryl, hydroxyl, halogen, nitro, In R7, the substituents of the substituted C1-C6 alkoxy and substituted C1-C6 alkyl are selected from halogen and 6-membered aryl; and R9 is selected from C1-C4 alkyl.

[0041] Most preferably, in the above compounds, in R7, the substituents of the substituted aryl and substituted heteroaryl are selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, 6-membered aryl, hydroxyl, chlorine, bromine, nitro, In R7, the substituents of the substituted C1-C4 alkoxy and substituted C1-C4 alkyl are selected from fluorine and 6-membered aryl; and R9 is selected from methyl.

[0042] Among them, in the above compounds, R7 is selected from substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, In R7, the number of heteroatoms of the 5- to 10-membered heteroaryl group is 1 to 2, and the heteroatoms are N, S, and O; in R7, W and Z are independently selected from O, NH, and CH; o=0 to 1; and p=1 to 2.

[0043] Preferably, in the above compounds, R7 is selected from substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, In R7, the number of heteroatoms of the 5- to 10-membered heteroaryl group is 1, and the heteroatom is N; in R7, W and Z are independently selected from O, NH, and CH; o=1; and p=1.

[0044] More preferably, in the above compounds, R7 is selected from substituted or unsubstituted phenyl or naphthyl, substituted or unsubstituted indolyl, In R7, W and Z are independently selected from O, NH, and CH; o=0-1; and p=1-2.

[0045] Most preferably, in the above compounds, R7 is selected from

[0046] The present invention also provides some specific compounds of the compound described in formula I, as follows:

[0047]

[0048]

[0049] The present invention also provides a pharmaceutical composition, which is composed of the above compound or a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable auxiliary ingredients.

[0050] The present invention also provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof, and the above-mentioned pharmaceutical composition in the preparation of NLRP3 small molecule inhibitors.

[0051] Definition of terms:

[0052] The compounds and derivatives provided by the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0053] The term "alkyl" is a group of a straight or branched saturated hydrocarbon group. Examples of C1-C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6).

[0054] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group containing no heteroatoms, which may be a monocyclic structure or a polycyclic structure (such as a bridged ring structure or a spirocyclic structure), for example: cyclopropane (3-membered), cyclohexane (6-membered).

[0055] The term "aryl" refers to an all-carbon monocyclic or condensed ring group with a conjugated π electron system. The aryl group may be a completely aromatic group, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, etc. The carbon atoms in the aryl group may be replaced by heteroatoms selected from sulfur, oxygen and / or nitrogen, such as thiophene, furan, pyrrole, pyridine, quinoline, indole, etc.

[0056] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

[0057] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or formed salt is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form and physiologically compatible with the receptor.

[0058] The term "pharmaceutically acceptable salt" refers to organic salts and inorganic salts of the compounds of the present invention, preferably inorganic salts, salts formed by pharmaceutically acceptable non-toxic acids, including but not limited to inorganic acid salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, nitrates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, hydrochlorides, oleates, stearates, ascorbic acid salts, formates, borates, camphorates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, malates, and the like.

[0059] The pharmaceutically acceptable auxiliary ingredients of the present invention refer to substances contained in the dosage form in addition to the active ingredients, such as cyclodextrin, arginine or meglumine. The cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, (C 1-4 Alkyl)-α-cyclodextrin, (C 1-4 Alkyl)-β-cyclodextrin, (C 1-4 Alkyl)-γ-cyclodextrin, (hydroxy-C 1-4 Alkyl)-α-cyclodextrin, (hydroxy-C 1-4 Alkyl)-β-cyclodextrin, (hydroxy-C 1-4 Alkyl)-γ-cyclodextrin, (carboxyl-C 1-4 Alkyl)-α-cyclodextrin, (carboxyl-C 1-4 Alkyl)-β-cyclodextrin, (carboxyl-C 1-4 The auxiliary components also include medically acceptable carriers, adjuvants or vehicles. Pharmaceutically acceptable pharmaceutical compositions also include ion exchangers, alumina, aluminum stearate, and egg gelatin; buffer substances include phosphates, glycine, arginine, sorbic acid, and the like.

[0060] Beneficial effects:

[0061] The present invention provides a class of amide phenol derivatives, which can inhibit the activity of NOD-like receptor protein 3 (NLRP3) inflammasomes, provide a new option for the development of NLRP3 small molecule inhibitors, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Compound 30 inhibits Nigericin-induced NLRP3 inflammasome activation.

[0063] Figure 2 Compound 30 inhibits NLRP3 inflammasome formation. DETAILED DESCRIPTION

[0064] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0065] Example 1

[0066] Preparation of intermediates (5), (6), (11), (12) and product 1

[0067]

[0068] The reaction conditions in the above synthetic route include: (i), concentrated sulfuric acid, methanol, 50°C, 3h; (ii), potassium carbonate, ethyl iodide (a) / benzyl bromide (b), acetonitrile, 80°C, overnight; (iii), sodium hydroxide, methanol, water, room temperature, overnight; (iv), sodium hydride, ethyl bromoacetate, DMF, 0°C, 3h; (v), HATU, DIEA, 3-aminophenol, DMF, room temperature, 3h.

[0069] Intermediate (5): Preparation of 4-ethoxyphenylacetic acid:

[0070]

[0071] Preparation of methyl 2-(4-hydroxyphenyl)acetate (2): 30 mL of methanol was added to 2-(4-hydroxyphenyl)acetic acid (raw material 1) (1 g, 6.57 mmol) and concentrated sulfuric acid (cat, 1 mL), and heated at 50°C for 3 h. TLC confirmed the completion of the reaction. The solvent was evaporated, and the residue was dissolved in ethyl acetate, washed with saturated aqueous NaHCO3, H2O, and brine, and dried over anhydrous sodium sulfate. The solvent was then evaporated to obtain methyl 2-(4-hydroxyphenyl)acetate (2) (1.07 g, 98%).

[0072] Preparation of methyl 2-(4-ethoxyphenyl)acetate (3): Methyl 2-(4-hydroxyphenyl)acetate (2) (1 g, 6.02 mmol), iodoethane (0.865 g, 6.1 mmol) and K2CO3 (1.65 g, 12 mmol) were stirred in acetonitrile overnight at 80°C. The solvent was evaporated under reduced pressure and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and evaporated to give methyl 2-(4-ethoxyphenyl)acetate (3), which was used in the next step without further purification.

[0073] A suspension of intermediate (3) (1 g) in methanol (30 ml) was treated with 2 M NaOH (15 ml) and stirred overnight at room temperature. The reaction was monitored by thin layer chromatography (TLC) to completion. The reaction mixture was concentrated under reduced pressure. The remainder was diluted with water and then 10% aqueous HCl was added to adjust the pH to 5-6. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo and purified by petroleum ether / ethyl acetate gradient chromatography to give 2-(4-ethoxyphenyl)acetic acid (5) as a white solid in a yield of 99%, 0.918 g. 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 7.16 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 3.99 (q, J = 7.0 Hz, 2H), 3.48 (s, 2H), 1.32 (t, J = 7.0 Hz, 3H). MS (ESI, positive ion) m / z: 181.07 [M+H] + .

[0074] Intermediate (6): Preparation of 4-benzyloxyphenylacetic acid:

[0075]

[0076] The synthetic route of intermediate (6) is similar to that of intermediate (5). 1 H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 7.45 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.35–7.31 (m, 1H), 7.20–7.14 (m, 2H), 6.99–6.93 (m, 2H), 5.09 (s, 2H), 3.49 (s, 2H). MS (ESI, positive ion) m / z: 243.09 [M+H] + .

[0077] Intermediate (11): Preparation of 2-(1-carbonylisoquinolin-2(1H)-yl)acetic acid:

[0078]

[0079] Dissolve isoquinolin-1(2H)-one (raw material 7) (1 g, 6.9 mmol) in DMF (20 mL) and cool to 0°C. Add NaH (0.55 g, 13.8 mmol) and stir at room temperature for 0.5 h, then add ethyl bromoacetate (1.14 mL, 10.35 mmol) and stir for 3 h. After monitoring the reaction by thin layer chromatography (TLC), treat with saturated aqueous ammonium chloride solution and extract with ethyl acetate. The organic phase is dried over anhydrous sodium sulfate. After filtration, the solvent is concentrated under reduced pressure to obtain ethyl 2-(1-oxyisoquinolin-2(1H)-yl)acetate (9) as a white solid with a yield of 1.51 g, 95%.

[0080] A suspension of intermediate (9) (1 g, 4.3 mmol) in methanol (30 ml) was treated with 2 M NaOH (15 ml), stirred at room temperature overnight, and the reaction mixture was concentrated under reduced pressure. After the reaction was completed by monitoring by thin layer chromatography (TLC), the remaining portion was diluted with water, and then 10% HCl aqueous solution was added to adjust the pH to 5-6. The aqueous layer was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated in vacuo, and purified by petroleum ether / ethyl acetate gradient chromatography to obtain 2-(1-carbonylisoquinolin-2(1H)-yl)acetic acid (11) as a white solid in a yield of 98%. 1 H NMR (400 MHz, DMSO-d6) δ8.21 (d, J = 8.0 Hz, 1H), 7.75–7.69 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.55–7.48 (m, 1H), 7.43 (d, J = 7.4 Hz, 1H), 6.62 (d, J = 7.4 Hz, 1H), 4.66 (s, 2H). MS (ESI, positive ion) m / z: 204.06 [M+H] + .

[0081] Intermediate (12): Preparation of indole-1-acetic acid:

[0082]

[0083] The synthetic route of intermediate (12) is similar to that of intermediate 11. 1 H NMR (400 MHz, DMSO-d6) δ12.94 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 3.1 Hz, 1H), 7.16–7.10 (m, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.45 (d, J = 3.1 Hz, 1H), 5.01 (s, 2H). MS (ESI, positive ion) m / z: 176.06 [M+H] + .

[0084] Product 1: N-(3-hydroxyphenyl)-2-(4-methoxyphenyl)acetamide

[0085]

[0086] 3-Aminophenol (56 mg, 0.5 mmol), HATU (247 mg, 0.65 mmol) and DIEA (200 μl, 1.14 mmol) were added to a solution of 2-(4-methoxyphenyl)acetic acid (83 mg, 0.5 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature overnight, DMF was evaporated, and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography to obtain the desired product as a white solid (109 mg, yield 85%). 1 H NMR (400 MHz, DMSO-d6) δ9.95 (s, 1H), 9.34 (s, 1H), 7.27–7.21 (m, 2H), 7.17 (t, J = 2.1 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.91–6.83 (m, 2H), 6.43 (dd, J = 8.0, 2.3 Hz, 1H), 3.72 (s, 3H), 3.53 (s, 2H); MS (ESI, positive ion) m / z: 258.1 [M+H] + .

[0087] Example 2

[0088] Preparation of intermediate (17) and product 35:

[0089]

[0090] The reaction conditions of the above synthetic route include: (i), concentrated sulfuric acid, methanol, 50°C, 3h; (ii), cesium carbonate, ethyl bromoacetate, DMF, 100°C, overnight; (iii), iron powder, glacial acetic acid, 80°C, 3h; (iv), sodium hydroxide, methanol, water, room temperature, overnight; (v), HATU, DIEA, 3-aminophenol, DMF, rt, 3h.

[0091] Intermediate (17): Preparation of 2-(3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acetic acid:

[0092]

[0093] Preparation of methyl 2-(4-hydroxy-3-nitrophenyl) acetate (14): 30 mL of methanol was added to 4-hydroxy-3-nitrophenylacetic acid (raw material 13) (1 g, 5.07 mmol) and concentrated sulfuric acid (cat, 1 mL), and heated at 50°C for 3 h. TLC confirmed the completion of the reaction. The solvent was evaporated, and the residue was dissolved in ethyl acetate, washed with saturated aqueous NaHCO3, H2O, and brine, and dried over anhydrous sodium sulfate. The solvent was then evaporated to obtain 2-(4-hydroxy-3-nitrophenyl) acetate (14) (1.05 g, 98%).

[0094] Preparation of ethyl 2-(4-(2-methoxy-2-carbonylethyl)-2-nitrophenoxy) acetate (15): Methyl 2-(4-hydroxy-3-nitrophenyl) acetate (14) (1.05 g, 5 mmol) and cesium carbonate (3.25 g, 10 mmol) were dissolved in DMF (30 mL). Ethyl bromoacetate (630 μL, 6 mmol) was added and stirred at 100°C overnight. After the reaction was completed, it was extracted with ethyl acetate after monitoring by thin layer chromatography (TLC). The organic phase was dried over anhydrous sodium sulfate and the crude product was purified by silica gel chromatography to obtain ethyl 2-(4-(2-methoxy-2-carbonylethyl)-2-nitrophenoxy) acetate (15) as a white solid with a yield of 1.32 g, 90%.

[0095] Preparation of methyl 2-(3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acetate (16):

[0096] Ethyl 2-(4-(2-methoxy-2-carbonylethyl)-2-nitrophenoxy) acetate (15) (1 g, 3.37 mmol) and iron powder (600 mg, 10.7 mmol) were stirred in glacial acetic acid for 3 h at 80°C. The reaction was monitored by thin layer chromatography (TLC) and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and the crude product was purified by silica gel chromatography to give methyl 2-(3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl) acetate (16) as a white solid in a yield of 560 mg, 80%.

[0097] A suspension of intermediate (16) (500 mg) in methanol (30 ml) was treated with 2M NaOH (15 ml) and stirred at room temperature overnight. The reaction was monitored by thin layer chromatography (TLC). The reaction mixture was concentrated under reduced pressure. The remainder was diluted with water and then 10% aqueous HCl was added to adjust the pH to 5-6. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo and purified by petroleum ether / ethyl acetate gradient chromatography to give 2-(3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acetic acid (17) as a white solid with a yield of 441 mg, 90%. 1 H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 10.66 (s, 1H), 6.87–6.79 (m, 2H), 6.68 (d, J = 2.1 Hz, 1H), 4.54 (d, J = 0.7 Hz, 2H), 3.69 (d, J = 6.4 Hz, 2H). MS (ESI, positive ion) m / z: 208.07 [M+H] + .

[0098] Product (35): Preparation of N-(3-hydroxyphenyl)-2-(3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acetamide:

[0099]

[0100] 3-Aminophenol (56 mg, 0.5 mmol), HATU (247 mg, 0.65 mmol) and DIEA (200 μl, 1.14 mmol) were added to a solution of 2-(3-carbonyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acetic acid (103 mg, 0.5 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature overnight, DMF was evaporated and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography to give the desired product 35 as a white solid (126 mg, 85% yield). 1 H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 10.00 (s, 1H), 9.37 (s, 1H), 7.17 (s, 1H), 7.06 (s, 1H), 6.92 (d, J = 28.6 Hz, 4H), 6.44 (d, J = 7.9 Hz, 1H), 4.54 (s, 2H), 3.52 (s, 2H); MS (ESI, positive ion) m / z: 299.09 [M+H] + .

[0101] Example 3

[0102] Preparation of intermediate (20) and product 39:

[0103]

[0104] The reaction conditions of the above synthetic route include: (i) DIEA, methyl iodide, DMF, 80°C, 4h; (ii) hydrogen, 10% palladium / carbon, methanol, 50°C, 3h; (iii) HATU, DIEA, 1H-indole-6-acetic acid, DMF, rt, 3h.

[0105] Intermediate (20): Preparation of 3-(methylamino)phenol:

[0106]

[0107] Preparation of 3-(Benzyloxy)-N-methylaniline (19): The raw material (18) 3-(Benzyloxy)aniline (1.0 g, 5 mmol), iodomethane (0.71 g, 5 mmol) and DIEA (1.73 mL, 10 mmol) were added to DMF (20 mL) and stirred at 80°C under nitrogen atmosphere for 4 h. The mixture was concentrated under reduced pressure and purified by column chromatography to obtain the intermediate (19) as a colorless oil (0.958 g, yield 90%).

[0108] Preparation of 3-(methylamino)phenol (20): 3-(benzyloxy)-N-methylaniline (19) (1 g, 4.7 mmol) was dissolved in methanol, 10% palladium / carbon (50 mg) was added, and stirred for 3 h under a hydrogen atmosphere at 50°C. TLC confirmed the completion of the reaction. The reaction mixture was then filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel chromatography to obtain 3-(methylamino)phenol (20) as a white solid (0.519 g, yield 90%). 1 H NMR (400 MHz, cdcl3) δ7.02 (t, J = 8.0 Hz, 1H), 6.20 (m, 2H), 6.08 (t, J = 2.3 Hz, 1H), 4.55 (s, 2H), 2.76 (s, 3H). MS (ESI, positive ion) m / z: 124.1 [M+H] + .

[0109] Product 39: Preparation of N-(3-hydroxyphenyl)-2-(1H-indol-6-yl)-N-methylacetamide:

[0110]

[0111] 3-(Methylamino)phenol (56 mg, 0.5 mmol), HATU (247 mg, 0.65 mmol) and DIEA (200 μl, 1.14 mmol) were added to a solution of 1H-indole-6-acetic acid (87 mg, 0.5 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature overnight, DMF was evaporated, and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography to obtain the desired product 35 as a white solid (119 mg, yield 85%). 1 HNMR (400MHz, DMSO-d6) δ10.95 (s, 1H), 9.73 (s, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.30–7.20 (m, 2H), 7.13 (s, 1H), 6.79 (d, J = 8.2 Hz, 1H), 6.75–6.62 (m, 3H), 6.35 (t, J = 2.5 Hz, 1H), 3.49 (s, 2H), 3.15 (s, 3H); MS (ESI, positive ion) m / z: 281.12 [M+H] + .

[0112] Example 4

[0113] Preparation of intermediate (23) and product 53:

[0114]

[0115] The reaction conditions of the above synthetic route include: (i), cesium carbonate, ethyl bromoacetate, DMF, 100°C, overnight; (ii), sodium hydroxide, methanol, water, room temperature, overnight; (iii), HATU, DIEA, 3-aminophenol, DMF, rt, 3h.

[0116] Intermediate (23): Preparation of (4-(trifluoromethyl)phenyl)glycine (y):

[0117]

[0118] Preparation of (4-(trifluoromethyl)phenyl)glycine ethyl ester (22): Dissolve the raw material (21) 4-(trifluoromethyl)aniline (805 mg, 5 mmol) and cesium carbonate (3.25 g, 10 mmol) in DMF (30 mL). Add ethyl bromoacetate (630 μL, 6 mmol) and stir at 100°C overnight. After monitoring the reaction by thin layer chromatography (TLC), extract with ethyl acetate. The organic phase is dried over anhydrous sodium sulfate and the crude product is purified by silica gel chromatography to obtain (4-(trifluoromethyl)phenyl)glycine ethyl ester (22) as a white solid with a yield of 985 mg, 90%.

[0119] Preparation of (4-(trifluoromethyl)phenyl)glycine (23): A suspension of intermediate (22) (500 mg) in methanol (30 ml) was treated with 2M NaOH (15 ml) and stirred at room temperature overnight. The reaction was monitored by thin layer chromatography (TLC). The reaction mixture was concentrated under reduced pressure. The remainder was diluted with water and then 10% aqueous HCl was added to adjust the pH to 5-6. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo and purified by petroleum ether / ethyl acetate gradient chromatography to give (4-(trifluoromethyl)phenyl)glycine (23) as a white solid with a yield of 398 mg, 90%. 1 H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 9.34 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 4.55 (d, J = 6.0 Hz, 2H); MS (ESI, positive ion) m / z: 208.07 [M+H] + .

[0120] Product 53: Preparation of N-(3-hydroxyphenyl)-2-((4-(trifluoromethyl)phenyl)amino)acetamide:

[0121] 3-Aminophenol (56 mg, 0.5 mmol), HATU (247 mg, 0.65 mmol) and DIEA (200 μl, 1.14 mmol) were added to a solution of (4-(trifluoromethyl)phenyl)glycine (109 mg, 0.5 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature overnight, DMF was evaporated and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography to give the desired product 53 as a white solid (139 mg, 90% yield). 1H NMR (400 MHz, DMSO-d6) δ9.92 (s, 1H), 9.38 (s, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 2.3 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.74–6.64 (m, 3H), 6.45 (dd, J = 8.1, 2.3 Hz, 1H), 3.93 (d, J = 6.1 Hz, 2H).; MS (ESI, positive ion) m / z: 311.09 [M+H]+.

[0122] Example 5

[0123] Preparation of intermediate (28) and product 69

[0124]

[0125] The reaction conditions of the above-mentioned synthetic route include: (i), potassium carbonate, benzyl bromide, acetonitrile, 80°C, 3h; (ii), iron powder, ammonium chloride, ethanol: water = 2: 1, 80°C, 3h; (iii), HATU, DIEA, 1H-indole-6-acetic acid, DMF, rt, overnight; (iv), 4-nitrobenzeneboric acid, 1,1-bis(diphenylphosphino)diferronichloridopalladium, cesium carbonate, 1,4-dioxane: water = 5: 1, 100°C, overnight; (v), hydrogen, 10% palladium / carbon, methanol, 50°C, 3h.

[0126] Intermediate (28): Preparation of N-(6-(benzyloxy)-4'-nitro-[1,1'-biphenyl]-2-yl)-2-(1H-indol-6-yl)acetamide:

[0127]

[0128] Preparation of 1-(Benzyloxy)-2-bromo-3-nitrobenzene (25): Dissolve the raw material (24) 2-bromo-3-nitrophenol (3 g, 13.76 mmol) and potassium carbonate (2.85 g, 20.64 mmol) in acetonitrile (50 mL). Add benzyl bromide (1.8 ml, 16.51 mmol) and stir at 80°C for 3 h. After the reaction is completed, monitor with thin layer chromatography (TLC) and extract with ethyl acetate. The organic phase is dried over anhydrous sodium sulfate and the crude product is purified by silica gel chromatography to obtain 1-(benzyloxy)-2-bromo-3-nitrobenzene (25) as a light yellow solid with a yield of 3.7 g, 87%.

[0129] Preparation of 3-(Benzyloxy)-2-bromoaniline (26): 1-(Benzyloxy)-2-bromo-3-nitrobenzene (25) (3.7 g, 12 mmol), iron powder (3.35 g, 60 mmol), and ammonium chloride (1.03 g, 19.2 mmol) were dissolved in a mixed solvent of 50 ml ethanol and 25 ml water. The mixture was heated to 80 °C and stirred for 3 h. After the reaction was completed by monitoring with thin layer chromatography (TLC), the reaction system was filtered and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and the crude product was purified by silica gel chromatography to obtain 3-(Benzyloxy)-2-bromoaniline (26) as a colorless oil with a yield of 2.95 g, 88%.

[0130] Preparation of N-(3-(Benzyloxy)-2-bromophenyl)-2-(1H-indol-6-yl)acetamide (27): 3-(Benzyloxy)-2-bromoaniline (26) (1.59 g, 5.71 mmol), HATU (3.26 g, 8.56 mmol) and DIEA (2.19 ml, 12.56 mmol) were added to a solution of 1H-indole-6-acetic acid (1 g, 5.71 mmol) in DMF (30 mL). The reaction mixture was stirred at room temperature overnight, DMF was evaporated, and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography to give N-(3-(benzyloxy)-2-bromophenyl)-2-(1H-indol-6-yl)acetamide (27) as a pale yellow solid (1.6 g, yield 64%).

[0131] Preparation of N-(6-(Benzyloxy)-4'-nitro-[1,1'-biphenyl]-2-yl)-2-(1H-indol-6-yl)acetamide (28): N-(3-(Benzyloxy)-2-bromophenyl)-2-(1H-indol-6-yl)acetamide (27) (100 mg, 0.23 mmol), 4-nitrophenylboric acid (58 mg, 0.34 mmol), 1,1-bis(diphenylphosphino)dichloropalladium iron (8.3 mg 0.0115 mmol), cesium carbonate (150 mg, 0.46 mmol) were dissolved in a mixed solvent of 10 ml 1,4-dioxane and 2 ml water. The reaction system was protected by inert gas, heated to 80°C and stirred overnight. After the reaction was completed by monitoring by thin layer chromatography (TLC), the reaction system was filtered and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and the crude product was purified by silica gel chromatography to give N-(6-(benzyloxy)-4'-nitro-[1,1'-biphenyl]-2-yl)-2-(1H-indol-6-yl)acetamide (28) as a yellow solid in a yield of 70 mg, 65%. 1H NMR (400MHz, DMSO-d6) δ10.95 (s, 1H), 8.81 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.35–7.24 (m, 9H), 7.22–7.19 (m, 2H), 7.14 (s, 1H), 7.04 (d, J = 8.1 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 6.33 (td, J = 2.1, 1.1 Hz, 1H), 5.06 (s, 2H), 3.49 (s, 2H). MS (ESI, positive ion) m / z: 478.1 [M+H] + .

[0132] Product (69): Preparation of N-(4'-amino-6-hydroxy-[1,1'-biphenyl]-2-yl)-2-(1H-indol-6-yl)acetamide: N-(6-(benzyloxy)-4'-nitro-[1,1'-biphenyl]-2-yl)-2-(1H-indol-6-yl)acetamide (28) (70 mg, 0.15 mmol) was dissolved in methanol, 10% palladium / carbon (50 mg) was added, and stirred for 3 h under a hydrogen atmosphere at 50°C. TLC confirmed the completion of the reaction. The reaction mixture was then filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel chromatography to obtain the product N-(4'-amino-6-hydroxy-[1,1'-biphenyl]-2-yl)-2-(1H-indol-6-yl)acetamide (28) as a white solid (47 g, yield 90%). 1 H NMR (400MHz, DMSO-d6) δ10.95(s,1H),9.06(s,1H),8.33(s,1H),7.42(d,J=8.1Hz,1 H),7.30(t,J=2.8Hz,1H),7.22(d,J=8.1Hz,1H),7.16(s,1H),7.01(t,J=8.1Hz,1H) ,6.77(d,J=8.0Hz,2H),6.72(d,J=7.8Hz,1H),6.67(d,J=8.1Hz,1H),6.54–6.48(m,2H),6.40(d,J=2.6Hz,1H),5.06(s,2H),3.52(s,2H).MS(ESI,positive ion)m / z:358.1[M+H] + .

[0133] Table 1 Some intermediates 1 H NMR

[0134]

[0135]

[0136] Table 2 Compounds of the present invention 1 H NMR

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] Evaluation of pharmacological activity of compounds

[0149] Experimental methods

[0150] 1. In vitro pyroptosis inhibition activity of phenylacetamide phenol derivatives

[0151] THP-1 was inoculated in a 96-well plate at a density of 105 / well. After PMA-induced differentiation into macrophages, RPMI-1640 medium containing 2μg / mL LPS was added for 4 hours. The drug was diluted with the medium to a specific concentration and added to the well plate for 30 minutes. At the same time, a drug-free group (i.e., modeling group, 3 to 6 replicates) was set up. Finally, Nigericin was added at a final concentration of 10μM. After continuing to incubate in a cell culture incubator for 3-4 hours, the cells in the modeling group should show obvious death under a microscope. 20μL MTT solution was added to each well, placed in a cell culture incubator for 1.5 to 2 hours, and then 50μL 20% SDS solution was added. It was placed in a cell culture incubator overnight, and the OD value at the absorption wavelength of 570nm was detected by an enzyme marker the next day. Relative cell survival rate % = 100% × (AX-A0) / (A1-A0). Among them, AX is the absorbance value of the drug treatment group or modeling group, A1 is the average absorbance value of the non-modeling group (the number of cells and the volume of culture medium are the same, but the cells are not stimulated by LPS and Nigericin), and A0 is the average absorbance value of the blank control group (no cells but the same volume of culture medium is added). The half effective concentration (IC50) of the drug was fitted using GraphPad Prism 6.0.

[0152] Experimental results: Inhibitory activity of the compounds of the present invention on pyroptosis

[0153] Table 4 IC of compounds of the present invention 50 value

[0154]

[0155]

[0156] 2. NLRP3 inflammasome activation and detection

[0157] THP-1 was 10 5 The cells were seeded at a density of 10 μg / well in a 6-well plate. After PMA-induced differentiation into macrophages, RPMI-1640 medium containing 2 μg / mL LPS was added for 4 h. The medium was aspirated, compound 30 was added for 30 min, and then Nigericin with a final concentration of 10 μM was added for 30 min. The supernatant was collected, and the LDH content and IL-1β, IL-18 and other inflammatory factors released by the cells into the supernatant were detected using an LDH kit (Promega) and an ELISA kit (Xinbosheng). The results are shown in Figure 1 AD.

[0158] At the same time, the supernatant protein sample (supernatant sample) was prepared by organic solvent precipitation method, the cells were lysed with RIPA lysis buffer, the intracellular protein (cytoplasm sample) was extracted, and the changes of NLRP3 inflammatory signaling pathway were detected by western blot. The results are shown in Figure 1 E.

[0159] Depend on Figure 1 It can be seen that LPS / nigericin induces NLRP3 inflammasome-dependent cell pyroptosis, which is accompanied by the release of lactate dehydrogenase (LDH) and inflammatory factors IL-1β and IL-18. The experimental results showed that the molecule can reduce the content of LDH, IL-1β and IL-18 in the cell culture supernatant in a concentration-dependent manner, but has no effect on the release of TNF-α, a cytokine that is independent of NLRP3. Western blot experimental results showed that the molecule can inhibit the activation of caspase-1 mediated by NLRP3 inflammasome activation, thereby affecting the cleavage of pyroptosis execution protein GSDMD and the maturation and release of inflammatory factor IL-1β.

[0160] 3. Co-immunoprecipitation experiment

[0161] THP-1 was 10 5 / well were inoculated in a 6-well plate. After PMA-induced differentiation into macrophages, RPMI-1640 medium containing 2μg / mL LPS was added for 4h, the medium was aspirated, compound 30 was added for 30min, and then Nigericin with a final concentration of 10μM was added for 30min. The supernatant was discarded, washed three times with PBS to remove dead cells, and 300μl pre-cooled NP40 was added for lysis on ice for 30min. Centrifuged at 13000rpm for 10min. The supernatant was taken to a new 1.5ml EP tube, 40μl was taken from it and added to 10μl sample buffer, and the metal bath was 100℃ for 10min as input sample for western blot detection. The remaining 250u1 was used for immunoprecipitation. Protein G beads were washed 3 times with 1ml NP40, then resuspended with 50ul, added to the remaining liquid in the previous step, and then ASC antibody was added, and incubated at 4℃ overnight. The next day, the beads were washed with NP40, centrifuged at 4°C, 5000 rpm, for 1 min, washed 3 times, discarded the supernatant for the last time, and then 30 μl sample buffer was added, and the beads were incubated at 100°C for 10 min as IP samples for western blot detection. The results are shown in Figure 2 .

[0162] Depend on Figure 2 It can be seen that compound 30 can reduce the interaction between NLRP3 and ASC, block the formation of NLRP3 inflammasome, and thus exert a pyroptosis inhibitory effect.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from:

2. A pharmaceutical composition, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable auxiliary ingredients.

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 cell pyroptosis inhibitor.

4. Use of a compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a cell pyroptosis inhibitor, characterized in that: The compound is selected from: The pharmaceutical composition is composed of the compound or a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable auxiliary ingredients.

5. Use of a compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a NLRP3 small molecule inhibitor, characterized in that: The compound is: The pharmaceutical composition comprises Or its pharmaceutically acceptable salt is used as the active ingredient, and pharmaceutically acceptable auxiliary ingredients are added to form the composition.

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