CN110759895A

CN110759895A - Novel RIP1/RIP3 dual-target inhibitor and application thereof in treatment of multi-target disease with one drug

Info

Publication number: CN110759895A
Application number: CN201810826696.1A
Authority: CN
Inventors: 夏宏光; 吴劼; 段树民; 杨淑颖; 岑旭峰; 何福生
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-07-25
Filing date: 2018-07-25
Publication date: 2020-02-07
Anticipated expiration: 2038-07-25
Also published as: CN112351979B; CN112351979A; CN110759895B; WO2020020119A1

Abstract

The invention provides a novel RIP1/RIP3 double-target inhibitor and application thereof in treating multi-target diseases with one medicine. Specifically, the RIP1/RIP3 double-target inhibitor can effectively prevent or treat RIP1 and/or RIP3, apoptosis necrosis, TDP25 protein and/or NLRP3 protein related diseases or liver injury.

Description

Novel RIP1/RIP3 dual-target inhibitor and application thereof in treatment of multi-target disease with one drug

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly provides a novel RIP1/RIP3 double-target inhibitor and application thereof in treating multi-target diseases with one medicine.

Background

The RIP1 protein has functions of regulating inflammation and cell death. Inhibitors of RIP1 protein kinase may be effective in inhibiting cell death, including programmed necrosis and RIP 1-dependent apoptosis. Meanwhile, the RIP1 protein kinase inhibitor can also inhibit various inflammatory reactions, but the mechanism is not clear at present, and the RIP3 protein can be regulated to play a role, and cell death can be inhibited to prevent the amplification of inflammatory signals. At present, RIP1 inhibitors of two companies are in clinical stage, the RIP1 inhibitor (Nec1) of Denali enters clinical stage 2, the indications are neurodegenerative diseases, the RIP1 inhibitor (GSK963) of GSK is in clinical stage 2, and the indications are autoimmune diseases, including rheumatoid arthritis, ulcerative enteritis, psoriasis and the like.

The inhibition activity of the kinase inhibitor of RIP3 on inflammation is probably higher than that of the kinase inhibitor of RIP1, but the RIP3 kinase inhibitor can trigger apoptosis and cannot enter clinic. For example, some inhibitors that inhibit RIP3 singularly: GSK872, GSK 843. The reason why RIP3 inhibitors induce apoptosis may be that only RIP3 kinase activity is inhibited, and RIP1 protein still functions to change the mode of cell death from necrosis to RIP 1-dependent apoptosis.

Therefore, the development of a drug capable of inhibiting both RIP1 and RIP3 is of great significance in inhibiting inflammation and cell death.

Disclosure of Invention

The invention aims to provide a novel RIP1/RIP3 inhibitor.

The invention aims to provide a preparation method and application of the RIP1/RIP3 inhibitor.

The invention provides in a first aspect the use of a compound of formula I,

the compounds are useful for the preparation of:

(a) drugs for preventing or treating RIP1 and/or RIP 3-related diseases;

(b) an agent for preventing or treating a disease associated with apoptosis;

(c) a medicament for preventing or treating a disease associated with TDP25 protein;

(d) a medicament for preventing or treating NLRP3 protein-related diseases;

(e) a medicament for preventing or treating liver injury.

In a second aspect the invention provides the use of a compound of formula I,

the compounds have one or more of the following uses:

(1) inhibiting apoptosis;

(2) inhibition of RIP1 kinase activity and RIP3 kinase activity;

(3) reducing the interaction of RIP1 and RIP 3;

(4) degrading TDP25 protein;

(5) reducing binding of RIP1 and RIP 3;

(6) degrading NLRP3 protein.

In another preferred embodiment, the cell is selected from the group consisting of: Jurkat-FADD-/-cells, U937 cells, BV2 cells, HT-29 cells.

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 and/or RIP 3-related disease is selected from the group consisting of: neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), etc.), ischemic injuries, autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.), atherosclerosis, psoriasis, gaucher's disease, pain (neuropathic pain), inflammation (pancreatitis, ulcerative enteritis, hepatitis), retinal detachment, tumors (e.g. melanoma, brain glioma, colon cancer, glia, lymphoma, T-cell leukemia).

In another preferred embodiment, the apoptosis-related disease is selected from the group consisting of: neurodegenerative diseases (ALS, AD, PD, MS, etc.), ischemic injury, autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.), atherosclerosis, psoriasis, gaucher's disease, pain (neuropathic pain), inflammation (pancreatitis, ulcerative enteritis, hepatitis), retinal detachment, tumors (e.g., melanoma, brain glioma, colon cancer, glioma, lymphoma, T cell leukemia).

In another preferred embodiment, the TDP25 protein-related disease is selected from the group consisting of: neurodegenerative diseases (such as ALS).

In another preferred embodiment, the NLRP3 protein-related disease is selected from the group consisting of: alzheimer's disease, systemic lupus erythematosus, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and tumors.

In another preferred embodiment, the compounds of formula I are administered at a concentration of 1-50. mu.M; preferably, it is 5 to 20. mu.M.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows that ZJU-37 inhibits apoptosis of Jurkat-FADD-/-cells; wherein A shows that at 10uM concentration, Nec1 and ZJU-37 inhibit apoptosis in Jurkat-FADD-/-cells; b shows the inhibition of apoptosis of Jurkat-FADD-/-cells by Nec1 and ZJU-37 at different concentrations; panel C and D show IC50 in which Nec1 and ZJU-37 inhibit programmed necrosis of Jurkat-FADD-/-cells.

FIG. 2 shows that ZJU-37 inhibits apoptosis of U937 cells; wherein A shows that at 10uM concentration, Nec1 and ZJU-37 inhibit apoptosis of U937 cells; b shows the inhibition of apoptosis of U937 cells by Nec1 and ZJU-37 at different concentrations; panel C and D show IC50 in which Nec1 and ZJU-37 inhibited apoptosis in U937 cells.

FIG. 3 shows that ZJU-37 inhibits apoptosis of BV2 cells; wherein A shows that at 10uM concentration, Nec1 and ZJU-37 can inhibit programmed necrosis of BV2 cells; b shows the inhibition of apoptosis of BV2 cells by Nec1 and ZJU-37 at different concentrations; panels C and D show IC50, where Nec1 and ZJU-37 inhibited apoptosis of BV2 cells.

FIG. 4 shows that ZJU-37 is effective in inhibiting the level of p-RIP1 in cells, thereby inhibiting kinase activity.

FIG. 5 shows that ZJU-37 is effective in degrading inflammation-induced RIP3 and reducing the interaction of RIP1 and RIP 3.

FIG. 6 shows that ZJU-37 is effective in degrading various misfolds.

FIG. 7 shows that ZJU-37 is effective in attenuating the interaction of RIP1/RIP3 proteins in mouse brain tissue.

FIG. 8 shows that ZJU-37 is effective in inhibiting LPS-induced acute liver injury in mice.

FIG. 9 shows that ZJU-37 is effective in degrading inflammation-induced NLRP3 protein.

Detailed Description

The inventors of the present invention have conducted extensive and intensive studies, and as a result, the inventors have unexpectedly found that the novel RIP1/RIP3 inhibitor ZJU37 developed in the present invention can inhibit the activities of RIP1 and RIP3 at the same time, and has a stronger ability to inhibit cell death and inflammation, but does not induce apoptosis. Meanwhile, the compound can pass through a blood brain barrier and has the function of promoting the proliferation of nerve cells. The compound has therapeutic effect on various inflammation and cell death related diseases, including but not limited to various neurodegenerative diseases, autoimmune diseases, liver diseases such as liver injury and liver failure, gastroenteritis and the like. The present invention has been completed based on this finding.

Compounds of the invention

The structure of the compound ZJU-37 is shown as follows:

term(s) for

NLRP3 protein

The NLRP3 protein is an important component of NLRP3 inflammasome and plays an important role in the immune response of a body and the occurrence process of diseases as an important component of innate immunity. NLRP 3-related diseases include: alzheimer's disease, systemic lupus erythematosus, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and malignancy.

TDP25 protein

TDP25 is a C-terminal fragment of TDP-43 with a molecular weight of 25kD found in brain regions of ALS patients. Research shows that TDP25 can promote the formation of inclusion body of TDP-43, has toxic effect on motor neuron, and can cause neuron degeneration and play an important role in the disease attack process.

Medicament and method of administration

As the compound of the formula I has one or more of the following excellent functions: (1) inhibiting apoptosis; (2) inhibition of RIP1 kinase activity and RIP3 kinase activity; (3) reducing the interaction of RIP1 and RIP 3; (4) degrading TDP25 protein; (5) reducing binding of RIP1 and RIP 3; (6) degrading NLRP3 protein; therefore, the compound of the formula I and the pharmaceutical composition containing the compound as the main active ingredient can be used for treating, preventing and relieving diseases related to RIP1 and RIP3 or diseases related to TDP25 protein or NLRP3 protein.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of formula I of the present invention in combination with a pharmaceutically acceptable excipient or carrier.

The safe and effective amount refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of formula I of the present invention per dose, more preferably, 5-200mg of a compound of formula I of the present invention per dose. Preferably, said "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 be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and between the compounds of formula I of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers

Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds of formula I of the present invention or of the medicaments containing them is not particularly restricted, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures 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 shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound in such compositions may be delayed in a certain portion of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound 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 compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

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

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, 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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof. Dosage forms for topical administration of the compounds of the present invention 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 which may be required if necessary.

The compounds of formula I 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 compound of formula I of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1-2000mg, preferably 5-500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention are:

the novel RIP1/RIP3 inhibitor ZJU37 can inhibit the activities of RIP1 and RIP3 at the same time, has stronger capacities of inhibiting cell death and inflammation, and does not trigger cell apoptosis.

The inhibitor can be used for treating various diseases by one medicine with multiple targets.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The experimental materials (e.g., various cells) and reagents used in the following examples are commercially available without specific reference.

Example 1 ZJU-37 inhibits Jurkat-FADD^-/-Programmed necrosis of cells

Raw materials:

Jurkat FADD^-/-cell (FADD)^-/-Knockout Jurkat cells specifically induce the onset of programmed necrotic death under TNF α -induced conditions), Jurkat FADD^-/-Cells were obtained by CRISPR technique.

TNFα，ZJU-37，Nec1。

The experimental method comprises the following steps:

Jurkat FADD^-/-cells were plated in 96-well white plates at concentrations of 10uM for both Nec1 and ZJU-37 by treatment with TNF α (30ng/ml) and drug (ZJU-37 or Nec1, where Nec1 is a positive control drug) for 18 hours, with DMSO in control wells where cell viability was determined by ATP-based viability assay₂Level (5%), constant pH value (pH value: 7.2-7.4), higher relative saturation humidity (95%).

The numbers represent the percentage of cell viability in the dosed wells compared to the control wells, with higher numbers representing greater cell viability (drug action viability calculation ═ drug treated wells/control wells x 100%).

The results are shown in FIG. 1, that in the case of TNF α exclusively inducing apoptosis, Nec1 and ZJU-37 inhibited apoptosis in Jurkat-FADD-/-cells (Nec1 is a positive control).

Example 2 ZJU-37 inhibits apoptosis of U937 cells

Raw materials:

u937 cells, TNF α, z-VAD-fmk (inhibitor of apoptosis), ZJU-37, Nec1

The experimental method comprises the following steps:

u937 cells were plated in 96-well white plates by treatment with TNF α (30ng/ml), z-VAD-fmk (40uM), and drugs (ZJU-37 or Nec1) for 72 hours, with Nec1 and ZJU-37 both at 10uM concentration, and control wells in DMSO, with cell viability determined by ATP-based viability assays₂Level (5%), constant pH value (pH value: 7.2-7.4), higher relative saturation humidity (95%).

The results of the experiment are shown in FIG. 2, that in the case of TNF α and z-VAD-fmk exclusively inducing apoptosis, Nec1 and ZJU-37 inhibited apoptosis of U937 cells (Nec1 is a positive control).

Example 3 ZJU-37 inhibits apoptosis of BV2 cells

Raw materials:

BV2 cells, z-VAD-fmk, ZJU-37, Nec1

The experimental method comprises the following steps:

BV2 cells were plated in 96-well plates by treatment with z-VAD-fmk (70uM) and drugs (ZJU-37 or Nec1) for 72 hours, both at 10uM for Nec1 and ZJU-37, and DMSO for control wells (since BV2 cells secrete TNF α themselves under z-VAD-fmk-induced conditions, no TNF α needs to be added) where cell viability was determined by ATP-based viability assays₂Level (5%), constant pH value (pH value: 7.2-7.4), higher relative saturation humidity (95%).

Values represent the percentage of cell viability in the drug-loaded wells versus the control wells, with higher values representing greater cell viability (drug-action viability calculated as drug-treated wells/control wells 100%).

The experimental results are shown in fig. 3: in the case of z-VAD-fmk exclusively inducing apoptosis, Nec1 and ZJU-37 inhibited apoptosis in BV2 cells (Nec1 was a positive control).

Example 4 ZJU-37 is effective in inhibiting RIP1 kinase Activity

Raw materials:

293T cells, Jurkat FADD-/-cells, Flag-RIP1 plasmid, LPS, ZJU-37, Nec1

The experimental method comprises the following steps:

FIG. 4A: 293T cells were plated in six-well plates and, 24 hours after plating, transfected with 1ug Flag-RIP1, 24 hours after transfection, cells were treated with LPS (100ng/ml) and drug (ZJU-37 or Nec1) for 6 hours to harvest protein (DMSO in control wells) and immunoblot hybridization was performed.

FIG. 4B Jurkat FADD-/-cells plated in six well plates 2 hours later, treated simultaneously with TNF α (30ng/ml) and drug (ZJU-37 or Nec1) (10uM) for 4 hours, control wells treated with DMSO, and cells collected separately for immunoblot hybridization with the indicated antibodies.

The conditions for cell culture and drug action are stable temperature (37 deg.C), stable CO₂Level (5%), constant pH value (pH value: 7.2-7.4), higher relative saturation humidity (95%).

The results of the experiment are shown in FIG. 4:

under the condition of being induced by LPS or TNF α alone, the level of P-RIPK1 is improved, and is particularly obvious in Jurkat FADD-/-cells, but the level of P-RIP1 can be effectively inhibited after drugs (ZJU-37 or Nec1) are added, so that the kinase activity of the cells is inhibited (Nec1 is a positive control) (see figures 4A and 4B), and figure 4C shows that the P-RIPK1 content is more effectively inhibited by the ZJU-37 of 10 um.

Example 5 ZJU-37 degrades inflammation-induced RIP3 and reduces the interaction of RIP1 and RIP3

Raw materials:

HT-29 cells, 293T cells, TNF α, z-VAD-fmk, SMAC, ZJU-37, Nec1, EGFP-RIP1, Flag-RIP 3;

the experimental method comprises the following steps:

FIG. 5A HT-29 cells were plated in six-well plates and 24h prior to treatment with TZS (TNF α (30ng/ml), z-VAD-fmk (20uM), SMAC (100nM)) and the corresponding compound (ZJU-37 or Nec1) for 72 h, after which the cells were individually harvested for immunoblot hybridization with the indicated antibodies, FIG. 5B is a quantitative illustration of FIG. 5A.

FIG. 5C HT-29 cells plated in 10cm dishes, plated 24h, treated with TZS (TNF α (30ng/ml), z-VAD-fmk (20uM), SMAC (100nM)) and the corresponding compound (ZJU-37 or Nec1) 72 h later (control wells in DMSO) washed twice with PBS, added RIPA lysate 1ml for 4 ℃ lysis for 30min to harvest protein, added RIPK1 antibody for incubation, centrifuged beads to harvest protein and immunoblotted with the corresponding antibody.

FIG. 5D: 293T cells are plated in 10cm culture dishes for 24h, and then 2ug of EGFP-RIP1 and Flag-RIP3 are transfected, and corresponding compounds (ZJU-37 or Nec1 and 10uM) are added at the same time for treatment for 24h (DMSO is used for control wells), and then PBS is washed twice, 1ml of RIPA lysate is added for 30min of 4-degree lysis for protein collection, EGFP antibody is added for incubation, and immunoblot hybridization is carried out on the collected proteins of centrifugal beads by using corresponding antibodies.

The conditions for cell culture and drug action were stable temperature (37 ℃), stable CO2 level (5%), constant pH (pH 7.2-7.4), higher relative saturation humidity (95%).

The results of the experiment are shown in FIG. 5:

fig. 5A and 5B: in HT-29 cells, ZJU-37 and Nec1 can not only reduce the protein level of p-RIP1, but also reduce the protein level of RIP3, and the effect of ZJU-37 is better than that of Nec 1.

FIG. 5C: in HT-29 cells, Nec1 and ZJU-37 reduced the interaction between RIP1 and RIP 3.

FIG. 5D: nec1 and ZJU-37 reduced the RIP1 and RIP3 interaction in 293T cells.

While a decrease in interaction means that programmed necrosis can be impaired to some extent.

Example 6 ZJU-37 is effective in degrading various misfolds with cytotoxic protein levels

Raw materials:

H4-TDP25 cell, Compound B3, ZJU-37, Nec1

The experimental method comprises the following steps:

H4-TDP25 cells were plated in six-well plates, and after 6 hours of treatment with B3, cells were changed and then treated with the corresponding compound (ZJU-37 or Nec1) for 6 hours (DMSO was used in control wells), and the cells were collected separately and subjected to immunoblot hybridization with the designated antibody. The conditions for cell culture and drug action are stable temperature (37 deg.C), stable CO₂Level (5%), constant pH value (pH value: 7.2-7.4), higher relative saturation humidity (95%).

The structure of the compound of B3 is shown in the specification,

the experimental results are shown in fig. 6: after compound B3 induced the expression of TDP25, ZJU-37 could degrade TDP25 (aggregation of TDP25 protein would induce a series of diseases such as ALS).

Example 7 ZJU-37 was effective in attenuating the binding of RIP1/RIP3 proteins in mouse brain tissue

Raw materials:

c57BL/6 Male mouse, LPS, ZJU-37, Nec1, mouse brain tissue

The experimental method comprises the following steps:

mice were treated with LPS and the corresponding compound (ZJU-37 or Nec1) by intraperitoneal injection for 6 hours before subjecting brain lysates to immunoblot hybridization.

The detailed steps are as follows: dissecting and taking brains of mice, adding RIPA lysate into 1/2 mass of brain tissues to grind for 1min, centrifuging at 12000rpm for 20min, taking supernatant, repeating the steps for three times, adding beads and antibodies into the supernatant to incubate, and collecting proteins to perform immunoblotting hybridization by using the specified antibodies.

The results of the experiment are shown in FIG. 7: ZJU-37 was effective in attenuating the interaction of RIP1/RIP3 in mouse brain tissue. From the IP bands we can see that the interaction between RIP3 and RIP1 is enhanced after LPS action, as can be seen from the protein level content of RIP3 band, while the level of RIP3 is reduced after ZJU37 action, demonstrating that the interaction between RIP3 and RIP1 is attenuated.

Example 8 ZJU-37 is effective in inhibiting LPS-induced acute liver injury in mice

Raw materials:

c57BL/6 Male mouse, LPS, ZJU-37, Nec1, liver tissue

The experimental method comprises the following steps:

mice were treated with LPS and the corresponding compound (ZJU-37) by intraperitoneal injection for 6 hours, then the mice were dissected to take out livers, and morphological observation was performed by He section staining. The main purposes of making a liver HE stained section are generally to observe whether the morphology of liver cells is complete, whether nuclei are enlarged, whether the morphology of liver lobules is complete, whether inflammation occurs or not so as to detect the damage effect of stimulation factors such as drugs on the liver.

The experimental results are shown in fig. 8: ZJU-37 can effectively inhibit mice acute liver injury induced by LPS. Compared with the cell morphology of a blank control group, the cell morphology of the LPS-added cell is obviously deteriorated and has an obvious inflammatory infiltration phenomenon, and after the ZJU-37 is added, the cell morphology is obviously improved and the inflammatory infiltration phenomenon is relieved.

Example 9 ZJU-37 is effective in degrading inflammation-induced NLRP3 protein

Raw materials:

HT-29 cells, Jurkat FADD-/-cells

TNFα，z-VAD-fmk，SMAC

ZJU-37，Nec1

The experimental method comprises the following steps:

FIG. 9A HT-29 cells were plated in six-well plates and 24 hours later, treated with TZS (TNF α (30ng/ml), z-VAD-fmk (20uM), SMAC (100nM)) and the corresponding compound (ZJU-37 or Nec1) for 72 hours (DMSO in control wells), and then cells were separately harvested for immunoblot hybridization with the indicated antibodies.

FIG. 9B Jurkat FADD-/-cells plated in six well plates 2 hours later, treated simultaneously with TNF α (30ng/ml) and drug (ZJU-37 or Nec1) (10uM) for 4 hours (control wells in DMSO), and cells were collected separately for immunoblot hybridization with the indicated antibodies.

The results of the experiment are shown in FIG. 9:

a, picture A: under the condition that NLRP3 is induced to rise by TZS, ZJU-37 can effectively degrade NLRP3 protein induced by inflammation and has better effect than the positive drug Nec 1.

B picture, under the condition that NLRP3 is induced to rise by TNF α, ZJU-37 can effectively degrade NLRP3 protein induced by inflammation, and the effect is better than that of the positive drug Nec 1.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. The use of a compound of formula I,

characterized in that said compound is used for the preparation of:

(a) drugs for preventing or treating RIP1 and/or RIP 3-related diseases;

(b) an agent for preventing or treating a disease associated with apoptosis;

(d) a medicament for preventing or treating NLRP3 protein-related diseases;

(e) a medicament for preventing or treating liver injury.

2. The use of a compound of formula I,

characterized in that the compound has one or more of the following uses:

(1) inhibiting apoptosis;

(2) inhibition of RIP1 kinase activity and RIP3 kinase activity;

(3) reducing the interaction of RIP1 and RIP 3;

(4) degrading TDP25 protein;

(5) reducing binding of RIP1 and RIP 3;

(6) degrading NLRP3 protein.

3. The use according to claim 1 or 2, wherein the cell is selected from the group consisting of: Jurkat-FADD-/-cells, U937 cells, BV2 cells, HT-29 cells.

4. The use of claim 1 or 2, wherein the liver injury is toxin-induced acute liver injury.

5. The use of claim 4, wherein the toxin is LPS.

6. The use of claim 1, where RIP1 and/or RIP 3-related disease is selected from the group consisting of: neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), etc.), ischemic injuries, autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.), atherosclerosis, psoriasis, gaucher's disease, pain (neuropathic pain), inflammation (pancreatitis, ulcerative enteritis, hepatitis), retinal detachment, tumors (e.g. melanoma, brain glioma, colon cancer, glia, lymphoma, T-cell leukemia).

7. The use of claim 1, wherein the diseases associated with apoptosis are selected from the group consisting of: neurodegenerative diseases (ALS, AD, PD, MS, etc.), ischemic injury, autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.), atherosclerosis, psoriasis, gaucher's disease, pain (neuropathic pain), inflammation (pancreatitis, ulcerative enteritis, hepatitis), retinal detachment, tumors (e.g., melanoma, brain glioma, colon cancer, glioma, lymphoma, T cell leukemia).

8. The use according to claim 1, wherein the TDP25 protein-related disease is selected from the group consisting of: neurodegenerative diseases (such as ALS).

9. The use according to claim 1, wherein the NLRP3 protein related disease is selected from the group consisting of: alzheimer's disease, systemic lupus erythematosus, inflammatory bowel disease, atherosclerosis, type 2 diabetes, gout, obesity, and tumors.

10. Use according to claim 1 or 2, wherein the compound of formula I is administered at a concentration of 1-50 μ Μ; preferably, it is 5 to 20. mu.M.