CN111228273A

CN111228273A - Necrotic apoptosis inhibitor GNF-7 and its use as a medicament

Info

Publication number: CN111228273A
Application number: CN202010133088.XA
Authority: CN
Inventors: 王中良
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-03-01
Filing date: 2020-03-01
Publication date: 2020-06-05
Anticipated expiration: 2040-03-01
Also published as: CN111228273B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a necrotic apoptosis inhibitor GNF-7 and application thereof as a medicine, in particular to application thereof in preparing necrotic apoptosis inhibitors and medicines for resisting tumors, ischemic or cisplatin-induced acute kidney injury, Alzheimer disease, ischemic cardiomyopathy, ischemic stroke, atherosclerosis, acute pancreatitis, inflammatory bowel disease, sepsis, salmonella infection, Listeria infection, vaccinia virus infection and other inflammation and infection related diseases.

Description

Necrotic apoptosis inhibitor GNF-7 and its use as a medicament

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a necrotic apoptosis inhibitor GNF-7 and application thereof as a medicine.

Background

Necrotic apoptosis is a recently discovered regulated mode of programmed cell death that is morphologically characterized by necrosis. Research shows that protein kinases RIPK1 and RIPK3 form a complex and activate MLKL protein, and MLKL is polymerized to be directly positioned on a cell membrane and cause the cell membrane to break, so that the key mechanism for inducing programmed cell necrosis is realized. Necrotic apoptosis can cause infiltration of a large number of inflammatory cells in the body due to the release of cellular contents, thereby inducing a severe inflammatory response. Necrotic apoptosis, as a new type of programmed cell death mode, plays an important role in the pathophysiological processes of various diseases such as ischemic injury, acute kidney injury, neurodegenerative disease, malignant tumor, viral infection, immunological disease and the like. The identification and discovery of small molecule inhibitors of necrotic apoptosis are of great importance in the clinical treatment of diseases associated with necrotic apoptosis.

Chinese alias of GNF-7: n- [3- [1, 4-dihydro-1-methyl-7- [ (6-methyl-3-pyridyl) amino group]-2-oxopyrimidino [4,5-D]Pyrimidin-3 (2H) -yl]-4-methylphenyl radical]-3- (trifluoromethyl) benzamide of formula C₂₈H₂₄F₃N₇O₂The inhibitor is a potent II-type kinase Bcr-Abl inhibitor, and the application of the inhibitor which is directly used as an inhibitor of RIPK1 and RIPK3 kinases to resist the necrosis and apoptosis is not reported at present.

Disclosure of Invention

In view of the existing problems, the invention provides a necrotic apoptosis inhibitor GNF-7 and application thereof as a medicament, and aims to provide a novel anti-necrotic apoptosis inhibitor, in particular to a compound GNF-7 and a pharmaceutically acceptable salt thereof, wherein the compound can be used as an inhibitor of RIPK1 and RIPK3 kinase to play an anti-necrotic apoptosis activity. The invention also provides a new application of the compound GNF-7 in preparing medicaments for treating inflammation and infection related diseases such as tumors, acute kidney injury, Alzheimer disease, ischemic cardiomyopathy, ischemic stroke, atherosclerosis, acute pancreatitis, inflammatory bowel disease in children, sepsis, salmonella infection, Listeria infection, vaccinia virus infection and the like.

The invention obtains the necrotic apoptosis inhibitor with a brand-new structure by screening the self compound library.

The specific technical scheme of the invention is as follows:

in a first aspect of the present invention, a compound GNF-7 and pharmaceutically acceptable salts thereof, having the structure shown in formula (i), is: use of N- [3- [1, 4-dihydro-1-methyl-7- [ (6-methyl-3-pyridinyl) amino ] -2-oxopyrimidino [4,5-D ] pyrimidin-3 (2H) -yl ] -4-methylphenyl ] -3- (trifluoromethyl) benzamide or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament against cell necrosis;

the compound of the invention can be prepared into a form of medicinal salt according to a conventional method, and comprises organic acid salt and inorganic acid salt thereof: inorganic acids include, but are not limited to, hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, nitric acids, and the like, and organic acids include, but are not limited to, acetic, maleic, fumaric, tartaric, succinic, lactic, p-toluenesulfonic, salicylic, oxalic, and the like.

In a preferred embodiment, the compound shown in the formula (I) or the isomer, solvate or precursor thereof, or the pharmaceutically acceptable salt thereof has a drug action target of RIPK1/RIPK3, and inhibits cell necrosis by inhibiting the kinase activity of RIPK1/RIPK 3.

In another aspect of the invention, there is provided the use of a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for RIPK1/RIPK3 inhibitors.

In another aspect of the invention there is provided the use of a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for a disease associated with a RIPK1/RIPK3 kinase disorder, overactivation or overactive interaction.

In a preferred embodiment, the RIPK1/RIPK3 kinase disorder, overactivation or overactive interaction-related disease is an inflammatory, infectious, ischemic and degenerative related disease or tissue injury.

In another preferred embodiment, the inflammatory, infectious, ischemic and degenerative related diseases are mediated by RIPK1/RIPK3 kinase, or are triggered by cellular necrosis (inhibition of inflammatory and infectious related diseases by inhibition of cellular necrosis).

In another preferred embodiment, the inflammatory, infectious, ischemic and degenerative related diseases include, but are not limited to: tumor, Alzheimer's disease, ischemic cardiomyopathy, ischemic stroke, atherosclerosis, acute pancreatitis, inflammatory bowel disease in children, sepsis, Salmonella infection, Listeria infection, or vaccinia virus infection.

In another preferred embodiment, the tumor includes cancers of the esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system, as well as thyroid cancer, leukemia, hodgkin's disease, lymphoma and myeloma.

In another aspect of the present invention, there is provided a method of inhibiting cell necrosis, comprising: treating cells with a compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the method of inhibiting cell necrosis is a method that does not aim at treating a disease; such as in vitro cell culture methods.

In a preferred embodiment, the compound shown in the formula (I) or the isomer, solvate or precursor thereof, or the pharmaceutically acceptable salt thereof has excellent anti-necrotic apoptosis effect when the cells are treated in vitro.

In a preferred embodiment, the compound shown in the formula (I) or an isomer, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inhibiting the systemic inflammatory syndrome (SIRS) caused by TNF- α, acetaminophen (APAP) -induced hepatotoxicity, cisplatin-induced acute kidney injury, ischemic kidney injury and breast cancer metastasis.

In another aspect of the present invention, there is provided a method for preventing, alleviating or treating inflammatory, infectious, ischemic and degenerative related diseases, comprising: a compound represented by the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof is administered to a subject in need of prevention, alleviation or treatment of inflammation and infectious related diseases.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 is a graph showing the results of screening an inhibitor against necrotic apoptosis using a self-constructed drug library in example 1 of the present invention;

FIG. 2 is a graph of the inhibitory effect of GNF-7 on HT-29 necrotic apoptosis after TSZ stimulation at various concentrations in example 2 of the present invention;

FIG. 3 is a graph showing the effect of GNF-7 on the inhibitory activity of necroptosis in different cells in example 3 of the present invention;

FIG. 4 is a graph showing the effect of GNF-7 on the inhibition of necrotic apoptosis by different stimulators in example 4 of the present invention;

FIG. 5 is a graph showing that GNF-7 inhibits phosphorylation of RIPK1 and RIPK3 and prevents interaction thereof in example 5 of the present invention;

FIG. 6 is a graph showing GNF-7 binding target confirmation in example 6 of the present invention;

FIG. 7 is a graph of GNF-7 inhibiting kinase activity of RIPK1 and RIPK3 in example 7 of the present invention;

FIG. 8 is a graph of global immune syndrome (SIRS) induced by TNF- α in mice protected by GNF-7 in example 8 of the present invention;

FIG. 9 is a graph of the in vivo protection of GNF-7 from acetaminophen (APAP) -induced hepatotoxicity in mice in example 9 of the present invention;

FIG. 10 is a graph showing that the inhibitor of necrotic apoptosis GNF-7 protects Cisplatin-induced acute kidney injury in vivo in example 10 of the present invention;

FIG. 11 is a graph showing that the necrotic apoptosis inhibitor GNF-7 inhibits lung metastasis of mouse breast cancer tumor in example 11 of the present invention.

Detailed Description

The following description is of preferred embodiments of the present invention, and it should be understood that the preferred embodiments described herein are for purposes of illustration only and are not intended to limit the invention.

Example 1

Screening of anti-necrotic apoptosis inhibitors from the own compound library:

each compound was dissolved in DMSO to 10mM stock solution, human colon cancer cells HT-29 were plated in 96-well plates and pre-stimulated with 10. mu.M of one compound for 30min per well, and then necrotic apoptotic stimulation was induced by TNF- α, Smac mimetic and z-VAD-fmk (TSZ) for 6 h, after which cell survival was determined by assay for activated cell ATP content (using Promega corporation, Inc.; see FIGS.; for 6 h)

Luminescab Cell vitality Assay kit test catalog number: g7570) In that respect

As shown in fig. 1, the results indicate that most of the 120 compounds screened have no protective effect on apoptosis induced by TSZ stimulation, and only GNF-7 significantly inhibited apoptosis induced by TSZ stimulation.

Example 2

GNF-7 has anti-HT-29 cellular necrosis activity:

we further examined the protective effect of GNF-7 on TNF- α -induced necroptosis in HT-29 cells at various concentrations We found that GNF-7 concentration-dependent inhibition of TSZ-induced necroptosis, with its EC₅₀0.1 μ M (FIG. 2A) while, at concentrations of 1-100 μ M, GNF-7 had no significant toxic effect on HT-29 cells (FIG. 2B). furthermore, GNF-7 had no protective effect on TNF- α and Smac mimetic (TS) induced apoptosis (FIG. 2C).

Example 3

GNF-7 has inhibitory activity against necrotic apoptosis in different cells:

the inhibitory activity of GNF-on necrotic apoptosis in different cell lines induced by TNF- α was examined using another human-derived cell line (U937) and a mouse-derived cell line (L929), wherein U937 cells were stimulated with TNF- α, Smac mimetic and z-VAD-fmk (TSZ) for 6 hours and L929 cell lines were stimulated with TNF- α and z-VAD-fmk (TZ) for 4 hours, the results are shown in FIG. 3, and GNF-7 concentration-dependently inhibited the occurrence of necrotic apoptosis in both cell lines.

Example 4

GNF-7 has inhibitory activity on necrotic apoptosis induced by different stimulators:

using mouse macrophage cell line (J774.1), the inhibitory activity of GNF-7 on different stimulators causing necrotic apoptosis was examined. Mouse J774.1 cells were stimulated for necrotic apoptosis with LPS (0.1. mu.g/ml) or PolyI: C (100ng/ml) plus z-VAD-fmk for 12 hours before cell survival was determined by measuring ATP content in viable cells (Promega corporation)

Luminescab Cell vitality Assay kit test catalog number: g7570) In that respect As shown in FIG. 4, GNF-7 inhibited the LPS-and PolyI: C-induced necroptosis of the cells.

Example 5

GNF-7 inhibits necrotic apoptosis by inhibiting phosphorylation of RIPK1 and RIPK 3:

HT-29 cells were treated with GNF-7 (1. mu.M) for 30min, and the expression of RIPK1, RIPK3, MLKL and its phosphorylated protein was examined by stimulating with TSZ at different time points, as shown in FIG. 5A, GNF-7 could significantly inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, but had no effect on the expression level of RIPK1, RIPK3 and MLKL protein itself. Further, using the co-immunoprecipitation method (IP), GNF-7 was examined to inhibit the interaction of RIPK1 and RIPK3 after TSZ necrosis activation, and the results showed that GNF-7 or KW-2449 could directly inhibit the interaction of intracellular RIPK1 and RIPK3 after 4h of TSZ treatment (FIG. 5B).

Example 6

Target validation of the necrotic apoptosis inhibitor GNF-7:

drug affinity response target stability assays (DARTS) were used to detect potential interactions between GNF-7 and RIPK1/3 kinase, relying on a decrease in protease sensitivity of the target protein to drug binding. As shown in FIG. 6, both RIPK1 and RIPK3 proteins were protected by digestion with 0.01% protease, whereas no protection of MLKL was detected in the same samples, in extracts of cells of GNF-7, suggesting that GNF-7 may interact with RIPK1 and RIPK 3.

Example 7

GNF-7 inhibits the kinase activity of RIPK1 and RIPK 3:

further in vitro by ligand kinase binding assay (KinomeScan, Ambit Co., Ltd.)^TM) The inhibitory effect of GNF-7 on the binding ability of RIPK1 and RIPK3 kinase to substrates was examined. As shown in fig. 7A and B, GNF-7 inhibited RIPK1 and RIPK3 kinase activity with Kd values of 60nm and 157nm, respectively.

Example 8

The necrotic apoptosis inhibitor GNF-7 protects mice in vivo against TNF-induced systemic immune syndrome (SIRS):

mice were given GNF-7(10mg/kg) by gavage 3 hours prior to model establishment and then induced to develop SIRS by intravenous injection of mTNF- α (400 μ g/kg) plus intraperitoneal injection of z-VAD-fmk (200 μ g), GNF-7 significantly protected mice from death (fig. 8A). furthermore, when these mice were examined at 6 hours, the concentration levels of IL-6 in serum were significantly reduced by GNF-7 treatment (fig. 8B). these results thus indicate that GNF-7 protected mice in vivo from TNF- α -induced SIRS.

Example 9

Necrotic apoptosis inhibitor GNF-7 protects against acetaminophen (APAP) -induced liver damage in vivo

Previous studies showed that RIPK1 is essential for APAP to induce hepatotoxicity, and thus the effect of GNF-7 on tissue damage and inflammation in this model was further investigated. Liver injury in mice was induced by injection of acetaminophen (APAP,300mg/kg) into the mice. As shown in fig. 9A, mouse liver cells were significantly necrotic and their serum levels of aspartate Aminotransferase (AST) were elevated (fig. 9B). The inventors determined that gavage of GNF-7(10mg/kg) in mice significantly reduced liver necrosis (fig. 9A) and plasma AST levels (fig. 9B) in mice. Overall, the results of this example demonstrate that GNF-7 is able to protect against acetaminophen (APAP) -induced liver injury and in vivo inflammation.

Example 10

The necrotic apoptosis inhibitor GNF-7 protects acute kidney injury induced by Cisplatin in vivo:

it has been reported in the literature that necrotic apoptosis is also involved in acute kidney injury induced by Cisplatin (cissplatin), a tumor chemotherapeutic drug. This example further examines the protective effect of the necrotic apoptosis inhibitor GNF-7 on cisplatin-induced acute kidney injury.

GNF-7(10mg/kg) was first intragastrically administered to mice, then 25mg/kg of cisplatin was intraperitoneally injected, and kidney tissue and serum were removed 48 hours later to examine the degree of loss. Wherein GNF-7 was gavaged every 24 hours. As shown in fig. 10A and B, cisplatin induced significant pathological necrosis of the kidney, while GNF-7 administration significantly inhibited the renal necrosis induced. Meanwhile, GNF-7 significantly inhibited the increase of Creatinine (Creatinine) and urea nitrogen (BUN) in mouse serum due to cisplatin stimulation (fig. 10C and D). Thus, the necrotic apoptosis inhibitor GNF-7 was able to protect against acute kidney injury induced by Cisplatin in vivo.

Example 11

The necrotic apoptosis inhibitor GNF-7 can inhibit lung metastasis of mouse breast cancer tumor:

it has been reported in the literature that necrotic apoptosis promotes lung metastasis of breast cancer tumors in mice. The embodiment of the invention further detects the inhibition effect of the necrotic apoptosis inhibitor GNF-7 on the lung metastasis of the mouse breast cancer tumor. We injected MVT-1 breast cancer cells into the mammary gland of FVB/N mice and examined the level of lung metastasis of breast cancer 4 weeks later. Among them, GNF-7(10mg/kg) was gavaged to mice 2 weeks after breast cancer cell injection and administered once every 2 days. And detecting the lung metastasis level of the breast cancer of the mouse at 4 weeks. As shown in fig. 11A and B, GNF-7 significantly inhibited lung metastasis of mouse breast cancer. Therefore, the necrotic apoptosis inhibitor GNF-7 can inhibit lung metastasis of mouse breast cancer tumors.

The experimental results show that the compound GNF-7 has excellent anti-cell necrosis activity, can be used as an RIPK1 or RIPK3 inhibitor, can resist TNF-induced systemic immune syndrome (SIRS) and acetaminophen (APAP) induced hepatotoxicity, and is used for preparing medicaments for resisting inflammation or infection related diseases such as tumors, Alzheimer diseases, ischemic cardiomyopathy, ischemic stroke, atherosclerosis, acute pancreatitis, inflammatory bowel disease of children, sepsis, Salmonella infection, Listeria infection and vaccinia virus infection.

Therefore, the compound and the salt thereof can be used for preparing the anti-necrotic apoptosis inhibitor.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The compound shown in the formula (I) is: use of N- [3- [1, 4-dihydro-1-methyl-7- [ (6-methyl-3-pyridinyl) amino ] -2-oxopyrimidino [4,5-D ] pyrimidin-3 (2H) -yl ] -4-methylphenyl ] -3- (trifluoromethyl) benzamide or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament against cell necrosis;

2. the use as claimed in claim 1, wherein the compound is prepared in the form of a pharmaceutically acceptable salt according to conventional methods, including organic and inorganic acid salts thereof: inorganic acids include, but are not limited to, hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, nitric acids, and the like, and organic acids include, but are not limited to, acetic, maleic, fumaric, tartaric, succinic, lactic, p-toluenesulfonic, salicylic, oxalic acids.

3. The use of claim 1, wherein the compound of formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, targets RIPK1/RIPK3 for inhibiting cellular necrosis by inhibiting RIPK1/RIPK3 kinase activity.

4. The application of the compound shown in the formula (I) or isomers, solvates or precursors thereof, or pharmaceutically acceptable salts of the compounds and the isomers, the solvates or the precursors in preparation of RIPK1/RIPK3 inhibitor medicines.

5. The application of the compound shown in the formula (I) or an isomer, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof in preparing medicines for treating diseases related to RIPK1/RIPK3 kinase disorder, over-activation or over-interaction.

6. The use of claim 5, wherein the RIPK1/RIPK3 kinase disorder, overactivation or overactive interaction related disease is an inflammatory, infectious, ischemic and degenerative related disease or tissue injury.

7. The use of claim 6, wherein the inflammatory, infectious, ischemic, and degenerative related diseases are mediated by RIPK1/RIPK3 kinase, or are triggered by cellular necrosis.

8. The use of claim 7, wherein the inflammatory, infectious, ischemic and degenerative related diseases include, but are not limited to: tumor, Alzheimer's disease, ischemic cardiomyopathy, ischemic stroke, atherosclerosis, acute pancreatitis, inflammatory bowel disease in children, sepsis, Salmonella infection, Listeria infection, or vaccinia virus infection.

9. The use of claim 8, wherein said tumor comprises cancer of the esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system, thyroid, leukemia, hodgkin's disease, lymphoma and myeloma.

10. The compound shown in the formula (I) or an isomer, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof, and the application of the compound shown in the formula (I) or the isomer, the solvate or the precursor thereof in preparing a medicament for inhibiting the systemic inflammatory syndrome (SIRS) caused by TNF- α, acetaminophen (APAP) -induced hepatotoxicity, cisplatin-induced acute kidney injury, ischemic kidney injury and breast cancer metastasis.