WO2020020119A1 - Rip1 inhibitor and use thereof in medicine - Google Patents

Abstract

An RIP1 inhibitor and use thereof in medicine. The RIP1 inhibitor can effectively inhibit RIP1 kinase activity, can also reduce the interaction between RIP1 and RIP3, inhibit programmed cell necrosis, and degrade NLRP3 protein and TDP25 protein, and can be used for preventing or treating a variety of diseases.

Description

RIP1 inhibitor and its use in medicine Technical field

The invention belongs to the field of medicinal chemistry. Specifically, the invention provides a RIP1 inhibitor and its use in medicine.

Background technique

Receptor-interacting protein kinase 1 (RIP1) 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. At the same time, 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. For example, GSK872, GSK843 and other inhibitors that unilaterally inhibit RIP3. 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.

Currently, there are few and few RIP1 inhibitors in the clinical stage. For example, 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.

Summary of the Invention

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;

among them,

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;

L is-(C = O)-,-(S = O)-, or-(SO ₂ )-.

In another preferred example, the C _1-6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and the like.

In another preferred example, the C _1-6 alkyl group is a C _1-3 alkyl group.

In another preferred example, the C _6-10 aryl is phenyl or naphthyl.

In another preferred example, 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.

In another preferred example, the compound is selected from the group consisting of:

In another preferred embodiment, the compound is a compound of formula (I):

In another preferred example, 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.

In another preferred example, 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.

In another preferred example, the compound or a derivative thereof can also be used to reduce the interaction between RIP1 and RIP3.

In another preferred example, 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.

In another preferred example, 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.

In another preferred embodiment, 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.

According to 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:

(1) Inhibit programmed cell necrosis;

(2) Inhibition of RIP1 kinase activity;

(3) reduce the interaction between RIP1 and RIP3;

(4) Degradation of TDP25 protein;

(5) Inhibit cellular RIP1 kinase-dependent coke death;

(6) Degradation of NLRP3 protein.

In another preferred example, the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.

In another preferred example, the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.

In another preferred example, the liver injury is acute liver injury.

In another preferred example, the liver injury is toxin-induced acute liver injury.

In another preferred example, the toxin is LPS.

In another preferred example, 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.

In another preferred example, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and the like.

In another preferred example, the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.

In another preferred example, the pain is neuropathic pain.

In another preferred example, the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.

In another preferred example, the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.

In another preferred example, the multiple sclerosis is induced by cuprizone.

In another preferred example, 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.

In another preferred example, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and the like.

In another preferred example, the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.

In another preferred example, the pain is neuropathic pain.

In another preferred example, the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.

In another preferred example, the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.

In another preferred example, the multiple sclerosis is induced by cuprizone.

In another preferred example, the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.

In another preferred example, the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.

In another preferred example, the TDP25 protein-related disease is a neurodegenerative disease.

In another preferred example, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

In another preferred example, 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.

In another preferred example, the neurodegenerative disease is Alzheimer's disease (AD).

In another preferred example, the autoimmune disease is systemic lupus erythematosus.

In another preferred example, 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.

In another preferred example, the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.

In another preferred example, the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.

In another preferred example, the cell is a cell treated with an inducer; the inducer is one or more selected from the group consisting of: TNFα, z-VAD-fmk, LPS, SMAC, compound B3, 5z -7.

In another preferred example, the compound or its derivative is applied at a concentration of 1-50 μM; preferably, 5-20 μM.

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.

In another preferred example, 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.

In another preferred example, the compound or a derivative thereof can also be used to reduce the interaction between RIP1 and RIP3.

In another preferred example, 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.

In another preferred example, 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.

In another preferred example, 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:

(1) Inhibit programmed cell necrosis;

(2) Inhibition of RIP1 kinase activity;

(3) reduce the interaction between RIP1 and RIP3;

(4) Degradation of TDP25 protein;

(5) Inhibit cellular RIP1 kinase-dependent coke death;

(6) Degradation of NLRP3 protein.

In another preferred example, the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.

In another preferred example, the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.

In another preferred example, the liver injury is acute liver injury.

In another preferred example, the liver injury is toxin-induced acute liver injury.

In another preferred example, the toxin is LPS.

In another preferred example, 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.

In another preferred example, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and the like.

In another preferred example, the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.

In another preferred example, the pain is neuropathic pain.

In another preferred example, the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.

In another preferred example, the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.

In another preferred example, the multiple sclerosis is induced by cuprizone.

In another preferred example, 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.

In another preferred example, the neurodegenerative disease is ALS, AD, PD, MS, and the like.

In another preferred example, the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, and the like.

In another preferred example, the pain is neuropathic pain.

In another preferred example, the inflammation is pancreatitis, ulcerative enteritis, hepatitis, and the like.

In another preferred example, the tumor is melanoma, glioma, colon cancer, glioma, lymphoma, T-cell leukemia, and the like.

In another preferred example, the TDP25 protein-related disease is a neurodegenerative disease.

In another preferred example, the neurodegenerative disease is ALS.

In another preferred example, 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.

In another preferred example, the neurodegenerative disease is Alzheimer's disease (AD).

In another preferred example, the autoimmune disease is systemic lupus erythematosus.

In another preferred example, 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;

In another preferred example, the cells are selected from the group consisting of leukemia T cells, lymphoma cells, microglia, and colon cancer cells.

In another preferred example, the cells are selected from the group consisting of Jurkat-FADD-/-cells, U937 cells, BV2 cells, and HT-29 cells.

In another preferred example, the cell is a cell treated with an inducer; the inducer is one or more selected from the group consisting of: TNFα, z-VAD-fmk, LPS, SMAC, compound B3, 5z -7.

In another preferred example, the compound of formula (I) or a derivative thereof is applied at a concentration of 1-50 μM; preferably, 5-20 μM.

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (such as the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that ZJU-37 inhibits programmed necrosis of Jurkat-FADD-/-cells.

Figure 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.

detailed description

Through in-depth research, the inventors have unexpectedly discovered that 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.

the term

As used herein, the term "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.

As used herein, the term "C _6-10 aryl" is an aryl group having 6 to 10 carbon atoms, such as phenyl or naphthyl.

As used herein, the term "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. Preferred 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.

NLRP3 protein

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

The 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.

Active ingredient

As used herein, the compound of the present invention is a compound of formula (A), and its structure is as follows:

In the formula, the definition of each group is as described above.

Preferably, the compound according to the present invention is a compound of formula (I), and its structure is as follows:

Herein, the compound "ZJU-37", the compound "ZJU37", and the "compound of formula (I)" may be used interchangeably.

As used herein, the active ingredient according to the present invention is a compound of formula (A) or a derivative thereof.

Preferably, 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.

As used herein, "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. In addition, if the compound described herein is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid addition salt. In contrast, 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. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. 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.

As used herein, "hydrate" refers to a complex formed by combining a compound of the invention and water.

As used herein, "solvate" refers to an association or complex of one or more solvent molecules and a compound of the invention. Examples of solvate-forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and ethanolamine.

As used herein, 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.

As used herein, "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. Mirror. "Diastereomers" are stereoisomers with at least two asymmetric atoms, but they are not mirror images of one another.

As used herein, "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. Higuchi and V. Stella, "Pro-drugs, Novel, Delivery Systems," ACS Academic Symposium Series Vol. 14; "Design of Prodrugs", edited by H. Bundgaard, Elsevier, 1985; And Bioreversible Carriers, Drug Design, edited Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, the references are hereby incorporated by reference in their entirety.

Preparation

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).

In the formula, 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. For example, DMF, DMSO, THF, etc. This solvent is generally not involved in the reaction. Of course, 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. Examples of 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).

Drugs and methods of administration

Studies have found that 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. Accordingly, 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. These 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. Generally, the pharmaceutical composition contains 1-2000 mg of active ingredient / dose, and more preferably, 5-200 mg of active ingredient / dose. Preferably, 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. Examples of 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.

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. In these solid dosage forms, 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 glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, hard Calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or a mixture thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

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. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers. For example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof.

In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.

In addition to the active ingredient, 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.

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.

When the pharmaceutical composition is used, 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 For humans, the daily dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, 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.

As used herein, "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).

As used herein, "prevention" means any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop. In certain embodiments, 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.

As used herein, 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. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

The main advantages of the invention are:

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.

Specifically, the compound of the present invention or a derivative thereof can also treat liver damage.

That is, 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.

The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions or conditions recommended by the manufacturer. Unless stated otherwise, percentages and parts are percentages by weight and parts by weight.

In the following examples, 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. Among them, cell viability was determined by ATP-based viability measurement.

In the following examples, 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%).

The experimental materials (such as various cells) and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

In the following examples, 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 TNFα. Jurkat FADD ^-/- cells in this experiment can be obtained by CRISPR technology.

In the following examples, U937 cells are human histiocyte lymphoma cells; purchased from Nanjing Kebai Cell Bank.

In the following examples, BV2 cells are mouse microglia; purchased from Nanjing Kebai Cell Bank. Since BV2 cells secrete TNFα by themselves under conditions induced by z-VAD-fmk, no additional TNFα is required for the induction conditions.

In the following examples, 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.

In the following examples, H4-TDP25 cells are H4 cells stably expressing TDP25; purchased from Nanjing Kebai Cell Bank.

In the following examples, HT-29 cells are human colon cancer cells; purchased from Nanjing Kebai Cell Bank.

In the following examples, BMDM cells are bone marrow-derived macrophages; purchased from Nanjing Kebai Cell Bank.

Preparation example 1 Preparation method of compound JIU-37

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. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and column chromatography to obtain ZJU-37 (38.7 mg, 60%) as a white solid . ¹ H 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 = 9.2 Hz, 1H), 4.74 (dd, J = 5.5, 2.2 Hz, 1H), 3.75 (dd, J = 15.0, 5.6 Hz, 1H), 3.52 (dd, J = 15.0, 2.1 Hz, 1H), 2.80 (s, 3H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ ¹³ C NMR (100 MHz, CDCl ₃ ) δ 171.3, 158.4, 155.0 (d, J = 242.1 Hz), 154.3, 134.0 (d , J = 4.2 Hz), 124.7 (d, J = 3.4 Hz), 124.4, 117.8 (d, J = 9.1 Hz), 109.6 (d, J = 23.7 Hz), 108.5, 103.5 (d, J = 22.3 Hz) , 57.9, 24.8, 24.3.

Preparation Example 2 Preparation Method of Compound (II)

Under nitrogen protection and ice bath conditions, Compound 2 (0.2 mmol, 59 mg) was dissolved in THF (1 mL), NaH (0.3 mmol, 7.2 mg) was added, and stirring was continued in the ice bath for 0.5 hours. Then, a solution of 2-pyridinesulfonyl chloride (0.3 mmol, 53 mg) in THF was added to the reaction solution, and the temperature was raised to room temperature for overnight reaction. An appropriate amount of water was added to quench the reaction, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain a white solid compound (II) (62 mg, 71%). . ¹ H NMR (400MHz, DMSO) δ 11.57 (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, 4.9 Hz, 1H), 7.48 (dd, J = 8.7, 4.8 Hz, 1H), 7.12 (d, J = 1.5 Hz, 1H), 7.10--7.04 (m, 1H ), 5.21--5.15 (m, 1H), 3.57--3.51 (m, 2H), 2.54 (s, 3H).

According to the experimental method of Preparation Example 2, the following compounds were prepared using the corresponding raw materials:

HRMS (ESI) calcd for C ₂₀ H ₁₅ ClFN ₃ O ₃ : 400.0866 (M + H ⁺ ), found: 400.0871.

HRMS (ESI) calcd for C ₁₉ H ₁₄ ClFN ₄ O ₃ : 401.0818 (M + H ⁺ ), found: 401.0826.

HRMS (ESI) calcd for C ₁₉ H ₁₅ ClFN ₃ O ₄ S: 436.0536 (M + H ⁺ ), found: 436.0540.

Example 1 ZJU-37 can inhibit Jurkat-FADD ^-/- programmed cell necrosis

Raw materials: TNFα (purchased from Sigma, USA); Nec1 (structure is

(Selleck; purchased as a positive control).

experimental method:

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 μM, 10 -6 μM,} 10 -5 μM, 10 -4 μM, 10 -3 μM , ^10-2 μM, 10 ^-1 μM, 1 μM, 10 μM) together;

ZJU-37 well: use TNFα (final action concentration is 30ng / ml) and drugs with different action concentrations ZJU-37 (final action concentration is 10 ^-7 μM, 10 ^-6 μM, 10 ^-5 μM, 10 ^-4 μM, 10 ^-3 μM, 10 ^-2 μM, 10 ^-1 μM, 1 μM, 10 μM) together;

TNFα well: treated with TNFα (30ng / ml) and DMSO with the same volume as the drug;

Control well: blank control without adding inducer and drug;

After 18 hours of treatment, the cell viability of each well was tested and the cell viability (%) after drug treatment was calculated.

The experimental results are shown in Figure 1. Among them, Figure A shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of Jurkat-FADD-/-cells at a concentration of 10 μM, 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.

It can be seen that in the case that TNFα specifically induces programmed necrosis, 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

Raw materials: TNFα (ibid.); Z-VAD-fmk (purchased from BioVision); Nec1 (ibid.).

experimental method:

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 (TNFα (final action concentration: 30ng / ml) and z-VAD-fmk (final action concentration: 40μM)) and Nec1 (final action concentration: 0.0001μM, 0.0005μM, 0.001μM, 0.005μM, 0.01μM, 0.1μM, 0.5μM, 5μM, 10μM) together;

ZJU-37 wells: TZ (TNFα (final action concentration: 30ng / ml) and z-VAD-fmk (final action concentration: 40μM)) and ZJU-37 (final action concentration: 0.0001μM, 0.0005μM) , 0.001 μM, 0.005 μM, 0.01 μM, 0.1 μM, 0.5 μM, 5 μM, 10 μM) together;

TZ well: treated with TZ (TNFα (final action concentration: 30ng / ml) and z-VAD-fmk (final action concentration: 40μM)) and DMSO with the same volume as the drug

Control well: blank control without adding inducer and drug;

After 72 hours of treatment, the cell viability of each well was tested and the cell viability (%) after drug treatment was calculated.

The experimental results are shown in Figure 2. Among them, A shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of U937 cells at a concentration of 10 μM, 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.

It can be seen that in the case that TNFα and z-VAD-fmk specifically induce programmed necrosis, 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

Raw materials: z-VAD-fmk (ibid.); Nec1 (ibid.).

experimental method:

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 μM) and Nec1 (final action concentration of 0 μM, 0.0001 μM, 0.0005 μM, 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 1 μM, 5 μM) , 10 μM) processing together;

ZJU-37 well: use z-VAD-fmk (final action concentration of 70 μM) and ZJU-37 (final action concentration of 0 μM, 0.0001 μM, 0.0005 μM, 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM , 1μM, 5μM, 10μM) together;

Z-VAD well: treated with z-VAD-fmk (final effect concentration: 70 μM) and DMSO with the same volume as the drug;

Control well: blank control without adding inducer and drug;

After 72 hours of treatment, the cell viability of each well was tested and the cell viability (%) after drug treatment was calculated.

The experimental results are shown in Figure 3. Among them, Figure A shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of BV2 cells at a concentration of 10 μM 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.

It can be seen that in the case that z-VAD-fmk specifically induces programmed necrosis, 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

Raw materials: Flag-RIP1 (purchased from Vitrus Bio); LPS (purchased from Sigma); Nec1 (ibid.).

experimental method:

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 wells: transfect 1 μg Flag-RIP1;

Flag + LPS wells: Transfect 1 μg 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 μg Flag-RIP1, and treat with LPS (final action concentration of 100 ng / ml) and Nec1 (final action concentration of 10 μM) 24 hours after transfection;

ZJU37-5 wells: transfect 1 μg Flag-RIP1, and treat with LPS (final action concentration: 100 ng / ml) and ZJU-37 (final action concentration: 5 μM) 24 hours after transfection;

ZJU37-10 wells: transfect 1 μg Flag-RIP1, and treat with LPS (final action concentration of 100 ng / ml) and ZJU-37 (final action concentration of 10 μM) after transfection for 24 hours

ZJU37-20 wells: transfect 1 μg Flag-RIP1, and treat with LPS (final action concentration: 100ng / ml) and ZJU-37 (final action concentration: 20 μM) 24 hours after transfection;

Control well: blank control without adding inducer and drug.

After 6 hours of treatment, cells were collected separately and immunoblotted with the corresponding antibodies. The results are shown in Figures A and C.

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:

TNFα well: treated with TNFα (final effect concentration 30ng / ml) and DMSO with the same volume as the drug;

Nec1 wells: treated with TNFα (final action concentration 30ng / ml) and Nec1 (final action concentration 10μM);

ZJU-37 wells: treated with TNFα (final action concentration 30ng / ml) and ZJU-37 (final action concentration 10μM);

Control well: blank control without adding inducer and drug.

After 4 hours of treatment, cells were collected separately and immunoblot hybridized with the corresponding antibodies. The results are shown in Figure 4B.

It can be seen that in the case of LPS or TNFα alone induction, the level of P-RIP1 has increased, especially in Jurkat FADD-/-cells. After adding ZJU-37 treatment, the level of P-RIP1 was effectively reduced, thereby inhibiting its kinase activity. And the inhibitory effect of ZJU-37 on P-RIP1 was better than that of the positive control drug Nec1. Figure 4C shows that 10 μM ZJU-37 is more effective in reducing P-RIP1 levels.

Example 5 ZJU-37 can effectively inhibit RIP1 kinase activity

Raw materials: pCMV-Flag (no load, purchased from Vitrus Bio); Flag-beads (purchased from Bimake); Flag-RIP1 (ibid.); Phosphatase inhibitor (purchased from Shenggong Biotechnology Co., Ltd.); Same as above); Kinase Buffer (20 mM-HEPES, pH = 7.5, ₂ mM DTT, 10 mM MnCl ₂ ).

experimental method:

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 μg 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.

At the same time of transfection, DMSO was added to System 2 and System 3; 50 μM of Nec1 was added to System 4; and 50 μM of ZJU-37 was added to System 5.

The specific systems of the kinase experiment are shown below:

Zh		System 1	System 2	System 3	System 4	System 5
ddH2O	+	+	+	+	+
Kinase buffer	+	+	+	+	+
ATP	+	-	+	+	+
Phosphatase inhibitor	+	+	+	+	+
DMSO	Zh	+	+	Zh	Zh
Nec1	Zh	Zh	Zh	+	Zh
ZJU37	Zh	Zh	Zh	Zh	+

After the cells were treated for 20 hours, the supernatant was discarded in each dish, washed twice with pre-chilled PBS, and then 1 ml of RIPA lysate was added to each dish, which was incubated with Flag-beads, placed on a shaker at 4 ° C for 30 minutes, and lysed at 4 ° C. Centrifuge at 12000 rpm for 10 min, incubate the supernatant and Flag-beads at 4 ° C, centrifuge after 4 h, and centrifuge at 2 rpm at 4 ° C for 2 min to leave Flag-beads and discard the supernatant. Repeatedly wash the Flag-beads three times with RIPA lysate to perform kinase experiments.

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.

The experimental results are shown in Figure 5. In the case of ATP, the level of P-RIP1 in sample 3 was increased compared to sample 2, but after adding the drug ZJU-37, it could effectively inhibit the content of P-RIP1. Thus inhibiting its kinase activity, its inhibitory effect is similar to that of the positive control drug Nec1.

Example 6 ZJU-37 can reduce the interaction of RIP1 and RIP3 in cells

Raw materials: TNFα (ibid.); Z-VAD-fmk (ibid.); SMAC (purchased from Selleck); Nec1 (ibid.); EGFP-RIP1 (purchased from Weizhen Bio); Flag-RIP3 (purchased from Weiwei) True creature);

experimental method:

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 (TNFα (final action concentration: 30ng / ml), z-VAD-fmk (final action concentration: 20μM) and SMAC (final action concentration: 100nM)) and DMSO with the same volume as the drug;

Nec1 wells: treated with TZS (TNFα (final action concentration: 30 ng / ml), z-VAD-fmk (final action concentration: 20 μM) and SMAC (final action concentration: 100 nM)) and Nec1 (final action concentration: 10 μM);

ZJU-37 wells: TZS (TNFα (final action concentration 30ng / ml), z-VAD-fmk (final action concentration 20μM) and SMAC (final action concentration 100nM)) and ZJU-37 (final action concentration is 10 μM) processing;

Control well: blank control without adding inducer and drug.

After 72 hours of treatment, wash with PBS twice, add RIPA lysate, and incubate with beads. After 1ml lysis at 4 ° C for 30min, collect protein, add RIP1 antibody to incubate, centrifuge beads to collect protein, and perform immunoblot hybridization with the corresponding antibody. The results are shown in Figure A in FIG. 6.

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 μg each of EGFP-RIP1 and Flag-RIP3, and add and treat with the same volume of DMSO as the drug;

Nec1 wells: transfect 2 μg each of EGFP-RIP1 and Flag-RIP3, and add Nec1 (final action concentration is 10 μM) at the same time;

ZJU-37 well: transfection with 2μg each of EGFP-RIP1 and Flag-RIP3, and add ZJU-37 (final action concentration: 10μM) at the same time;

Control well: blank control without adding inducer and drug.

After 24 hours of treatment, wash with PBS twice, add RIPA lysate, and incubate with beads. After 1ml lysing at 4 ° C for 30min, collect protein, add EGFP antibody to incubate, centrifuge beads to collect protein, and perform immunoblot hybridization with the corresponding antibody. This is shown in B in FIG. 6.

When the RIP1 content is equivalent, the lower the RIP3 content, the smaller the interaction between the two. The weakened interaction means that programmed cell necrosis can be weakened to a certain extent. The experimental results are shown in Figure 6:

Panel A: In HT-29 cells, both Nec1 and ZJU-37 can reduce the interaction between RIP1 and RIP3, and the effect of ZJU-37 is better than Nec1.

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.).

experimental method:

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 μM) 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 μM) for 6 hours, and then the solution was changed, and then treated with Nec1 (final effect concentration 10 μM);

ZJU37-1 well: cells were treated with compound B3 (final action concentration: 10 μM) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration: 1 μM);

ZJU37-5 wells: cells were treated with compound B3 (final action concentration 10 μM) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration 5 μM);

ZJU37-10 wells: cells were treated with compound B3 (final action concentration 10 μM) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration 10 μM);

ZJU37-20 wells: cells were treated with compound B3 (final action concentration 10 μM) for 6 hours, and then the solution was changed, and then treated with ZJU-37 (final action concentration 20 μM);

Control well: blank control without adding inducer and drug.

After 6 hours of treatment, cells were collected separately and immunoblotted with the corresponding antibodies. The experimental results are shown in Figure 7.

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.).

experimental method:

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);

After 6 hours of treatment, the 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.

The experimental results are shown in Figure 8. ZJU-37 can effectively attenuate the interaction of RIP1 / RIP3 proteins in mouse brain tissue. From the IP bands, we can see that the interaction between RIP3 and RIP1 is enhanced after the action of LPS, which can be seen from the protein level content of the RIP3 band, and the level of RIP3 after ZJU37 is reduced, which proves that RIP3 and RIP1 The interaction is weakened. And Nec1 cannot weaken the interaction between RIP3 and RIP1.

Example 9 ZJU-37 can effectively inhibit LPS-induced acute liver injury in mice

Raw materials: C57BL / 6 male mice (ibid.); LPS (ibid.); Nec1 (ibid.).

experimental method:

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);

After 6 hours of treatment, the mice were dissected and livers were taken for morphological observation of HE sections staining. The main purpose of 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.

The experimental results are shown in Figure 9. ZJU-37 can effectively inhibit LPS-induced acute liver injury in mice. Compared with the blank cell control group, it was found that the cell morphology was significantly worsened with the addition of LPS, and there was obvious inflammatory infiltration. After treatment with ZJU-37, the cell morphology was significantly improved, and the inflammatory infiltration was relieved.

Example 10 ZJU-37 can effectively degrade NLRP3 protein induced by inflammation

Raw materials: TNFα (ibid.); Z-VAD-fmk (ibid.); SMAC (ibid.); Nec1 (ibid.).

experimental method:

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:

Control well: blank control without adding inducer and drug;

TZS well: treated with TZS (TNFα (final action concentration: 30ng / ml), z-VAD-fmk (final action concentration: 20μM) and SMAC (final action concentration: 100nM)) and DMSO with the same volume as the drug;

Nec1 wells: treated with TZS (TNFα (final action concentration: 30 ng / ml), z-VAD-fmk (final action concentration: 20 μM) and SMAC (final action concentration: 100 nM)) and Nec1 (final action concentration: 10 μM);

ZJU-37 wells: TZS (TNFα (final action concentration 30ng / ml), z-VAD-fmk (final action concentration 20μM) and SMAC (final action concentration 100nM)) and ZJU-37 (final action concentration is 10 μM) processing;

After 72 hours of treatment, cells were collected separately and immunoblot hybridized with the corresponding antibodies. The results are shown in Fig. 10A.

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:

Control well: blank control without adding inducer and drug;

TNFα well: treated with TNFα (final effect concentration 30ng / ml) and DMSO with the same volume as the drug;

Nec1 wells: treated with TNFα (final effect concentration 30ng / ml) and Nec1 (final effect concentration 10μM);

ZJU-37 well: treated with TNFα (final action concentration 30ng / ml) and ZJU-37 (final action concentration 10μM);

After 4 hours of treatment, cells were collected separately and immunoblot hybridized with the corresponding antibodies. The results are shown in Figure B.

The experimental results are shown in Figure 10:

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 TNFα, 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

Raw material: 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).

experimental method:

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: 10μM);

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: 10μM);

After the cells were treated for 5 hours, the cells were removed to measure cell viability and immunoblot hybridized with the corresponding antibodies. The results are shown in Figures A and B, respectively.

The experimental results are shown in Figure 11:

Mouse BMDM cells induced RIP1-dependent scorch death under the induction of LPS and 5z-7, and the cell viability decreased. At the same time, the levels of P-RIP1 and GSDMD increased, but it was able to effectively inhibit cell viability after adding the drug ZJU-37. Decreased P-RIP1 and GSDMD protein levels, thereby inhibiting its kinase activity and inhibiting the occurrence of RIP1-dependent scorch. And its inhibitory effect is similar to the positive control drug Nec1.

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).

experimental method:

6-8 week old male C57 mice were divided into two groups to make the MS model, namely group one and group two. Among them, 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. Among them, 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; In the second group, 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).

The experimental results are shown in Figure 12:

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

Raw materials: BALB / c Nude mice (source: Shanghai Slyco Corporation, grade: SPF, age: 6 to 8 weeks, gender: male); Nec1 (ibid.).

experimental method:

Seven BALB / c Nude mice were divided into two groups (drug treatment group and solvent group):

Drug treatment group:

The 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.

Solvent group:

Mice were injected intraperitoneally with 2% DMSO + 30% peg400 + water; then samples were taken for analysis one hour after intraperitoneal injection of the solvent.

When sampling for analysis, mice were first injected intraperitoneally with a 4% aqueous solution of chloral hydrate, 200 μl / 20 g of body weight, and anesthetized. The plasma and brain drug concentrations were then analyzed separately.

Carotid blood was taken 300 μl into heparin tube. After mixing, 100 μl of the supernatant was centrifuged at 4000 rpm for 5 min, and 700 μl of acetonitrile was added. analysis.

Take about 0.35g of whole brain, add 1ml of acetonitrile homogenate (PRIMA PB100, 35000rpm, 1min), sonicate for 20min, centrifuge at 12000rpm for 10min, leave the supernatant at 4 ° C overnight, centrifuge at 12000rpm for 10min, and take the supernatant for HPLC analysis.

The experimental results are shown in Figure 13. ZJU-37 drug reached its maximum concentration in the brain for 30 minutes and was subsequently metabolized.

Example 14 Test of half-inhibition concentration on RIPK1 enzyme

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.

The structure of Nec-1s is

Purchased from Selleck;

RIPK1 (Medicilon);

ATP 10mM (Cat.PV3227, Invitrogen);

DTT 1M (Cat.D5545, Sigma);

MgCl ₂ 1M (Cat. M8266, Sigma).

Reagent preparation

Components and concentrations of the reaction system of RIPK1 enzyme:

Table 1

1 × enzyme buffer: 1 mL of 1 × kinase buffer contains 200 μL 5 × enzyme buffer, 5 μL 1M MgCl ₂ , 1 μL 1M MnCl ₂ , 1 μL 1M DTT, 793 μL ddH2O.

5 × 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.

2. Experimental process

After all the reagents were prepared according to the above method, except for the enzyme, equilibrate to room temperature, and then start to add samples.

First, use the configured 1 × kinase buffer to prepare 2.5% DMSO solution (control the final DMSO concentration to 1%), and then dilute the test compound with 2.5% DMSO solution. The final concentration of the compound from 10 μM , 4 times the dilution ratio, 10 concentration points. In addition to the control wells (including the positive control well and the negative control well), 2 μL of the diluted test compound solution was added to the reaction wells, and 2 μL of the 2.5% DMSO solution previously prepared was added to the control wells.

Add 1 μL of the prepared STK-biotin substrate solution to all reaction wells (see Table 1 for the amount of substrate during enzyme screening).

Add 1 μL of the previously prepared enzyme solution to all reaction wells except the negative control wells (see Table 1 for the amount of enzyme used). The negative control wells were made up to volume with 1 × kinase buffer. Seal the plate with a sealing plate and incubate for several minutes at room temperature after mixing to allow the compound and enzyme to fully combine.

Add 1 μL of ATP solution to all reaction wells to start the kinase reaction. The RIPK1 enzyme reaction time is 180 minutes (see Table 1 for the corresponding ATP concentration and reaction time).

After the kinase reaction is completed, 5 μL of ADP-Glo reagent is added to all reaction wells, and the mixture is reacted at room temperature for 40 minutes.

Add 10 μL of kinase detection reagent to all reaction wells, seal the plate and mix well. After 30 minutes of reaction at room temperature, use an ENVISION (Perkinelmer) instrument to detect the fluorescence signal.

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%

The experimental results are shown in Figure 14. It was found that the IC50 of ZJU-37 for RIPK1 enzyme was 406.3 nM, which was only 36% of Nec-1s (IC50 for RIPK1 enzyme was 1142 nM).

Example 15 Compound (II) inhibits Jurkat-FADD ^-/- programmed cell necrosis

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 TNFα specifically induces programmed necrosis, compound (II) can effectively inhibit programmed necrosis of Jurkat-FADD-/-cells.

All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (15)

1. A compound represented by formula (A) or a derivative thereof;

   

   among them,

   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; said 5- to 10-membered One or more ring atoms in the heteroaryl group are each independently a heteroatom selected from nitrogen, sulfur, or oxygen; L is-(C = O)-,-(S = O)-, or-(SO ₂ )- .
2. The compound or derivative thereof according to claim 1, wherein the compound is selected from the group consisting of:

   
3. The compound or derivative thereof according to claim 1, wherein the compound is a compound of formula (I):

   
4. The use of a compound or a derivative thereof according to claim 1 or 3, wherein 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.
5. The use of a compound or a derivative thereof according to claim 1 or 3, wherein the compound or a derivative thereof is used to prepare a medicament for preventing or treating a disease related to programmed cell necrosis.
6. The use of a compound or a derivative thereof according to claim 1 or 3, wherein the compound or a derivative thereof is used for preparing a medicament for preventing or treating a TDP25 protein-related disease.
7. The use of a compound or a derivative thereof according to claim 1 or 3, wherein the compound or a derivative thereof is used for preparing a medicament for preventing or treating a disease related to the NLRP3 protein.
8. The use of a compound or a derivative thereof according to claim 1 or 3, wherein the compound or a derivative thereof is used for preparing a medicament for preventing or treating liver injury.
9. The use of the compound or its derivative according to claim 1 or 3, wherein the compound or its derivative has one or more of the following uses:

   (1) Inhibit programmed cell necrosis;

   (2) Inhibition of RIP1 kinase activity;

   (3) reduce the interaction between RIP1 and RIP3;

   (4) Degradation of TDP25 protein;

   (5) Inhibit cellular RIP1 kinase-dependent coke death;

   (6) Degradation of NLRP3 protein.
10. The use according to claim 4, wherein the RIP1-related diseases are selected from the group consisting of: neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher's disease , Pain, inflammation, retinal detachment, tumor.
11. The use according to claim 5, wherein the programmed cell necrosis-related disease is selected from the group consisting of: neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, high Snow's disease, pain, inflammation, retinal detachment, tumor.
12. The use according to claim 6, wherein the TDP25 protein-related disease is a neurodegenerative disease.
13. The use according to claim 7, wherein the NLRP3 protein-related diseases are selected from the group consisting of neurodegenerative diseases, autoimmune diseases, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity , Tumor.
14. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound or a derivative thereof according to claim 1 or 3 and a pharmaceutically acceptable carrier.
15. A method for preventing or treating a disease, characterized in that the method comprises the step of administering a compound according to claim 1 or 3 or a derivative thereof or a pharmaceutical composition according to claim 14 to a subject in need. 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.

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