CN112351979B - RIP1 inhibitors and their use in medicine - Google Patents

Abstract

A RIP1 inhibitor and its use in medicine, which can effectively inhibit RIP1 kinase activity, can also reduce the interaction between RIP1 and RIP3, inhibit programmed cell necrosis, degrade NLRP3 protein and TDP25 protein, and can be used to prevent or treat multiple a disease.

Description

RIP1 inhibitors and their use in medicine

technical field

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

Background technique

Receptor-interacting protein kinase 1 (RIP1) regulates inflammation and cell death. RIP1 kinase inhibitors can effectively inhibit cell death, including programmed necrosis and RIP1-dependent apoptosis. At the same time, RIP1 kinase inhibitors can also inhibit a variety of inflammatory responses. The mechanism is still unclear. It may play a role by regulating receptor-interacting protein kinase 3 (RIP3), or it may prevent the amplification of inflammatory signals by inhibiting cell death. . 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 the drug from entering the clinic. For example, GSK872, GSK843 and other inhibitors that inhibit RIP3 singly. RIP3 kinase inhibitor induces apoptosis probably because it only inhibits RIP3 kinase activity but not RIP1 activity, which leads to the pattern of cell death changing to RIP1-dependent apoptosis.

There are few RIP1 inhibitors currently in clinical stage, such as Denali's RIP1 inhibitor (Nec1), which has entered clinical phase 2 and is indicated for neurodegenerative diseases; and GSK's RIP1 inhibitor (GSK963) , has been in clinical phase 2, indications are autoimmune diseases, including rheumatoid arthritis, ulcerative colitis, psoriasis, etc. Therefore, the development of efficient RIP1 inhibitors is of great significance in the art, especially in inhibiting inflammation and cell death.

SUMMARY OF THE INVENTION

The purpose 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;

in,

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 groups (eg 1, 2 3 or 4) ring atoms are each independently a heteroatom selected from nitrogen, sulphur 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 embodiment, the C _1-6 alkyl group is a C _1-3 alkyl group.

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

In another preferred embodiment, the 5- to 10-membered heteroaryl group is a 5- to 6-membered heteroaryl group or a 6- to 10-membered heteroaryl group; for example, furan, thiophene, pyrrole, thiazole, imidazole, pyridine, Pyrazine, pyrimidine, pyridazine, indole, quinoline.

In another preferred embodiment, the compound is selected from the following group:

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

In another preferred embodiment, the derivative is a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, stereoisomer or mixture of stereoisomers.

The second aspect of the present invention provides the use of the compound or its derivative according to the first aspect, said compound or its derivative being used as a RIP1 inhibitor or for preparing a medicament for preventing or treating RIP1-related diseases.

In another preferred example, the compound or its derivative can also be used as a necroptosis inhibitor or a medicament for preventing or treating necroptosis-related diseases.

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

In another preferred example, the compound or its derivatives can also be used to degrade TDP25 protein or to prepare a drug for preventing or treating TDP25 protein-related diseases.

In another preferred example, the compound or its derivatives can also be used to degrade NLRP3 protein or to prepare a drug for preventing or treating NLRP3 protein-related diseases.

In another preferred example, the compound or its derivative can also be used to prepare a medicament for preventing or treating liver injury.

The third aspect of the present invention provides the use of the compound or its derivative in the first aspect, wherein the compound or its derivative is used to prepare 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 or its derivative in the first aspect, wherein the compound or its derivative is used to prepare a medicament for preventing or treating TDP25 protein-related diseases.

The fifth aspect of the present invention provides the use of the compound or its derivative according to the first aspect, wherein the compound or its derivative is used to prepare a medicament for preventing or treating NLRP3 protein-related diseases.

The sixth aspect of the present invention provides the use of the compound or its derivative according to the first aspect, for preparing a medicament for preventing or treating liver damage.

The seventh aspect of the present invention provides the use of the compound or its derivative according to the first aspect, and the compound or its derivative has one or more of the following uses:

(1) Inhibit programmed cell necrosis;

(2) Inhibit RIP1 kinase activity;

(3) Reduce the interaction between RIP1 and RIP3;

(4) Degrade TDP25 protein;

(5) Inhibition of cellular RIP1 kinase-dependent pyroptosis;

(6) Degrade NLRP3 protein.

In another preferred embodiment, the cells are selected from the group consisting of leukemia T cells, lymphoid cancer cells, microglia cells, 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 embodiment, the liver injury is toxin-induced acute liver injury.

In another preferred embodiment, the toxin is LPS.

In another preferred embodiment, the RIP1-related disease is selected from the group consisting of neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher's 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 nerve pain.

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

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

In another preferred embodiment, the multiple sclerosis is induced by dicyclohexanone oxalyl dihydrazone (Cuprizone).

In another preferred embodiment, the disease associated with programmed cell necrosis is selected from the group consisting of neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher's 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 nerve pain.

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

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

In another preferred embodiment, the multiple sclerosis is induced by dicyclohexanone oxalyl dihydrazone (Cuprizone).

In another preferred embodiment, the cells are selected from the group consisting of leukemia T cells, lymphoid cancer cells, microglia cells, 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 embodiment, the TDP25 protein-related disease is a neurodegenerative disease.

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

In another preferred embodiment, the NLRP3 protein-related disease is selected from the group consisting of neurodegenerative diseases, autoimmune diseases, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and tumors.

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

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

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

The eighth aspect of the present invention provides a pharmaceutical composition comprising the compound or its derivative of the first aspect 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 steps of: administering the compound or its derivative of the first aspect or the pharmaceutical composition of the eighth aspect to a subject in need; The disease is one or more selected from the group consisting of RIP1-related diseases, programmed cell necrosis-related diseases, TDP25 protein-related diseases, NLRP3 protein-related diseases, and liver damage.

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 derivative thereof of the first aspect or the pharmaceutical composition of the eighth aspect.

In another preferred embodiment, the cells are selected from the group consisting of leukemia T cells, lymphoid cancer cells, microglia cells, 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 cells are cells 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 embodiment, the administration concentration of the compound or its derivatives is 1-50 μM; preferably, it is 5-20 μM.

The present invention further provides the use of a compound of formula (I) or a derivative thereof,

The compounds or derivatives thereof are used as RIP1 inhibitors or for the preparation of medicaments for preventing or treating RIP1-related diseases.

In another preferred example, the compound or its derivative can also be used as an inhibitor of programmed necrosis or used to prepare a medicament for preventing or treating diseases related to programmed necrosis.

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

In another preferred example, the compound or its derivatives can also be used to degrade TDP25 protein or to prepare a drug for preventing or treating TDP25 protein-related diseases.

In another preferred example, the compound or its derivatives can also be used to degrade NLRP3 protein or to prepare a drug for preventing or treating NLRP3 protein-related diseases.

In another preferred example, the compound or its derivative can also be used to prepare a medicament for preventing or treating liver injury.

The present invention further provides the use of a compound of formula (I) or a derivative thereof,

The compounds or derivatives thereof are used for the preparation of medicaments for preventing or treating diseases related to programmed cell necrosis.

The present invention further provides the use of a compound of formula (I) or a derivative thereof,

The compounds or derivatives thereof are used for preparing medicines for preventing or treating TDP25 protein-related diseases.

The present invention further provides the use of a compound of formula (I) or a derivative thereof,

The compounds or derivatives thereof are used for preparing medicines for preventing or treating NLRP3 protein-related diseases.

The present invention further provides the use of a compound of formula (I) or a derivative thereof,

The compound or its derivative is used for preparing a medicament for preventing or treating liver injury.

The present invention further provides the use of a compound of formula (I) or a derivative thereof,

The compounds or derivatives thereof have one or more of the following uses:

(1) Inhibit programmed cell necrosis;

(2) Inhibit RIP1 kinase activity;

(3) Reduce the interaction between RIP1 and RIP3;

(4) Degrade TDP25 protein;

(5) Inhibition of cellular RIP1 kinase-dependent pyroptosis;

(6) Degrade NLRP3 protein.

In another preferred embodiment, the cells are selected from the group consisting of leukemia T cells, lymphoid cancer cells, microglia cells, 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 embodiment, the liver injury is toxin-induced acute liver injury.

In another preferred embodiment, the toxin is LPS.

In another preferred embodiment, the RIP1-related disease is selected from the group consisting of neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher's 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 nerve pain.

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

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

In another preferred embodiment, the multiple sclerosis is induced by dicyclohexanone oxalyl dihydrazone (Cuprizone).

In another preferred embodiment, the disease associated with programmed cell necrosis is selected from the group consisting of neurodegenerative disease, ischemic injury, autoimmune disease, atherosclerosis, psoriasis, Gaucher's 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 nerve pain.

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

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

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

In another preferred embodiment, the neurodegenerative disease is ALS.

In another preferred embodiment, the NLRP3 protein-related disease is selected from the group consisting of neurodegenerative diseases, autoimmune diseases, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and tumors.

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

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

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

The present invention further provides a pharmaceutical composition comprising 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 steps of: administering a compound of formula (I) or a derivative thereof or comprising a compound of formula (I) or a derivative thereof and a pharmacy to a subject in need thereof The pharmaceutical composition of the above acceptable carrier; the disease is one or more selected from the group consisting of RIP1-related diseases, programmed cell necrosis-related diseases, TDP25 protein-related diseases, NLRP3 protein-related diseases, and liver damage;

The present invention further provides a method for inhibiting RIP1 activity in a cell, comprising the steps of: combining the 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 contacting the pharmaceutical composition with the carrier;

In another preferred embodiment, the cells are selected from the group consisting of leukemia T cells, lymphoid cancer cells, microglia cells, 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 cells are cells 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 administration concentration of the compound of formula (I) or its derivatives is 1-50 μM; preferably, it is 5-20 μM.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to constitute new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows that ZJU-37 inhibits the 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 between 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 between 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 can effectively degrade inflammation-induced NLRP3 protein.

Figure 11 shows that ZJU-37 can effectively improve the occurrence of RIPK1-dependent pyroptosis.

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 the _IC50 of ZJU-37 for RIP1 inhibition.

Figure 15 shows that compound (II) inhibits necroptosis in Jurkat-FADD-/- cells.

Detailed ways

Through in-depth research, the inventor unexpectedly discovered a compound with a novel structure that can be used as a highly efficient RIP1 inhibitor. This kind of inhibitor compound can effectively inhibit the activity of RIP1, and can also effectively reduce the interaction between RIP1 and RIP3. Programmed necrosis, improve cell viability; can also effectively degrade TDP25 protein and NLRP3 protein, etc., thus having preventive and therapeutic effects on various diseases. These compounds can quickly pass through the blood-brain barrier and have the function of promoting the proliferation of nerve cells. 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 chain alkyl group having 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl , isobutyl, tert-butyl, n-pentyl, n-hexyl, etc. Preferably it is C _1-3 alkyl.

As used herein, the term " _C6-10 aryl" is an aryl group having 6-10 carbon atoms, such as phenyl or naphthyl.

As used herein, the term "5- 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 is independently a heteroatom selected from nitrogen, sulfur or oxygen. Preferred are 5- to 6-membered heteroaryl groups or 6- to 10-membered heteroaryl groups; for example, furan, thiophene, pyrrole, thiazole, imidazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, quinoline.

NLRP3 protein

NLRP3 protein is an important part of NLRP3 inflammasome, and as an important component of innate immunity, it plays an important role in the body's immune response and disease occurrence. NLRP3 protein-related diseases are diseases mediated by (or at least partially mediated by) NLRP3 protein, for example 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 region of ALS patients. Studies have found that TDP25 protein can promote the formation of TDP-43 inclusion bodies, has a toxic effect on motor neurons, causes neuronal degeneration, and plays an important role in the pathogenesis of the disease. TDP25 protein-related diseases are diseases mediated (or at least partially mediated by) TDP25 protein, such as ALS.

Active ingredient

As used herein, the compounds of the present invention are compounds of formula (A), the structure of which is shown below:

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

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

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

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

Preferably, the active ingredient of the present invention is a compound of formula (I) or a derivative thereof.

A derivative of a compound described in the present invention is any derivative of the compound, for example, a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, stereoisomer or stereoisomer of the compound A mixture of isomers, etc.

As used herein, "pharmaceutically acceptable salts" or "physiologically acceptable salts" include, for example, salts of the compounds of the present invention with inorganic acids and salts with organic acids. Alternatively, if a 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. Conversely, if the product is a free base, it can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with an acid according to conventional methods for the preparation of acid addition salts from basic compounds There are addition salts, especially pharmaceutically acceptable addition salts. Those skilled in the art will recognize various synthetic methods available for the preparation of 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 hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, 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, salicylates, etc.

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

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

As used herein, the compounds of the present invention may exist as tautomers. Tautomers are in equilibrium with each other. For example, the carbonyl moiety can exist in the enol form. Regardless of which tautomer is displayed and regardless of the nature of the equilibrium between the tautomers, one of ordinary skill in the art understands that the compounds contain both keto and enol tautomers.

As used herein, "stereoisomers" refer to compounds composed of the same atoms bonded by the same bonds, but with different three-dimensional structures, which are not interchangeable. "Stereoisomers" as used herein encompasses various stereoisomers of the compounds of the present invention and mixtures thereof and includes "enantiomers", which refer to two stereoisomers whose molecules are non-superimposable to each other mirror. "Diastereomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror images of each other.

As used herein, "prodrug" means any compound that releases the active parent drug in vivo according to the structure of the compounds of the present invention when the prodrug is administered to a subject. The prodrugs can be prepared by modifying any functional group present in the compounds of the present invention in such a way 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 to the parent compound in routine manipulation or in vivo. The prodrugs of the present invention include groups such as amino groups in the compounds of the present invention bonded to any group that can be cleaved in vivo to regenerate the free amino groups, respectively. The preparation, selection, and use of prodrugs are discussed in T. Higuchi and V. Stella, "Pro-drugsas Novel Delivery Systems" A.C.S. Symposium Series Vol. 14; "Design of Prodrugs" edited by H. Bundgaard, Elsevier, 1985; and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, all of which are hereby incorporated by reference in their entirety.

Preparation

The compounds of the present invention can be prepared according to conventional methods in the art, or can be prepared according to the following methods (the methods shown in the following reaction formulas).

The preparation method of the present invention includes the steps of: reacting compound 2 with R ³ -LX to obtain the compound of formula (A).

In the formula, R ¹ , R ² , R ³ and L are as defined above; X is N(CH ₃ ) ₂ or halogen (eg fluorine, chlorine, bromine or iodine).

Compound 2 was prepared by referring to the operation method of Example 1 of WO2016094848A1 and using corresponding raw materials.

The reaction is carried out in a solvent. For example DMF, DMSO, THF, etc. The solvent generally does not participate in the reaction. Of course, some solvents can also be used as the reagent R ³ -LX to participate in the reaction, such as when DMF is used as the solvent.

The reaction can be carried out under basic or acidic conditions. Basic conditions are carried out, for example, in the presence of a base. The bases are alkaline reagents such as sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide and the like. Acidic conditions are carried out, for example, in the presence of an acid. The acid is, for example, an acidic reagent such as phosphorus oxychloride.

The reaction can be carried out under conventional conditions. For example, it is carried out at a certain temperature (eg -10°C-30°C; preferably 0°C-30°C) for a period of time (eg 0.1-10 hours; preferably 0.1-5 hours).

Medications and methods of administration

Studies have found that the compounds of the present invention have one or more of the following functions: inhibiting RIP1 kinase activity; reducing the interaction between RIP1 and RIP3; inhibiting programmed cell necrosis; degrading TDP25 protein; degrading NLRP3 protein and the like. Accordingly, the compounds of the present invention or their derivatives and pharmaceutical compositions containing the compounds or their derivatives as main active ingredients can be used for the treatment, prevention and remission of 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 damage, liver failure, gastroenteritis, and the like.

The pharmaceutical composition of the present invention contains the active ingredient in a safe and effective amount and a pharmacologically acceptable excipient or carrier.

The "safe and effective amount" refers to: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg active ingredient/dose, more preferably 5-200 mg active ingredient/dose. Preferably, the "one dose" is a capsule or tablet.

)、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。The "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gel substances, which are suitable for human use, and which must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be blended with the active ingredient and with each other without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the active ingredient of the present invention or the medicament containing the active ingredient is not particularly limited, and representative modes of administration 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 (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato 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 fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active ingredient in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active ingredient may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally employed 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 of these substances.

Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

In addition to the active ingredient, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

Compositions for parenteral injection may comprise 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 carriers, 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 using the pharmaceutical composition, a safe and effective amount of the active ingredient of the present invention is suitable for a subject in need of treatment, such as a mammal (such as a human), wherein the dose is a pharmaceutically effective dose when administered, for a body weight of 60kg For humans, the daily dose is usually 1-2000 mg, preferably 5-500 mg. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

As used herein, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical outcomes may include one or more of the following: a) inhibition of the disease or condition (eg, reduction of one or more symptoms caused by the disease or condition and/or alleviation of the extent of the disease or condition) ; b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the exacerbation or progression of the disease or condition and/or preventing or delaying the spread of the disease or condition (e.g., metastasis); and/or c) ameliorating the disease, i.e., causing regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or complete relief of the disease or condition, enhancing the effect of another drug, delaying 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 results in the absence of clinical symptoms of the disease or condition. In certain embodiments, the active ingredient can be administered to subjects (including humans) who are at risk or have a family history of a disease or condition.

As used herein, "subject" refers to an animal, such as a mammal (including a human), that has been or will be the subject of treatment, observation, or experimentation. 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 present invention are:

The compounds of the present invention or their derivatives can effectively inhibit the activity of RIP1, and can be used as an effective RIP1 inhibitor to prevent or treat diseases mediated by RIP1.

The compounds of the present invention or their derivatives can effectively reduce programmed cell necrosis and improve cell viability.

The compounds of the present invention or derivatives thereof also have the ability to inhibit inflammation.

The compounds of the present invention or derivatives thereof can also degrade NLRP3 protein and TDP25 protein.

In particular, the compounds of the present invention or derivatives thereof can also treat liver injury.

That is, the compounds of the present invention or derivatives thereof can be used as a single drug to treat various diseases (eg, diseases mediated by RIP1 protein, diseases mediated by NLRP3 protein or diseases mediated by TDP25 protein).

The present invention will be further described below in conjunction with 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. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise specified.

In the following examples, cell viability (%) after drug treatment = cell viability in drug-treated wells/cell viability in control wells*100%; the higher the cell viability (%), the stronger the cell viability after drug treatment. Among them, cell viability was determined by an ATP-based viability assay.

In the following examples, the conditions of cell culture and drug treatment are: stable temperature (37°C), stable CO ₂ level (5%), constant pH (pH value: 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 refer to FADD-/- knockout Jurkat cells. Jurkat cells are commonly used to study acute T-cell leukemia. Jurkat cells FADD-/- knockout Jurkat cells specifically induced programmed necroptosis under TNFα-induced conditions. Jurkat FADD ^-/- cells in this experiment can be obtained by CRISPR technology.

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

In the following examples, BV2 cells were mouse microglia cells; purchased from Nanjing Kebai Cell Bank. Since BV2 cells secreted TNFα under the condition induced by z-VAD-fmk, no additional TNFα was required under the induction condition.

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 were 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 according to the operation method of Example 1 of WO2016094848A1. Under nitrogen protection and ice bath, POCl ₃ (0.4 mmol, 60 mg) was added dropwise to DMF (1 mL), and stirring was continued in ice bath for 0.5 h. Then, a DMF solution of compound 2 (0.2 mmol, 59 mg) was added to the reaction solution, and the reaction was continued for 5 hours at room temperature. An appropriate amount of water was added to quench the reaction, and 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 white solid ZJU-37 (38.7 mg, 60%) . ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.06 (s, 1H), 8.31 (s, 1H), 7.32 (dd, J=8.7, 4.5 Hz, 1H), 6.99 (d, J=2.2 Hz, 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, 2.1 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=4.2Hz), 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=22.3 Hz), 57.9, 24.8, 24.3.

Preparation Example 2 Preparation method of compound (II)

Under nitrogen protection and ice bath, 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 ice bath for 0.5 h. Then, a solution of 2-pyridinesulfonyl chloride (0.3 mmol, 53 mg) in THF was added to the reaction solution, and the reaction was warmed to room temperature overnight. An appropriate amount of water was added to quench the reaction, and 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 compound (II) as a white solid (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, 4.9Hz, 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).

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

HRMS (ESI) calcd for _{C20H15ClFN3O3} : _400.0866 ( _M +H ⁺ ), found: _400.0871 .

HRMS (ESI) calcd for _{C19H14ClFN4O3} : _401.0818 ( _{M +} H ⁺ ), found: 401.0826.

HRMS (ESI) calcd for _{C19H15ClFN3O4S} : _436.0536 ( _{M +} H ⁺ ), found: 436.0540.

Example 1 ZJU-37 can inhibit the programmed necrosis of Jurkat-FADD ^-/- cells

购自Selleck公司；作为阳性对照药物)。Raw materials: TNFα (purchased from Sigma, USA); Nec1 (structure is

purchased from Selleck; as a positive control drug).

experimental method:

Jurkat FADD ^-/- cells were plated on 96-well white plates, and the medium used was 1640 medium (purchased from Southern Model Biotechnology Co., Ltd., the same below), and the plating density was 20,000 cells per well. After 2-4 hours of cell plating, the following treatments were performed:

Nec1 well: with TNFα (final effect concentration of 30ng/ml) and different concentration of drug Nec1 (final effect concentration 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: with TNFα (final concentration of 30ng/ml) and different concentration of drug ZJU-37 (final concentration 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;

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

Control well: no inducer and drug are added, it is a blank control;

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 the necrosis of Jurkat-FADD-/- cells at different concentrations, respectively. - Inhibition of necroptosis of cells; Panel C represents the EC50 of Nec1 inhibiting necroptosis of Jurkat-FADD-/- cells; Panel D represents the EC50 of ZJU-37 inhibiting necroptosis of Jurkat-FADD-/- cells.

It can be seen that under the condition that TNFα specifically induces programmed necrosis, ZJU-37 can inhibit the 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 (same as above); z-VAD-fmk (purchased from BioVision); Nec1 (same as before).

experimental method:

U937 cells were plated on 96-well white plates, and the medium used was 1640 medium, and the plating density was 20,000 cells per well. After 2-4 hours of cell plating, the following treatments were performed:

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

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

TZ wells: treated with TZ (TNFα (final effect concentration of 30 ng/ml) and z-VAD-fmk (final effect concentration of 40 μM)) and an equivalent volume of DMSO to the drug;

Control well: no inducer and drug are added, it is a blank control;

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, Figure A shows that Nec1 and ZJU-37 can inhibit the programmed necrosis of U937 cells at a concentration of 10 μM; Figure B shows that Nec1 and ZJU-37 inhibit the programmed necrosis of U937 cells at different concentrations; C Panel shows the EC50 of Nec1 inhibiting apoptosis of U937 cells; Panel D shows the EC50 of ZJU-37 inhibiting apoptosis of U937 cells.

It can be seen that when TNFα and z-VAD-fmk specifically induce programmed necrosis, ZJU-37 can inhibit the 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 the programmed necrosis of BV2 cells

Starting materials: z-VAD-fmk (as before); Nec1 (as before).

experimental method:

BV2 cells were plated on 96-well white plates, and the medium used was DMEM medium (purchased from Thermo Fisher Scientific, the same below), and the plating density was 8,000 thousand cells per well. The cells were plated for 1 day and the following treatments were performed:

Nec1 wells: Nec1 with z-VAD-fmk (final effect concentration of 70 μM) and different effect concentrations of Nec1 (final effect 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) together;

ZJU-37 well: with z-VAD-fmk (final concentration of 70 μM) and different concentration of ZJU-37 (final 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) together;

Z-VAD wells: treated with z-VAD-fmk (final effect concentration of 70 μM) and an equivalent volume of DMSO to the drug;

Control well: no inducer and drug are added, it is a blank control;

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 inhibit the programmed necrosis of BV2 cells at different concentrations, respectively; C Panel shows the EC50 of Nec1 inhibiting the programmed necrosis of BV2 cells; Panel D represents the EC50 of ZJU-37 inhibiting the programmed necrosis of BV2 cells.

It can be seen that under the condition that z-VAD-fmk specifically induces programmed necrosis, ZJU-37 can inhibit the 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 Weizhen Biotechnology); LPS (purchased from Sigma Company); Nec1 (same as above).

experimental method:

293T cells were plated in six-well plates, the medium used was DMEM medium, and the plating density was 500,000 cells per well; 24 hours after plating, the following treatments were performed:

Flag well: transfected with 1 μg Flag-RIP1;

Flag+LPS wells: transfected with 1 μg Flag-RIP1, and treated with LPS (final concentration of 100 ng/ml) and DMSO of the same volume as the drug 24 hours after transfection;

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

ZJU37-5 well: transfected with 1 μg Flag-RIP1, and treated with LPS (final concentration of 100 ng/ml) and ZJU-37 (final concentration of 5 μM) 24 hours after transfection;

ZJU37-10 well: transfected with 1 μg Flag-RIP1, and treated with LPS (final concentration of 100 ng/ml) and ZJU-37 (final concentration of 10 μM) 24 hours after transfection;

ZJU37-20 well: transfected with 1 μg Flag-RIP1, and treated with LPS (final concentration of 100 ng/ml) and ZJU-37 (final concentration of 20 μM) 24 hours after transfection;

Control well: no inducer and drug are added, it is a blank control.

After 6 hours of treatment, the cells were collected and subjected to immunoblot hybridization with the corresponding antibodies. The results are shown in panels A and C of FIG. 4 .

Jurkat FADD-/- cells were plated in six-well plates, the medium used was 1640 medium, and the plating density was 1.5 million cells per well; 2 hours after plating, the following treatments were performed:

TNFα wells: treated with TNFα (final effect concentration of 30 ng/ml) and an equivalent volume of DMSO to the drug;

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

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

Control well: no inducer and drug are added, it is a blank control.

After 4 hours of treatment, the cells were collected and subjected to immunoblot hybridization with the corresponding antibodies. The results are shown in panel B of FIG. 4 .

It can be seen that the level of P-RIP1 was increased in the case of LPS or TNFα induction alone, especially in Jurkat FADD-/- cells. The addition of ZJU-37 treatment effectively reduced the level of P-RIP1, 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 of ZJU-37 was more effective in reducing P-RIP1 levels.

Example 5 ZJU-37 can effectively inhibit RIP1 kinase activity

Raw materials: pCMV-Flag (empty, purchased from Weizhen Bio); Flag-beads (purchased from Bimake); Flag-RIP1 (same as above); phosphatase inhibitor (purchased from Sangon Bioengineering Co., Ltd.); Nec1 ( Ibid); Kinase Buffer (20 mM-HEPES, pH=7.5, 2 mM DTT, 10 mM MnCl ₂ ).

experimental method:

293 T cells were plated in a 10cm culture dish, the medium used was DMEM medium, and the plating density was 3 million cells per dish; 24 hours after plating, 6 μg pCMV-Flag (system 1) or Flag-RIP1 (system 2-system was transfected) 5). ATP was added in system 1, system 3 to system 5, and ATP was not added in system 2.

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

The specific systems of the kinase assay are as follows:

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

After the cells were treated for 20 hours, the supernatant was discarded from each dish, washed twice with pre-cooled PBS, and then 1 ml of RIPA lysis solution was added to each dish, incubated with Flag-beads, and placed on a shaker at 4 °C for 30 min. Centrifuge at 12000rpm for 10min, take the supernatant and Flag-beads and incubate at 4°C, centrifuge after 4h, centrifuge at 2000rpm for 2min at 4°C, leave the Flag-beads discard the supernatant, wash the Flag-beads with RIPA lysis solution three times, and then do the kinase assay.

The five systems were added to the centrifuge tubes of each sample, placed in a metal bath at 30°C for 30 min, and the beads were collected and subjected to immunoblotting hybridization with the corresponding antibodies.

The experimental results are shown in Figure 5: in the case of adding ATP, the level of P-RIP1 in sample No. 3 increased compared with that in sample No. 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 between RIP1 and RIP3 in cells

Raw materials: TNFα (same as before); z-VAD-fmk (same as before); SMAC (purchased from Selleck Company); Nec1 (same as before); EGFP-RIP1 (purchased from Weizhen Biotechnology); true creature);

experimental method:

HT-29 cells were plated in a 10cm culture dish, and the medium used was 1640 medium; the plating density was 2 million cells per dish; 24h after plating, the following treatments were performed:

TZS wells: treated with TZS (TNFα (final effect concentration of 30 ng/ml), z-VAD-fmk (final effect concentration of 20 μM) and SMAC (final effect concentration of 100 nM)) and an equivalent volume of DMSO to the drug;

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

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

Control well: no inducer and drug are added, it is a blank control.

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

293T cells were plated in a 10cm culture dish, and the medium used was DMEM medium; the plating density was 4 million cells per dish; 24h after plating, the following treatments were performed:

EGFP-RIP1+Flag-RIP3 wells: transfect 2μg of EGFP-RIP1 and Flag-RIP3, and add DMSO with the same volume as the drug at the same time;

Nec1 well: transfect EGFP-RIP1 and Flag-RIP3 with 2 μg each, and add Nec1 (final concentration of 10 μM) for treatment;

ZJU-37 wells: transfected with 2μg of EGFP-RIP1 and Flag-RIP3, and added ZJU-37 (final concentration of 10μM) for treatment;

Control well: no inducer and drug are added, it is a blank control.

After 24 hours of treatment, wash twice with PBS, add RIPA lysis buffer, incubate with beads, 1 ml was lysed at 4 °C for 30 min, and then collect protein, add EGFP antibody to incubate, centrifuge beads to collect protein, and use corresponding antibody for immunoblot hybridization. shown in Figure B in Figure 6.

In the case of equivalent RIP1 content, 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 that of 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 that of Nec1.

Example 7 ZJU-37 can effectively degrade the level of TDP25 protein

)；Nec1(同前)。Raw material: compound B3 (structure is

); Nec1 (ibid.).

experimental method:

H4-TDP25 cells were plated in six-well plates, the medium used was DMEM medium, and the plating density was 200,000 cells per well; 12 hours after plating, the following treatments were performed:

Well B3: cells were treated with compound B3 (final concentration of 10 μM) for 6 hours, then the medium was changed, and then treated with the same volume of DMSO as the drug;

Nec1-10 wells: cells were treated with compound B3 (final concentration of 10 μM) for 6 hours, then the medium was changed, and then treated with Nec1 (final concentration of 10 μM);

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

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

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

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

Control well: no inducer and drug are added, it is a blank control.

After 6 hours of treatment, the cells were collected and subjected to immunoblot hybridization 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 was increased. After treatment with ZJU-37, the expression of intracellular TDP25 protein was significantly decreased. 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 attenuate the binding of RIP1/RIP3 proteins in mouse brain tissue

Materials: C57BL/6 male mice (purchased from Shanghai Nanmo Organisms); LPS (same as above); Nec1 (same as before).

experimental method:

C57BL/6 male mice were treated as follows:

Control group: blank control, no inducer and drug added;

LPS group: treated with intraperitoneal injection of LPS (final effect concentration of 100 ng/kg) and DMSO of the same volume as the drug;

Nec1 group: treated with LPS (final concentration of 100ng/kg) and Nec1 (final concentration of 5mg/kg) by intraperitoneal injection;

ZJU-37 group: treated with LPS (final effect concentration of 100ng/kg) and ZJU-37 (final effect concentration of 5mg/kg) by intraperitoneal injection;

After 6 hours of treatment, the mice were dissected and their brains were taken, 1/2 of the brain tissue was taken and ground with RIPA lysis buffer for 1 min, centrifuged at 12,000 rpm for 20 min, and the supernatant was taken. This step was repeated three times. Finally, the supernatant was combined and incubated with beads and antibodies. Finally, the protein was collected and the corresponding body was used for immunoblot hybridization.

The experimental results are shown in Figure 8. ZJU-37 can effectively attenuate the interaction between RIP1/RIP3 proteins in mouse brain tissue. From the IP band, we can see that the interaction between RIP3 and RIP1 is enhanced after the action of LPS, as can be seen from the protein level content of the RIP3 band, while the level of RIP3 after the action of ZJU37 is reduced, which proves that the interaction between RIP3 and RIP1 Interactions are weakened. Nec1 did not attenuate 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 (same as before); LPS (same as before); Nec1 (same as before).

experimental method:

C57BL/6 male mice were treated as follows:

Control group: blank control, no inducer and drug added;

LPS group: treated with intraperitoneal injection of LPS (final action concentration 100ng/kg) and DMSO of the same volume as the drug;

LPS+ZJU-37 group: treated with LPS (final concentration of 100ng/kg) and ZJU-37 (final concentration of 5mg/kg) by intraperitoneal injection;

After 6 hours of treatment, the mice were dissected and the livers were taken out for HE section staining and morphological observation. The main purpose of doing liver HE staining is generally to observe whether the shape of liver cells is complete, whether the nucleus is enlarged, whether the shape of the hepatic lobule is complete, and whether inflammation occurs to detect the damage of drugs and other stimuli to 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 cell morphology of the blank control group, it was found that after adding LPS, the cell morphology was significantly worse, and there was obvious inflammatory infiltration; after ZJU-37 treatment, the cell morphology was significantly improved, and the inflammatory infiltration phenomenon was alleviated.

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

Raw materials: TNFα (as before); z-VAD-fmk (as before); SMAC (as before); Nec1 (as before).

experimental method:

HT-29 cells were plated in six-well plates, the medium used was 1640 medium, and the plating density was 700,000 cells per well; 24 hours after plating, the following treatments were performed:

Control well: no inducer and drug are added, it is a blank control;

TZS wells: treated with TZS (TNFα (final effect concentration of 30 ng/ml), z-VAD-fmk (final effect concentration of 20 μM) and SMAC (final effect concentration of 100 nM)) and an equivalent volume of DMSO to the drug;

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

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

After 72 hours of treatment, the cells were collected and subjected to immunoblot hybridization with corresponding antibodies. The results are shown in panel A of FIG. 10 .

Jurkat FADD-/- cells were plated in six-well plates, the medium used was 1640 medium, and the plating density was 1.5 million cells per well; 2 hours after plating, the following treatments were performed:

Control well: no inducer and drug are added, it is a blank control;

TNFα wells: treated with TNFα (final effect concentration of 30 ng/ml) and an equivalent volume of DMSO to the drug;

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

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

After 4 hours of treatment, the cells were collected and subjected to immunoblot hybridization with the corresponding antibodies. The results are shown in panel B of FIG. 10 .

The experimental results are shown in Figure 10:

Panel A: When NLRP3 is induced to rise by TZS, ZJU-37 can effectively degrade inflammation-induced NLRP3 protein, and the effect is better than the positive drug Nec1.

Panel B: When NLRP3 is induced to rise by TNFα, ZJU-37 can effectively degrade inflammation-induced NLRP3 protein, and the effect is better than the positive drug Nec1.

Example 11 ZJU-37 can effectively inhibit RIP1-dependent pyroptosis

Raw material: Mouse BMDM cells: Mice were purchased from Southern Model Organisms; BMDM extraction method: Take mouse thigh bones, cut both ends of the thigh bones, and flush the inside of the thigh bones with a needle to flush out the cells, the cells obtained were 1000 rpm, 4 Centrifuge for 5 min. The supernatant was discarded, an appropriate amount of ACK was added to lyse the red blood cells, and PBS was added for neutralization after 3-5 min. Centrifuge at 1000 rpm, 4 degrees for 5 min. The plate can be resuspended and counted in medium.

CAS：66018-38-0)。LPS (same as above); Nec1 (same as above); 5z-7 (structure is:

CAS: 66018-38-0).

experimental method:

The mouse BMDM cells were plated in six-well plates, and the medium used was 1640 medium (purchased from Southern Model Biological Company; 10% FBS--56 ℃ fire for 30 minutes + M-CSF-20ng/ml + 1% double antibody), The plating density was 2 million cells per well, that is, 10 ⁶ /ml. The medium was changed after 3 days of cell plating, and the following treatments were performed after 5 days:

Control group: blank control, no inducer and drug added;

LPS+5z-7 well: add pyroptosis inducers (LPS (final concentration of 10ng/ml) and 5z-7 (final concentration of 125nM)) and the same volume of DMSO as the drug;

Nec1 well: add pyroptosis inducers (LPS (final concentration of 10 ng/ml) and 5z-7 (final concentration of 125 nM)), and add Nec1 (final concentration of 10 μM) for treatment;

ZJU-37 well: add pyroptosis inducers (LPS (final concentration of 10ng/ml) and 5z-7 (final concentration of 125nM)), and at the same time add ZJU-37 (final concentration of 10 μM);

After treating the cells for 5 hours, cells were removed to measure cell viability and subjected to immunoblotting with corresponding antibodies. The results are shown in panels A and B of Figure 11, respectively.

The experimental results are shown in Figure 11:

Mouse BMDM cells undergo RIP1-dependent pyroptosis under the induction of LPS and 5z-7, and cell viability decreases. At the same time, the levels of P-RIP1 and GSDMD are increased, but the addition of drug ZJU-37 can effectively inhibit cell viability The decrease of P-RIP1 and GSDMD protein levels, thereby inhibiting their kinase activities, and inhibiting the occurrence of RIP1-dependent pyroptosis. And its inhibitory effect was similar to that of 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 Southern Model Organisms); conventional feed (purchased from Hangzhou Sailuojin Biotechnology Co., Ltd.); 0.2% Cuprizone feed (purchased from Hangzhou Sailuojin Biotechnology Co., Ltd., for the addition of 0.2% Cuprizone to conventional feed); Nec1 (ibid.).

experimental method:

The 6-8 week old male C57 mice were divided into two groups for the MS model, group one and group two. Among them, group 1 and group 2 are each divided into the following four groups:

The first group: blank control group;

The second group: Cup group (Cuprizone diet 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: The mice in the first group were fed regular chow from the first day, and the brain tissue was obtained by perfusion after feeding for 6 weeks; the mice in the second, third or fourth groups were fed 0.2% Cuprizone chow from the first day, Brain tissue was collected by perfusion after 6 weeks of feeding. Among them, the third group simultaneously started intraperitoneal injection of drugs (Nec1, the concentration of action was 5 mg/kg) from the first day; the fourth group simultaneously began to inject drugs (ZJU-37, the concentration of action was 5 mg/kg) from the first day; The second group was intraperitoneally injected with solvent (solvent formula: 2% DMSO+30% PEG400+68% ddH2O) from the first day.

Group 2: Four groups of mice were fed 0.2% Cuprizone chow from the first day to the thirtieth day and then regular chow from the thirty-first day to the forty-fifth day. After 6 weeks, the brain tissue was collected by perfusion. Among them, the third group started to inject the drug (Nec1, the effect concentration was 5mg/kg) from the thirty-first day at the same time, and the fourth group started to inject the drug (ZJU-37, the effect concentration was 5mg/kg) from the thirty-first day at the same time. ), the second group started to inject solvent (solvent formula: 2% DMSO+30% PEG400+68% ddH2O) from the thirty-first day.

The experimental results are shown in Figure 12:

Figure A: The mouse brain slice was fixed with 4% glutaraldehyde and the corpus callosum was excised for electron microscope observation. The electron microscope results showed that the cup group exhibited partial demyelination, which was characterized by loose myelin sheath and decreased g-ratio (g-ratio). g-ratio is the inner diameter of the myelin sheath compared to the outer diameter of the myelin sheath, and a lower value indicates that the myelin sheath is more loose), but after intraperitoneal injection of the two drugs, the myelin sheath is less demyelinated and the myelin sheath is more compact.

Panel B: The brain slices of each group of mice were stained with eleganic blue (normal myelin sheaths appear blue, and the demyelinated areas disappear in blue and become white). From the figure, we can see that the cup group showed partial demyelination, but after intraperitoneal injection of the two drugs, the demyelination 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 Slack Company, grade: SPF grade, age: 6 to 8 weeks, sex: male); Nec1 (same as before).

experimental method:

7 BALB/c Nude mice were divided into two groups (drug-treated group and solvent group):

Drug treatment group:

Mice were intraperitoneally injected with 4 mg/kg ZJU37; the drug treatment components were divided into two groups:

0.5h group: samples were taken 0.5 hours after intraperitoneal injection of ZJU37 for analysis.

1h group: samples were taken 1 hour after intraperitoneal injection of ZJU37 for analysis.

Solvent group:

Mice were injected ip with 2% DMSO + 30% peg400 + water; samples were then taken for analysis 1 hour after the solvent was injected ip.

When sampling for analysis, mice were first anesthetized by intraperitoneal injection of 4% aqueous chloral hydrate solution, 200 μl/20 g body weight. Plasma and brain drug concentrations were then analyzed separately.

Take 300 μl of blood from the carotid artery into a heparin tube. After mixing, centrifuge at 4000 rpm for 5 min to take 100 μl of the supernatant, add 700 μl of acetonitrile, mix for 20 min, and centrifuge at 12,000 rpm for 10 min. analyze.

About 0.35 g of whole brain was taken, added 1 ml of acetonitrile homogenate (PRIMA PB100, 35000rpm, 1min), ultrasonicated for 20min, centrifuged at 12000rpm for 10min, the supernatant was left at 4°C overnight, centrifuged at 12000rpm for 10min, and the supernatant was taken for HPLC analysis.

The experimental results are shown in Figure 13. The ZJU-37 drug reached a maximum concentration in the brain at 30 min and was subsequently metabolized.

Example 14 Half-inhibitory concentration test for RIPK1 enzyme

HTRF kinEASE STK kit (Cat.62STKOPEC, Cisbio company) and ADP-Glo Kinase kit (Cat.V6930, Promega company) were used to detect the median inhibitory concentration (IC50) of ZJU-37 and Nec-1s on RIPK1 kinase.

购自Selleck公司；The structure of Nec-1s is

Purchased from Selleck;

RIPK1 (Medicilon Corporation);

ATP 10mM (Cat.PV3227, Invitrogen);

DTT 1M (Cat. D5545, Sigma);

MgCl ₂ 1M (Cat. M8266, Sigma).

1. Reagent preparation

RIPK1 enzyme reaction system components and concentrations:

Table 1

1× Enzyme Buffer: 1 mL of 1× Kinase Buffer contains 200 μL 5× Enzyme Buffer, 5 μL 1M MgCl ₂ , 1 μL 1 M MnCl ₂ , 1 μL 1 M DTT, 793 μL ddH2O.

5×STK-biotin substrate working solution: The specific concentration of STK-biotin substrate is shown in Table 1. Dilute the STK-biotin substrate to 5-fold the reaction concentration with 1X Kinase Buffer.

5×ATP working solution: see Table 1 for the specific concentration of ATP. Dilute ATP to 5-fold the reaction concentration with 1X Kinase Buffer.

5× enzyme working solution: see Table 1 for enzyme concentration. Make up a 5x enzyme working solution of the enzyme in 1x kinase buffer.

2. Experimental procedure

After all the reagents are prepared according to the above method, except the enzyme, after equilibrating to room temperature, start adding samples.

First, use the prepared 1× kinase buffer to prepare 2.5% DMSO solution respectively (the final concentration of control DMSO is 1%), then dilute the test compound with 2.5% DMSO solution, and the final concentration of the compound starts from 10 μM , 4-fold dilution, 10 concentration points. In addition to the control wells (including positive control wells and negative control wells), add 2 μL of the diluted test compound solution to the reaction wells used, and add 2 μL of the previously prepared 2.5% DMSO solution 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 for enzyme screening).

1 μL of previously prepared enzyme solution (see Table 1 for the amount of enzyme) was added to all reaction wells except the negative control wells, and the negative control wells were supplemented with 1× kinase buffer. Seal the plate with a sealing film, and incubate at room temperature for a few minutes after mixing to allow the compound and the enzyme to fully bind.

1 μL of ATP solution was added to all reaction wells to initiate the kinase reaction, and the RIPK1 enzyme reaction time was 180 minutes (see Table 1 for the corresponding ATP concentration and reaction time).

After the kinase reaction was completed, add 5 μL of ADP-Glo reagent to all reaction wells, mix well, and react at room temperature for 40 min.

10 μL of kinase detection reagent was added to all reaction wells, the plate was sealed and mixed, and after 30 min of reaction at room temperature, the fluorescence signal was detected by ENVISION (Perkinelmer) instrument.

The inhibition rate of each well was calculated from the reaction wells and control wells, and the average value was calculated in the duplicate wells, and the inhibitory activity of the tested compounds was analyzed by the analysis software PRISM5.0.

Inhibition rate=(fluorescence signal of positive control well-fluorescence signal of dosing well)/(fluorescence signal of positive control well-fluorescence signal of 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 that of Nec-1s (IC50 for RIPK1 enzyme was 1142 nM).

Example 15 Compound (II) can inhibit the programmed necrosis of Jurkat-FADD ^-/- cells

The procedure of Example 1 was repeated, except that compound ZJU37 was replaced by compound (II) and the treatment time was 12 hours. The results are shown in Figure 15. It can be seen that under the condition that TNFα specifically induces programmed necrosis, compound (II) can effectively inhibit the programmed necrosis of Jurkat-FADD-/- cells.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above 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 appended claims of the present application.

Claims (13)

1. A compound represented by the formula (II):

(II)。

2. a compound of formula (I):

(I)。

3. use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of a RIP 1-related disease.

4. Use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of diseases associated with apoptosis.

5. Use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of a disease associated with TDP25 protein.

6. Use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of NLRP3 protein related diseases.

7. Use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of liver damage.

8. Use of a compound according to claim 1, wherein the compound has one or more of the following uses:

(1) used for preparing medicines for inhibiting apoptosis;

(2) for preparing a medicament for inhibiting the activity of RIP1 kinase;

(3) for use in the preparation of a medicament for reducing the interaction of RIP1 and RIP 3;

(4) the preparation method is used for preparing a medicament for degrading TDP25 protein;

(5) for preparing a medicament for inhibiting cell RIP1 kinase-dependent apoptosis;

(6) is used for preparing medicines for degrading NLRP3 protein.

9. The use of claim 3, wherein the RIP 1-associated disease is selected from the group consisting of: neurodegenerative diseases, ischemic injury, autoimmune diseases, atherosclerosis, psoriasis, gaucher's disease, pain, inflammation, retinal detachment, tumors.

10. The use of claim 4, wherein the apoptosis-related disease is selected from the group consisting of: neurodegenerative diseases, ischemic injury, autoimmune diseases, atherosclerosis, psoriasis, gaucher's disease, pain, inflammation, retinal detachment, tumors.

11. The use of claim 5, wherein said TDP25 protein-related disease is a neurodegenerative disease.

12. The use of claim 6, wherein the NLRP3 protein related disease is selected from the group consisting of: neurodegenerative diseases, autoimmune diseases, inflammatory bowel diseases, atherosclerosis, type 2 diabetes, gout, obesity, tumors.

13. A pharmaceutical composition comprising a compound of claim 1 or 2 and a pharmaceutically acceptable carrier.

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