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WO2020020119A1 - Rip1抑制剂及其在医药中的用途 - Google Patents

Rip1抑制剂及其在医药中的用途 Download PDF

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Publication number
WO2020020119A1
WO2020020119A1 PCT/CN2019/097202 CN2019097202W WO2020020119A1 WO 2020020119 A1 WO2020020119 A1 WO 2020020119A1 CN 2019097202 W CN2019097202 W CN 2019097202W WO 2020020119 A1 WO2020020119 A1 WO 2020020119A1
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Prior art keywords
compound
disease
derivative
rip1
cells
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French (fr)
Inventor
夏宏光
杨淑颖
何福生
段树民
陈智
岑旭峰
楼国华
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Zhejiang University ZJU
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Zhejiang University ZJU
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Priority to CN201980043726.4A priority Critical patent/CN112351979B/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention belongs to the field of medicinal chemistry. Specifically, the invention provides a RIP1 inhibitor and its use in medicine.
  • Receptor-interacting protein kinase 1 has functions of regulating inflammation and cell death.
  • RIP1 kinase inhibitors are effective in inhibiting cell death, including programmed cell necrosis and RIP1-dependent apoptosis.
  • RIP1 kinase inhibitors can also inhibit a variety of inflammatory reactions. The current mechanism is not clear. It may be through regulating the receptor-interacting protein kinase 3 (RIP3). It may also be through inhibiting cell death to prevent the amplification of inflammatory signals .
  • the inhibitory activity of RIP3 kinase inhibitors on inflammation may be higher than that of RIP1 kinase inhibitors, but RIP3 kinase inhibitors can cause apoptosis and prevent drugs from entering the clinic.
  • RIP3 kinase inhibitors can cause apoptosis and prevent drugs from entering the clinic.
  • RIP3 kinase inhibitor-induced apoptosis may be because it only inhibits RIP3 kinase activity and not RIP1 activity, leading to the pattern of cell death becoming RIP1-dependent apoptosis.
  • RIP1 inhibitors there are few and few RIP1 inhibitors in the clinical stage.
  • Denali's RIP1 inhibitor (Nec1) has entered clinical phase 2 and its indication is neurodegenerative disease; and GSK's RIP1 inhibitor (GSK963) , Has been in clinical Phase 2, the indications are autoimmune diseases, including rheumatoid arthritis, ulcerative enteritis, psoriasis and so on. Therefore, the development of highly effective RIP1 inhibitors is of great significance to the art, especially in terms of inhibiting inflammation and cell death.
  • the object of the present invention is to provide a RIP1 inhibitor with novel structure and its use in medicine.
  • the first aspect of the present invention provides a compound represented by formula (A) or a derivative thereof;
  • R 1 is C 1-6 alkyl
  • R 2 is hydrogen
  • R 3 is hydrogen, C 1-6 alkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl; one or more of the 5- to 10-membered heteroaryl (for example, 1, 2 , Three, or four) ring atoms are each independently a heteroatom selected from nitrogen, sulfur, or oxygen;
  • the C 1-6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and the like.
  • the C 1-6 alkyl group is a C 1-3 alkyl group.
  • the C 6-10 aryl is phenyl or naphthyl.
  • the 5- to 10-membered heteroaryl is 5- to 6-membered heteroaryl or 6- to 10-membered heteroaryl; for example, furan, thiophene, pyrrole, thiazole, imidazole, pyridine, Pyrazine, pyrimidine, pyridazine, indole, quinoline.
  • the compound is selected from the group consisting of:
  • the compound is a compound of formula (I):
  • the derivative is a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, stereoisomer, or a mixture of stereoisomers.
  • the second aspect of the present invention provides the use of the compound or derivative thereof according to the first aspect, which is used as a RIP1 inhibitor or for preparing a medicament for preventing or treating RIP1-related diseases.
  • the compound or a derivative thereof can also be used as an inhibitor of programmed cell necrosis or a medicament for preventing or treating a disease related to programmed cell necrosis.
  • the compound or a derivative thereof can also be used to reduce the interaction between RIP1 and RIP3.
  • the compound or its derivative can also be used to degrade TDP25 protein or to prepare a medicament for preventing or treating diseases related to TDP25 protein.
  • the compound or a derivative thereof can also be used to degrade the NLRP3 protein or to prepare a medicament for preventing or treating a disease related to the NLRP3 protein.
  • the compound or a derivative thereof can also be used for preparing a medicament for preventing or treating liver injury.
  • the third aspect of the present invention provides the use of the compound of the first aspect or a derivative thereof for preparing a medicament for preventing or treating a disease related to programmed cell necrosis.
  • the fourth aspect of the present invention provides the use of the compound described in the first aspect or a derivative thereof for preparing a medicament for preventing or treating a TDP25 protein-related disease.
  • a fifth aspect of the present invention provides the use of the compound of the first aspect or a derivative thereof for preparing a medicament for preventing or treating a disease related to the NLRP3 protein.
  • a sixth aspect of the present invention there is provided the use of the compound or the derivative thereof according to the first aspect, which is used for preparing a medicament for preventing or treating liver injury.
  • a seventh aspect of the present invention provides the use of the compound or its derivative according to the first aspect, the compound or its derivative having one or more of the following uses:
  • the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.
  • the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.
  • the liver injury is acute liver injury.
  • the liver injury is toxin-induced acute liver injury.
  • the toxin is LPS.
  • the RIP1-related disease is selected from the group consisting of: neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher disease, pain, inflammation, retina Shedding, tumor.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and the like.
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • MS multiple sclerosis
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.
  • the pain is neuropathic pain.
  • the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.
  • the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.
  • the multiple sclerosis is induced by cuprizone.
  • the programmed cell necrosis-related disease is selected from the group consisting of neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher disease, pain, Inflammation, retinal detachment, tumor.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and the like.
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • MS multiple sclerosis
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.
  • the pain is neuropathic pain.
  • the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.
  • the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.
  • the multiple sclerosis is induced by cuprizone.
  • the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.
  • the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.
  • the TDP25 protein-related disease is a neurodegenerative disease.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • the NLRP3 protein-related disease is selected from the group consisting of neurodegenerative disease, autoimmune disease, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and tumor.
  • the neurodegenerative disease is Alzheimer's disease (AD).
  • the autoimmune disease is systemic lupus erythematosus.
  • the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.
  • An eighth aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect or a derivative thereof and a pharmaceutically acceptable carrier.
  • a ninth aspect of the present invention provides a method for preventing or treating a disease, the method comprising the step of administering to a subject in need the compound described in the first aspect or a derivative thereof or the pharmaceutical composition described in the eighth aspect;
  • the disease is one or more selected from the group consisting of RIP1-related disease, programmed cell necrosis-related disease, TDP25 protein-related disease, NLRP3 protein-related disease, and liver injury.
  • a tenth aspect of the present invention provides a method for inhibiting RIP1 activity in a cell, comprising the step of: contacting the cell with the compound or a derivative thereof according to the first aspect or the pharmaceutical composition according to the eighth aspect.
  • the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.
  • the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.
  • the cell is a cell treated with an inducer; the inducer is one or more selected from the group consisting of: TNF ⁇ , z-VAD-fmk, LPS, SMAC, compound B3, 5z -7.
  • the compound or its derivative is applied at a concentration of 1-50 ⁇ M; preferably, 5-20 ⁇ M.
  • the invention further provides the use of a compound of formula (I) or a derivative thereof,
  • the compound or a derivative thereof is used as a RIP1 inhibitor or for preparing a medicament for preventing or treating a RIP1-related disease.
  • the compound or a derivative thereof can also be used as an inhibitor of programmed cell necrosis or a medicament for preparing or treating a disease related to programmed cell necrosis.
  • the compound or a derivative thereof can also be used to reduce the interaction between RIP1 and RIP3.
  • the compound or its derivative can also be used to degrade TDP25 protein or to prepare a medicament for preventing or treating diseases related to TDP25 protein.
  • the compound or a derivative thereof can also be used to degrade the NLRP3 protein or to prepare a medicament for preventing or treating a disease related to the NLRP3 protein.
  • the compound or a derivative thereof can also be used for preparing a medicament for preventing or treating liver injury.
  • the invention further provides the use of a compound of formula (I) or a derivative thereof,
  • the compound or a derivative thereof is used for preparing a medicament for preventing or treating a disease related to programmed cell necrosis.
  • the invention further provides the use of a compound of formula (I) or a derivative thereof,
  • the compound or a derivative thereof is used for preparing a medicament for preventing or treating a TDP25 protein-related disease.
  • the invention further provides the use of a compound of formula (I) or a derivative thereof,
  • the compound or a derivative thereof is used for preparing a medicament for preventing or treating a disease related to the NLRP3 protein.
  • the invention further provides the use of a compound of formula (I) or a derivative thereof,
  • the compound or a derivative thereof is used for preparing a medicament for preventing or treating liver injury.
  • the invention further provides the use of a compound of formula (I) or a derivative thereof,
  • the compound or its derivative has one or more of the following uses:
  • the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.
  • the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.
  • the liver injury is acute liver injury.
  • the liver injury is toxin-induced acute liver injury.
  • the toxin is LPS.
  • the RIP1-related disease is selected from the group consisting of: neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher disease, pain, inflammation, retina Shedding, tumor.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and the like.
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • MS multiple sclerosis
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.
  • the pain is neuropathic pain.
  • the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.
  • the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.
  • the multiple sclerosis is induced by cuprizone.
  • the programmed cell necrosis-related disease is selected from the group consisting of neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher disease, pain, Inflammation, retinal detachment, tumor.
  • the neurodegenerative disease is ALS, AD, PD, MS, and the like.
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.
  • the pain is neuropathic pain.
  • the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.
  • the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.
  • the TDP25 protein-related disease is a neurodegenerative disease.
  • the neurodegenerative disease is ALS.
  • the NLRP3 protein-related disease is selected from the group consisting of neurodegenerative disease, autoimmune disease, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and tumor.
  • the neurodegenerative disease is Alzheimer's disease (AD).
  • the autoimmune disease is systemic lupus erythematosus.
  • the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.
  • the invention further provides a pharmaceutical composition, which comprises a compound of formula (I) or a derivative thereof and a pharmaceutically acceptable carrier.
  • the present invention further provides a method for preventing or treating a disease, the method comprising the step of administering to a subject in need thereof a compound of formula (I) or a derivative thereof or comprising a compound of formula (I) or a derivative thereof and a pharmaceutical A pharmaceutical composition of an acceptable carrier;
  • the disease is one or more selected from the group consisting of: RIP1-related disease, programmed cell necrosis-related disease, TDP25 protein-related disease, NLRP3 protein-related disease, liver damage;
  • the invention further provides a method for inhibiting RIP1 activity in a cell, comprising the steps of: combining a cell with a compound of formula (I) or a derivative thereof or comprising a compound of formula (I) or a derivative thereof and a pharmaceutically acceptable Exposure of the pharmaceutical composition of the received carrier;
  • the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.
  • the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.
  • the cell is a cell treated with an inducer; the inducer is one or more selected from the group consisting of: TNF ⁇ , z-VAD-fmk, LPS, SMAC, compound B3, 5z -7.
  • the compound of formula (I) or a derivative thereof is applied at a concentration of 1-50 ⁇ M; preferably, 5-20 ⁇ M.
  • Figure 1 shows that ZJU-37 inhibits programmed necrosis of Jurkat-FADD-/-cells.
  • FIG. 2 shows that ZJU-37 inhibits programmed necrosis of U937 cells.
  • Figure 3 shows that ZJU-37 inhibits programmed necrosis of BV2 cells.
  • Figure 4 shows that ZJU-37 can effectively inhibit the content of P-RIP1 in cells, thereby inhibiting its kinase activity.
  • Figure 5 shows that ZJU-37 can effectively inhibit the content of P-RIP1 in cells, thereby inhibiting its kinase activity.
  • Figure 6 shows that ZJU-37 can effectively reduce the interaction of RIP1 and RIP3.
  • Figure 7 shows that ZJU-37 can effectively degrade TDP25 protein.
  • Figure 8 shows that ZJU-37 can effectively attenuate the interaction of RIP1 / RIP3 proteins in mouse brain tissue.
  • Figure 9 shows that ZJU-37 can effectively inhibit acute liver injury in mice.
  • Figure 10 shows that ZJU-37 is effective in degrading the NLRP3 protein induced by inflammation.
  • Figure 11 shows that ZJU-37 can effectively improve the occurrence of RIPK1-dependent coke death.
  • Figure 12 shows that ZJU-37 is effective in relieving multiple sclerosis.
  • Figure 13 shows that ZJU-37 can quickly cross the blood-brain barrier.
  • Figure 14 shows a pair of RIP1 ZJU-37 Inhibition IC 50.
  • Figure 15 shows that compound (II) inhibits programmed necrosis of Jurkat-FADD-/-cells.
  • a novel compound can be used as a highly effective RIP1 inhibitor.
  • Such inhibitor compounds can effectively inhibit RIP1 activity, can also effectively reduce the interaction between RIP1 and RIP3, and effectively inhibit cells.
  • Programmed necrosis, improve cell viability; can also effectively degrade TDP25 protein and NLRP3 protein, etc., thereby preventing and treating various diseases.
  • These compounds can quickly cross the blood-brain barrier and have the function of promoting nerve cell proliferation. The present invention has been completed on this basis.
  • C 1-6 alkyl is a straight or branched alkyl group having 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl , Isobutyl, tert-butyl, n-pentyl, n-hexyl and the like. C 1-3 alkyl is preferred.
  • C 6-10 aryl is an aryl group having 6 to 10 carbon atoms, such as phenyl or naphthyl.
  • 5-membered to 10-membered heteroaryl is a heteroaryl group having 5 to 10 ring atoms, wherein one or more (eg, 1, 2, 3, or 4) ring atoms each It is independently a heteroatom selected from nitrogen, sulfur, or oxygen.
  • 5- to 6-membered heteroaryl or 6- to 10-membered heteroaryl for example, furan, thiophene, pyrrole, thiazole, imidazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, quinoline.
  • NLRP3 protein is an important part of NLRP3 inflammatory bodies, and as an important component of innate immunity plays an important role in the body's immune response and disease development.
  • NLRP3 protein-related diseases are diseases mediated by (or at least partially mediated by) NLRP3 protein, including, but not limited to: Alzheimer's disease, systemic lupus erythematosus, inflammatory bowel disease, atherosclerosis Sclerosis, type 2 diabetes, gout, obesity, malignancy.
  • TDP25 protein is a C-terminal fragment of TDP-43 with a molecular weight of 25 kD found in the brain regions of ALS patients. Studies have found that TDP25 protein can promote the formation of TDP-43 inclusion bodies, have toxic effects on motor neurons, cause neuron degeneration, and play an important role in the pathogenesis of disease. TDP25 protein related diseases are diseases mediated by (or at least partially mediated by) TDP25 protein, such as ALS.
  • the compound of the present invention is a compound of formula (A), and its structure is as follows:
  • the compound according to the present invention is a compound of formula (I), and its structure is as follows:
  • the active ingredient according to the present invention is a compound of formula (A) or a derivative thereof.
  • the active ingredient according to the present invention is a compound of formula (I) or a derivative thereof.
  • the derivative of the compound according to the present invention is any derivative of the compound, for example, a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, stereoisomer or stereo Mixtures of isomers and the like.
  • pharmaceutically acceptable salt or “physiologically acceptable salt” includes, for example, salts of the compounds of the present invention with inorganic acids and salts with organic acids.
  • the free base can be obtained by basifying a solution of the acid addition salt.
  • the product if the product is a free base, it can be prepared according to a conventional method for preparing an acid addition salt from a basic compound by dissolving the free base in a suitable organic solvent and treating the solution with an acid. Addition salts are obtained, especially pharmaceutically acceptable addition salts.
  • Those skilled in the art will recognize various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable addition salts.
  • Salts derived from inorganic acids include hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like.
  • Salts derived from organic acids include acetate, propionate, glycolate, pyruvate, oxalate, malate, malonate, succinate, maleate, fumarate, Tartrate, citrate, benzoate, cinnamate, mandelate, mesylate, ethanesulfonate, p-toluenesulfonate, salicylate, etc.
  • hydrate refers to a complex formed by combining a compound of the invention and water.
  • solvate refers to an association or complex of one or more solvent molecules and a compound of the invention.
  • solvate-forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine.
  • compounds of the invention may exist as tautomers. Tautomers are in equilibrium with each other. For example, the carbonyl moiety may exist in the enol form. Regardless of which tautomer is displayed and regardless of the nature of the equilibrium between the tautomers, those of ordinary skill in the art understand that the compounds include both keto and enol tautomers.
  • stereoisomer refers to a compound composed of the same atoms bonded by the same bond, but with different three-dimensional structures, which are not interchangeable.
  • the “stereoisomers” in the present invention encompass various stereoisomers and mixtures of the compounds of the present invention and include “enantiomers”, which refer to two stereoisomers whose molecules are non-overlapping with each other.
  • enantiomers which refer to two stereoisomers whose molecules are non-overlapping with each other.
  • “Diastereomers” are stereoisomers with at least two asymmetric atoms, but they are not mirror images of one another.
  • prodrug means any compound that releases the active parent drug in vivo in accordance with the compound structure of the invention when said prodrug is administered to a subject.
  • the prodrug can be prepared by modifying any functional group present in a compound of the invention, such that the modification can be cleaved in vivo to release the parent compound.
  • Prodrugs can be prepared by modifying functional groups present in a compound in a manner that cleaves the modification into the parent compound in routine manipulations or in vivo.
  • the prodrug according to the present invention includes a group such as an amino group in the compound of the present invention and any group which can be cleaved in vivo to regenerate a free amino group respectively. Prodrug preparation, selection, and use are discussed in T.
  • the compound of the present invention can be prepared according to a conventional method in the art, and can also be prepared according to the following method (the method shown in the following reaction formula).
  • the preparation method of the present invention includes the steps of reacting Compound 2 and R 3 -LX to obtain a compound of formula (A).
  • R 1 , R 2 , R 3 , and L have the same definitions as above;
  • X is N (CH 3 ) 2 or halogen (for example, fluorine, chlorine, bromine, or iodine).
  • Compound 2 was prepared by following the method of Example 1 of WO2016094848A1 and using corresponding raw materials.
  • the reaction is performed in a solvent.
  • a solvent for example, DMF, DMSO, THF, etc.
  • This solvent is generally not involved in the reaction.
  • some solvents can also participate in the reaction as the reaction reagent R 3 -LX, such as when DMF is used as a solvent.
  • the reaction may be performed under basic conditions or acidic conditions.
  • Basic conditions are performed, for example, in the presence of a base.
  • the base include alkaline agents such as sodium hydride, potassium hydride, sodium hydroxide, and potassium hydroxide.
  • Acidic conditions are performed, for example, in the presence of an acid.
  • the acid is, for example, an acidic reagent such as phosphorus oxychloride.
  • the reaction can be performed under conventional conditions. For example, it is performed at a certain temperature (for example, -10 ° C to 30 ° C; preferably 0 ° C to 30 ° C) for a period of time (for example, 0.1 to 10 hours; preferably 0.1 to 5 hours).
  • a certain temperature for example, -10 ° C to 30 ° C; preferably 0 ° C to 30 ° C
  • a period of time for example, 0.1 to 10 hours; preferably 0.1 to 5 hours.
  • the compounds of the present invention have one or more of the following functions: inhibition of RIP1 kinase activity; reduction of RIP1 and RIP3 interactions; inhibition of programmed cell necrosis; degradation of TDP25 protein; degradation of NLRP3 protein, and so on.
  • the compound of the present invention or a derivative thereof and a pharmaceutical composition containing the compound or a derivative thereof as a main active ingredient can be used to treat, prevent, and alleviate diseases related to RIP1, diseases related to RIP1 and RIP3, diseases related to TDP25 protein, or NLRP3 Protein-related diseases.
  • diseases include, but are not limited to, various neurodegenerative diseases, autoimmune diseases, liver diseases such as liver injury, liver failure, gastroenteritis, and the like.
  • the pharmaceutical composition of the present invention contains an active ingredient in a safe and effective amount range and a pharmacologically acceptable excipient or carrier.
  • the “safe and effective amount” means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of active ingredient / dose, and more preferably, 5-200 mg of active ingredient / dose.
  • the "one dose” is a capsule or tablet.
  • the “pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and low enough toxicity. "Compatibility” here means that the components of the composition and the active ingredient can blend with each other without significantly reducing the efficacy of the active ingredient.
  • pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid).
  • Magnesium stearate calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as Tween
  • Wetting agents such as sodium lauryl sulfate
  • the administration method of the active ingredient of the present invention or the medicine containing the active ingredient is not particularly limited, and representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration medicine.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerol; (d) disintegrating agents, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glycerol, such
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as casings and other materials known in the art. They may contain opaque agents and the release of the active ingredient in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient may also be in micro-encapsulated form with one or more of the aforementioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers.
  • inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these, and the like.
  • compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous vehicles, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the active ingredients of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the active ingredient of the present invention is applied to a subject in need of treatment, such as a mammal (such as a human), wherein the dose when administered is a pharmaceutically considered effective dose for 60 kg body weight
  • the daily dose is usually 1 to 2000 mg, preferably 5 to 500 mg.
  • the specific dosage should also consider factors such as the route of administration, the patient's health, etc., which are all within the skill of a skilled physician.
  • treatment is a method for obtaining beneficial or desired results, including clinical results.
  • beneficial or required clinical results may include one or more of the following: a) Inhibition of a disease or condition (e.g., reduction of one or more symptoms caused by the disease or condition and / or reduction of the degree of the disease or condition) B) slow or prevent the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilize the disease or condition, prevent or delay the deterioration or progression of the disease or condition, and / or prevent or delay the spread of the disease or condition (E.g., metastasis); and / or c) alleviate the disease, i.e., cause the regression of clinical symptoms (e.g., improve the state of the disease, provide partial or total relief of the disease or condition, enhance the effect of another drug, delay the progression of the disease, Improve quality of life and / or prolong survival).
  • a disease or condition e.g., reduction of one or more symptoms caused by the disease or condition and / or reduction of the
  • prevention means any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop.
  • the active ingredient may be administered to a subject (including a human) who is at risk or has a family history of the disease or condition.
  • a "subject” refers to an animal, such as a mammal (including a human), that has been or will be the subject of a treatment, observation, or experiment.
  • the methods described herein can be used in human therapy and / or veterinary applications.
  • the subject is a mammal.
  • the subject is a human.
  • the compound of the present invention or its derivative can effectively inhibit RIP1 activity, and can be used as an effective RIP1 inhibitor, so as to prevent or treat RIP1-mediated diseases.
  • the compound of the present invention or its derivative can effectively reduce programmed cell necrosis of cells and improve cell viability.
  • the compounds of the present invention or their derivatives also have the ability to inhibit inflammation.
  • the compound of the present invention or its derivative can also degrade the NLRP3 protein and the TDP25 protein.
  • the compound of the present invention or a derivative thereof can also treat liver damage.
  • the compound of the present invention or a derivative thereof can be used for treating multiple diseases (such as RIP1-mediated diseases, NLRP3 protein-mediated diseases, or TDP25 protein-mediated diseases) with one drug and multiple targets.
  • multiple diseases such as RIP1-mediated diseases, NLRP3 protein-mediated diseases, or TDP25 protein-mediated diseases
  • cell viability (%) after drug treatment cell viability of drug-treated wells / cell viability of control wells * 100%; the higher the cell viability (%), the stronger the cell viability after drug treatment.
  • cell viability was determined by ATP-based viability measurement.
  • the conditions for cell culture and drug treatment are: stable temperature (37 ° C), stable CO 2 level (5%), constant pH (pH: 7.2-7.4), high relative saturation Humidity (95%).
  • experimental materials such as various cells
  • reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
  • Jurkat FADD-/-cells represent FADD-/-knockout Jurkat cells.
  • Jurkat cells are commonly used to study acute T-cell leukemia.
  • Jurkat cells FADD-/-knockout Jurkat cells specifically induce the occurrence of programmed necrosis under the conditions induced by TNF ⁇ .
  • Jurkat FADD -/- cells in this experiment can be obtained by CRISPR technology.
  • U937 cells are human histiocyte lymphoma cells; purchased from Nanjing Kebai Cell Bank.
  • BV2 cells are mouse microglia; purchased from Nanjing Kebai Cell Bank. Since BV2 cells secrete TNF ⁇ by themselves under conditions induced by z-VAD-fmk, no additional TNF ⁇ is required for the induction conditions.
  • 293T cells were derived from 293 cells; purchased from Nanjing Kebai Cell Bank.
  • 293 cells are a human renal epithelial cell line transfected with the adenovirus E1A gene.
  • H4-TDP25 cells are H4 cells stably expressing TDP25; purchased from Nanjing Kebai Cell Bank.
  • HT-29 cells are human colon cancer cells; purchased from Nanjing Kebai Cell Bank.
  • BMDM cells are bone marrow-derived macrophages; purchased from Nanjing Kebai Cell Bank.
  • Compound 2 was prepared by following the method of Example 1 of WO2016094848A1. Under nitrogen protection and ice bath conditions, POCl 3 (0.4 mmol, 60 mg) was added dropwise to DMF (1 mL), and stirring was continued in the ice bath for 0.5 hours. Then, a DMF solution of compound 2 (0.2 mmol, 59 mg) was added to the reaction solution, and the temperature was raised to room temperature to continue the reaction for 5 hours. An appropriate amount of water was added to quench the reaction, and the mixture was extracted three times with ethyl acetate.
  • Example 1 ZJU-37 can inhibit Jurkat-FADD -/- programmed cell necrosis
  • TNF ⁇ purchased from Sigma, USA
  • Nec1 structure is (Selleck; purchased as a positive control).
  • Jurkat FADD -/- cells were plated on a 96-well white plate.
  • the medium used was 1640 medium (purchased from Nanmo Biological Company, the same below).
  • the plated density was 20,000 cells per well.
  • NEc1 hole with TNF [alpha] (final concentration effect of 30ng / ml) and different concentrations of the drug action NEc1 (final concentration effect of 10 -7 ⁇ M, 10 -6 ⁇ M, 10 -5 ⁇ M, 10 -4 ⁇ M, 10 -3 ⁇ M , 10-2 ⁇ M, 10 -1 ⁇ M, 1 ⁇ M, 10 ⁇ M) together;
  • ZJU-37 well use TNF ⁇ (final action concentration is 30ng / ml) and drugs with different action concentrations ZJU-37 (final action concentration is 10 -7 ⁇ M, 10 -6 ⁇ M, 10 -5 ⁇ M, 10 -4 ⁇ M, 10 -3 ⁇ M, 10 -2 ⁇ M, 10 -1 ⁇ M, 1 ⁇ M, 10 ⁇ M) together;
  • TNF ⁇ well treated with TNF ⁇ (30ng / ml) and DMSO with the same volume as the drug;
  • Figure 1 shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of Jurkat-FADD-/-cells at a concentration of 10 ⁇ M, respectively.
  • Figure B shows that Nec1 and ZJU-37 can inhibit Jurkat-FADD- / at different concentrations, respectively.
  • -Inhibition of programmed necrosis of cells Panel C represents the EC50 of Nec1 inhibiting the programmed necrosis of Jurkat-FADD-/-cells
  • Panel D represents the EC50 of the programmed necrosis of Jurkat-FADD-/-cells by ZJU-37.
  • ZJU-37 can inhibit programmed necrosis of Jurkat-FADD-/-cells, and its inhibitory effect is similar to that of the positive control drug Nec1.
  • Example 2 ZJU-37 can inhibit programmed necrosis of U937 cells
  • TNF ⁇ (ibid.); Z-VAD-fmk (purchased from BioVision); Nec1 (ibid.).
  • U937 cells were plated on a 96-well white plate, the medium used was 1640 medium, the plate density was 20,000 cells per well, and the cells were plated for 2-4 hours and then subjected to the following processing:
  • Nec1 wells TZ (TNF ⁇ (final action concentration: 30ng / ml) and z-VAD-fmk (final action concentration: 40 ⁇ M)) and Nec1 (final action concentration: 0.0001 ⁇ M, 0.0005 ⁇ M, 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 5 ⁇ M, 10 ⁇ M) together;
  • ZJU-37 wells TZ (TNF ⁇ (final action concentration: 30ng / ml) and z-VAD-fmk (final action concentration: 40 ⁇ M)) and ZJU-37 (final action concentration: 0.0001 ⁇ M, 0.0005 ⁇ M) , 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 5 ⁇ M, 10 ⁇ M) together;
  • TZ well treated with TZ (TNF ⁇ (final action concentration: 30ng / ml) and z-VAD-fmk (final action concentration: 40 ⁇ M)) and DMSO with the same volume as the drug
  • FIG. 2 The experimental results are shown in Figure 2.
  • A shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of U937 cells at a concentration of 10 ⁇ M, respectively.
  • Figure B shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of U937 cells at different concentrations;
  • C The figure shows that Nec1 inhibits the EC50 of U937 cells;
  • the figure D shows that ZJU-37 inhibits the EC50 of U937 cells.
  • ZJU-37 can inhibit programmed necrosis of U937 cells, and its inhibitory effect is similar to that of the positive control drug Nec1.
  • Example 3 ZJU-37 can inhibit programmed necrosis of BV2 cells
  • BV2 cells were plated on a 96-well white plate.
  • the medium used was DMEM medium (purchased from Thermo Fisher Scientific, the same below).
  • the plated density was 8000 thousand cells per well. After the cells were plated for 1 day, the following treatment was performed:
  • Nec1 well use z-VAD-fmk (final action concentration of 70 ⁇ M) and Nec1 (final action concentration of 0 ⁇ M, 0.0001 ⁇ M, 0.0005 ⁇ M, 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M) , 10 ⁇ M) processing together;
  • ZJU-37 well use z-VAD-fmk (final action concentration of 70 ⁇ M) and ZJU-37 (final action concentration of 0 ⁇ M, 0.0001 ⁇ M, 0.0005 ⁇ M, 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M , 1 ⁇ M, 5 ⁇ M, 10 ⁇ M) together;
  • Z-VAD well treated with z-VAD-fmk (final effect concentration: 70 ⁇ M) and DMSO with the same volume as the drug;
  • Figure 3 shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of BV2 cells at a concentration of 10 ⁇ M respectively;
  • Figure B shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of BV2 cells at different concentrations;
  • C The figure shows that Nec1 inhibits the EC50 of programmed necrosis of BV2 cells;
  • the figure D shows that ZJU-37 inhibits the EC50 of programmed necrosis of BV2 cells.
  • ZJU-37 can inhibit programmed necrosis of BV2 cells, and its inhibitory effect is similar to that of the positive control drug Nec1.
  • Example 4 ZJU-37 can effectively inhibit RIP1 kinase activity
  • Flag-RIP1 purchased from Vitrus Bio
  • LPS purchased from Sigma
  • Nec1 ibid.
  • 293T cells were plated in six-well plates using DMEM medium with a plate density of 500,000 cells per well; after plated for 24 hours, the following treatments were performed:
  • Flag + LPS wells Transfect 1 ⁇ g Flag-RIP1, and treat with LPS (final action concentration 100ng / ml) and DMSO with the same volume as the drug 24 hours after transfection;
  • Nec1-10 wells transfect 1 ⁇ g Flag-RIP1, and treat with LPS (final action concentration of 100 ng / ml) and Nec1 (final action concentration of 10 ⁇ M) 24 hours after transfection;
  • ZJU37-5 wells transfect 1 ⁇ g Flag-RIP1, and treat with LPS (final action concentration: 100 ng / ml) and ZJU-37 (final action concentration: 5 ⁇ M) 24 hours after transfection;
  • ZJU37-10 wells transfect 1 ⁇ g Flag-RIP1, and treat with LPS (final action concentration of 100 ng / ml) and ZJU-37 (final action concentration of 10 ⁇ M) after transfection for 24 hours
  • ZJU37-20 wells transfect 1 ⁇ g Flag-RIP1, and treat with LPS (final action concentration: 100ng / ml) and ZJU-37 (final action concentration: 20 ⁇ M) 24 hours after transfection;
  • Control well blank control without adding inducer and drug.
  • Jurkat FADD-/-cells were plated on a six-well plate, the medium used was 1640 medium, and the plate density was 1.5 million cells per well; after the plate was plated for 2 hours, the following treatment was performed:
  • TNF ⁇ well treated with TNF ⁇ (final effect concentration 30ng / ml) and DMSO with the same volume as the drug;
  • Nec1 wells treated with TNF ⁇ (final action concentration 30ng / ml) and Nec1 (final action concentration 10 ⁇ M);
  • ZJU-37 wells treated with TNF ⁇ (final action concentration 30ng / ml) and ZJU-37 (final action concentration 10 ⁇ M);
  • Control well blank control without adding inducer and drug.
  • Example 5 ZJU-37 can effectively inhibit RIP1 kinase activity
  • 293T cells were plated in a 10 cm culture dish using DMEM medium with a plate density of 3 million cells per dish; 24 hours after plating, 6 ⁇ g of pCMV-Flag (System 1) or Flag-RIP1 (System 2-System 5) was transfected. ). ATP was added to System 1, System 3 to System 5, and ATP was not added to System 2.
  • DMSO was added to System 2 and System 3; 50 ⁇ M of Nec1 was added to System 4; and 50 ⁇ M of ZJU-37 was added to System 5.
  • the 5 systems were added to the centrifuge tube of each sample, and then placed in a metal bath at 30 ° C for 30 minutes.
  • the beads were collected and immunoblot hybridized with the corresponding antibodies.
  • Example 6 ZJU-37 can reduce the interaction of RIP1 and RIP3 in cells
  • TNF ⁇ ibid.
  • Z-VAD-fmk ibid.
  • SMAC purchased from Selleck
  • Nec1 ibid.
  • EGFP-RIP1 purchasedd from Weizhen Bio
  • Flag-RIP3 purchased from Weiwei
  • HT-29 cells were plated in a 10cm culture dish, the medium used was 1640 medium; the plating density was 2 million cells per dish; after plating for 24h, the following treatment was performed:
  • TZS well treated with TZS (TNF ⁇ (final action concentration: 30ng / ml), z-VAD-fmk (final action concentration: 20 ⁇ M) and SMAC (final action concentration: 100nM)) and DMSO with the same volume as the drug;
  • Nec1 wells treated with TZS (TNF ⁇ (final action concentration: 30 ng / ml), z-VAD-fmk (final action concentration: 20 ⁇ M) and SMAC (final action concentration: 100 nM)) and Nec1 (final action concentration: 10 ⁇ M);
  • ZJU-37 wells TZS (TNF ⁇ (final action concentration 30ng / ml), z-VAD-fmk (final action concentration 20 ⁇ M) and SMAC (final action concentration 100nM)) and ZJU-37 (final action concentration is 10 ⁇ M) processing;
  • Control well blank control without adding inducer and drug.
  • 293T cells were plated in a 10 cm culture dish, the medium used was DMEM medium; the plate density was 4 million cells per dish; after the plate was plated for 24 h, the following treatment was performed:
  • EGFP-RIP1 + Flag-RIP3 wells transfect 2 ⁇ g each of EGFP-RIP1 and Flag-RIP3, and add and treat with the same volume of DMSO as the drug;
  • Nec1 wells transfect 2 ⁇ g each of EGFP-RIP1 and Flag-RIP3, and add Nec1 (final action concentration is 10 ⁇ M) at the same time;
  • ZJU-37 well transfection with 2 ⁇ g each of EGFP-RIP1 and Flag-RIP3, and add ZJU-37 (final action concentration: 10 ⁇ M) at the same time;
  • Control well blank control without adding inducer and drug.
  • Panel B In 293T cells, both Nec1 and ZJU-37 can reduce the interaction between RIP1 and RIP3, and the effect of ZJU-37 is better than Nec1.
  • Example 7 ZJU-37 can effectively degrade the level of TDP25 protein
  • Raw material Compound B3 (structure is ); Nec1 (ibid.).
  • H4-TDP25 cells were plated in a six-well plate, the medium used was DMEM medium, and the plate density was 200,000 cells per well; after the plate was plated for 12 hours, the following treatment was performed:
  • Well B3 cells are treated with compound B3 (final action concentration 10 ⁇ M) for 6 hours, and then the solution is changed, and then treated with the same volume of DMSO as the drug;
  • Nec1-10 wells cells were treated with compound B3 (final effect concentration 10 ⁇ M) for 6 hours, and then the solution was changed, and then treated with Nec1 (final effect concentration 10 ⁇ M);
  • ZJU37-1 well cells were treated with compound B3 (final action concentration: 10 ⁇ M) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration: 1 ⁇ M);
  • ZJU37-5 wells cells were treated with compound B3 (final action concentration 10 ⁇ M) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration 5 ⁇ M);
  • ZJU37-10 wells cells were treated with compound B3 (final action concentration 10 ⁇ M) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration 10 ⁇ M);
  • ZJU37-20 wells cells were treated with compound B3 (final action concentration 10 ⁇ M) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration 20 ⁇ M);
  • Control well blank control without adding inducer and drug.
  • TDP25 After H4-TDP25 cells were induced by compound B3, the expression of TDP25 increased. After treatment with ZJU-37, the expression of TDP25 protein in the cells decreased significantly. It can be seen that ZJU-37 can effectively degrade TDP25 protein, and its inhibitory effect is better than that of the positive control drug Nec1 (while TDP25 protein aggregation can induce a series of diseases, such as ALS).
  • Example 8 ZJU-37 can effectively weaken the binding of RIP1 / RIP3 proteins in mouse brain tissue
  • Raw materials C57BL / 6 male mice (purchased from Shanghai Nanmo Biological); LPS (ibid.); Nec1 (ibid.).
  • mice C57BL / 6 male mice were treated as follows:
  • Control group blank control, without adding inducers and drugs
  • LPS group intraperitoneal injection treatment with LPS (final effect concentration 100ng / kg) and DMSO with the same volume as the drug;
  • Nec1 group intraperitoneal injection treatment with LPS (final effect concentration 100ng / kg) and Nec1 (final effect concentration 5mg / kg);
  • ZJU-37 group intraperitoneal injection treatment with LPS (final action concentration: 100ng / kg) and ZJU-37 (final action concentration: 5mg / kg);
  • mice were dissected to take out the brain, 1/2 of the brain tissue was added and RIPA lysate was ground for 1 min, and the supernatant was centrifuged for 20 min at 12000 rpm. This step was repeated three times. Finally, the supernatants were combined and beads and antibodies were added for incubation. Finally, the proteins were collected and used for immunoblot hybridization.
  • Example 9 ZJU-37 can effectively inhibit LPS-induced acute liver injury in mice
  • mice C57BL / 6 male mice were treated as follows:
  • Control group blank control, without adding inducers and drugs
  • LPS group intraperitoneal injection treatment with LPS (final effect concentration 100ng / kg) and DMSO with the same volume as the drug;
  • LPS + ZJU-37 group intraperitoneal injection treatment with LPS (final action concentration 100ng / kg) and ZJU-37 (final action concentration 5mg / kg);
  • liver HE staining section is generally to observe whether the liver cell morphology is complete, whether the nucleus is enlarged, whether the liver lobular morphology is intact, and whether inflammation occurs to detect the damaging effects of drugs and other stimulating factors on the liver.
  • Example 10 ZJU-37 can effectively degrade NLRP3 protein induced by inflammation
  • TNF ⁇ (ibid.); Z-VAD-fmk (ibid.); SMAC (ibid.); Nec1 (ibid.).
  • HT-29 cells were plated in a six-well plate using 1640 medium and a plate density of 700,000 cells per well; after plated for 24 hours, the following treatments were performed:
  • TZS well treated with TZS (TNF ⁇ (final action concentration: 30ng / ml), z-VAD-fmk (final action concentration: 20 ⁇ M) and SMAC (final action concentration: 100nM)) and DMSO with the same volume as the drug;
  • Nec1 wells treated with TZS (TNF ⁇ (final action concentration: 30 ng / ml), z-VAD-fmk (final action concentration: 20 ⁇ M) and SMAC (final action concentration: 100 nM)) and Nec1 (final action concentration: 10 ⁇ M);
  • ZJU-37 wells TZS (TNF ⁇ (final action concentration 30ng / ml), z-VAD-fmk (final action concentration 20 ⁇ M) and SMAC (final action concentration 100nM)) and ZJU-37 (final action concentration is 10 ⁇ M) processing;
  • Jurkat FADD-/-cells were plated on a six-well plate, the medium used was 1640 medium, and the plate density was 1.5 million cells per well; after the plate was plated for 2 hours, the following treatment was performed:
  • TNF ⁇ well treated with TNF ⁇ (final effect concentration 30ng / ml) and DMSO with the same volume as the drug;
  • Nec1 wells treated with TNF ⁇ (final effect concentration 30ng / ml) and Nec1 (final effect concentration 10 ⁇ M);
  • ZJU-37 well treated with TNF ⁇ (final action concentration 30ng / ml) and ZJU-37 (final action concentration 10 ⁇ M);
  • Figure A In the case that NLRP3 is induced to increase by TTZ, ZJU-37 can effectively degrade the NLRP3 protein induced by inflammation, and the effect is better than that of the positive drug Nec1.
  • Panel B In the case that NLRP3 is induced to increase by TNF ⁇ , ZJU-37 can effectively degrade the NLRP3 protein induced by inflammation, and the effect is better than that of the positive drug Nec1.
  • Example 11 ZJU-37 can effectively suppress RIP1-dependent coke death
  • Mouse BMDM cells Mice were purchased from Nanmo Biological; BMDM extraction method: Take the femur of the mouse, cut the two ends of the leg bone, wash the inside of the leg bone with a needle suction PBS to wash out the cells, the obtained cells are 1000rpm, 4 Centrifuge for 5 min. Discard the supernatant, add appropriate amount of ACK to lyse red blood cells, and add PBS to neutralize after 3-5min. Centrifuge at 1000 rpm for 5 min at 4 degrees. That is, the medium can be resuspended to count and plate.
  • LPS ibid.
  • Nec1 ibid.
  • 5z-7 structure is: CAS: 66018-38-0.
  • the mouse BMDM cells were spread on a six-well plate, and the medium used was 1640 medium (purchased from Nanmo Biological Co., Ltd .; 10% FBS--56 ° C fire extinguishing for 30 minutes + M-CSF-20ng / ml + 1% double antibody)
  • the plating density is 2 million cells per well, which is 10 6 / ml.
  • the cells are exchanged after 3 days of plating, and the following treatment is performed after 5 days:
  • Control group blank control, without adding inducers and drugs
  • LPS + 5z-7 wells add coke death inducer (LPS (final action concentration: 10ng / ml) and 5z-7 (final action concentration: 125nM)) and DMSO with the same volume as the drug
  • Nec1 well add coke death inducer (LPS (final action concentration: 10ng / ml) and 5z-7 (final action concentration: 125nM)), and add Nec1 (final action concentration: 10 ⁇ M);
  • ZJU-37 well add coke death inducer (LPS (final action concentration: 10ng / ml) and 5z-7 (final action concentration: 125nM)), and add ZJU-37 (final action concentration: 10 ⁇ M);
  • LPS final action concentration: 10ng / ml
  • 5z-7 final action concentration: 125nM
  • Example 12 ZJU-37 can alleviate multiple sclerosis (MS) in mice
  • Raw materials 6-8 week old male C57 mice (purchased from Nanmo Biological); conventional feed (purchased from Hangzhou Siluojin Biotechnology Co., Ltd.); 0.2% Cuprizone feed (purchased from Hangzhou Siluojin Biotechnology Co., Ltd., In order to add 0.2% Cuprizone to conventional feed); Nec1 (same as above).
  • group one and group two are each divided into the following four groups:
  • the first group blank control group
  • the second group Cup group (Cuprizone feed can induce the occurrence of MS in mice);
  • the third group Cup + Nec1 group (Cuprizone feed + Nec1);
  • the fourth group Cup + ZJU-37 group (Cuprizone feed + ZJU-37)
  • Group 1 Mice in the first group were fed with conventional feed from the first day, and brain tissue was perfused after 6 weeks of feeding. Mice in the second, third or fourth group were fed with 0.2% Cuprizone feed from the first day. After 6 weeks of feeding, brain tissue was perfused.
  • the third group started intraperitoneal injection of drugs (Nec1, effective concentration of 5mg / kg) from the first day; the fourth group started intraperitoneal injection of drugs (ZJU-37, effective concentration of 5mg / kg) from the first day;
  • the solvent was injected intraperitoneally from the first day (solvent formula: 2% DMSO + 30% PEG400 + 68% ddH2O).
  • Group 2 Four groups of mice were fed with 0.2% Cuprizone feed from the first day to the 30th day and then fed with conventional feed from the 31st to the 45th day, and the drugs were injected intraperitoneally from the 31st day. After 6 weeks, brain tissue was perfused. Among them, the third group started to inject drugs at the 31st day (Nec1, with an effective concentration of 5mg / kg), and the fourth group started to inject drugs at the same time from the 31st day (ZJU-37, with an effective concentration of 5mg / kg) ), The second group started to inject the solvent from the 31st day (solvent formulation: 2% DMSO + 30% PEG400 + 68% ddH2O).
  • A Mouse brain slices were fixed with 4% glutaraldehyde and the carcass part was observed under electron microscopy. The electron microscopy results showed that the cup group showed partial demyelination, with loose myelin sheaths and lower g-ratio values ( The g-ratio is the inner diameter of the myelin sheath than the outer diameter of the myelin sheath. A lower value indicates that the myelin sheath is more loose.) However, after intraperitoneal injection of two drugs, the myelin sheath is less demyelinated and the myelin sheath is more compact.
  • Panel B The brain slices of each group were stained with elechroman blue (the normal myelin sheath shows blue, and the demyelination site disappears to be white). From the figure we can see that the cup group showed partial demyelination, but after the two drugs were injected intraperitoneally, the demyelination phenomenon was improved, and the effect of ZJU-37 was better than that of Nec1.
  • Example 13 ZJU-37 can pass through the blood-brain barrier of mice
  • mice Seven BALB / c Nude mice were divided into two groups (drug treatment group and solvent group):
  • mice were intraperitoneally injected with 4 mg / kg ZJU37; the drug treatment components were divided into two groups:
  • 0.5h group 0.5 hours after intraperitoneal injection of ZJU37, samples were taken for analysis.
  • Group 1h Samples were taken for analysis one hour after intraperitoneal injection of ZJU37.
  • mice were injected intraperitoneally with 2% DMSO + 30% peg400 + water; then samples were taken for analysis one hour after intraperitoneal injection of the solvent.
  • mice When sampling for analysis, mice were first injected intraperitoneally with a 4% aqueous solution of chloral hydrate, 200 ⁇ l / 20 g of body weight, and anesthetized. The plasma and brain drug concentrations were then analyzed separately.
  • Carotid blood was taken 300 ⁇ l into heparin tube. After mixing, 100 ⁇ l of the supernatant was centrifuged at 4000 rpm for 5 min, and 700 ⁇ l of acetonitrile was added. analysis.
  • HTRFkinEASESTK kit (Cat.62STKOPEC, Cisbio) and ADP-Glo Kinase kit (Cat.V6930, Promega) were used to detect the half inhibitory concentration (IC50) of ZJU-37 and Nec-1s on RIPK1 kinase.
  • Nec-1s The structure of Nec-1s is Purchased from Selleck;
  • 1 ⁇ enzyme buffer 1 mL of 1 ⁇ kinase buffer contains 200 ⁇ L 5 ⁇ enzyme buffer, 5 ⁇ L 1M MgCl 2 , 1 ⁇ L 1M MnCl 2 , 1 ⁇ L 1M DTT, 793 ⁇ L ddH2O.
  • STK-biotin substrate working solution The specific concentration of STK-biotin substrate is shown in Table 1. STK-biotin substrate was diluted to 5 times the reaction concentration with 1 ⁇ kinase buffer.
  • 5 ⁇ ATP working solution The specific concentration of ATP is shown in Table 1. Dilute ATP to 5 times the reaction concentration with 1x kinase buffer.
  • 5 ⁇ enzyme working solution See Table 1 for enzyme concentration.
  • a 5 ⁇ enzyme working solution of the enzyme was prepared with 1 ⁇ kinase buffer.
  • the RIPK1 enzyme reaction time is 180 minutes (see Table 1 for the corresponding ATP concentration and reaction time).
  • the inhibition rate of each well was calculated from the reaction well and the control well, and the average value of the duplicate wells was used to analyze the inhibitory activity of the test compound using the analysis software PRISM 5.0.
  • Inhibition rate (fluorescence signal from positive control well-fluorescence signal from dosing well) / (fluorescence signal from positive control well-fluorescence signal from negative control well) * 100%
  • Example 15 Compound (II) inhibits Jurkat-FADD -/- programmed cell necrosis
  • Example 1 The method of Example 1 was repeated, except that the compound ZJU37 was replaced by the compound (II), and the treatment time was 12 hours. The results are shown in Figure 15. It can be seen that in the case where TNF ⁇ specifically induces programmed necrosis, compound (II) can effectively inhibit programmed necrosis of Jurkat-FADD-/-cells.

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Abstract

一种RIP1抑制剂及其在医药中的用途，其可有效抑制RIP1激酶活性，还可以减少RIP1和RIP3的相互作用，抑制细胞程序性坏死，降解NLRP3蛋白和TDP25蛋白，可用于预防或治疗多种疾病。

Description

RIP1抑制剂及其在医药中的用途 技术领域
本发明属于药物化学领域，具体地，本发明提供了一种RIP1抑制剂及其在医药中的用途。
背景技术
受体相互作用蛋白激酶1(RIP1)具有调控炎症和细胞死亡的功能。RIP1激酶抑制剂可以有效抑制细胞死亡，包括细胞程序性坏死和RIP1依赖的细胞凋亡。同时RIP1激酶抑制剂还可以抑制多种炎症反应，目前机制尚不清楚，有可能是通过调控受体相互作用蛋白激酶3(RIP3)发挥作用，也有可能是通过抑制细胞死亡阻止了炎症信号的放大。RIP3激酶抑制剂对炎症的抑制活性可能高于RIP1的激酶抑制剂，但RIP3激酶抑制剂会引发细胞凋亡而导致药物无法进入临床。例如GSK872、GSK843等单一性抑制RIP3的抑制剂。RIP3激酶抑制剂诱导细胞凋亡可能是因为其仅仅抑制RIP3激酶活性而不抑制RIP1的活性从而导致细胞死亡的模式变为RIP1依赖的细胞凋亡。
目前处于临床阶段的RIP1抑制剂少而又少，例如有Denali公司的RIP1抑制剂(Nec1)，已进入临床2期，适应症为神经退行性疾病；还有GSK公司的RIP1抑制剂(GSK963)，已处在临床2期，适应症是自身免疫性疾病，包括类风湿关节炎，溃疡性肠炎，银屑病等。因此，研发出高效的RIP1抑制剂对于本领域而言具有重要意义，尤其是在抑制炎症和细胞死亡等方面。
发明内容
本发明的目的是提供一种结构新颖的RIP1抑制剂及其在医药方面的用途。
本发明第一方面提供了式(A)所示化合物或其衍生物；
Figure PCTCN2019097202-appb-000001
其中，
R 1为C 1-6烷基；
R 2为氢；
R 3为氢、C 1-6烷基、C 6-10芳基或5元至10元杂芳基；所述5元至10元杂芳基中的一个或多个(例如1个、2个、3个或4个)环原子各自独立地为选自氮、 硫或氧的杂原子；
L为-(C＝O)-、-(S＝O)-或-(SO 2)-。
在另一优选例中，所述C 1-6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等。
在另一优选例中，所述C 1-6烷基为C 1-3烷基。
在另一优选例中，所述C 6-10芳基为苯基或萘基。
在另一优选例中，所述5元至10元杂芳基为5元至6元杂芳基或6元至10元杂芳基；例如，呋喃、噻吩、吡咯、噻唑、咪唑、吡啶、吡嗪、嘧啶、哒嗪、吲哚、喹啉。
在另一优选例中，所述化合物选自下组：
Figure PCTCN2019097202-appb-000002
在另一优选例中，所述化合物为式(I)化合物：
Figure PCTCN2019097202-appb-000003
在另一优选例中，所述衍生物为药学上可接受的盐、水合物、溶剂合物、前药、互变异构体、立体异构体或立体异构体的混合物。
本发明第二方面提供了第一方面所述化合物或其衍生物的用途，所述化合物或其衍生物用作RIP1抑制剂或用于制备预防或治疗RIP1相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用作细胞程序性坏死抑制剂或用于制备预防或治疗细胞程序性坏死相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用于降低RIP1和RIP3的相互作用。
在另一优选例中，所述化合物或其衍生物还可以用于降解TDP25蛋白或用于制备预防或治疗TDP25蛋白相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用于降解NLRP3蛋白或用于制备预防或治疗NLRP3蛋白相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用于制备预防或治疗肝损 伤的药物。
本发明第三方面提供了第一方面所述化合物或其衍生物的用途，所述化合物或其衍生物用于制备预防或治疗细胞程序性坏死相关疾病的药物。
本发明第四方面提供了第一方面所述化合物或其衍生物的用途，所述化合物或其衍生物用于制备预防或治疗TDP25蛋白相关疾病的药物。
本发明第五方面提供了第一方面所述化合物或其衍生物的用途，所述化合物或其衍生物用于制备预防或治疗NLRP3蛋白相关疾病的药物。
本发明第六方面提供了第一方面所述化合物或其衍生物的用途，所述化合物或其衍生物用于制备预防或治疗肝损伤的药物。
本发明第七方面提供了第一方面所述化合物或其衍生物的用途，所述化合物或其衍生物具有如下一种或多种用途：
(1)抑制细胞程序性坏死；
(2)抑制RIP1激酶活性；
(3)降低RIP1和RIP3的相互作用；
(4)降解TDP25蛋白；
(5)抑制细胞RIP1激酶依赖的焦亡；
(6)降解NLRP3蛋白。
在另一优选例中，所述细胞选自下组：白血病T细胞、淋巴癌细胞、小胶质细胞、结肠癌细胞。
在另一优选例中，所述细胞选自下组：Jurkat-FADD-/-细胞、U937细胞、BV2细胞、HT-29细胞。
在另一优选例中，所述肝损伤为急性肝损伤。
在另一优选例中，所述肝损伤为毒素诱导的急性肝损伤。
在另一优选例中，所述毒素为LPS。
在另一优选例中，所述RIP1相关疾病选自下组：神经退行性疾病、缺血性损伤、自身免疫疾病、动脉粥样硬化、银屑病、高雪氏症、疼痛、炎症、视网膜脱落、肿瘤。
在另一优选例中，所述神经退行性疾病为肌萎缩侧索硬化(ALS)、阿尔茨海默病(AD)、帕金森病(PD)、多发性硬化症(MS)等。
在另一优选例中，所述自身免疫疾病为系统性红斑狼疮、类风湿性关节炎等。
在另一优选例中，所述疼痛为神经疼痛。
在另一优选例中，所述炎症为胰腺炎、溃疡性肠炎、肝炎等。
在另一优选例中，所述肿瘤为黑色素瘤、脑胶质瘤、结肠癌、神经胶质、淋巴瘤、T细胞白血病等。
在另一优选例中，所述多发性硬化症由双环己酮草酰双腙(Cuprizone)诱导引起。
在另一优选例中，所述细胞程序性坏死相关疾病选自下组：神经退行性疾病、缺血性损伤、自身免疫疾病、动脉粥样硬化、银屑病、高雪氏症、疼痛、炎症、视网膜脱落、肿瘤。
在另一优选例中，所述神经退行性疾病为肌萎缩侧索硬化(ALS)、阿尔茨海默病(AD)、帕金森病(PD)、多发性硬化症(MS)等。
在另一优选例中，所述自身免疫疾病为系统性红斑狼疮、类风湿性关节炎等。
在另一优选例中，所述疼痛为神经疼痛。
在另一优选例中，所述炎症为胰腺炎、溃疡性肠炎、肝炎等。
在另一优选例中，所述肿瘤为黑色素瘤、脑胶质瘤、结肠癌、神经胶质、淋巴瘤、T细胞白血病等。
在另一优选例中，所述多发性硬化症由双环己酮草酰双腙(Cuprizone)诱导引起。
在另一优选例中，所述细胞选自下组：白血病T细胞、淋巴癌细胞、小胶质细胞、结肠癌细胞。
在另一优选例中，所述细胞选自下组：Jurkat-FADD-/-细胞、U937细胞、BV2细胞、HT-29细胞。
在另一优选例中，所述TDP25蛋白相关疾病为神经退行性疾病。
在另一优选例中，所述神经退行性疾病为肌萎缩侧索硬化(ALS)。
在另一优选例中，所述NLRP3蛋白相关疾病选自下组：神经退行性疾病、自身免疫疾病、炎症性肠病、动脉粥样硬化、2型糖尿病、痛风、肥胖、肿瘤。
在另一优选例中，所述神经退行性疾病为阿尔茨海默病(AD)。
在另一优选例中，所述自身免疫疾病为系统性红斑狼疮。
在另一优选例中，所述肿瘤为黑色素瘤、脑胶质瘤、结肠癌、神经胶质、淋巴瘤、T细胞白血病等。
本发明第八方面提供了一种药物组合物，所述药物组合物包含第一方面所述化合物或其衍生物和药学上可接受的载体。
本发明第九方面提供了一种预防或治疗疾病的方法，所述方法包括步骤：给需要的受试者施用第一方面所述化合物或其衍生物或第八方面所述的药物组合物；所述疾病为选自下组的一种或多种：RIP1相关疾病、细胞程序性坏死相关疾病、TDP25蛋白相关疾病、NLRP3蛋白相关疾病、肝损伤。
本发明第十方面提供了一种用于抑制细胞中的RIP1活性的方法，其包括步骤：将细胞与第一方面所述化合物或其衍生物或第八方面所述的药物组合物接 触。
在另一优选例中，所述细胞选自下组：白血病T细胞、淋巴癌细胞、小胶质细胞、结肠癌细胞。
在另一优选例中，所述细胞选自下组：Jurkat-FADD-/-细胞、U937细胞、BV2细胞、HT-29细胞。
在另一优选例中，所述细胞为经诱导剂处理的细胞；所述诱导剂为选自下组的一种或多种：TNFα、z-VAD-fmk、LPS、SMAC、化合物B3、5z-7。
在另一优选例中，所述化合物或其衍生物的施用浓度为1-50μM；较佳地，为5-20μM。
本发明进一步还提供了式(I)化合物或其衍生物的用途，
Figure PCTCN2019097202-appb-000004
所述化合物或其衍生物用作RIP1抑制剂或用于制备预防或治疗RIP1相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用作细胞程序性坏死的抑制剂或用于制备预防或治疗细胞程序性坏死相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用于降低RIP1和RIP3的相互作用。
在另一优选例中，所述化合物或其衍生物还可以用于降解TDP25蛋白或用于制备预防或治疗TDP25蛋白相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用于降解NLRP3蛋白或用于制备预防或治疗NLRP3蛋白相关疾病的药物。
在另一优选例中，所述化合物或其衍生物还可以用于制备预防或治疗肝损伤的药物。
本发明进一步还提供了式(I)化合物或其衍生物的用途，
Figure PCTCN2019097202-appb-000005
所述化合物或其衍生物用于制备预防或治疗细胞程序性坏死相关疾病的药物。
本发明进一步还提供了式(I)化合物或其衍生物的用途，
Figure PCTCN2019097202-appb-000006
所述化合物或其衍生物用于制备预防或治疗TDP25蛋白相关疾病的药物。
本发明进一步还提供了式(I)化合物或其衍生物的用途，
Figure PCTCN2019097202-appb-000007
所述化合物或其衍生物用于制备预防或治疗NLRP3蛋白相关疾病的药物。
本发明进一步还提供了式(I)化合物或其衍生物的用途，
Figure PCTCN2019097202-appb-000008
所述化合物或其衍生物用于制备预防或治疗肝损伤的药物。
本发明进一步还提供了式(I)化合物或其衍生物的用途，
Figure PCTCN2019097202-appb-000009
所述化合物或其衍生物具有如下一种或多种用途：
(1)抑制细胞程序性坏死；
(2)抑制RIP1激酶活性；
(3)降低RIP1和RIP3的相互作用；
(4)降解TDP25蛋白；
(5)抑制细胞RIP1激酶依赖的焦亡；
(6)降解NLRP3蛋白。
在另一优选例中，所述细胞选自下组：白血病T细胞、淋巴癌细胞、小胶 质细胞、结肠癌细胞。
在另一优选例中，所述细胞选自下组：Jurkat-FADD-/-细胞、U937细胞、BV2细胞、HT-29细胞。
在另一优选例中，所述肝损伤为急性肝损伤。
在另一优选例中，所述肝损伤为毒素诱导的急性肝损伤。
在另一优选例中，所述毒素为LPS。
在另一优选例中，所述RIP1相关疾病选自下组：神经退行性疾病、缺血性损伤、自身免疫疾病、动脉粥样硬化、银屑病、高雪氏症、疼痛、炎症、视网膜脱落、肿瘤。
在另一优选例中，所述神经退行性疾病为肌萎缩侧索硬化(ALS)、阿尔茨海默病(AD)、帕金森病(PD)、多发性硬化症(MS)等。
在另一优选例中，所述自身免疫疾病为系统性红斑狼疮、类风湿性关节炎等。
在另一优选例中，所述疼痛为神经疼痛。
在另一优选例中，所述炎症为胰腺炎、溃疡性肠炎、肝炎等。
在另一优选例中，所述肿瘤为黑色素瘤、脑胶质瘤、结肠癌、神经胶质、淋巴瘤、T细胞白血病等。
在另一优选例中，所述多发性硬化症由双环己酮草酰双腙(Cuprizone)诱导引起。
在另一优选例中，所述细胞程序性坏死相关疾病选自下组：神经退行性疾病、缺血性损伤、自身免疫疾病、动脉粥样硬化、银屑病、高雪氏症、疼痛、炎症、视网膜脱落、肿瘤。
在另一优选例中，所述神经退行性疾病为ALS、AD、PD、MS等。
在另一优选例中，所述自身免疫疾病为系统性红斑狼疮、类风湿性关节炎等。
在另一优选例中，所述疼痛为神经疼痛。
在另一优选例中，所述炎症为胰腺炎、溃疡性肠炎、肝炎等。
在另一优选例中，所述肿瘤为黑色素瘤、脑胶质瘤、结肠癌、神经胶质、淋巴瘤、T细胞白血病等。
在另一优选例中，所述TDP25蛋白相关疾病为神经退行性疾病。
在另一优选例中，所述神经退行性疾病为ALS。
在另一优选例中，所述NLRP3蛋白相关疾病选自下组：神经退行性疾病、自身免疫疾病、炎症性肠病、动脉粥样硬化、2型糖尿病、痛风、肥胖、肿瘤。
在另一优选例中，所述神经退行性疾病为阿尔茨海默病(AD)。
在另一优选例中，所述自身免疫疾病为系统性红斑狼疮。
在另一优选例中，所述肿瘤为黑色素瘤、脑胶质瘤、结肠癌、神经胶质、淋巴瘤、T细胞白血病等。
本发明进一步还提供了一种药物组合物，所述药物组合物包含式(I)化合物或其衍生物和药学上可接受的载体。
本发明进一步还提供了一种预防或治疗疾病的方法，所述方法包括步骤：给需要的受试者施用式(I)化合物或其衍生物或包含式(I)化合物或其衍生物和药学上可接受的载体的药物组合物；所述疾病为选自下组的一种或多种：RIP1相关疾病、细胞程序性坏死相关疾病、TDP25蛋白相关疾病、NLRP3蛋白相关疾病、肝损伤；
Figure PCTCN2019097202-appb-000010
本发明进一步还提供了一种用于抑制细胞中的RIP1活性的方法，其包括步骤：将细胞与式(I)化合物或其衍生物或包含式(I)化合物或其衍生物和药学上可接受的载体的药物组合物接触；
Figure PCTCN2019097202-appb-000011
在另一优选例中，所述细胞选自下组：白血病T细胞、淋巴癌细胞、小胶质细胞、结肠癌细胞。
在另一优选例中，所述细胞选自下组：Jurkat-FADD-/-细胞、U937细胞、BV2细胞、HT-29细胞。
在另一优选例中，所述细胞为经诱导剂处理的细胞；所述诱导剂为选自下组的一种或多种：TNFα、z-VAD-fmk、LPS、SMAC、化合物B3、5z-7。
在另一优选例中，式(I)化合物或其衍生物的施用浓度为1-50μM；较佳地，为5-20μM。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了ZJU-37抑制Jurkat-FADD-/-细胞的程序性坏死。
图2显示了ZJU-37抑制U937细胞的程序性坏死。
图3显示了ZJU-37抑制BV2细胞的程序性坏死。
图4显示了ZJU-37能有效抑制细胞中P-RIP1含量，从而抑制其激酶活性。
图5显示了ZJU-37能有效抑制细胞中P-RIP1含量，从而抑制其激酶活性。
图6显示了ZJU-37能有效降低RIP1和RIP3的相互作用。
图7显示了ZJU-37能有效降解TDP25蛋白。
图8显示了ZJU-37能有效削弱小鼠脑组织中RIP1/RIP3两种蛋白的相互作用。
图9显示了ZJU-37能有效抑制小鼠急性肝损伤。
图10显示了ZJU-37能有效降解炎症诱导的NLRP3蛋白。
图11显示了ZJU-37能有效改善RIPK1依赖的焦亡的发生。
图12显示了ZJU-37能有效缓解多发性硬化症。
图13显示了ZJU-37能快速通过血脑屏障。
图14显示了ZJU-37对RIP1抑制作用的IC 50。
图15显示了化合物(II)抑制Jurkat-FADD-/-细胞的程序性坏死。
具体实施方式
发明人通过深入研究，意外发现了一种结构新颖的化合物可作为高效的RIP1抑制剂，这类抑制剂化合物能有效抑制RIP1活性，还可以有效减少RIP1和RIP3的相互作用，还有有效抑制细胞程序性坏死、提高细胞活力；还可以有效降解TDP25蛋白和NLRP3蛋白等，从而对于多种疾病具有预防和治疗作用。这类化合物能快速通过血脑屏障，并且有促进神经细胞增殖的功能。在此基础上完成了本发明。
术语
如本文所用，术语“C 1-6烷基”为具有1-6个碳原子的直链或支链的烷基，例如为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等。优选为C 1-3烷基。
如本文所用，术语“C 6-10芳基”为具有6-10个碳原子的芳基，例如为苯基或萘基。
如本文所用，术语“5元至10元杂芳基”为具有5至10个环原子的杂芳基，其中一个或多个(例如1个、2个、3个或4个)环原子各自独立地为选自氮、硫或氧的杂原子。优选为5元至6元杂芳基或6元至10元杂芳基；例如，呋喃、噻吩、吡咯、噻唑、咪唑、吡啶、吡嗪、嘧啶、哒嗪、吲哚、喹啉。
NLRP3蛋白
NLRP3蛋白是NLRP3炎性小体的重要组成部分，作为固有免疫的重要组分在机体免疫反应和疾病发生过程中具有重要作用。NLRP3蛋白相关疾病为由NLRP3蛋白介导(或至少部分由NLRP3蛋白介导)的疾病，例如包括(但不限于)：阿尔兹海默病、系统性红斑狼疮、炎症性肠病、动脉粥样硬化、2型糖尿病、痛风、肥胖、恶性肿瘤。
TDP25蛋白
TDP25蛋白是在ALS患者的大脑区域中发现的分子量为25kD的TDP-43的C末端片段。研究发现，TDP25蛋白可以促进TDP-43包涵体的形成，对运动神经元具有毒性作用，引起神经元变性，在疾病的发病过程中起重要作用。TDP25蛋白相关疾病为由TDP25蛋白介导(或至少部分由TDP25蛋白介导)的疾病，例如ALS。
活性成分
如本文所用，本发明所述的化合物为式(A)化合物，其结构如下所示：
Figure PCTCN2019097202-appb-000012
式中，各基团的定义如前所述。
优选地，本发明所述的化合物为式(I)化合物，其结构如下所示：
Figure PCTCN2019097202-appb-000013
在本文中，化合物“ZJU-37”、化合物“ZJU37”和“式(I)化合物”可以互换使用。
如本文所用，本发明所述的活性成分为式(A)化合物或其衍生物。
优选地，本发明所述的活性成分为式(I)化合物或其衍生物。
本发明所述的化合物的衍生物为该化合物的任意衍生物，例如，化合物的药学上可接受的盐、水合物、溶剂合物、前药、互变异构体、立体异构体或立体异构体的混合物等。
如本文所用，“药学上可接受的盐”或“生理学上可接受的盐”包括例如本发明化合物与无机酸形成的盐和与有机酸形成的盐。另外，如果本文所述的化合物作为酸加成盐获得，则游离碱可通过碱化所述酸加成盐的溶液而获得。相反地，如果所述产物是游离碱，则可根据用于从碱性化合物制备酸加成盐的常规方法，通过将所述游离碱溶解于适当的有机溶剂中并且用酸处理所述溶液制得加成盐，特别是药学上可接受的加成盐。本领域的技术人员将会认识到可用于制备无毒的药学上可接受的加成盐的各种合成方法。可以由无机酸和有机酸制备药学上可接受的酸加成盐。衍生自无机酸的盐包括盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等。衍生自有机酸的盐包括乙酸盐、丙酸盐、乙醇酸盐、丙酮酸盐、草酸盐、苹果酸盐、丙二酸盐、琥珀酸盐、马来酸盐、富马酸盐、酒石酸盐、柠檬酸盐、苯甲酸盐、肉桂酸盐、扁桃酸盐、甲磺酸盐、乙磺酸盐、对甲苯磺酸盐、水杨酸盐等。
如本文所用，“水合物”是指通过组合本发明化合物和水而形成的复合物。
如本文所用，“溶剂合物”是指一种或多种溶剂分子与本发明化合物的缔合物或复合物。形成溶剂合物的溶剂的实例包括但不限于水、异丙醇、乙醇、甲醇、二甲基亚砜、乙酸乙酯、乙酸和乙醇胺。
如本文所用，本发明化合物可以互变异构体存在。互变异构体彼此处于平衡状态。例如，羰基部分可以烯醇式存在。不管显示哪种互变异构体并且不管互变异构体之间平衡的性质如何，本领域普通技术人员均理解所述化合物包含酮式和烯醇式互变异构体两者。
如本文所用，“立体异构体”是指由相同原子通过相同键键合而组成的化合物，但具有不同的三维结构，其为不可互换的。本发明所述“立体异构体”涵盖了本发明化合物的各种立体异构体及其混合物并且包括“对映体”，其是指两种立体异构体，其分子为彼此不可重叠的镜像。“非对映异构体”是具有至少两个不对称原子的立体异构体，但其彼此不为镜像。
如本文所用，“前药”意指当将所述前药向受试者施用时，根据本发明化合物结构在体内释放活性母体药物的任何化合物。所述前药可以通过修饰存在于本发明化合物中的任意官能团来制备，方式为使得所述修饰可在体内裂解以释放母体化合物。可通过以在常规操作或体内使修饰裂解为母体化合物的方式修饰存在于化合物中的官能团来制备前药。本发明所述前药包括本发明化合物中的氨基等基团与可在体内裂解以分别再产生游离的氨基的任何基团键合。前药的制备、选择和用途讨论于T.Higuchi和V.Stella,“Pro-drugs as Novel Delivery Systems”A.C.S.学术讨论会丛刊第14卷；“Design of Prodrugs”编著H.Bundgaard,Elsevier,1985；和Bioreversible Carriers in Drug Design,编著Edward B.Roche,American Pharmaceutical Association and Pergamon Press,1987中，所述参考文献 均特此以引用的方式整体并入。
制备方法
本发明化合物可以按照本领域常规方法制备，也可以按照如下方法(如下反应式所示的方法)进行制备。
本发明的制备方法包括步骤：化合物2和R 3-L-X进行反应，从而得到式(A)化合物。
Figure PCTCN2019097202-appb-000014
式中，R 1、R 2、R 3、L定义同前；X为N(CH 3) 2或卤素(例如氟、氯、溴或碘)。
化合物2参照WO2016094848A1的实施例1的操作方法和采用相应的原料进行制备。
所述反应在溶剂中进行。例如DMF、DMSO、THF等。该溶剂一般不参与反应。当然有些溶剂也可以作为反应试剂R 3-L-X参与反应，例如DMF作为溶剂时。
所述反应可以在碱性条件或酸性条件下进行。碱性条件例如在碱存在下进行。所述碱例如氢化钠、氢化钾、氢氧化钠、氢氧化钾等碱性试剂。酸性条件例如在酸存在下进行。所述酸例如三氯氧磷等酸性试剂。
所述反应可以在常规条件下进行。例如在一定温度(例如-10℃-30℃；优选0℃-30℃)下进行一段时间(例如0.1-10小时；优选0.1-5小时)。
药物和施用方法
研究发现本发明化合物具有以下一种或多种功能：抑制RIP1激酶活性；减少RIP1和RIP3的相互作用；抑制细胞程序性坏死；降解TDP25蛋白；降解NLRP3蛋白等等。据此，本发明化合物或其衍生物以及含有该化合物或其衍生物为主要活性成分的药物组合物可用于治疗、预防以及缓解与RIP1相关疾病、RIP1和RIP3相关疾病、TDP25蛋白相关疾病或NLRP3蛋白相关疾病。这些疾病包括且不限于各种神经退行性疾病，自身免疫性疾病，肝损伤、肝衰竭等肝脏疾病，肠胃炎等。
本发明的药物组合物包含安全有效量范围内的活性成分及药理上可以接受的赋形剂或载体。
所述的“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg活性成分/剂，更佳地，含有5-200mg活性成分/剂。较佳地，所述的“一剂”为一个胶囊或药片。
所述的“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组分能和活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2019097202-appb-000015
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明活性成分或含有该活性成分的药物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。用于局部给药的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明活性成分可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明活性成分适用于需要治疗的受试者，例如哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
如本文所用，“治疗”是用于获得有益或所需结果(包括临床结果)的方法。有益的或所需的临床结果可包括以下中的一个或多个：a)抑制疾病或病状(例如，减少由疾病或病状导致的一种或多种症状和/或减轻疾病或病状的程度)；b)减缓或阻止与疾病或病状相关的一种或多种临床症状的发展(例如，稳定疾病或病状、预防或延迟疾病或病状的恶化或进展和/或预防或延迟疾病或病状的扩散(例如，转移)；和/或c)缓解疾病，即引起临床症状的消退(例如，改善疾病状态、提供疾病或病状的部分或全部缓解、增强另一种药物的作用、延迟疾病的进展、提高生活质量和/或延长存活)。
如本文所用，“预防”意指导致疾病或病状的临床症状不发展的疾病或病状的任何治疗。在某些实施方案中，活性成分可施用于处于风险或者具有疾病或病状家族史的受试者(包括人)。
如本文所用，“受试者”是指已经或将要成为治疗、观察或实验对象的动物，诸如哺乳动物(包括人)。本文所述的方法可用于人疗法和/或兽医应用。在某些实施方案中，受试者是哺乳动物。在某些实施方案中，受试者是人。
本发明的主要优点在于：
本发明化合物或其衍生物可以有效抑制RIP1活性，可以作为一种有效的 RIP1抑制剂，从而用于预防或治疗RIP1介导的疾病。
本发明化合物或其衍生物能有效减少细胞程序性坏死、提高细胞活力。
本发明化合物或其衍生物还具有抑制炎症的能力。
本发明化合物或其衍生物还可以降解NLRP3蛋白和TDP25蛋白。
具体地，本发明化合物或其衍生物还可以治疗肝损伤。
也就是说，本发明化合物或其衍生物可以一药多靶地用来治疗多种疾病(例如RIP1介导的疾病、NLRP3蛋白介导的疾病或TDP25蛋白介导的疾病)。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
以下实施例中，药物处理后的细胞活力(％)＝药物处理孔的细胞活力/对照孔的细胞活力*100％；细胞活力(％)越高，代表药物处理后的细胞活力越强。其中，细胞活力通过基于ATP的生存力测定来确定。
以下实施例中，细胞培养以及药物处理的条件均为：稳定的温度(37℃)、稳定的CO 2水平(5％)、恒定的酸碱度(pH值：7.2-7.4)、较高的相对饱和湿度(95％)。
以下实施例中所用的实验材料(如各种细胞)和试剂如无特别说明均可从市售渠道获得。
以下实施例中，Jurkat FADD-/-细胞表示FADD-/-敲除的Jurkat细胞。Jurkat细胞常用于研究急性T细胞白血病。Jurkat细胞FADD-/-敲除的Jurkat细胞在TNFα诱导的条件下专一诱导程序性坏死死亡的发生。本实验中的Jurkat FADD -/-细胞可以通过CRISPR技术得到。
以下实施例中，U937细胞为人组织细胞淋巴瘤细胞；购自南京科佰细胞库。
以下实施例中，BV2细胞为小鼠小胶质细胞；购自南京科佰细胞库。由于BV2细胞在z-VAD-fmk诱导的条件下自身会分泌TNFα，所以诱导条件不需要再加TNFα。
以下实施例中，293T细胞由293细胞派生；购自南京科佰细胞库。293细胞是转染腺病毒E1A基因的人肾上皮细胞系。
以下实施例中，H4-TDP25细胞是稳定表达TDP25的H4细胞；购自南京科佰细胞库。
以下实施例中，HT-29细胞为人结肠癌细胞；购自南京科佰细胞库。
以下实施例中，BMDM细胞为骨髓来源的巨噬细胞；购自南京科佰细胞库。
制备例1 化合物JIU-37的制备方法
Figure PCTCN2019097202-appb-000016
化合物2参照WO2016094848A1的实施例1的操作方法制得。在氮气保护及冰浴条件下，将POCl 3(0.4mmol,60mg)逐滴加入到DMF(1mL)中，继续在冰浴中搅拌0.5小时。然后将化合物2(0.2mmol,59mg)的DMF溶液加入到反应液中，升至室温继续反应5小时。加入适量的水淬灭反应，并用乙酸乙酯萃取3次，合并有机相经饱和食盐水洗涤，无水硫酸钠干燥后，浓缩并柱层析得白色固体ZJU-37(38.7mg，60％)。 1H NMR(400MHz,CDCl 3)δ9.06(s,1H),8.31(s,1H),7.32(dd,J＝8.7,4.5Hz,1H),6.99(d,J＝2.2Hz,1H),6.95(t,J＝9.2Hz,1H),4.74(dd,J＝5.5,2.2Hz,1H),3.75(dd,J＝15.0,5.6Hz,1H),3.52(dd,J＝15.0,2.1Hz,1H),2.80(s,3H). 13C NMR(100MHz,CDCl 3)δ 13C NMR(100MHz,CDCl 3)δ171.3,158.4,155.0(d,J＝242.1Hz),154.3,134.0(d,J＝4.2Hz),124.7(d,J＝3.4Hz),124.4,117.8(d,J＝9.1Hz),109.6(d,J＝23.7Hz),108.5,103.5(d,J＝22.3Hz),57.9,24.8,24.3.
制备例2 化合物(II)的制备方法
Figure PCTCN2019097202-appb-000017
在氮气保护及冰浴条件下，将化合物2(0.2mmol,59mg)溶于THF(1mL)中，加入NaH(0.3mmol,7.2mg)，继续在冰浴中搅拌0.5小时。然后将2-吡啶磺酰氯(0.3mmol,53mg)的THF溶液加入到反应液中，升至室温过夜反应。加入适量的水淬灭反应，并用乙酸乙酯萃取3次，合并有机相经饱和食盐水洗涤，无水硫酸钠干燥后，浓缩并柱层析得白色固体化合物(II)(62mg，71％)。 1H NMR(400MHz,DMSO)δ11.57(s,1H),9.17(d,J＝2.2Hz,1H),8.93(d,J＝4.8Hz,1H),8.42(d,J＝8.1Hz,1H),7.71(dd,J＝8.1,4.9Hz,1H),7.48(dd,J＝8.7,4.8Hz,1H),7.12(d,J＝1.5Hz,1H),7.10–7.04(m,1H),5.21–5.15(m,1H),3.57–3.51(m,2H),2.54(s,3H).
按制备例2的实验方法，采用相应的原料分别制备如下化合物：
Figure PCTCN2019097202-appb-000018
HRMS(ESI)calcd for C 20H 15ClFN 3O 3:400.0866(M+H +),found:400.0871。
Figure PCTCN2019097202-appb-000019
HRMS(ESI)calcd for C 19H 14ClFN 4O 3:401.0818(M+H +),found:401.0826。
Figure PCTCN2019097202-appb-000020
HRMS(ESI)calcd for C 19H 15ClFN 3O 4S:436.0536(M+H +),found:436.0540。
实施例1 ZJU-37能抑制Jurkat-FADD -/-细胞程序性坏死
原料：TNFα(购自美国Sigma公司)；Nec1(结构为
Figure PCTCN2019097202-appb-000021
购自Selleck公司；作为阳性对照药物)。
实验方法：
Jurkat FADD -/-细胞铺于96孔白板，所用培养基为1640培养基(购自南模生物公司，下同)，铺板密度为2万细胞每孔，细胞铺板2-4h后进行以下处理：
Nec1孔：用TNFα(最终作用浓度为30ng/ml)和不同作用浓度的药物Nec1(最终作用浓度为10 -7μM、10 -6μM、10 -5μM、10 -4μM、10 -3μM、10 -2μM、10 -1μM、1μM、10μM)一起处理；
ZJU-37孔：用TNFα(最终作用浓度为30ng/ml)和不同作用浓度的药物 ZJU-37(最终作用浓度为10 -7μM、10 -6μM、10 -5μM、10 -4μM、10 -3μM、10 -2μM、10 -1μM、1μM、10μM)一起处理；
TNFα孔：用TNFα(30ng/ml)和与药物等同体积的DMSO处理；
对照孔：不添加诱导剂和药物，为空白对照；
处理18小时后，分别测试各孔的细胞活力并计算药物处理后的细胞活力(％)。
实验结果如图1所示。其中，A图表示在10μM浓度下，Nec1和ZJU-37分别都能抑制Jurkat-FADD-/-细胞的程序性坏死；B图表示Nec1和ZJU-37分别在不同浓度下对Jurkat-FADD-/-细胞的程序性坏死的抑制；C图表示Nec1抑制Jurkat-FADD-/-细胞的程序性坏死的EC50；D图表示ZJU-37抑制Jurkat-FADD-/-细胞的程序性坏死的EC50。
可见，在TNFα专一诱导程序性坏死的情况下，ZJU-37能抑制Jurkat-FADD-/-细胞的程序性坏死，其抑制效果与阳性对照药物Nec1相似。
实施例2 ZJU-37能抑制U937细胞程序性坏死
原料：TNFα(同前)；z-VAD-fmk(购自BioVision公司)；Nec1(同前)。
实验方法：
U937细胞铺于96孔白板，所用培养基为1640培养基，铺板密度为2万细胞每孔，细胞铺板2-4h后进行以下处理：
Nec1孔：用TZ(TNFα(最终作用浓度为30ng/ml)和z-VAD-fmk(最终作用浓度为40μM))和不同作用浓度的Nec1(最终作用浓度为0.0001μM、0.0005μM、0.001μM、0.005μM、0.01μM、0.1μM、0.5μM、5μM、10μM)一起处理；
ZJU-37孔：用TZ(TNFα(最终作用浓度为30ng/ml)和z-VAD-fmk(最终作用浓度为40μM))和不同作用浓度的ZJU-37(最终作用浓度为0.0001μM、0.0005μM、0.001μM、0.005μM、0.01μM、0.1μM、0.5μM、5μM、10μM)一起处理；
TZ孔：用TZ(TNFα(最终作用浓度为30ng/ml)和z-VAD-fmk(最终作用浓度为40μM))和与药物等同体积的DMSO处理；
对照孔：不添加诱导剂和药物，为空白对照；
处理72小时后，分别测试各孔的细胞活力并计算药物处理后的细胞活力(％)。
实验结果如图2所示。其中，A图表示在10μM浓度下，Nec1和ZJU-37分别都能抑制U937细胞的程序性坏死；B图表示Nec1和ZJU-37分别在不同浓度下对U937细胞的程序性坏死的抑制；C图表示Nec1抑制U937细胞的程 序性坏死的EC50；D图表示ZJU-37抑制U937细胞的程序性坏死的EC50。
可见，在TNFα和z-VAD-fmk专一诱导程序性坏死的情况下，ZJU-37能抑制U937细胞的程序性坏死，其抑制效果与阳性对照药物Nec1相似。
实施例3 ZJU-37能抑制BV2细胞程序性坏死
原料：z-VAD-fmk(同前)；Nec1(同前)。
实验方法：
BV2细胞铺于96孔白板，所用培养基为DMEM培养基(购自赛默飞公司，下同)，铺板密度为8000千细胞每孔，细胞铺板1天后进行以下处理：
Nec1孔：用z-VAD-fmk(最终作用浓度为70μM)和不同作用浓度的Nec1(最终作用浓度为0μM、0.0001μM、0.0005μM、0.001μM、0.005μM、0.01μM、0.5μM、1μM、5μM、10μM)一起处理；
ZJU-37孔：用z-VAD-fmk(最终作用浓度为70μM)和不同作用浓度的ZJU-37(最终作用浓度为0μM、0.0001μM、0.0005μM、0.001μM、0.005μM、0.01μM、0.5μM、1μM、5μM、10μM)一起处理；
Z-VAD孔：用z-VAD-fmk(最终作用浓度为70μM)和与药物等同体积的DMSO处理；
对照孔：不添加诱导剂和药物，为空白对照；
处理72小时后，分别测试各孔的细胞活力并计算药物处理后的细胞活力(％)。
实验结果如图3所示。其中，A图表示在10μM浓度下，Nec1和ZJU-37分别都能抑制BV2细胞的程序性坏死；B图表示Nec1和ZJU-37分别在不同浓度下对BV2细胞的程序性坏死的抑制；C图表示Nec1抑制BV2细胞的程序性坏死的EC50；D图表示ZJU-37抑制BV2细胞的程序性坏死的EC50。
可见，在z-VAD-fmk专一诱导程序性坏死的情况下，ZJU-37能抑制BV2细胞的程序性坏死，其抑制效果与阳性对照药物Nec1相似。
实施例4 ZJU-37能有效抑制RIP1激酶活性
原料：Flag-RIP1(购自维真生物)；LPS(购自Sigma公司)；Nec1(同前)。
实验方法：
293T细胞铺于六孔板，所用培养基为DMEM培养基，铺板密度为50万细胞每孔；铺板24小时后进行以下处理：
Flag孔：转染1μg Flag-RIP1；
Flag+LPS孔：转染1μg Flag-RIP1，转染24小时后用LPS(最终作用浓度为100ng/ml)和与药物等同体积的DMSO处理；
Nec1-10孔：转染1μg Flag-RIP1，转染24小时后用LPS(最终作用浓度为100ng/ml)和Nec1(最终作用浓度为10μM)一起处理；
ZJU37-5孔：转染1μg Flag-RIP1，转染24小时后用LPS(最终作用浓度为100ng/ml)和ZJU-37(最终作用浓度为5μM)一起处理；
ZJU37-10孔：转染1μg Flag-RIP1，转染24小时后用LPS(最终作用浓度为100ng/ml)和ZJU-37(最终作用浓度为10μM)一起处理；
ZJU37-20孔：转染1μg Flag-RIP1，转染24小时后用LPS(最终作用浓度为100ng/ml)和ZJU-37(最终作用浓度为20μM)一起处理；
对照孔：不添加诱导剂和药物，为空白对照。
处理6小时后，分别收集细胞用相应抗体进行免疫印迹杂交。结果如图4的A图和C图所示。
Jurkat FADD-/-细胞铺于六孔板，所用培养基为1640培养基，铺板密度为150万细胞每孔；铺板2小时后进行以下处理：
TNFα孔：用TNFα(最终作用浓度为30ng/ml)和与药物等同体积的DMSO处理；
Nec1孔：用TNFα(最终作用浓度为30ng/ml)和Nec1(最终作用浓度为10μM)一起处理；
ZJU-37孔：用TNFα(最终作用浓度为30ng/ml)和ZJU-37(最终作用浓度为10μM)一起处理；
对照孔：不添加诱导剂和药物，为空白对照。
处理4小时后，分别收集细胞用相应抗体进行免疫印迹杂交。结果如图4的B图所示。
可见，在LPS或TNFα单独诱导的情况下，P-RIP1水平均有所提高，Jurkat FADD-/-细胞中尤为明显。在加入ZJU-37处理后有效降低了P-RIP1的水平，从而抑制了其激酶活性。且ZJU-37对P-RIP1的抑制效果优于阳性对照药物Nec1。图4C表明10μM的ZJU-37降低P-RIP1水平更有效。
实施例5 ZJU-37能有效抑制RIP1激酶活性
原料：pCMV-Flag(空载，购自维真生物)；Flag-beads(购自Bimake)；Flag-RIP1(同前)；磷酸酶抑制剂(购自生工生物工程股份有限公司)；Nec1(同前)；激酶缓冲液(Kinase Buffer)(20mM-HEPES，pH＝7.5，2mM DTT，10mM MnCl 2)。
实验方法：
293T细胞铺于10cm培养皿，所用培养基为DMEM培养基，铺板密度为300万细胞每皿；铺板24小时后，转染6μg pCMV-Flag(体系1)或Flag-RIP1(体系2-体系5)。在体系1、体系3至体系5中添加了ATP，体系2中不添加ATP。
转染的同时，在体系2和体系3中加入DMSO；在体系4中加入50μM的Nec1；在体系5中加入50μM的ZJU-37。
激酶实验各体系具体如下所示：
  体系1 体系2 体系3 体系4 体系5
ddH2O + + + + +
激酶缓冲液 + + + + +
ATP + - + + +
磷酸酶抑制剂 + + + + +
DMSO   + +    
Nec1       +  
ZJU37         +
处理细胞20小时后，每皿弃掉上清，用预冷的PBS洗两遍，然后每皿加入1ml RIPA裂解液，与Flag-beads一起孵育，放于摇床上4℃裂解30min后，4℃12000rpm离心10min，取上清和Flag-beads于4℃孵育，4h后离心，4℃2000rpm离心2min留Flag-beads弃上清，反复用RIPA裂解液洗三遍Flag-beads即可做激酶实验。
将5种体系分别加入每个样本的离心管后，放于30℃金属浴30min后，收集beads用相应抗体进行免疫印迹杂交。
实验结果如图5所示：在加入ATP的情况下，3号样本相比于2号样本P-RIP1水平有所提高，但在加入药物ZJU-37后，能有效抑制P-RIP1的含量，从而抑制其激酶活性，其抑制效果与阳性对照药物Nec1相似。
实施例6 ZJU-37能降低细胞中RIP1和RIP3的相互作用
原料：TNFα(同前)；z-VAD-fmk(同前)；SMAC(购自Selleck公司)；Nec1(同前)；EGFP-RIP1(购自维真生物)；Flag-RIP3(购自维真生物)；
实验方法：
HT-29细胞铺于10cm培养皿中，所用培养基为1640培养基；铺板密度为200万细胞每皿；铺板24h后进行以下处理：
TZS孔：用TZS(TNFα(最终作用浓度为30ng/ml)、z-VAD-fmk(最终作用浓度为20μM)和SMAC(最终作用浓度为100nM))和与药物等同体积的DMSO处理；
Nec1孔：用TZS(TNFα(最终作用浓度为30ng/ml)、z-VAD-fmk(最终作用浓度为20μM)和SMAC(最终作用浓度为100nM))和Nec1(最终作用浓度为10μM)处理；
ZJU-37孔：用TZS(TNFα(最终作用浓度为30ng/ml)、z-VAD-fmk(最终作用浓度为20μM)和SMAC(最终作用浓度为100nM))和ZJU-37(最终作用浓度为10 μM)处理；
对照孔：不添加诱导剂和药物，为空白对照。
处理72小时后用PBS洗两遍，加入RIPA裂解液，与beads一起孵育，1ml于4℃裂解30min后收蛋白，加入RIP1抗体孵育，离心beads收蛋白后用相应抗体进行免疫印迹杂交。结果如图6中的A图所示。
293T细胞铺于10cm培养皿中，所用培养基为DMEM培养基；铺板密度为400万细胞每皿；铺板24h后进行以下处理：
EGFP-RIP1+Flag-RIP3孔：转染EGFP-RIP1、Flag-RIP3各2μg，同时加入和与药物等同体积的DMSO处理；
Nec1孔：转染EGFP-RIP1、Flag-RIP3各2μg，同时加入Nec1(最终作用浓度为10μM)处理；
ZJU-37孔：转染EGFP-RIP1、Flag-RIP3各2μg，同时加入ZJU-37(最终作用浓度为10μM)处理；
对照孔：不添加诱导剂和药物，为空白对照。
处理24h后用PBS洗两遍，加入RIPA裂解液，与beads一起孵育，1ml于4℃裂解30min后收蛋白，加入EGFP抗体孵育，离心beads收蛋白后用相应抗体进行免疫印迹杂交。图6中的B图所示。
在RIP1含量相当的情况下，RIP3含量越低，两者相互作用越小。而相互作用减弱意味着能一定程度上削弱细胞程序性坏死。实验结果如图6所示：
A图：HT-29细胞中，Nec1和ZJU-37都能降低RIP1和RIP3的相互作用，并且ZJU-37的效果优于Nec1。
B图：293T细胞中，Nec1和ZJU-37都能降低RIP1和RIP3的相互作用，并且ZJU-37的效果优于Nec1。
实施例7 ZJU-37能有效降解TDP25蛋白的水平
原料：化合物B3(结构为
Figure PCTCN2019097202-appb-000022
)；Nec1(同前)。
实验方法：
H4-TDP25细胞铺于六孔板，所用培养基为DMEM培养基，铺板密度为20万细胞每孔；铺板12小时后进行以下处理：
B3孔：用化合物B3(最终作用浓度10μM)处理细胞6小时后换液，再用与药 物等同体积的DMSO处理；
Nec1-10孔：用化合物B3(最终作用浓度10μM)处理细胞6小时后换液，再用Nec1(最终作用浓度为10μM)处理；
ZJU37-1孔：用化合物B3(最终作用浓度10μM)处理细胞6小时后换液，再用ZJU-37(最终作用浓度为1μM)处理；
ZJU37-5孔：用化合物B3(最终作用浓度10μM)处理细胞6小时后换液，再用ZJU-37(最终作用浓度为5μM)处理；
ZJU37-10孔：用化合物B3(最终作用浓度10μM)处理细胞6小时后换液，再用ZJU-37(最终作用浓度为10μM)处理；
ZJU37-20孔：用化合物B3(最终作用浓度10μM)处理细胞6小时后换液，再用ZJU-37(最终作用浓度为20μM)处理；
对照孔：不添加诱导剂和药物，为空白对照。
处理6小时后，分别收集细胞用相应抗体进行免疫印迹杂交。实验结果如图7所示。
H4-TDP25细胞经化合物B3诱导后，TDP25表达提高，经ZJU-37处理，细胞内TDP25蛋白表达明显下降。可见，ZJU-37能有效降解TDP25蛋白，且其抑制效果较阳性对照药物Nec1更好(而TDP25蛋白聚集会诱发一系列疾病，例如ALS)。
实施例8 ZJU-37能有效削弱小鼠脑组织中RIP1/RIP3两种蛋白的结合
原料：C57BL/6雄性小鼠(购自上海南模生物)；LPS(同前)；Nec1(同前)。
实验方法：
C57BL/6雄性小鼠进行以下处理：
对照组：为空白对照，不添加诱导剂和药物；
LPS组：用LPS(最终作用浓度为100ng/kg)和与药物等同体积的DMSO的腹腔注射处理；
Nec1组：用LPS(最终作用浓度为100ng/kg)和Nec1(最终作用浓度为5mg/kg)腹腔注射处理；
ZJU-37组：用LPS(最终作用浓度为100ng/kg)和ZJU-37(最终作用浓度为5mg/kg)腹腔注射处理；
处理6小时后，将小鼠解剖取脑，取脑组织1/2量加RIPA裂解液研磨1min，12000rpm离心20min，取上清，此步骤重复三次，最后合并上清并加入beads以及抗体孵育，最后收集蛋白用相应体进行免疫印迹杂交。
实验结果如图8所示。ZJU-37能有效削弱小鼠脑组织中RIP1/RIP3两种蛋白的相互作用。从IP条带中我们可以看到，在LPS作用后，RIP3与RIP1的相 互作用增强，从RIP3条带的蛋白水平含量即可看出，而ZJU37作用后的RIP3水平降低，证明RIP3与RIP1的相互作用是减弱的。而Nec1并不能减弱RIP3与RIP1的相互作用。
实施例9 ZJU-37能有效抑制LPS诱导的小鼠急性肝损伤
原料：C57BL/6雄性小鼠(同前)；LPS(同前)；Nec1(同前)。
实验方法：
C57BL/6雄性小鼠进行以下处理：
对照组：为空白对照，不添加诱导剂和药物；
LPS组：用LPS(最终作用浓度100ng/kg)和与药物等同体积的DMSO的腹腔注射处理；
LPS+ZJU-37组：用LPS(最终作用浓度100ng/kg)和ZJU-37(最终作用浓度5mg/kg)腹腔注射处理；
处理6小时后，解剖小鼠取肝脏，进行HE切片染色形态学观察。做肝脏HE染色切片主要目的一般都是为了观察肝细胞形态是否完整、胞核有无增大、肝小叶形态是否完整，是否发生炎症以检测药物等刺激因素对肝脏的损伤作用。
实验结果如图9所示。ZJU-37能有效抑制LPS诱导的小鼠急性肝损伤。与空白对照组细胞形态进行比较，发现添加LPS后，细胞形态明显变差，有明显的炎性浸润现象；而经ZJU-37处理后，细胞形态有明显改善，并且炎性浸润现象得以缓解。
实施例10 ZJU-37能有效降解炎症诱导的NLRP3蛋白
原料：TNFα(同前)；z-VAD-fmk(同前)；SMAC(同前)；Nec1(同前)。
实验方法：
HT-29细胞铺于六孔板，所用培养基为1640培养基，铺板密度为70万细胞每孔；铺板24小时后进行以下处理：
对照孔：不添加诱导剂和药物，为空白对照；
TZS孔：用TZS(TNFα(最终作用浓度为30ng/ml)、z-VAD-fmk(最终作用浓度为20μM)和SMAC(最终作用浓度为100nM))和与药物等同体积的DMSO处理；
Nec1孔：用TZS(TNFα(最终作用浓度为30ng/ml)、z-VAD-fmk(最终作用浓度为20μM)和SMAC(最终作用浓度为100nM))和Nec1(最终作用浓度为10μM)处理；
ZJU-37孔：用TZS(TNFα(最终作用浓度为30ng/ml)、z-VAD-fmk(最终作用浓度为20μM)和SMAC(最终作用浓度为100nM))和ZJU-37(最终作用浓度为10 μM)处理；
处理72小时后，分别收集细胞用相应抗体进行免疫印迹杂交。结果如图10的A图所示。
Jurkat FADD-/-细胞铺于六孔板，所用培养基为1640培养基，铺板密度为150万细胞每孔；铺板2小时后进行以下处理：
对照孔：不添加诱导剂和药物，为空白对照；
TNFα孔：用TNFα(最终作用浓度为30ng/ml)和与药物等同体积的DMSO处理；
Nec1孔：用TNFα(最终作用浓度为30ng/ml)和Nec1(最终作用浓度为10μM)处理；
ZJU-37孔：用TNFα(最终作用浓度为30ng/ml)和ZJU-37(最终作用浓度为10μM)处理；
处理4小时后，分别收集细胞用相应抗体进行免疫印迹杂交。结果如图10的B图所示。
实验结果如图10所示：
A图：在NLRP3被TZS诱导上升的情况下，ZJU-37能有效降解炎症诱导的NLRP3蛋白，并且效果优于阳性药物Nec1。
B图：在NLRP3被TNFα诱导上升的情况下，ZJU-37能有效降解炎症诱导的NLRP3蛋白，并且效果优于阳性药物Nec1。
实施例11 ZJU-37能有效抑制RIP1依赖的焦亡
原料：小鼠BMDM细胞：小鼠购自南模生物；BMDM提取方法：取小鼠大腿骨，将腿骨两端剪开，用针头吸取PBS冲洗腿骨内部冲出细胞，所得细胞1000rpm，4度离心5min。弃去上清，加入适量ACK裂解红细胞，3-5min后加入PBS中和。在1000rpm，4度离心5min。即可用培养基重悬计数铺板。
LPS(同前)；Nec1(同前)；5z-7(结构为：
Figure PCTCN2019097202-appb-000023
CAS:66018-38-0)。
实验方法：
小鼠BMDM细胞铺于六孔板，所用培养基为1640培养基(购自南模生物公司；10％FBS--56℃灭火30分钟+M-CSF-20ng/ml+1％双抗)，铺板密度为200万细胞每孔，即10 6/ml，细胞铺板3天后换液，5天后进行如下处理：
对照组：为空白对照，不添加诱导剂和药物；
LPS+5z-7孔：加入焦亡诱导剂(LPS(最终作用浓度为10ng/ml)和5z-7(最终作用浓度为125nM))和与药物等同体积的DMSO处理；
Nec1孔：加入焦亡诱导剂(LPS(最终作用浓度为10ng/ml)和5z-7(最终作用浓度为125nM))，同时加入Nec1(最终作用浓度为10μM)处理；
ZJU-37孔：加入焦亡诱导剂(LPS(最终作用浓度为10ng/ml)和5z-7(最终作用浓度为125nM))，同时加入ZJU-37(最终作用浓度为10μM)处理；
处理细胞5小时后，取出细胞测细胞活力并用相应抗体进行免疫印迹杂交。结果分别如图11的A图和B图所示。
实验结果如图11所示：
小鼠BMDM细胞在LPS和5z-7诱导的情况下发生RIP1依赖的焦亡，细胞活力下降，同时P-RIP1和GSDMD水平均有所提高，但在加入药物ZJU-37后能有效抑制细胞活力的下降，降低P-RIP1和GSDMD蛋白水平，从而抑制其激酶活性，以及抑制RIP1依赖的焦亡的发生。而且其抑制效果与阳性对照药物Nec1相似。
实施例12 ZJU-37能缓解小鼠多发性硬化症(MS)
原料：6-8周龄雄性C57小鼠(购自南模生物)；常规饲料(购自杭州赛洛进生物科技有限公司)；0.2％Cuprizone饲料(购自杭州赛洛进生物科技有限公司，为在常规饲料中添加0.2％Cuprizone)；Nec1(同前)。
实验方法：
6-8周龄雄性C57小鼠分两组做MS模型，分别是组一和组二。其中，组一和组二又各分为如下四组：
第一组：空白对照组；
第二组：Cup组(Cuprizone饲料可以诱导小鼠MS的发生)；
第三组：Cup+Nec1组(Cuprizone饲料+Nec1)；
第四组：Cup+ZJU-37组(Cuprizone饲料+ZJU-37)
组一：第一组小鼠从第一天开始喂常规饲料，喂6周后灌流取脑组织；第二组、第三组或第四组小鼠从第一天开始喂0.2％Cuprizone饲料，喂6周后灌流取脑组织。其中，第三组同时从第一天开始腹腔注射药物(Nec1，作用浓度为5mg/kg)；第四组同时从第一天开始腹腔注射药物(ZJU-37，作用浓度为5mg/kg)；第二组从第一天开始腹腔注射溶剂(溶剂配方为：2％DMSO+30％PEG400+68％ddH2O)。
组二：四组小鼠从第一天到第三十天喂0.2％Cuprizone饲料然后从第三十一天到四十五天喂常规饲料，并且从第三十一天开始腹腔注射药物，喂6周后 灌流取脑组织。其中，第三组同时从第三十一天开始注射药物(Nec1，作用浓度为5mg/kg)，第四组同时从第三十一天开始注射药物(ZJU-37，作用浓度为5mg/kg)，第二组从第三十一天开始注射溶剂(溶剂配方为：2％DMSO+30％PEG400+68％ddH2O)。
实验结果如图12所示：
A图：小鼠脑片用4％戊二醛固定后切胼胝体部分电镜观察，电镜结果显示cup组呈现部分脱髓鞘现象，表现为髓鞘松散，g-比值(g-ratio)数值降低(g-ratio为髓鞘内径比髓鞘外径，此数值低表示髓鞘更为松散)，但是腹腔注射两种药物后，髓鞘较少出现脱髓鞘现象，髓鞘更为紧实。
B图：每组小鼠脑片用依来铬氰蓝染色(正常髓鞘显示蓝色，脱髓鞘部位蓝色消失偏于白色)。从图中我们可以看到，cup组呈现部分脱髓鞘现象，但是腹腔注射两种药物后，脱髓鞘现象得到改善，并且ZJU-37的效果优于Nec1。
实施例13 ZJU-37能通过小鼠的血脑屏障
原料：BALB/c Nude小鼠(来源:上海斯莱科公司，级别:SPF级，周龄:6至8周性别：雄性)；Nec1(同前)。
实验方法：
7只BALB/c Nude小鼠，将小鼠分成为两组(药物处理组和溶剂组)：
药物处理组：
小鼠腹腔注射4mg/kg ZJU37；药物处理组分又为两组：
0.5h组：在腹腔注射ZJU37后0.5小时取样进行分析。
1h组：在腹腔注射ZJU37后1小时取样进行分析。
溶剂组：
小鼠进行腹腔注射2％DMSO+30％peg400+水；然后在腹腔注射溶剂后1小时取样进行分析。
在取样进行分析时，首先对小鼠腹腔注射4％水合氯醛水溶液，200μl/20g体重，进行麻醉。然后分别对血浆和脑部药物浓度进行分析。
颈动脉取血300μl至肝素管，混匀后，4000rpm5min离心取上清100μl，加入700μl乙腈，混匀超声20min，12000rpm离心10min，上清4℃静置过夜，12000rpm离心10min，取上清进行HPLC分析。
取全脑约0.35g，加1ml乙腈匀浆(PRIMA PB100，35000rpm，1min)，超声20min，12000rpm离心10min，上清4℃静置过夜，12000rpm离心10min，取上清进行HPLC分析。
实验结果如图13所示。ZJU-37药物在脑中30min达到最大浓度，随后被代谢掉。
实施例14 对RIPK1酶的半数抑制浓度测试
应用HTRF kinEASE STK试剂盒(Cat.62STKOPEC，Cisbio公司)和ADP-Glo Kinase试剂盒(Cat.V6930，Promega公司)检测ZJU-37和Nec-1s对RIPK1激酶的半数抑制浓度(IC50)。
Nec-1s的结构为
Figure PCTCN2019097202-appb-000024
购自Selleck公司；
RIPK1(Medicilon公司)；
ATP 10mM(Cat.PV3227,Invitrogen公司)；
DTT 1M(Cat.D5545,Sigma公司)；
MgCl 2 1M(Cat.M8266,Sigma公司)。
1.试剂配制
RIPK1酶的反应体系各组分及浓度：
表1
Figure PCTCN2019097202-appb-000025
1×酶缓冲液：1mL 1×激酶缓冲液中含有200μL 5×酶缓冲液,5μL 1M MgCl 2,1μL 1M MnCl 2,1μL 1M DTT,793μL ddH2O。
5×STK-biotin底物工作液：STK-biotin底物的具体浓度见表1。用1×激酶缓冲液稀释STK-biotin底物至反应浓度的5倍。
5×ATP工作液：ATP的具体浓度见表1。用1×激酶缓冲液稀释ATP至反应浓度的5倍。
5×酶工作液：酶浓度见表1。用1×激酶缓冲液配制酶的5×酶工作液。
2.实验流程
所有试剂按照上述方法配好后，除酶外，平衡到室温以后，开始进行加样。
首先，使用配置好的1×激酶缓冲液，分别配制2.5％的DMSO溶液(控制DMSO的终浓度为1％)，然后用2.5％的DMSO溶液稀释待测化合物，化合物 的作用终浓度从10μM起，4倍倍比稀释，10个浓度点。除对照孔(包括阳性对照孔和阴性对照孔)外，向所用反应孔中加入2μL稀释好的待测化合物溶液，向对照孔中加入2μL先前配制的2.5％的DMSO溶液。
向所有反应孔中加入1μL配制好的STK-biotin底物溶液(酶筛选时底物的用量见表1)。
向除阴性对照孔外的所有反应孔中加入1μL先前配制好的酶溶液(酶的用量见表1)，阴性对照孔用1×激酶缓冲液补足体积。用封板膜封板，混匀后室温孵育数分钟，让化合物和酶充分作用结合。
向所有反应孔中加入1μL ATP溶液来启动激酶反应，RIPK1酶反应时间为180分钟(对应的ATP浓度和反应时间见表1)。
待激酶反应结束后，向所有反应孔中加入5μL ADP-Glo试剂，混匀后室温反应40min。
向所有反应孔中加入10μL激酶检测试剂，封板混匀，室温反应30min后，使用ENVISION(Perkinelmer)仪器检测荧光信号。
通过反应孔和对照孔计算出每个孔的抑制率，复孔取平均值，用分析软件PRISM 5.0分析待测化合物抑制活性。
抑制率＝(阳性对照孔的荧光信号-加药孔的荧光信号)/(阳性对照孔的荧光信号-阴性对照孔的荧光信号)*100％
实验结果如图14所示。结果发现，ZJU-37对RIPK1酶的IC50为406.3nM，仅为Nec-1s(对RIPK1酶的IC50为1142nM)的36％。
实施例15 化合物(II)能抑制Jurkat-FADD -/-细胞程序性坏死
重复实施例1的方法，不同之处在于用化合物(II)代替化合物ZJU37，处理时间为12小时。结果如图15所示。可见，在TNFα专一诱导程序性坏死的情况下，化合物(II)能有效抑制Jurkat-FADD-/-细胞的程序性坏死。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 式(A)所示化合物或其衍生物；
    Figure PCTCN2019097202-appb-100001
    其中，
    R 1为C 1-6烷基；R 2为氢；R 3为氢、C 1-6烷基、C 6-10芳基或5元至10元杂芳基；所述5元至10元杂芳基中的一个或多个环原子各自独立地为选自氮、硫或氧的杂原子；L为-(C＝O)-、-(S＝O)-或-(SO 2)-。
  2. 如权利要求1所述的化合物或其衍生物，其特征在于，所述化合物选自下组：
    Figure PCTCN2019097202-appb-100002
  3. 如权利要求1所述的化合物或其衍生物，其特征在于，所述化合物为式(I)化合物：
    Figure PCTCN2019097202-appb-100003
  4. 如权利要求1或3所述的化合物或其衍生物的用途，其特征在于，所述化合物或其衍生物用作RIP1抑制剂或用于制备预防或治疗RIP1相关疾病的药物。
  5. 如权利要求1或3所述的化合物或其衍生物的用途，其特征在于，所述 化合物或其衍生物用于制备预防或治疗细胞程序性坏死相关疾病的药物。
  6. 如权利要求1或3所述的化合物或其衍生物的用途，其特征在于，所述化合物或其衍生物用于制备预防或治疗TDP25蛋白相关疾病的药物。
  7. 如权利要求1或3所述的化合物或其衍生物的用途，其特征在于，所述化合物或其衍生物用于制备预防或治疗NLRP3蛋白相关疾病的药物。
  8. 如权利要求1或3所述的化合物或其衍生物的用途，其特征在于，所述化合物或其衍生物用于制备预防或治疗肝损伤的药物。
  9. 如权利要求1或3所述的化合物或其衍生物的用途，其特征在于，所述化合物或其衍生物具有如下一种或多种用途：
    (1)抑制细胞程序性坏死；
    (2)抑制RIP1激酶活性；
    (3)降低RIP1和RIP3的相互作用；
    (4)降解TDP25蛋白；
    (5)抑制细胞RIP1激酶依赖的焦亡；
    (6)降解NLRP3蛋白。
  10. 如权利要求4所述的用途，其特征在于，所述RIP1相关疾病选自下组：神经退行性疾病、缺血性损伤、自身免疫疾病、动脉粥样硬化、银屑病、高雪氏症、疼痛、炎症、视网膜脱落、肿瘤。
  11. 如权利要求5所述的用途，其特征在于，所述细胞程序性坏死相关疾病选自下组：神经退行性疾病、缺血性损伤、自身免疫疾病、动脉粥样硬化、银屑病、高雪氏症、疼痛、炎症、视网膜脱落、肿瘤。
  12. 如权利要求6所述的用途，其特征在于，所述TDP25蛋白相关疾病为神经退行性疾病。
  13. 如权利要求7所述的用途，其特征在于，所述NLRP3蛋白相关疾病选自下组：神经退行性疾病、自身免疫疾病、炎症性肠病、动脉粥样硬化、2型糖尿病、痛风、肥胖、肿瘤。
  14. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1或3所述的化合物或其衍生物和药学上可接受的载体。
  15. 一种预防或治疗疾病的方法，其特征在于，所述方法包括步骤：给需要的受试者施用如权利要求1或3所述的化合物或其衍生物或权利要求14所述的药物组合物；所述疾病为选自下组的一种或多种：RIP1相关疾病、细胞程序性坏死相关疾病、TDP25蛋白相关疾病、NLRP3蛋白相关疾病、肝损伤。
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