RIPK1 inhibitor and use thereof

Abstract

The present invention belongs to the technical field of medicinal chemistry, and specifically relates to an RIPK1 inhibitor and the use thereof. Specifically provided are compounds represented by formula (I), formula (II), formula (III) or formula (IV), isomers thereof and pharmaceutically acceptable salts thereof. The RIPK1 inhibitor provided by the present invention has good in vitro activity, can significantly increase the survival rate of mice in an LPS-induced sepsis model, can significantly increase the body temperature of mice in an LPS-induced inflammation model, and reduce the level of TNF-αin plasma. In addition, the compounds provided by the present invention have good pharmacokinetic properties.

Description

RIPK1 inhibitors and their uses Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to an RIPK1 inhibitor and its uses, including but not limited to systemic inflammatory syndrome, sepsis, lung injury, kidney injury, and autoimmune diseases. Background Technology

[0002] Receptor-interacting protein 1 (RIPK1) kinase, originally called RIP, is a TKL family serine / threonine protein kinase involved in innate immune signaling. RIPK1 kinase is a protein containing an RHIM domain, which has an N-terminal kinase domain and a C-terminal death domain. The RIPK1 death domain mediates interactions with other proteins containing death domains, including Fas and TNFR-1, TRAIL-R1 and TRAIL-R2, and TRADD. The RHIM domain is crucial for binding to other RHIM domain-containing proteins, such as TRIF, DAI, and RIP3, and enables them to perform numerous functions through these interactions.

[0003] RIPK1-mediated necroptosis plays a significant pathophysiological role in clinical diseases such as neurodegenerative diseases, inflammation, hematologic and solid organ malignancies, bacterial and viral infections, and lysosomal storage disorders. Therefore, selective inhibitors of RIPK1 kinase activity are expected to be potential therapeutic options for diseases mediated by this pathway and associated with inflammation and / or necrotizing cell death. Summary of the Invention

[0004] The main objective of this invention is to provide a RIPK1 inhibitor and its use, in order to at least partially solve at least one of the above-mentioned technical problems.

[0005] As a first aspect of the present invention, the present invention provides a compound of formula (I), its isomers, and a pharmaceutically acceptable salt:

[0006] Among them, one of X1, X2, X3, and X4 is N, and the rest are CH;

[0007] Preferably, X1 is N, and X2, X3, and X4 are all CH;

[0008] R1 is selected from H, halogens, Or C1-3 alkyl, where Rc is selected from C3-6 cycloalkyl or C1-3 alkyl;

[0009] Preferably, R1 is selected from H;

[0010] R2 is selected from H, C1-3 alkyl, or one or more deuterated C1-3 alkyl groups;

[0011] Preferably, R2 is selected from C1-3 alkyl groups or one or more deuterium-substituted C1-3 alkyl groups;

[0012] More preferably, R2 is selected from methyl, ethyl, propyl, deuterated methyl, deuterated ethyl, or deuterated propyl;

[0013] More preferably, R2 is selected from methyl or deuterated methyl;

[0014] Ring A is selected from 5-6 membered heterocyclic groups or 8-9 membered fused heterocyclic groups, wherein the 5-6 membered heterocyclic group or 8-9 membered fused heterocyclic group is optionally substituted by 1-2 Re, wherein the Re is independently selected from halogens or C1-3 alkyl groups;

[0015] Preferably, A is selected from a 5-membered nitrogen heterocyclic group or an 8-9-membered fused heterocyclic group, wherein the 5-membered nitrogen heterocyclic group or the 8-9-membered fused heterocyclic group is optionally substituted by 1-2 Re, wherein the Re is independently selected from halogens or C1-3 alkyl groups;

[0016] More preferably, ring A is selected from...

[0017] More preferably, ring A is selected from

[0018] More preferably, ring A is selected from...

[0019] L1 is selected from -(CH2)n-, where n is selected from 0, 1, or 2;

[0020] Preferably, L1 is selected from -(CH2)n-, and n is selected from 0 or 1;

[0021] More preferably, when ring A is selected from When L1 is selected from -(CH2)n-, n is selected from 0; when A is selected from In this case, L1 is selected from -(CH2)n-, and n is selected from 1;

[0022] Ring B is selected from phenyl, and the phenyl is optionally substituted with halogen, C1-3 alkyl, C1-3 alkoxy or phenyl;

[0023] Preferably, ring B is selected from phenyl.

[0024] In some embodiments, the compound of formula (I) is selected from:

[0025] Preferably, the compound of formula (I) is selected from: compound 1, compound 58, compound 79, compound 80, compound 81, compound 82, compound 80-1, compound 81-1 or compound 82-1;

[0026] More preferably, the compound of formula (I) is selected from: compound 1, compound 80, compound 81, compound 82, compound 80-1, compound 81-1 or compound 82-1.

[0027] As a second aspect of the present invention, the present invention provides compounds of formula (II), isomers thereof, and pharmaceutically acceptable salts:

[0028] R2 is selected from H, C1-3 alkyl, or one or more deuterated C1-3 alkyl groups;

[0029] Preferably, R2 is selected from C1-3 alkyl groups or one or more deuterium-substituted C1-3 alkyl groups;

[0030] More preferably, R2 is selected from methyl, ethyl, propyl, deuterated methyl, deuterated ethyl, or deuterated propyl;

[0031] More preferably, R2 is selected from methyl or deuterated methyl;

[0032] Ring A is selected from:

[0033] Preferably, ring A is selected from

[0034] More preferably, ring A is selected from...

[0035] L1 is selected from -(CH2)n-, where n is selected from 0, 1, or 2;

[0036] Preferably, L1 is selected from -(CH2)n-, and n is selected from 0;

[0037] Ring B is selected from phenyl, and the phenyl is optionally substituted with halogen, C1-3 alkyl, C1-3 alkoxy or phenyl;

[0038] Preferably, ring B is selected from phenyl.

[0039] In some embodiments, the compound of formula (II) is selected from:

[0040] Preferably, the compound of formula (II) is selected from: compound 64, compound 85, compound 87, compound 88, compound 64-1, compound 85-1 or compound 87-1;

[0041] More preferably, the compound of formula (II) is selected from: compound 64, compound 85, compound 87, compound 64-1, compound 85-1 or compound 87-1.

[0042] As a third aspect of the present invention, the present invention provides compounds of formula (III), isomers thereof, and pharmaceutically acceptable salts:

[0043] R2 is selected from H, C1-3 alkyl, or one or more deuterated C1-3 alkyl groups;

[0044] Preferably, R2 is selected from C1-3 alkyl groups or one or more deuterium-substituted C1-3 alkyl groups;

[0045] More preferably, R2 is selected from methyl, ethyl, propyl, deuterated methyl, deuterated ethyl, or deuterated propyl;

[0046] More preferably, R2 is selected from deuterated methyl;

[0047] Ring A is selected from:

[0048] Preferably, ring A is selected from

[0049] More preferably, ring A is selected from...

[0050] L1 is selected from -(CH2)n-, where n is selected from 0, 1, or 2;

[0051] Preferably, L1 is selected from -(CH2)n-, and n is selected from 0;

[0052] Ring B is selected from phenyl, and the phenyl is optionally substituted with halogen, C1-3 alkyl, C1-3 alkoxy or phenyl;

[0053] Preferably, ring B is selected from phenyl, and the phenyl is optionally substituted with halogen or C1-3 alkoxy group;

[0054] More preferably, ring B is selected from phenyl, and the phenyl is optionally substituted with a halogen;

[0055] More preferably, ring B is selected from phenyl;

[0056] The condition is that when A is selected from... In this case, R2 cannot be selected from C1-3 alkyl groups.

[0057] In some embodiments, the compound of formula (III) is selected from:

[0058] Preferably, the compound of formula (III) is selected from: compound 40, compound 68, compound 72, compound 73, compound 74, compound 75, compound 76 or compound 77;

[0059] More preferably, the compound of formula (III) is selected from: compound 40, compound 72, compound 73, compound 74, compound 75 or compound 76.

[0060] As a fourth aspect of the present invention, the present invention provides compounds of formula (IV), isomers thereof, and pharmaceutically acceptable salts:

[0061] R2 is selected from H, C1-3 alkyl, or one or more deuterated C1-3 alkyl groups;

[0062] Preferably, R2 is selected from C1-3 alkyl groups or one or more deuterium-substituted C1-3 alkyl groups;

[0063] More preferably, R2 is selected from methyl, ethyl, propyl, deuterated methyl, deuterated ethyl, or deuterated propyl;

[0064] More preferably, R2 is selected from methyl;

[0065] Y1 is selected from O, S, or NH;

[0066] Preferably, Y1 is selected from O;

[0067] L1 is selected from -(CH2)n-, where n is selected from 0, 1, or 2;

[0068] Preferably, L1 is selected from -(CH2)n-, and n is selected from 1 or 2;

[0069] More preferably, L1 is selected from -(CH2)n-, and n is selected from 1;

[0070] Ring B is selected from phenyl or heterocyclic groups, and the phenyl or heterocyclic group is optionally substituted with halogen, C1-3 alkyl, C1-3 alkoxy or phenyl.

[0071] Preferably, ring B is selected from phenyl or five-membered heterocyclic groups, and the phenyl or five-membered heterocyclic group is optionally substituted with halogen or C1-3 alkyl.

[0072] More preferably, ring B is selected from phenyl or thiophene group, and the phenyl or thiophene group is optionally substituted with halogen or C1-3 alkyl group;

[0073] More preferably, ring B is selected from phenyl or thiophene, and the phenyl or thiophene group is optionally substituted with a halogen.

[0074] In some embodiments, the compound of formula (IV) is selected from:

[0075] Preferably, the compound of formula (IV) is selected from: compound 11, compound 44, compound 45, compound 46, compound 47 or compound 84;

[0076] More preferably, the compound of formula (IV) is selected from: compound 11, compound 44, compound 45, compound 46 or compound 84.

[0077] As a fifth aspect of the present invention, the present invention provides a pharmaceutical composition comprising a compound of formula (I), an isomer thereof and a pharmaceutically acceptable salt thereof, a compound of formula (II), an isomer thereof and a pharmaceutically acceptable salt thereof, a compound of formula (III), an isomer thereof and a pharmaceutically acceptable salt thereof, or a compound of formula (IV), an isomer thereof and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

[0078] As a sixth aspect of the present invention, the present invention provides the use of the above-described compound (I), its isomers and pharmaceutically acceptable salts, the above-described compound (II), its isomers and pharmaceutically acceptable salts, the above-described compound (III), its isomers and pharmaceutically acceptable salts, the above-described compound (IV), its isomers and pharmaceutically acceptable salts, or the above-described pharmaceutical compositions in the preparation of medicaments for treating RIPK1-mediated diseases;

[0079] Preferably, the RIPK1-mediated related diseases are inflammatory diseases; more preferably, the RIPK1-mediated related diseases are systemic inflammatory syndromes, sepsis, lung injury, kidney injury, and autoimmune diseases.

[0080] Compared with the prior art, the present invention has the following beneficial effects:

[0081] The RIPK1 inhibitor provided by this invention exhibits good in vitro activity. In an LPS-induced sepsis model, it significantly improves mouse survival rate, showing a significant difference compared to the model group. Furthermore, in an LPS-induced inflammation model, it significantly increases mouse body temperature and reduces plasma TNF-α levels. In addition, the compound provided by this invention possesses favorable pharmacokinetic properties. Detailed Implementation

[0082] Explanation and Definition

[0083] Unless otherwise stated, the following terms and phrases used in this invention are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense.

[0084] The term "C1-3 alkyl" refers to any straight-chain or branched group containing 1-3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, etc.

[0085] The term “deuterated C1-3 alkyl” refers to a C1-3 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, etc.) in which at least one H has been replaced by D (e.g., -CD3 or -C2D5, etc.).

[0086] The term "C3-6 cycloalkyl" refers to a monovalent ring in a saturated hydrocarbon containing 3-6 carbon atoms. Cycloalkyl groups can exist in monocyclic, fused, or bridged ring forms. Exemplary examples of cycloalkyl groups include, but are not limited to, the following:

[0087] Examples include wait.

[0088] The term "fused heterocyclic group" refers to an aromatic cyclic structure composed of two or more rings, with adjacent rings sharing two adjacent ring atoms. The fused heterocyclic group includes cyclic structures formed by the fusion of a monoheterocyclic group (e.g., a 5-membered nitrogen heterocycle) with an aryl or heteroaryl group (e.g., pyridine). Exemplary examples of fused heterocyclic groups include, but are not limited to, the following:

[0089] wait.

[0090] The term “C1-3 alkoxy” refers to any of the above-mentioned alkyl groups (e.g., C1-3 alkyl groups, etc.) that are attached to the rest of the molecule by an oxygen atom -O-, such as C1-3 alkoxy groups, specifically methoxy, ethoxy, propoxy, etc.

[0091] The term "heterocyclic group" refers to a monovalent group that is fully saturated or partially unsaturated (but not fully unsaturated, such as having one or two double bonds) of a monocyclic, bridged, or spirocyclic ring, wherein at least one (e.g., 1, 2, 3, or 4) of the ring atoms is a heteroatom selected from N, O, and S, and the remaining ring atoms are C. For example, 5- or 6-membered heterocyclic groups include heterocyclic alkyl or heterocyclic alkenyl groups containing 1 to 3 heteroatoms (selected from N, O, and S).

[0092] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0093] The terms “optional” or “optionally” mean that the event or circumstance described below may, but is not necessarily, occur, and the description includes the circumstances under which the event or circumstance may or may not occur. For example, “optionally replaced” includes both replacement and non-replacement, and “optionally included” covers both inclusion and non-inclusion.

[0094] In this invention, "treatment" generally refers to achieving the desired pharmacological and / or physiological effects. This effect may be preventative, based on the complete or partial prevention of a disease or its symptoms; and / or therapeutic, based on the partial or complete stabilization or cure of a disease and / or side effects resulting from the disease. The term "treatment" as used in this invention covers any treatment of a patient's disease, including: (a) preventing a disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., preventing its progression; or (c) alleviating the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0095] The compounds described in this invention may optionally be used in combination with one or more other active ingredients, and the dosage and ratio of each ingredient may be adjusted by those skilled in the art according to the specific symptoms, the patient's condition, and clinical needs.

[0096] Mass spectrometry: AB SCIEX TripleTOF 4600;

[0097] NMR: Bruker AVANCE NEO 400MHz;

[0098] LC-MS: Shimadzu LC20AD tandem AB SCIEX TripleTOF 4600 mass spectrometer;

[0099] High performance liquid chromatograph: Waters e2695, chromatographic column: Shim-pack GIS C18 (4.6*250nm, 5um).

[0100] It should be noted that in this invention, DMF is N,N-dimethylformamide, DIEA is N,N-diisopropylethylamine, DMSO is dimethyl sulfoxide, HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, THF is tetrahydrofuran, Et3N is triethylamine, DMAP is 4-dimethylaminopyridine, Boc2O is ditert-butyl dicarbonate, Pd(PPh3)4 is tetrakis(triphenylphosphine)palladium, CDI is N,N′-carbonyldiimidazole, and (Ph3P)2PdCl2 is bis(triphenylphosphine)palladium dichloride.

[0101] Synthesis Examples

[0102] Preparation of intermediates in Example 1:

[0103] Synthesis of intermediate I-6:

[0104] Step 1:

[0105] At 0°C, a DMF solution of (s)-2-((tert-butyloxycarbonyl)amino)-3-aminopropionic acid (10 g, 49.0 mmol) in 100 mL was added dropwise to a DMF suspension of sodium hydride (3.9 g, 97.5 mmol, 60%). After the efflux stopped, a DMF solution of 3-fluoro-2-nitropyridine (7.0 g, 49.0 mmol) was added dropwise. After the addition was complete, the reaction was stirred at room temperature. After the reaction was monitored by TLC until it was complete, ethyl acetate (500 mL) and 0.5 M hydrochloric acid solution were added to the reaction solution, and the aqueous phase was weakly acidic. The mixture was separated, washed with brine (100 mL x 3), dried and concentrated to obtain crude product I-7 (16 g), which could be used directly in the next step without purification.

[0106] MS m / z (ESI): 327.20 [M+H] + .

[0107] Step 2:

[0108] Intermediate I-7 (16 g, 49 mmol) was added to methanol (200 mL) at room temperature, and palladium on carbon (1.6 g, 10% by weight) was added under nitrogen protection. The reaction was carried out at room temperature under hydrogen atmosphere. After the reaction was completed by TLC monitoring, the mixture was filtered, washed with methanol, and the filtrate was evaporated to dryness to obtain intermediate I-8 (14.5 g), which could be used directly for the next step without purification.

[0109] MS m / z (ESI): 297.21 [M+H] + .

[0110] Step 3:

[0111] Intermediate I-8 (14.5 g, 49 mmol) and DIEA (18.9 g, 147 mmol) were added to DMSO (200 mL) at room temperature, followed by the addition of HATU (22.4 g, 58.8 mmol) in portions. The reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, water (400 mL) was added, and a solid precipitated. The solid was filtered and dried to obtain the crude product. The crude product was then subjected to column chromatography to obtain intermediate I-9 (5.1 g, yield: 37.5%).

[0112] MS m / z (ESI): 279.18 [M+H] + .

[0113] Step 4:

[0114] At 0°C, Et3N (3.6 g, 36 mmol) and DMAP (0.22 g, 1.8 mmol) were added to a THF (50 mL) solution of intermediate I-9 (5 g, 18 mmol), and the mixture was stirred for 10 minutes. Boc2O (4.7 g, 21.6 mmol) was added in portions. After the addition was complete, the reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered, and the filtrate was evaporated to dryness. The crude product was then subjected to column chromatography to give intermediate I-10 (6.2 g, yield: 91.6%).

[0115] MS m / z (ESI): 379.2 [M+H] + .

[0116] Step 5:

[0117] At 0°C, cesium carbonate (10.4 g, 31.8 mmol) was added to a DMF (50 mL) solution of intermediate I-10 (6 g, 15.9 mmol), and the mixture was stirred for 10 minutes. Iodomethane (2.5 g, 17.5 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered, and the filtrate was added to cold water (100 mL), precipitating a solid, which was then filtered again. The solid was washed three times with water and dried under vacuum to obtain the crude product. The crude product was subjected to column chromatography to obtain I-11 (3.5 g, yield: 56.3%).

[0118] MS m / z (ESI): 393.2 [M+H] + .

[0119] Step 6:

[0120] Intermediate I-11 (3.5 g, 8.9 mmol) was added in portions to a solution of dioxane in hydrogen chloride at room temperature, and the reaction was stirred at room temperature. After the reaction was completed by TLC monitoring, the mixture was filtered and washed with ethyl ester. The solution was dried under vacuum to give intermediate I-6 (1.5 g, yield: 63.6%).

[0121] MS m / z (ESI): 193.1 [M+H] + .

[0122] Synthesis of intermediate VI-12:

[0123] Intermediate VI-12 was synthesized by replacing iodomethane with deuterated iodomethane, following the same synthesis method as intermediate I-6. Its structure is as follows:

[0124] MS m / z (ESI): 196.10 [M+H] + .

[0125] Synthesis of intermediate VI-8:

[0126] Intermediate VI-8 was synthesized by replacing 3-fluoro-2-nitropyridine with 3-fluoro-4-nitropyridine, following the same synthesis method as intermediate I-6. Its structure is as follows:

[0127] MS m / z (ESI): 193.12 [M+H] + .

[0128] Synthesis of intermediate IV-2:

[0129] Step 1:

[0130] At 0°C, a DMF (100 mL) solution of (s)-2-((tert-butyloxycarbonyl)amino)-3-hydroxypropionic acid (10 g, 48.7 mmol) was added dropwise to a DMF (100 mL) suspension of sodium hydride (3.9 g, 97.5 mmol, 60%). After the efflux stopped, a DMF solution of 2-nitrobenzene (6.9 g, 48.7 mmol) was added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature. After the reaction was monitored by TLC, the reaction solution was quenched with ice water, followed by the addition of ethyl acetate (600 mL) and 0.5 M hydrochloric acid solution, resulting in a weakly acidic aqueous phase. The mixture was separated, washed with 100 mL of brine (3 times), dried, and concentrated to obtain crude IV-3 (16 g), which could be used directly in the next step without further purification.

[0131] MS m / z (ESI): 327.20 [M+H] + .

[0132] Step 2:

[0133] Intermediate IV-3 (16 g, 49 mmol) was added to methanol (160 mL) at room temperature. Palladium on carbon (1.6 g, 10% by weight) was added under nitrogen protection, and the reaction was carried out at room temperature under hydrogen atmosphere. After the reaction was completed by TLC monitoring, the mixture was filtered and washed with methanol. The filtrate was evaporated to dryness to obtain intermediate IV-4 (14.5 g), which could be used directly for the next step without purification.

[0134] MS m / z (ESI): 297.21 [M+H] + .

[0135] Step 3:

[0136] Intermediate IV-4 (14.5 g, 49 mmol) and DIEA (12.6 g, 98 mmol) were added to DMSO (200 mL) at room temperature, followed by the addition of HATU (22.4 g, 58.8 mmol) in portions. The reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, water (400 mL) was added, and a solid precipitated. The solid was filtered and dried to obtain the crude product. The crude product was then subjected to column chromatography to obtain intermediate IV-5 (4.5 g, yield: 33.1%).

[0137] MS m / z (ESI): 279.18 [M+H] + .

[0138] Step 4:

[0139] At 0°C, cesium carbonate (7.0 g, 21.6 mmol) was added to a DMF (50 mL) solution of intermediate IV-5 (4 g, 14.4 mmol), and the mixture was stirred for 10 minutes. Deuterated iodomethane (2.3 g, 15.8 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered, and the filtrate was added to cold water (100 mL), precipitating a solid, which was then filtered again. The solid was washed three times with water and dried under vacuum to obtain the crude product. The crude product was then subjected to column chromatography to obtain intermediate IV-6 (2.1 g, yield: 49.5%).

[0140] MS m / z (ESI): 296.18 [M+H] + .

[0141] Step 5:

[0142] Intermediate IV-6 (2 g, 6.8 mmol) was added in portions to a 2 M, 10 mL solution of dioxane hydrogen chloride at room temperature, and the mixture was stirred at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered and washed with ethyl ester. The solution was dried under vacuum to give intermediate IV-2 (1.2 g, yield: 76.4%).

[0143] MS m / z (ESI): 196.20 [M+H] + .

[0144] Synthesis of intermediate VI-10:

[0145] Intermediate VI-10 was synthesized by replacing 2-nitrofluorobenzene with 2-nitro-4-bromofluorobenzene and replacing deuterated iodomethane with iodomethane, following the same synthesis method as intermediate IV-2. Its structure is as follows:

[0146] MS m / z (ESI): 271.05 [M+H] + .

[0147] Synthesis of intermediate VI-13:

[0148] Intermediate VI-13 was synthesized by replacing 2-nitrofluorobenzene with 2-nitro-4-bromofluorobenzene, following the same synthetic method as intermediate IV-2. Its structure is as follows:

[0149] MS m / z (ESI): 274.05 [M+H] + .

[0150] Example 2: Preparation of the compound:

[0151] Method 1:

[0152] Synthesis of compound 80:

[0153] Step 1:

[0154] At room temperature, N-BOC-O-(2,4,6-trimethylbenzenesulfonyl)hydroxylamine (10 g, 31.7 mmol) was added to 30 mL of dichloromethane, followed by 30 mL of trifluoroacetic acid. The reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the dichloromethane and trifluoroacetic acid were removed by vacuum distillation. The solution was adjusted to a weakly alkaline state with saturated sodium bicarbonate solution, extracted with ethyl acetate (50 mL x 3), washed with saturated brine (50 mL x 2), and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to obtain intermediate I-1 (8 g), which was used directly in the next step without further purification.

[0155] MS m / z (ESI): 216.10 [M+H] + .

[0156] Step 2:

[0157] Intermediate I-1 (8.0 g, 37.1 mmol) was added to 50 mL of dichloromethane at room temperature, followed by 6-bromopyridin-2-amine (8.0 g, 46.4 mmol). The reaction was carried out at room temperature under a nitrogen atmosphere. After the reaction was completed as monitored by TLC, the mixture was filtered, the filter cake was washed with dichloromethane, and dried to give intermediate I-2 (12 g, yield: 83.3%).

[0158] MS m / z (ESI): 388.11 [M+H] + .

[0159] Step 3:

[0160] Intermediate I-2 (12.0 g, 30.9 mmol) was added to 60 mL of ethanol at room temperature, followed by KOH (2.1 g, 37.1 mmol). The mixture was stirred for 10 minutes, and then diethyl oxalate (13.5 g, 92.7 mmol) was added. The reaction was carried out at room temperature under a nitrogen atmosphere. After the reaction was completed as monitored by TLC, the mixture was concentrated under reduced pressure. The crude product was then subjected to column chromatography to obtain intermediate I-3 (3.8 g, yield: 45.5%).

[0161] MS m / z (ESI): 270.11 [M+H] + .

[0162] Step 4:

[0163] Under nitrogen protection at room temperature, intermediate I-3 (3 g, 11.1 mmol), potassium carbonate (4.6 g, 33.3 mmol), Pd(PPh3)4 (0.38 g, 0.33 mmol), and phenylboronic acid (1.6 g, 13.3 mmol) were added to 30 mL of dioxane and heated to reflux. After the reaction was completed by TLC monitoring, the temperature was lowered. The mixture was concentrated under reduced pressure, and the crude product was subjected to column chromatography to obtain intermediate I-4 (1.5 g, yield 50.5%).

[0164] MS m / z (ESI): 268.12 [M+H] + .

[0165] Step 5:

[0166] Intermediate I-4 (1.0 g, 3.74 mmol) was added to 12 mL of methanol / water (3:1) at room temperature, followed by sodium hydroxide (0.3 g, 7.48 mmol). The reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the methanol was removed under reduced pressure, 10 mL of water was added, and the solution was adjusted to a weakly acidic state with 6 M hydrochloric acid. A solid precipitated, which was filtered, the filter cake was washed with water, and dried to obtain intermediate I-5 (0.70 g, yield 78.2%).

[0167] MS m / z (ESI): 240.12 [M+H] + .

[0168] Step 6:

[0169] Intermediate I-5 (100 mg, 0.42 mmol) and HATU (190 mg, 0.50 mmol) were added to DMSO (3 mL) at room temperature and stirred for 10 minutes. Then, DIEA (108 mg, 0.84 mmol) and (S)-3-amino-5-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-b][1,4]diazazo-4-one hydrochloride (111 mg, 0.42 mmol) were added and the reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, water (10 mL) was added, and the mixture was extracted with ethyl acetate and purified by column chromatography to give compound 80 (100 mg, yield 57.8%).

[0170] MS m / z (ESI): 414.12 [M+H] + .

[0171] 1 H-NMR(DMSO-d6)δ:8.74-8.75(1H,d),8.00-8.02(1H,m),7.95-7.98(3H,m),7.85-7.89(1H,m),7.58-7.62(3H,m),7.46-7.48(1H, m),7.37-7.39(1H,m),7.11-7.14(1H,m),5.83-5.85(1H,d),4.65-4.71(1H,m),3.75-3.81(1H,m),3.55-3.61(1H,m),3.34(3H,s).

[0172] Synthesis of compound 80-1:

[0173] Compound 80-1 was synthesized by replacing iodomethane with deuterated iodomethane, following the same synthetic method as compound 80. Its structure is as follows:

[0174] MS m / z (ESI): 417.20 [M+H] + .

[0175] Method 2:

[0176] Synthesis of compound 81:

[0177] Step 1:

[0178] At room temperature, ethyl 5-amino-1H-1,2,4-triazol-3-carboxylate (5 g, 32.0 mmol) was added to 50 mL of glacial acetic acid, followed by 3-(dimethylamino)-1-(2-phenyl)-2-propen-1-one (5.6 g, 32.0 mmol), and the mixture was heated to reflux. After the reaction was complete as monitored by TLC, the temperature was lowered. The mixture was concentrated under reduced pressure, and the crude product was washed with ice water and dried to give intermediate II-1 (4.2 g, yield: 48.9%).

[0179] MS m / z (ESI): 269.10 [M+H] + .

[0180] Step 2:

[0181] At room temperature, intermediate II-1 (1.0 g, 3.73 mmol) was added to 12 mL of methanol / water (3:1), followed by sodium hydroxide (0.3 g, 7.46 mmol), and the reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, the methanol was removed under reduced pressure, 10 mL of water was added, and the solution was adjusted to a weakly acidic state with 6 M hydrochloric acid. A solid precipitated, which was filtered, the filter cake was washed with water, and dried to obtain intermediate II-2 (0.75 g, yield 83.8%).

[0182] MS m / z (ESI): 241.12 [M+H] + .

[0183] Step 3:

[0184] Intermediate II-2 (100 mg, 0.42 mmol) and HATU (190 mg, 0.50 mmol) were added to DMSO (3 mL) at room temperature and stirred for 10 minutes. Then, DIEA (108 mg, 0.84 mmol) and (S)-3-amino-5-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-b][1,4]diazazo-4-one hydrochloride (111 mg, 0.42 mmol) were added and the reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, water (10 mL) was added, and the mixture was extracted with ethyl acetate and purified by column chromatography to give compound 81 (110 mg, yield 63.8%).

[0185] MS m / z (ESI): 415.15 [M+H] + .

[0186] 1H-NMR(DMSO-d6)δ:9.03-9.05(1H,d),8.91-8.92(1H,d),8.16-8.18(2H,m),8.01-8.03(1H,m),7.63-7.71(4H,m),7.37- 7.40(1H,m),7.12-7.15(1H,m),5.81-5.83(1H,d),4.67-4.73(1H,m),3.75-3.80(1H,m),3.61-3.67(1H,m),3.35(3H,s).

[0187] Synthesis of compound 81-1:

[0188] Compound 81-1 was synthesized by replacing iodomethane with deuterated iodomethane, following the same synthetic method as compound 81. Its structure is as follows:

[0189] MS m / z (ESI): 418.20 [M+H] + .

[0190] Method 3:

[0191] Synthesis of compound 82:

[0192] Step 1:

[0193] At room temperature, 6-bromopyridine-2-carboxaldehyde (5 g, 26.9 mmol) was added to 40 mL of dioxane / water (3:1), followed by ethyl acrylate (5.4 g, 53.8 mmol) and triethylenediamine (0.3 g, 5.4 mmol). The reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, the solution was concentrated under reduced pressure. The crude product was then subjected to column chromatography to give intermediate III-1 (5.2 g, yield: 67.6%).

[0194] MS m / z (ESI): 286.01 [M+H] + .

[0195] Steps 2-3:

[0196] At room temperature, intermediate III-1 (5 g, 17.5 mmol) was added to 50 mL of acetic anhydride and heated to 100 °C for 1 hour under a nitrogen atmosphere to obtain a solution of intermediate III-2, which was then refluxed without further treatment. After the reaction was completed as monitored by TLC, the solution was concentrated under reduced pressure, and the residue was poured into a large amount of ice water, neutralized with an aqueous sodium bicarbonate solution, and a solid precipitated. The solid was filtered and dried to obtain intermediate III-3 (3.5 g, yield: 74.6%).

[0197] MS m / z (ESI): 268.05 [M+H]+ .

[0198] Step 4:

[0199] Under nitrogen protection at room temperature, intermediate III-3 (3 g, 11.2 mmol), potassium carbonate (4.6 g, 33.6 mmol), Pd(PPh3)4 (0.38 g, 0.33 mmol), and phenylboronic acid (1.6 g, 13.4 mmol) were added to 30 mL of dioxane and heated to reflux. After the reaction was completed by TLC monitoring, the temperature was lowered. The mixture was concentrated under reduced pressure, and the crude product was subjected to column chromatography to obtain intermediate III-4 (1.8 g, yield 60.6%).

[0200] MS m / z (ESI): 266.15 [M+H] + .

[0201] Step 5:

[0202] Intermediate III-4 (1.0 g, 3.77 mmol) was added to 12 mL of methanol / water (3:1) at room temperature, followed by sodium hydroxide (0.3 g, 7.54 mmol). The reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, the methanol was removed under reduced pressure, 10 mL of water was added, and the solution was adjusted to a weakly acidic state with 6 M hydrochloric acid. A solid precipitated, which was filtered, the filter cake was washed with water, and dried to obtain intermediate III-5 (0.60 g, yield 67.1%).

[0203] MS m / z (ESI): 238.10 [M+H] + .

[0204] Step 6:

[0205] Intermediate III-5 (100 mg, 0.42 mmol) and HATU (190 mg, 0.50 mmol) were added to DMSO (3 mL) and stirred at room temperature for 10 minutes. Then, DIEA (108 mg, 0.84 mmol) and (S)-3-amino-5-methyl-1,2,3,5-tetrahydro-4H-pyrido[2,3-b][1,4]diazazo-4-one hydrochloride (111 mg, 0.42 mmol) were added, and the reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, water (10 mL) was added, and the mixture was extracted with ethyl acetate and purified by column chromatography to give compound 82 (115 mg, yield 66.3%).

[0206] MS m / z (ESI): 412.21 [M+H] + .

[0207] 1H-NMR(DMSO-d6)δ:8.45-8.48(1H,d),8.03-8.05(1H,m),7.88(1H,s),7 .65-7.68(2H,m),7.55-7.63(3H,m),7.49-7.51(1H,d),7.36-7.39(1H, m),7.10-7.13(1H,m),6.92(1H,s),6.84-6.88(1H,m),6.57-6.59(1H,m ),5.56-5.57(1H,m),4.64-4.71(1H,q),3.58-3.61(2H,m),3.31(3H,m).

[0208] Synthesis of compound 82-1:

[0209] Compound 82-1 was synthesized by replacing iodomethane with deuterated iodomethane, following the same method used for compound 82. Its structure is as follows:

[0210] MS m / z (ESI): 415.21 [M+H] + .

[0211] Method 4:

[0212] Synthesis of compound 40:

[0213] Step 1:

[0214] At room temperature, 7-phenyl-2-pyrazole[1,5-A]pyrimidine carboxylic acid (100 mg, 0.42 mmol) and HATU (190 mg, 0.50 mmol) were added to DMSO (3 mL) and stirred at room temperature for 10 minutes. Then, DIEA (108 mg, 0.84 mmol) and (S)-3-amino-5-(methyl-d3)-1,2,3,5-tetrahydro-pyrido[2,3-b][1,4]diazazo-4-one hydrochloride (97 mg, 0.42 mmol) were added, and the reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, water (10 mL) was added, and the mixture was extracted with ethyl acetate and purified by column chromatography to give compound 40 (100 mg, yield 57.4%).

[0215] MS m / z (ESI): 417.15 [M+H] + .

[0216] 1H-NMR(DMSO-d6)δ:8.69-8.70(1H,d),8.45-8.47(1H,d),8.16-8.19(2H,m),7.61-7.67(3H,m),7.49-7.51(1H, m),7.38-7.39(1H,m),7.23-7.35(3H,m),7.17(1H,s),4.87-4.94(1H,m),4.55-4.60(1H,m),4.45-4.50(1H,m).

[0217] Following the synthetic method of compound 40, the following compounds were obtained, and their structures are shown in the table below:

[0218] Method 5:

[0219] Synthesis of compound 64:

[0220] Step 1:

[0221] At room temperature, 7-phenyl-2-pyrazole[1,5-A]pyrimidine carboxylic acid (100 mg, 0.42 mmol) and HATU (190 mg, 0.50 mmol) were added to DMSO (3 mL) and stirred at room temperature for 10 minutes. Then, DIEA (108 mg, 0.84 mmol) and (S)-3-amino-7-bromo-5-methyl-2,3-dihydrobenzo[b][1,4]oxazapyro-4(5H)-one hydrochloride (129 mg, 0.42 mmol) were added, and the reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, water (10 mL) was added, and a solid precipitated. The solid was filtered and dried to give intermediate IV-3 (150 mg, yield 72.9%).

[0222] MS m / z (ESI): 492.17 [M+H] + .

[0223] Step 2:

[0224] Intermediate IV-3 (0.15 g, 0.30 mmol), cyclopropanethyne (99 mg, 1.50 mmol), cuprous iodide (29 mg, 0.15 mmol), (Ph3P)2PdCl2 (21 mg, 0.03 mmol), triethylamine (4 mL), and DMF (2 mL) were mixed at room temperature, substituted with N2, and reacted at 100 °C. After the reaction was complete as monitored by TLC, 20 mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (20 mL * 3), washed with saturated brine (20 mL * 2), and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure, and the crude product was subjected to column chromatography to give compound 64 (50 mg, yield 34.4%).

[0225] MS m / z (ESI): 478.16 [M+H] + .

[0226] 1 H-NMR(DMSO-d6)δ:8.69-8.70(1H,d),8.44-8.46(1H,d),8.16-8.18(2H,d ),7.61-7.67(3H,m),7.49-7.50(1H,m),7.38-7.39(1H,m),7.25-7.28(1H, m),7.17-7.19(2H,m),4.86-4.93(1H,m),4.55-4.60(1H,m),4.45-4.49(1H ,m),3.32(3H,m),1.51-1.58(1H,m),0.87-0.93(2H,m),0.72-0.75(2H,m).

[0227] Following the synthetic method of compound 64, the following compounds were obtained, and their structures are shown in the table below:

[0228] Method 6:

[0229] Synthesis of compound 11:

[0230] Step 1:

[0231] At room temperature, D-phenylalanine (10.0 g, 60.5 mmol) was added to 200 mL of a water / tetrahydrofuran mixture (H₂O / THF = 1:1), followed by Boc₂O (19.8 g, 90.8 mmol) and sodium bicarbonate (10.2 g, 121 mmol). The reaction was allowed to proceed at room temperature. After the reaction was complete as monitored by TLC, most of the tetrahydrofuran was evaporated, and the mixture was washed with n-hexane (100 mL * 3), collecting the aqueous phase. The aqueous phase was adjusted to pH 4 with saturated citric acid, extracted with ethyl acetate (100 mL * 3), and washed with saturated brine (100 mL * 3). The organic phase was dried over anhydrous sodium sulfate and concentrated to give intermediate VII-1 (16 g, yield: 99.8%).

[0232] MS m / z (ESI): 266.20 [M+H] + .

[0233] Step 2:

[0234] Intermediate VII-1 (16.0 g, 60.3 mmol) was added to 200 mL of dichloromethane at room temperature, followed by HATU (27.5 g, 72.4 mmol). The mixture was stirred at room temperature for 10 minutes, then DIEA (15.6 g, 120.6 mmol) and p-methoxybenzylamine (8.3 g, 60.3 mmol) were added, and the mixture was allowed to react at room temperature. After the reaction was complete as monitored by TLC, the mixture was washed with saturated brine (100 mL x 3) and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure, and the crude product was slurried in hexane / ethyl acetate at a 1:1 ratio to obtain intermediate VII-2 (16 g, 68.9%).

[0235] MS m / z (ESI): 385.24 [M+H] + .

[0236] Step 3:

[0237] Intermediate VII-2 (16 g, 41.6 mmol) was added to 40 mL of ethyl acetate at room temperature, followed by the slow addition of ethyl acetate-hydrochloride solution (2 M, 83 mL, 166.4 mmol) under stirring at room temperature. After the reaction was complete as monitored by TLC, the solution was concentrated under reduced pressure. The crude product was then added to 100 mL of water and adjusted to alkaline with saturated sodium bicarbonate. Extraction was performed with ethyl acetate (100 mL x 3), followed by washing with saturated brine (100 mL x 3), and drying over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to give intermediate VII-3 (10.5 g, 89%).

[0238] MS m / z (ESI): 285.20 [M+H] + .

[0239] Step 4:

[0240] At room temperature, intermediate VII-3 (10 g, 35.2 mmol) was added to 50 mL of tetrahydrofuran, followed by the addition of a borane tetrahydrofuran solution (1 M, 100 mL, 100 mmol). The mixture was heated to reflux. After the reaction was completed as monitored by TLC, the reaction solution was quenched with 6 M hydrochloric acid, and most of the tetrahydrofuran was concentrated under reduced pressure. The mixture was extracted with ethyl acetate (100 mL * 3), and the aqueous phase was adjusted to alkalinity with saturated sodium bicarbonate to precipitate a solid. The solid was filtered, washed with water, and dried to obtain intermediate VII-4 (8 g, 84.2%).

[0241] MS m / z (ESI): 271.20 [M+H] + .

[0242] Step 5:

[0243] Intermediate VII-4 (8 g, 29.6 mmol) was added to 100 mL of tetrahydrofuran at room temperature, followed by CDI (5.7 g, 35.5 mmol). The reaction was allowed to proceed at room temperature. After the reaction was completed as monitored by TLC, most of the tetrahydrofuran was concentrated under reduced pressure. 50 mL each of water and ethyl acetate were added, and the mixture was extracted with ethyl acetate (50 mL x 2). The extract was washed with saturated brine (50 mL x 2) and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure, and column chromatography yielded intermediate VII-5 (4.7 g, 53.6%).

[0244] MS m / z (ESI): 297.14 [M+H] + .

[0245] Step 6:

[0246] Intermediate VII-5 (4.7 g, 15.9 mmol) was added to 30 mL of trifluoroacetic acid at room temperature and reacted at 50 °C. After the reaction was completed as monitored by TLC, most of the trifluoroacetic acid was concentrated under reduced pressure, adjusted to alkaline with saturated sodium bicarbonate solution, extracted with ethyl acetate (50 mL * 3), washed with saturated brine (50 mL * 2), and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure, and column chromatography yielded intermediate VII-6 (0.93 g, 33.3%).

[0247] MS m / z (ESI): 177.10 [M+H] + .

[0248] Step 7:

[0249] At room temperature, intermediate VII-6 (0.18 g, 1.02 mmol) was added to 3 mL of tetrahydrofuran, and triphosgene (0.1 g, 0.34 mmol) was added. The mixture was reacted at 30 °C for 4 hours. The mixture was concentrated under reduced pressure to obtain a solid, which was dissolved in 2 mL of tetrahydrofuran for later use. Under ice-water bath conditions, (S)-7-bromo-3-amino-5-methyl-2,3-dihydrobenzo[b][1,4]oxazazepine-4(5H)-one hydrochloride (314 mg, 1.02 mmol) and triethylamine (0.3 g, 2.97 mmol) were added to a 100 mL reaction flask, and the dissolved tetrahydrofuran solution was added dropwise. After the addition was complete, the reaction mixture was allowed to return to room temperature. After the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was then subjected to column chromatography to obtain intermediate VII-7 (0.38 g, 78.7%).

[0250] MS m / z (ESI): 473.04 [M+H] + .

[0251] Step 8:

[0252] At room temperature, intermediate VII-7 (0.29 g, 0.61 mmol), cyclopropylacetylene (0.12 g, 1.83 mmol), cuprous iodide (35 mg, 0.18 mmol), (Ph3P)2PdCl2 (21 mg, 0.03 mmol), and triethylamine (4 mL) were added to 2 mL of LDM, substituted with N2, and reacted at 100 °C. After the reaction was complete as monitored by TLC, 20 mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (20 mL x 3), washed with saturated brine (20 mL x 2), and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure, and column chromatography of the crude product yielded compound 11 (0.2 g, 71.4%).

[0253] MS m / z (ESI): 459.22 [M+H] + .

[0254] 1H-NMR(DMSO-d6)δ:8.65-8.67(1H,d),7.94(1H,s),7.42-7.45(1H,m),7.12- 7.30(7H,m),4.55-4.63(1H,m),4.38-4.44(1H,m),4.15-4.22(1H,m),3.88-3. 91(1H,m),3.58-3.63(1H,m),3.33-3.35(1H,m),3.28(3H,s),2.80-2.83(1H,m ),2.66-2.71(1H,m),1.50-1.57(1H,m),0.84-0.91(2H,m),0.73-0.74(2H,m).

[0255] Following the synthetic method of compound 11, the following compounds were obtained, and their structures are shown in the table below:

[0256] Example 3: Preparation of the control compound:

[0257] Reference compound 1:

[0258] Reference compound 2:

[0259] Pharmacological experiments

[0260] Pharmacological Example 1: In vitro enzyme activity test

[0261] Experimental instruments: Envision microplate reader (PerkinElmer), Echo (LABCYTE).

[0262] Experimental method: The ADP-Glo ​​method was used in this experiment to test the inhibitory effect of the compound on RIPK1 kinase.

[0263] Experimental Procedure: Dissolve the compound in DMSO to a storage concentration of 10 mM. Then dilute the compound with DMSO to prepare compound dilutions at different concentration gradients (100×). Transfer 50 nL of the 100× final concentration compound dilutions to a 384-well plate using an Echo instrument. Replace the low-signal control and high-signal control groups with the same volume of DMSO. Prepare the following 1× working solutions: HEPES pH 7.5 final concentration 50 mM, NaCl final concentration 50 mM, MgCl2 final concentration 30 mM, DTT final concentration 1 mM, CHAPS final concentration 0.02%, BSA final concentration 500 μg / mL. Prepare 2× final concentrations of RIPK1 kinase (final concentration 5 nM) and ATP (final concentration 10 μM) using the 1× working solution. Add 2.5 μL of RIPK1 kinase to each well of the 384-well plate. Replace the low-signal control group with the same volume of 1× working solution. Centrifuge at 1000 rpm for 30 seconds and incubate at 25°C for 15 minutes. Add 2.5 μL of ATP to each well of a 384-well plate, centrifuge at 1000 rpm for 30 seconds, and incubate at 25°C for 4 hours. Add ADP-Glo ​​to each well. TM Add 5 μL of reagent to each well of a 384-well plate, centrifuge at 1000 rpm for 30 seconds, and incubate at 25°C for 1 hour. Add 10 μL of kinase detection reagent to each well of the 384-well plate, centrifuge at 1000 rpm for 30 seconds, and incubate at 25°C for 30 minutes. Finally, read the signal values ​​using an Envision microplate reader (PerkinElmer).

[0264] Experimental data processing method: XLfit software, developed by IDBS and integrated into the Microsoft Excel environment, was used for test data processing and analysis. First, the average response signals of the high-signal control group and the low-signal control group were calculated separately. Then, the inhibition rate of each compound well was calculated using the formula: "Inhibition rate per well % = 100 - (Single-well signal value - Average value of low-signal control group) / (Average value of high-signal control group - Average value of low-signal control group)". Next, the concentration and corresponding inhibition rate data were imported into XLfit software. Using the Dose Response One Site 205 model in the software, a four-parameter method was employed to fit the inhibition rate-concentration curve, and the IC50 of the compound was calculated. 50 value. Appendix: A is less than or equal to 10 nM; B is greater than 10 nM and less than or equal to 100 nM; C is greater than 100 nM.

[0265] Pharmacological Example 2: In vitro cell activity test

[0266] Experimental instruments: Envision microplate reader (PerkinElmer), Echo (LABCYTE)

[0267] Experimental method: The Cell Titer-Glo method was used in this experiment to test the inhibitory effect of the compound on cell necrosis.

[0268] Experimental Procedure: U937 cells were resuscitated and cultured in RPMI 1640 complete medium at 37℃ with 5% CO2, passaged every 2-3 days. The compound was dissolved in DMSO to a storage concentration of 10 mM, and then diluted with DMSO to prepare compound dilutions at different concentration gradients (333×). 120 nL of the final 333× concentration compound dilution was transferred to a 384 experimental plate using an Echo instrument. The low-signal control and high-signal control groups were replaced with the same volume of DMSO. 1× Q-VD-OPh solution was prepared: using RPMI 1640 complete medium, Q-VD-OPh to a final concentration of 25 μM, 0.2% DMSO. 4× TNFα solution was prepared: using 1× Q-VD-OPh solution, TNFα to a final concentration of 100 ng / mL. U937 cells were resuspended in 1×Q-VD-OPh solution, seeded into plates (10,000 cells per well), and centrifuged at 1000 rpm for 30 seconds using 30 μL of the solution. Then, 10 μL of 4×TNFα solution was added to each well. The high-signal control group was replaced with the same volume of 1×Q-VD-OPh solution, and centrifuged at 1000 rpm for 30 seconds. The plates were incubated at 37°C with 5% CO2 for 48 hours. After incubation, 20 μL of CTG detection reagent was added to each well, and the plates were incubated at 25°C in the dark for 15 minutes before the signal was read using Envision.

[0269] Experimental data processing method: XLfit software, developed by IDBS and integrated into the Microsoft Excel environment, was used for test data processing and analysis. First, the average response signals of the high-signal and low-signal control groups were calculated separately. Then, the inhibition rate of each compound well was calculated using the formula: "Inhibition rate per well % = 100 - (Average signal value of high-signal control group - Signal value per well) / (Average signal value of high-signal control group - Average signal value of low-signal control group)". Next, the concentration and corresponding inhibition rate data were imported into XLfit software. Using the Dose Response One Site 205 model in the software, a four-parameter method was employed to fit the inhibition rate-concentration curve, and the IC50 of the compound was calculated. 50 value. Appendix: A is less than or equal to 10 nM; B is greater than 10 nM and less than or equal to 100 nM; C is greater than 100 nM.

[0270] Biological testing evaluation

[0271] The present invention will be further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present invention.

[0272] Test Example 1: In vivo mouse LPS-induced sepsis model:

[0273] Septicemia models were established in mice by intraperitoneal injection of 0.2 ml LPS (7.5 mg / kg). The drugs were administered once at 15 min and 7 h after model establishment. The test drug groups (compounds 80, 81, and 82) were administered intravenously at a dose of 5 mg / kg. The positive control group (ulinastatin) received an intraperitoneal injection of 100,000 U / kg ulinastatin. The blank control group and model groups received intravenous injections of the corresponding volumes of the test drug solvent (5% DMSO + 5% Tween 80 + 20% PEG400 + 70% double-distilled water). The drug administration details for each group are shown in the table below.

[0274] Observe the condition of mice after modeling and count the 24-hour survival rate.

[0275] After intraperitoneal injection of LPS to establish a sepsis model in mice, the 24-hour survival rate of the model group was 10%. After administration of ulinastatin to the sepsis model mice, the survival rate of the mice was 30%. After administration of the test drugs (compound 80, compound 81, and compound 82) to the sepsis model mice, the survival rates of the mice in each group were 60%, 50%, and 70%, respectively, and the survival rates of the mice in each test drug group were significantly improved.

[0276] The 24-hour survival of different compounds in an LPS-induced mouse sepsis model is shown in the table below:

[0277] Experimental conclusions: Compounds 80, 81, and 82 of this invention can significantly improve the survival rate of mice in the LPS-induced sepsis model, showing a significant difference compared with the model group, and are superior to the positive control drug ulinastatin.

[0278] According to the above method, compounds 1, 80-1, 81-1, and 82-1 of the present invention significantly improved the survival rate of mice in the LPS-induced sepsis model, showing a significant difference compared with the model group.

[0279] Test Example 2: In vivo mouse LPS-induced sepsis model:

[0280] Septicemia models were established in mice by intraperitoneal injection of 0.2 ml LPS (7.5 mg / kg). The drugs were administered once at 15 min and 7 h after model establishment. The test drug groups (compounds 11, 47, 64, and 74) and the control compound 2 group were administered intravenously at a dose of 5 mg / kg. The positive control group (ulinastatin) received an intraperitoneal injection of 100,000 U / kg ulinastatin. The blank control group and model groups received intravenous injections of the corresponding volume of the test drug solvent (5% DMSO + 5% Tween 80 + 20% PEG 400 + 70% double-distilled water). The drug administration details for each group are shown in the table below.

[0281] Observe the condition of mice after modeling and count the 24-hour survival rate.

[0282] After intraperitoneal injection of LPS to establish a sepsis model in mice, all animals in the model group died within 24 hours, with a survival rate of 0%. After administration of ulinastatin and control compound 2 to the sepsis model mice, the survival rates of the mice were 20% and 25%, respectively. After administration of the test drugs (compound 11, compound 47, compound 64, and compound 74) to the sepsis model mice, the survival rates of the mice in each group were 40%, 30%, 60%, and 50%, respectively, with a significant increase in the survival rate of the mice in each test drug group.

[0283] The 24-hour survival of different compounds in an LPS-induced mouse sepsis model is shown in the table below:

[0284] Experimental conclusions: Compounds 11, 47, 64, and 74 of this invention significantly improved the survival rate of mice in the LPS-induced sepsis model, showing a significant difference compared with the model group and being superior to the positive control drug ulinastatin.

[0285] According to the above method, compounds 85, 87, 64-1, 85-1, 87-1, 44, 45, 46, 84, 40, 72, 73, 75, and 76 of the present invention significantly improved the survival rate of mice in the LPS-induced sepsis model, showing a significant difference compared with the model group.

[0286] Test Example 3: In vivo mouse LPS-induced inflammation model:

[0287] Inflammation models were established in mice by intraperitoneal injection of 0.2 ml LPS (3.5 mg / kg). The drugs were administered once every 15 minutes after model establishment. The test drug groups (compounds 80, 81, and 82) were administered intravenously at a dose of 5 mg / kg. The positive control group (ulinastatin) received an intraperitoneal injection of 100,000 U / kg ulinastatin. The blank control group and model groups received intravenous injections of the corresponding volume of the test drug solvent (5% DMSO + 5% Tween 80 + 20% PEG400 + 70% double-distilled water). The drug administration details for each group are shown in the table below.

[0288] The body temperature of mice in each group was measured for 6 hours using a thermometer. After the body temperature measurement, the mice were sacrificed and blood was collected. Plasma was collected after centrifugation at 3000 rpm and the TNF-α content in the plasma was detected by ELISA kit.

[0289] After intraperitoneal injection of LPS to establish an inflammation model in mice, the body temperature was significantly lower than that of the blank control group, with a mean decrease of 5.51℃ at 6 h (P<0.001). After administration of ulinastatin to the inflammation model mice, the decrease in body temperature was reduced, but there was no significant difference compared with the model group (P>0.05). After administration of the test drugs (compound 80, compound 81, and compound 82) to the inflammation model mice, the decrease in body temperature was significantly reduced, with decreases of 54.1% (P<0.01), 46.3% (P<0.05), and 60.8% (P<0.01) respectively compared with the model group.

[0290] The changes in body temperature caused by different compounds in an LPS-induced mouse inflammation model are shown in the table below: Note: Compared with the blank control group, P ### <0.001; compared with the model group, P * <0.05, P ** <0.01, P *** <0.001.

[0291] After intraperitoneal injection of LPS to establish an inflammation model in mice, the plasma TNF-α level was significantly increased compared with the blank control group (P<0.001). After administration of ulinastatin to the inflammation model mice, the TNF-α level was significantly decreased, by 17.9% compared with the model group (P<0.05). After administration of the test drugs (compound 80, compound 81, and compound 82) to the inflammation model mice, the TNF-α level was significantly decreased, by 42.1% (P<0.001), 35.5% (P<0.01), and 60.8% (P<0.001) respectively compared with the model group.

[0292] The plasma TNF-α levels of different compounds in an LPS-induced mouse inflammation model are shown in the table below: Note: Compared with the blank control group, P ### <0.001; compared with the model group, P * <0.05, P ** <0.01, P *** <0.001.

[0293] Experimental conclusions: Compounds 80, 81, and 82 of this invention can significantly increase body temperature and reduce plasma TNF-α levels in mice in an LPS-induced inflammation model, which is superior to the positive control drug ulinastatin.

[0294] According to the above method, compounds 1, 80-1, 81-1, and 82-1 of the present invention can significantly increase the body temperature of mice and reduce the plasma TNF-α level in an LPS-induced inflammation model.

[0295] Test Example 4: In vivo mouse LPS-induced inflammation model:

[0296] Inflammation models were established in mice by intraperitoneal injection of 0.2 ml LPS (3.5 mg / kg). The drugs were administered once every 15 minutes after model establishment. The test drug groups (compounds 11, 47, 64, and 74) and the control compound 2 group were administered intravenously at a dose of 5 mg / kg. The positive control group (ulinastatin) received an intraperitoneal injection of 100,000 U / kg ulinastatin. The blank control group and model groups received intravenous injections of the corresponding volume of the test drug solvent (5% DMSO + 5% Tween 80 + 20% PEG 400 + 70% double-distilled water). The drug administration details for each group are shown in the table below.

[0297] The body temperature of mice in each group was measured for 6 hours using a thermometer. After the body temperature measurement, the mice were sacrificed and blood was collected. Plasma was collected after centrifugation at 3000 rpm and the TNF-α content in the plasma was detected by ELISA kit.

[0298] After intraperitoneal injection of LPS to establish an inflammation model in mice, the body temperature was significantly lower than that of the blank control group, with a mean decrease of 6.18℃ at 6 h (P<0.001). Administration of ulinastatin and control compound 2 to the inflammation model mice reduced the decrease in body temperature, but there was no significant difference compared to the model group (P>0.05). Administration of the test drugs (compounds 11, 47, 64, and 74) to the inflammation model mice significantly reduced the decrease in body temperature, with reductions of 41.9% (P<0.001), 35.4% (P<0.01), 53.4% ​​(P<0.001), and 47.9% (P<0.001) respectively compared to the model group.

[0299] The changes in body temperature caused by different compounds in an LPS-induced mouse inflammation model are shown in the table below: Note: Compared with the blank control group, P ### <0.001; compared with the model group, P * <0.05, P ** <0.01, P *** <0.001.

[0300] After intraperitoneal injection of LPS to establish an inflammation model in mice, the plasma TNF-α level was significantly increased compared with the blank control group (P<0.001). After administration of ulinastatin and control compound 2 to the inflammation model mice, the TNF-α level decreased, but there was no significant difference compared with the model group (P>0.05). After administration of the test drugs (compound 11, compound 47, compound 64, and compound 74) to the inflammation model mice, the TNF-α level was significantly reduced, decreasing by 37.6% (P<0.01), 33.4% (P<0.05), 48.8% (P<0.01), and 40.4% (P<0.01), respectively, compared with the model group.

[0301] The plasma TNF-α levels of different compounds in an LPS-induced mouse inflammation model are shown in the table below: Note: Compared with the blank control group, P ### <0.001; compared with the model group, P * <0.05, P ** <0.01, P *** <0.001.

[0302] Experimental conclusions: Compounds 11, 47, 64, and 74 of this invention can significantly increase body temperature and reduce plasma TNF-α levels in mice in an LPS-induced inflammation model, which is superior to the positive control drug ulinastatin.

[0303] According to the above method, compounds 85, 87, 64-1, 85-1, 87-1, 44, 45, 46, 84, 40, 72, 73, 75, and 76 of the present invention can significantly increase body temperature and reduce plasma TNF-α levels in mice in an LPS-induced inflammation model.

[0304] In addition, the compounds of this invention also have good pharmacokinetic properties.

[0305] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound of Formula (I), isomers, and pharmaceutically acceptable salts thereof: ###00001### (I). wherein one of X1, X2, X3, X4is N, and the others are CH; Preferably, X1is N, and X2, X3, X4are all CH; R1is selected from H, halogen, or C1-3alkyl, and Rc is selected from C3-6cycloalkyl or C1-3alkyl; Preferably, R1is selected from H; R2is selected from H, C1-3alkyl or one or more deuterium substituted C1-3alkyl; Preferably, R2is selected from C1-3alkyl or one or more deuterium substituted C1-3alkyl; Further preferably, R2is selected from methyl, ethyl, propyl, deuterated methyl, deuterated ethyl or deuterated propyl; More further preferably, R2is selected from methyl or deuterated methyl; Ring A is selected from 5-6 membered heterocyclyl or 8-9 membered fused heterocyclyl, wherein said 5-6 membered heterocyclyl or 8-9 membered fused Preferably, A is selected from 5 membered nitrogen heterocyclyl or 8-9 membered fused heterocyclyl, and wherein said 5 membered nitrogen heterocyclyl or 8-9 membered fused hetercyclyl is optionally substituted with 1-2 Re, and said Re is independently selected from halogen or C1-3alkyl; Further preferably, ring A is selected from Still more preferably, ring A is selected from Still further preferably, ring A is selected from L1is selected from -(CH2)n-, and n is selected from 0, 1 or 2; Preferably, L1is selected from -(CH2)n-, and n is selected from 0 or 1; Further preferably, when ring A is selected from L1is selected from -(CH2)n-, n is selected from 0; when A is selected from L1is selected from -(CH2)n-, and n is selected from 1; Ring B is selected from phenyl, and said phenyl is optionally substituted with halogen, C1-3alkyl, C1-3alkoxy or phenyl; Preferably, Ring B is selected from phenyl.

2. The compound of formula (I) according to claim 1, isomers and pharmaceutically acceptable salts thereof, characterized in that, The compound of formula (I) is selected from: Preferably, the compound of formula (I) is selected from: Compound 1, Compound 58, Compound 79, Compound 80, Compound 81, Compound 82, Compound 80-1, Compound 81-1 or Compound 82-1; Further preferably, the compound of formula (I) is selected from: Compound 1, Compound 80, Compound 81, Compound 82, Compound 80-1,Compound 81-1 or Compound 82-1.

3. A compound of formula (II), isomers, and pharmaceutically acceptable salts thereof: wherein, R2is selected from H, C1-3alkyl or one or more deuterated C1-3alkyl; Preferably, R2is selected from C1- 3alkyl or one or more deuterated C1-3alkyl; Further preferably, R2is selected from methyl, ethyl or deuterated methyl; More further preferably, R2is selected from methyl or deuterated methyl; Ring A is selected from: Preferably, ring A is selected from Further preferably, ring A is selected from L1is selected from -(CH2)n-, and n is selected from 0,1 or 2; Preferably, L1is selected from -(CH2)n-, n is selected from 0; Ring B is selected from phenyl, and said phenyl is optionally substituted with halogen, Preferably, Ring B is selected from phenyl.

4. The compound of formula (II) according to claim 3, isomers and pharmaceutically acceptable salts thereof, characterized in that, The compound of formula (II) is selected from: Preferably, the compound of Formula (II) is selected from: Compound 64, Compound 85, Compound 87, Compound 88, Compound 64-1, Compound 85-1 or Compound 87-1; Further preferably, the compound of Formula (II) is selected from: Compound 64, Compound 85 or Compound 87.

5. A compound of formula (III), isomers, and pharmaceutically acceptable salts thereof: wherein R2is selected from H, C1-3alkyl or one or more deuteated C1-3alkyl; Preferably, R2is selected from C1 -3alkyl or one or more deuterated C1-3alkyl; Further preferably, R2is selected from methyl, ethyl, propyl, deuterated methyl, deuterated ethyl or deuterated propyl; More preferably, R2is selected from deuterated methyl; Ring A is selected from: Preferably, ring A is selected from Further preferably, ring A is selected from L1is selected from -(CH2)n-, n is selected from 0, 1 or 2; Preferably, L1is selected from -(CH2)n-, n is selected from 0; Ring B is selected from phenyl, and said phenyl is optionally substituted with halogen, C1-3alkyl, C1-3alkoxy or phenyl; Preferably, ring B is selected from phenyl, and said phenyl is optionally substituted with halogen, C^alkoxy; Further preferably, ring B is selected from phenyl, and said phenyl is optionally substituted with halogen; More preferably, ring B is selected from phenyl; provided that when A is selected from When R2is selected from C1-3alkyl, R2cannot be selected from C1-3alkyl.

6. The compound of formula (III) according to claim 5, isomers and pharmaceutically acceptable salts thereof, characterized in that, The compound of formula (III) is selected from: Preferably, the compound of formula (III) is selected from: compound 40, compound 68, compound 72, compound 73, compound 74, compound 75, compound 76 or compound 77; Further preferably, the compound of formula (III) is selected from: compound 40, compound 72, compound 73, compound 74, compound 75 or compound 76.

7. A compound of formula (IV), isomers, and pharmaceutically acceptable salts thereof: wherein R2is selected from H, C1-3alkyl or 1 or more deuterium substituted C1-3alkyl; Preferably, R2is selected from C1-3alkyl or 1 or more deuterium substituted C1-3 alkyl; Further preferably, R2is selected from methyl, ethyl, propyl, deuterated methy l, deuterated ethyl or deuterated propyl; More preferably, R2is methyl; Y1is selected from O, S or NH; Preferably, Y1is O; L1is selected from -(CH2)n-, n is selected from 0, 2 or 3; Preferably, L1is selected from -(CH2)n-, n is selected from l or 2; Further preferably, L1is selected from -(CH2)n-, n is selected from 1; Ring B is selected from phenyl or heterocyclyl, and said phenyl or heterocyclyl is optionally substituted with halogen, C1-3alkyl, C1-3alkyloxy or phenyl; Preferably, ring B is selected from phenyl or five-membered heterocyclyl, and said phenyl or five-membered heterocyclyl is optionally substituted with halogen or C1-3alkyl; Further preferably, ring B is selected from phenyl or thienyl, and said phenyl or thienyl is optionally substituted with halogen or C1-3alkyl; More preferably, ring B is selected from phenyl or thienyl, and said phenyl or thi enyl is optionally substituted with halogen.

8. The compound of formula (IV) according to claim 7, isomers and pharmaceutically acceptable salts thereof, characterized in that, The compound of formula (IV) is selected from: Preferably, the compound of formula (IV) is selected from: compound 11, compound 44, compound 45, compound 46, compound 47 or compound 84; Further preferably, the compound of formula (IV) is selected from: compound 11, compound 44, compound 45, compound 46 or compound 84.

9. A pharmaceutical composition, characterized by, A compound of formula (I), isomers and pharmaceutically acceptable salts thereof according to claim 1 or 2, a compound of formula (II), isomers and pharmaceutically acceptable salts thereof according to claim 3 or 4, a compound of formula (III), isomers and pharmaceutically acceptable salts thereof according to claim 5 or 6 or a compound of formula (IV), isomers and pharmaceutically acceptable salts thereof according to claim 7 or 8, and a pharmaceutically acceptable carrier.

10. Use of the compound of formula (I), isomers and pharmaceutically acceptable salts thereof according to claim 1 or 2, the compound of formula (II), isomers and pharmaceutically acceptable salts thereof according to claim 3 or 4, the compound of formula (III), isomers and pharmaceutically acceptable salts thereof according to claim 5 or 6, the compound of formula (IV), isomers and pharmaceutically acceptable salts thereof according to claim 7 or 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating a RIPK1-mediated disease. Preferably, the RIPK1-mediated related disease is an inflammatory disease; further preferably, the RIPK1-mediated related disease is a systemic inflammatory syndrome, sepsis, lung injury, kidney injury, an auto-immune disease.

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