A compound of marine origin, Cpd-8, and its use in the preparation of a medicament for the treatment of TNF-induced cell death

Abstract

The application discloses a marine-derived compound Cpd-8 and application thereof in preparation of a medicine for resisting TNF-induced cell death. Through screening of an extract library of marine-derived sponge symbiotic fungi of a research group, a small-molecule inhibitor with a novel structure is obtained, which can resist TNF-induced cell death, and specifically, the marine-derived sesquiterpene lactone nitrobenzene analogue Cpd-8 and a pharmaceutically acceptable salt thereof. The compound can inhibit TNF-induced cell death and formation of a complex II in a signal pathway, thereby exerting an anti-cell death activity. In-vitro cell experiment results show that the compound of the application can significantly inhibit TNF-induced cell death. In-vivo animal experiment results show that the compound of the application can inhibit TNF-alpha / D-GaIN (D-galactosamine)-induced acute liver injury, and significantly prolong the survival period of mice.

Description

Technical Field

[0001] This invention relates to a marine-derived bioactive small molecule compound, specifically to a marine-derived compound Cpd-8 and its application in the preparation of drugs against TNF-induced cell death. It belongs to the field of pharmaceutical technology. Background Technology

[0002] Apoptosis is programmed cell death that does not induce an inflammatory response, and it is induced by two pathways: endogenous and exogenous. The endogenous pathway is induced by various intracellular stress stimuli and activated by oligomers of B-cell lymphoma-2 (BCL-2) family proteins BAK and BAX. BAK / BAX oligomers form pores in the outer mitochondrial membrane, promoting the release of cytochrome c into the cytoplasm and recruiting procaspase-9 to form apoptotic bodies, which in turn activate effectors caspase-3 and -7 to induce apoptosis. The exogenous pathway is induced by cell membrane death receptors (such as TNF-R1) or Toll-like receptors (TLRs) to form the death-inducing signaling complex (DISC), including the tumor necrosis factor receptor-associated death domain (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIPK1), and procaspase-8. Activated caspase-8 can also activate effectors caspase-3 and -7 to induce apoptosis, or participate in the endogenous apoptosis pathway by hydrolyzing BID protein.

[0003] Necroptosis, also known as programmed cell death, is a caspase-independent mode of programmed cell death that can be triggered by members of the tumor necrosis factor (TNF) receptor superfamily: TNF-R1, FAS, DR3, TRAILR1, TRAILR2, and DR6, Toll-like receptors 3 / 4, interferon receptors, and Z-DNA binding protein (ZBP1). Among these, TNF-α-induced necroptosis is the most prevalent: TNF-α binds to TNFR1 on the cell membrane surface and induces a conformational change, recruiting TRADD, RIPK1, and TNF-associated factor (TRAF) to form complex I. When ubiquitination of the components in complex I is inhibited, they are released from the cell membrane into the cytoplasm to form complex II, triggering caspase-8-induced apoptosis. When caspase-8 is inactivated, RIPK1 in complex II binds to and phosphorylates RIPK3. Phosphorylated RIPK3 recruits and phosphorylates activated mixed lineage kinase domain-like proteins (MLKL), and the three form a necrosome. After MLKL polymerizes, it translocates to the inner side of the cell membrane, leading to necrotic apoptosis through ion influx, cell swelling, and membrane lysis, and releasing a series of damage-associated signaling molecules (DAMPs), thereby inducing a severe inflammatory response in vivo.

[0004] Studies have shown that apoptosis and necroptosis signals are intertwined and interconnected. Their overactivation plays a crucial role in the pathophysiology of various diseases, including cardiovascular diseases (such as atherosclerosis and ischemic heart disease), ischemia-reperfusion injury (such as myocardial infarction and stroke), neurological diseases (such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis), acute organ injuries (such as acute lung injury, acute kidney injury, and acute liver injury), intestinal diseases (such as ulcerative colitis, inflammatory bowel disease, and Crohn's disease), liver diseases (such as non-alcoholic fatty liver disease and chronic hepatitis), and inflammatory dysregulation diseases (such as systemic inflammatory response syndrome and sepsis).

[0005] The marine environment, characterized by high salinity, high pressure, low temperature, and low nutrient levels, gives marine organisms distinct features in metabolism, survival strategies, information transmission, and adaptation mechanisms. Marine microorganisms, as a vital component of marine biodiversity, have, through long-term biological evolution, developed genetic and metabolic diversity significantly different from terrestrial organisms. This diversity significantly increases the likelihood of marine microorganisms producing drug prodrug compounds with unique structures and excellent activities. Therefore, marine microorganisms are a natural treasure trove for new drug development. However, no studies have yet reported on marine-derived bioactive substances targeting and inhibiting multiple TNF-induced death pathways. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a marine-derived compound Cpd-8 and its application in the preparation of drugs against TNF-induced cell death.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] 1. A marine-derived compound Cpd-8 and its pharmaceutically acceptable salt, wherein the compound Cpd-8 is named 9α,14-dihydroxy-6β-p-nitrobenzoylcinnamyl lactone, and its structural formula is shown in Formula (I):

[0009]

[0010] Preferably, the pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt.

[0011] More preferably, the organic acids include, but are not limited to: acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, and oxalic acid.

[0012] More preferably, the inorganic acid includes, but is not limited to: hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, and nitric acid.

[0013] 2. The use of compound Cpd-8 or a pharmaceutically acceptable salt thereof in the preparation of drugs against TNF-induced cell death.

[0014] 3. Application of compound Cpd-8 or a pharmaceutically acceptable salt thereof in the preparation of therapeutic drugs for TNF-induced cell death-related diseases.

[0015] Preferably, TNF-induced cell death-related diseases include, but are not limited to: cardiovascular diseases, ischemia-reperfusion injury, nervous system diseases, acute organ injury, intestinal diseases, liver diseases, and inflammatory dysregulations.

[0016] Further preferred options include cardiovascular diseases such as atherosclerosis and ischemic heart disease; ischemia-reperfusion injury such as myocardial infarction and stroke; neurological diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis; acute organ injuries such as acute lung injury, acute kidney injury, and acute liver injury; intestinal diseases such as ulcerative colitis, inflammatory bowel disease, and Crohn's disease; liver diseases such as non-alcoholic fatty liver disease and chronic hepatitis; and inflammatory disorders such as systemic inflammatory response syndrome and sepsis.

[0017] 4. A composition comprising the aforementioned compound Cpd-8 or a pharmaceutically acceptable salt thereof.

[0018] 5. Application of the aforementioned composition in the preparation of drugs against TNF-induced cell death.

[0019] 6. Application of the aforementioned composition in the preparation of therapeutic drugs for TNF-induced cell death-related diseases.

[0020] 7. A drug for inhibiting TNF-induced cell death, wherein the active ingredient is the aforementioned compound Cpd-8 or a pharmaceutically acceptable salt thereof.

[0021] 8. A therapeutic agent for TNF-induced cell death-related diseases, wherein the active ingredient is the aforementioned compound Cpd-8 or a pharmaceutically acceptable salt thereof.

[0022] 9. A drug for inhibiting TNF-induced cell death, comprising the aforementioned composition.

[0023] 10. A therapeutic agent for TNF-induced cell death-related diseases, comprising the aforementioned composition.

[0024] The beneficial effects of this invention are:

[0025] This invention obtains active substances through fermentation, extraction, and purification by marine symbiotic fungi, and discovers and identifies small molecule inhibitors targeting TNF-induced cell death, which has important research significance and social value for the clinical treatment of related diseases.

[0026] This invention, through screening an extract library of marine sponge epiphytic fungi from our research group, yielded a novel small-molecule inhibitor capable of combating TNF-induced cell death. Specifically, it is the marine-derived sesquiterpene lactone nitrobenzene analog Cpd-8 and its pharmaceutically acceptable salt. This compound inhibits TNF-induced cell death and the formation of complex II in the signaling pathway, thereby exerting anti-cell death activity. In vitro cell experiments showed that the compound significantly inhibited TNF-induced cell death. In vivo animal experiments showed that the compound inhibited TNF-α / D-GaIN (D-galactosamine)-induced acute liver injury and significantly prolonged the survival of mice.

[0027] The compounds and their salts of this invention exhibit excellent anti-TNF-induced cell death activity. Therefore, the compounds and their salts of this invention can be used to treat diseases related to TNF-induced cell death, such as cardiovascular diseases (e.g., atherosclerosis and ischemic heart disease), ischemia-reperfusion injury (e.g., myocardial infarction and stroke), nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis), acute organ injuries (e.g., acute lung injury, acute kidney injury, acute liver injury), intestinal diseases (e.g., ulcerative colitis, inflammatory bowel disease, Crohn's disease), liver diseases (e.g., non-alcoholic fatty liver disease and chronic hepatitis), and inflammatory disorders (e.g., systemic inflammatory response syndrome and sepsis). Attached Figure Description

[0028] Figure 1 Structural identification of Cpd-8, wherein (A) Cpd-8 in deuterated methanol 1 H-NMR spectrum. (B) Cpd-8 in deuterated methanol. 13 C-NMR and DEPT spectra.

[0029] Figure 2 The screening of the necroptosis inhibitor Cpd-8 included the following steps: (A) Schematic diagram of the extraction of crude metabolic extracts from sponge symbiotic fungi. Sponge symbiotic fungi were isolated from sponges and cultured in solid culture medium to obtain 88 crude metabolic extracts. (B) Schematic diagram of the screening process for necroptosis inhibitors from the crude metabolic extracts. (C) HT29 cells were pretreated with each crude extract (10 mg / mL) for 30 min, and then stimulated with TSZ (20 ng / mL TNF-α, 10 nM Smac analog, 20 μM Z-VAD-FMK) for 6 h to induce necroptosis. (D) Crude extract No. 70 was fractionated into 11 fractions (F1-F11) by silica gel column chromatography. Each fraction was pretreated with HT29 cells for 30 min, and then stimulated with TSZ for 6 h to induce necroptosis. (E) Nine compounds were isolated and purified from fraction F5. Each compound was pretreated with HT29 cells for 30 min, and then stimulated with TSZ for 6 h to induce necroptosis. (F)HT29 cells were pretreated with Cpd-8 (10 μM) for 30 min, followed by TSZ stimulation for 6 h to induce necrosis and apoptosis. Cell viability was determined using a cell viability assay kit. The EC50 for Cpd-8 inhibition of necrosis and apoptosis was 6.252 μM. Significance level: *, P < 0.05; ***, P < 0.001.

[0030] Figure 3To demonstrate the protective effect of Cpd-8 against TNF-α-induced cell death, the following methods were employed: (A) HT29 cells were pretreated with different concentrations of Cpd-8 and then stimulated with TCZ (20 ng / mL TNF-α, 5 μg / mL actinomycin, and 20 μM Z-VAD-FMK) for 6 h. (B) HT29 cells were pretreated with different concentrations of Cpd-8 for 30 min and then stimulated with TS (20 ng / mL TNF-α, 100 nM Smac mimic) or (C) TC (20 ng / mL TNF-α, 5 μM CHX) for 16 h. (D) MEF cells were pretreated with different concentrations of Cpd-8 and then stimulated with TSZ or (E) TCZ for 4 h. (F) MEF cells were pretreated with different concentrations of Cpd-8 for 30 min and then stimulated with TS or (G) TC for 8 h. Cell viability was determined using a cell viability assay kit. Significance levels: *, P<0.05; **, P<0.01 and ***, P<0.001.

[0031] Figure 4 To inhibit cell death induced by death receptors and Toll-like receptors induced by Cpd-8, the following methods were used: (A) HT29 cells were pretreated with different concentrations of Cpd-8 and then stimulated with TRAIL (100 ng / mL) + SZ for 6 h; or (B) TRAIL plus Smac mimicry was used for 12 h. (C) MDA-MB-231 cells were pretreated with different concentrations of Cpd-8 for 30 min and then stimulated with TRAIL plus Smac mimicry for 12 h. (D) HT29 cells were pretreated with different concentrations of Cpd-8 and then stimulated with FasL (100 ng / mL) + SZ for 6 h. (E) BMDM cells were pretreated with 5 μM Cpd-8 and then treated with the corresponding stimulant for 6 h. Cell viability was determined using a cell viability assay kit. Significance levels: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

[0032] Figure 5Cpd-8 showed no protective effect against endogenous apoptosis, necroptosis, or ferroptosis. The following cell types were tested: (A) HT-29 cells were pretreated with different concentrations of Cpd-8 for 30 min, then stimulated with cisplatin (300 μM) or (B) STS (0.2 μM) for 8 h. (C) HeLa cells were pretreated with different concentrations of Cpd-8 for 30 min, then stimulated with ABT737 (50 μM) for 8 h. (D) HT-29 cells were pretreated with different concentrations of Cpd-8 for 30 min, then stimulated with RSL3 (10 μM) for 4 h. (E) MEF cells were pretreated with different concentrations of Cpd-8 for 30 min, then stimulated with cisplatin (300 μM) or (F) STS (0.2 μM) for 8 h. (G) MEF cells were pretreated with different concentrations of Cpd-8 for 30 min, then stimulated with RSL3 (10 μM) for 4 h. (H)MEF cells were pretreated with different concentrations of Cpd-8 for 30 min, and then stimulated with CBL0137 (50 μM) for 20 h. Cell viability was determined using a cell viability assay kit. Significance level: NS indicates no significance.

[0033] Figure 6 The protective effect of Cpd-8 was independent of the NF-κB signaling pathway, in which (A) HT29 cells were treated with Cpd-8 (10 μM) at different time points. Cells were lysed and immunoblotted with IκBα antibody. (B) HT29 or (C) MEF cells were pretreated with Cpd-8 (10 μM) for 30 min, and then stimulated with TNF-α (20 ng / mL) at different time points. Cells were lysed and immunoblotted with IκBα antibody. (D) MEF cells were pretreated with Cpd-8 (10 μM) for 30 min, and then stimulated with TS at different time points. Cells were lysed and immunoblotted with the antibody shown. (E) p65- / -MEF cells were pretreated with Cpd-8 (10 μM) for 30 min, and then stimulated with TNF-α or TZ for 6 h. Cell viability was determined by a cell viability assay kit. Significance level: ***, P < 0.001.

[0034] Figure 7 To inhibit the TNF-α-induced cell death signaling pathway, Cpd-8 was used to pretreat (A) HT29 cells or (B) MEF cells with Cpd-8 (10 μM) for 30 min, followed by stimulation with TSZ at different time points. Cells were lysed and immunoblotted with the antibodies shown. (C) MEF cells were pretreated with Cpd-8 (10 μM) for 30 min, followed by stimulation with TS or (D) TC at different time points. Cells were lysed and immunoblotted with the antibodies shown.

[0035] Figure 8To investigate the effect of Cpd-8 on the formation of the necroptotic complex, the following methods were used: (A) HT29 cells were pretreated with Cpd-8 (10 μM) for 30 min, followed by stimulation with TSZ for 4 h. Cell lysates were immunoprecipitated using RIPK1 antibody (IP: RIPK1), and Western blot analysis was performed using different antibodies. (B) HT29 cells were pretreated with Cpd-8 (10 μM) for 30 min, followed by stimulation with TSZ for 2 and 4 h. Cell lysates were immunoprecipitated using caspase-8 antibody (IP: caspase-8), and Western blot analysis was performed using different antibodies. (C) HT29 cells were pretreated with Cpd-8 (10 μM) for 30 min, followed by stimulation with TSZ for 1 and 2 h. Cell lysates were immunoprecipitated using TNFR1 antibody (IP: TNFR1), and Western blot analysis was performed using different antibodies. (D) HT29 cells were pretreated with Cpd-8 (10 μM) for 30 min, followed by stimulation with TSZ for 2.5 h. Cell lysates were subjected to serial immunoprecipitation assays. First IP: TNFR1 complex I was immunoprecipitated using TNFR1 antibody. Second IP: The remaining lysates were immunoprecipitated again using TNFR1 antibody. Third IP: The remaining lysates were immunoprecipitated using TRADD antibody. The immunoprecipitated complexes were detected using the indicated antibodies.

[0036] Figure 9 To protect mice from TNF-α / D-GalN-induced acute liver injury using Cpd-8, C57BL / 6J mice were pretreated with Cpd-8 (15 mg / kg) intraperitoneally for 1 h, followed by acute liver failure induced by D-GalN (600 mg / kg) and mTNF-α (15 μg / kg). Body temperature (mean ± SD) was recorded, and survival curves were plotted (B). (C) Representative images of dissected mouse livers (n=5) 4 h after induction of acute liver failure. (D) Representative images of H&E staining of mouse liver sections (n=5). (E) TUNEL staining of mouse liver sections (n=5). (F) Statistical graph of the percentage of TUNEL-positive cells to total cells in the same field of view (n=5). (G) Serum AST and (H) ALT levels were measured using the corresponding detection kits (n=5). Significance level: ***, P<0.001.

[0037] Figure 10 The preparation method and mechanism of action of Cpd-8. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0039] Experimental materials:

[0040] Cells, culture media, and animals: HT-29 (American Cell Culture Collection Center ATCC), MDA-MB-231 (American Cell Culture Collection Center ATCC), HeLa (American Cell Culture Collection Center ATCC), HEK293T (American Cell Culture Collection Center ATCC), MEF, MEF RIPK1 - / - (Provided by Professor Zhang Haibin, Chinese Academy of Sciences), MEF p65 - / - (Provided courtesy of Professor Zhenggang Liu, American Cancer Institute). Fungal culture medium was *VillaNatura kraftnahrung Biomalt mitEisen im Preisvergleich* (pharmarissano Arzneimittel GmbH), and cell culture medium was high-glucose DMEM (Shanghai Yuanpei Biotechnology Co., Ltd.). C57BL / 6J mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.).

[0041] Reagents: TNF-α (R&D System), FasL (R&D System), Z-VAD-FMK (R&D System), Smacmimetic (SM-164) (MedChemExpress MCE), 5Z-7-Oxozeaenol (MedChemExpress MCE), ABT-737 (MedChemExpress MCE), LPS (Beyotime Biotechnology Co., Ltd.), Staurosporine (STS) (MedChemExpress MCE), Cisplatin (MedChemExpress MCE), RSL3 (MedChemExpress MCE), CBL0137 (MedChemExpress MCE). Cell viability kit (MedChemExpress MCE), AST and ALT test kits (Nanjing Jiancheng Bioengineering Institute).

[0042] Antibodies: phospho-RIPK1 S166 (Cell Signaling Technology Cat#65746, RRID: AB_2799693); phospho-RIPK1 S321 (Cell Signaling Technology Cat#83613, RRID: AB_2800023); RIPK3 (Abcam Cat#ab56164, RRID: AB_2178667); human phospho-RIPK3 (Abcam Cat#ab209384, RRID: AB_2714035); mouse phospho-RIPK3 (Abcam Cat#ab222320, RRID: AB_2868434); human MLKL (Abcam Cat#ab184718, RRID: AB_2755030); MLKL(Abcam,Cat#ab243142);human Phospho-MLKL (Abcam Cat#ab187091, RRID: AB_2619685); Mousephospho-MLKL (Abcam Cat#ab196436, RRID: AB_2687465); Actin (Sigma-Aldrich, Cat#A3853); Caspase-3 (Abcam Biotech, Cat#A19654); Caspase-3 (Cell Signaling Technology, Cat#1329664); Caspase-8 (Abcam Biotech, Cat#A11324); Caspase-8 (Cell Signaling... Technology, Cat#8592); FADD (Aibotek Biotechnology, Cat#A5819); TRAF2 (Aibotek Biotechnology, Cat#A0962); TNFR1 (Aibotek Biotechnology, Cat#A1540); TRADD (Aibotek Biotechnology, Cat#A1145); IκBα (Cell Signaling Technology, Cat#48143); FLAG (Aibotek Biotechnology, Cat#AE004); V5 (Aibotek Biotechnology, Cat#AE089); HA (Aibotek Biotechnology, Cat#AE008); GFP (Aibotek Biotechnology, Cat#AE012).

[0043] Example 1: Preparation of Cpd-8, a sesquiterpene lactone nitrobenzene analog

[0044] like Figure 10As shown, a Tedaniasp. sponge was collected from a depth of 15 meters in the Dongsha Islands area of ​​the South China Sea in December 2015. A symbiotic fungus was isolated from this sponge tissue and identified as *Aspergillus ochraceopetaliformis* (gene bank number MH109740) by intergenic spacer sequence (ITS) analysis of its rDNA. The strain was inoculated into Biomalt solid medium (20 L) for fermentation and cultured at 28°C for 28 days. After extraction with ethyl acetate four times by ultrasonication (2 L / time, 30 min), the extracts were combined and concentrated under reduced pressure to obtain a total extract. A small amount of the extract was analyzed by silica gel thin-layer chromatography (TLC), using a dichloromethane / methanol system as the eluent. The extract was then subjected to normal-phase silica gel column chromatography (200-300 mesh), eluted with a gradient of dichloromethane / methanol (volume ratio 100:1 to 1:1), and the fractions were collected and analyzed by TLC to obtain 11 fractions (F1–11). Fraction F5 was purified by Sephadex LH-20 gel column chromatography (dichloromethane / methanol 1:1) and separated into 9 fractions (F5a~F5i). Fraction F5h was subjected to normal-phase silica gel column chromatography (petroleum ether / ethyl acetate 6:1, v / v), followed by high-performance liquid chromatography (YMC Pack ODS-A column (5μm, 250×10mm); MeOH / H2O, v / v 1:1, 2.0mL / min, tR=50.0min) to give compound Cpd-8, which was identified by NMR and compared with literature data as 9α,14-dihydroxy-6β-p-nitrobenzoylcinnamyl lactone (Cpd-8). Figure 1 (A, B)

[0045] Example 2: Screening for anti-necrosis and apoptosis inhibitors from an extract library of sponge symbiotic fungi

[0046] Sponge-associated epiphytes are a valuable resource for discovering novel active pharmaceutical ingredients. The applicant cultured sponge-associated epiphytes in solid agar medium for 28 days and extracted secondary metabolites produced in each medium using ethyl acetate, obtaining crude extracts from a total of 88 sponge-associated epiphytes. Figure 2 (A). Then, the applicant constructed a necroptosis-induced apoptosis model in classic necroptosis-sensitive cells HT29 (human colorectal cancer cells) induced by TNF-α (20 ng / mL), Smac mimetic (10 nM), and Z-VAD-FMK (20 μM) (TSZ) (Nat Cell Biol. 2014 Jan; 16(1):55-65). In the MTS experiment, the applicant measured the effect of each extract pretreatment on cell viability after 30 min, thereby screening potential necroptosis-inhibiting inhibitors ( Figure 2 (B) For example Figure 2As shown in Figure C, the applicant found that extract No. 70 significantly inhibited necroptosis in HT29 cells, with an inhibition rate exceeding 90%. Subsequently, the applicant fractionated extract No. 70 using a silica gel column chromatography method, in which fraction 5 showed inhibitory activity against necroptosis. Figure 2 (D). The applicant further separated fraction 5 using Sephadex LH-20 and RP-HPLC columns, purifying it to obtain 12 compounds, among which compound 8 (Cpd-8) effectively protected cells from TSZ-induced necrosis and apoptosis. Figure 2 (E). For example Figure 2 As shown in Figure F, Cpd-8 concentration gradient-dependently inhibited TSZ-induced necrosis and apoptosis in HT29 cells, EC 50 It is 6.252 μM.

[0047] Example 3: Cpd-8 inhibits TNF-α-induced cell death

[0048] To further confirm the anti-necrosis and apoptosis activity of Cpd-8 and to investigate its effect on cell apoptosis. For example... Figure 3 As shown in Figure A, the applicant, using a TCZ (TNF-α, cycloheximide, and Z-VAD-FMK)-induced necroptosis model (Nat Cell Biol. 2014 Jan; 16(1):55-65), found that 1-10 μM Cpd-8 concentration gradient-dependently inhibited necroptosis in HT29 cells, and this concentration of Cpd-8 had no significant toxicity to HT29 cells. Subsequently, in TS (TNF-α and Smac mimetic) and TC (TNF-α and cycloheximide)-induced apoptosis models, Cpd-8 also significantly inhibited apoptosis in HT29 cells. Figure 3 (B, C). Furthermore, the applicant also found that Cpd-8 could inhibit TSZ and TCZ-induced necroptosis in mouse embryonic fibroblasts (MEF) cells. Figure 3 D, E), and TS and TC-induced apoptosis ( Figure 3 (F, G). In summary, Cpd-8 can inhibit TNF-α-induced cell death in a variety of cell types.

[0049] Example 4: Cpd-8 inhibits cell death induced by death receptor and Toll-like receptor.

[0050] Since other death receptor pathways (such as FasL / Fas and TRAIL / TRAIL-R) and Toll-like receptor pathways (such as LPS / TLR-4) can also induce apoptosis and necroptosis, the applicant further investigated whether Cpd-8 could inhibit cell death induced by these exogenous stimuli. Figure 4 As shown in Figures A and B, pretreatment of HT29 cells with Cpd-8 for 30 min inhibited, in a concentration gradient-dependent manner, TRAIL+SZ (TRAIL, Smac mimetic, and Z-VAD-FMK)-induced necrosis and apoptosis induced by TRAIL+S (TRAIL and Smac mimetic). In MDA-MB-231 breast cancer cells, the applicant further verified that Cpd-8 inhibited TRAIL+S-induced apoptosis. Figure 4 In HT29 cells, Cpd-8 also inhibited FasL+SZ (FasL, Smac mimetic, and Z-VAD-FMK)-induced necrosis and apoptosis. Figure 4 (D). Subsequently, the applicant extracted mouse bone marrow-derived macrophages (BMDM), which, upon stimulation, could spontaneously secrete TNF-α to induce cell death. Figure 4 As shown in Figure E, Cpd-8 can inhibit lipopolysaccharide (LPS) and 5Z-7-Oxozeaenol (TAK1 inhibitor)-induced apoptosis and pyroptosis, necrotizing apoptosis induced by 5Z-7-Oxozeaenol+Z (5Z-7-Oxozeaenol and Z-VAD-FMK), and 5Z-7-Oxozeaenol-induced apoptosis. In summary, Cpd-8 can inhibit cell death induced by death receptors and Toll-like receptors in various cell types.

[0051] Example 5: Cpd-8 has no protective effect against endogenous apoptosis, necroptosis, and ferroptosis.

[0052] Besides exogenous pathways such as death receptors and Toll-like receptors, endogenous pathways (including mitochondrial and endoplasmic reticulum pathways) can also induce apoptosis. Studies have shown that cisplatin and staurosporine (STS) can promote apoptosis in both mitochondrial and endoplasmic reticulum pathways by inducing DNA damage. Figure 5 As shown in Figures A and B, Cpd-8 had no effect on cisplatin (300 μM) and STS (0.2 μM)-induced apoptosis. Further evidence from the application of stimulating HeLa cervical cancer cells with ABT-737 (a Bcl-2, Bcl-xL, and Bcl-w inhibitor, 50 μM) demonstrated that Cpd-8 had no effect on mitochondrial pathway apoptosis. Figure 5 In HT29 cells, Cpd-8 also failed to inhibit RSL3 (glutathione peroxidase 4 inhibitor, 10 μM)-induced ferroptosis. Figure 5 (D). For example Figure 5 As shown in the EG diagram, Cpd-8 also failed to protect MEF cells from cisplatin, STS, and RSL3-induced cell death. Studies have shown that Z-DNA binding protein 1 (ZBP1) in cells binds to Z-DNA or Z-RNA and induces endogenous necrotizing apoptosis. Figure 5 As shown in Figure H, CBL0137 (5 μM) can stimulate MEF cells to produce Z-DNA, thereby activating Z-DNA binding protein 1 (ZBP1)-dependent necroptosis, while Cpd-8 cannot inhibit CBL0137-induced cell death. In conclusion, Cpd-8 has no protective effect against endogenous apoptosis, necroptosis, or ferroptosis.

[0053] Example 6: The anti-cell death activity of Cpd-8 is independent of the NF-κB signaling pathway

[0054] TNF-α stimulation not only mediates cell death signaling pathways but also activates the NF-κB signaling pathway and promotes cell survival. For example... Figure 6 As shown in Figure A, Western blot experiments indicated that Cpd-8 (10 μM) treatment for 10–60 min did not affect the expression of IκBα, a key protein in the NF-κB signaling pathway. Figure 6 As shown in Figures B and C, pretreatment of HT29 or MEF cells with Cpd-8 for 30 min, followed by TNF-α stimulation for 10-60 min, activated the NF-κB signaling pathway. The results indicate that Cpd-8 did not affect the protein change trend of IκBα. Furthermore, Cpd-8 did not affect the protein change trends of IκBα and NF-κB in the TS model. Figure 6 (D). For example Figure 6 As shown in Figure E, knocking out the NF-κB p65 subunit in MEF cells inhibits NF-κB activation, while Cpd-8 still inhibits TNF-α and TNF-α+Z-VAD-FMK-mediated cell death. In conclusion, Cpd-8 does not inhibit cell death by regulating the NF-κB signaling pathway.

[0055] Example 7: Cpd-8 inhibits TNF-α-induced cell death signaling pathway

[0056] Subsequently, the applicant used Western blot experiments to examine the effects of Cpd-8 on apoptosis and necroptosis signaling pathways. In TSZ-induced HT29 and MEF cells, pretreatment with Cpd-8 (10 μM) for 30 min regulated the phosphorylation of key regulators in the necroptosis signaling pathway, including inhibiting phosphorylation of RIPK1 at S166 (a pro-death site), and inhibiting phosphorylation of RIPK3 (human S227, mouse S232) and MLKL (human S358, mouse S345). Figure 7 (A, B). For example Figure 7 As shown in C and D, Cpd-8 can inhibit TS-induced phosphorylation of RIPK1 at S166 in MEF cells and inhibit TS- or TC-induced cleavage activation of caspase 8 and caspase 3. In summary, Cpd-8 can block the TNF-α-induced cell death signaling pathway, thereby inhibiting cell death.

[0057] Example 8: Cpd-8 inhibits the formation of the necroptosis-apoptosis complex, thereby exerting anti-necrosis activity.

[0058] TNF-α-induced necroptosis requires the sequential formation of complex I, complex II, and necrosomes to signal cell death. In a co-immunoprecipitation (coIP) experiment, necrosomes were enriched using RIPK1 antibody after 4 hours of TSZ stimulation of HT29 cells. The results showed that Cpd-8 (10 μM) significantly inhibited the interaction between RIPK1 and RIPK3 proteins to form necrosomes. Figure 8 (A). Subsequently, HT29 cells were stimulated with TSZ for 2-4 h, and complex II was enriched via caspase 8. The results showed that Cpd-8 (10 μM) could significantly inhibit the interaction between caspase 8 and RIPK1 and FADD proteins. Figure 8 (B). Next, TSZ stimulation of HT29 cells for 1-2 hours resulted in the enrichment of TNFR1 complex I. The results showed that Cpd-8 (10 μM) did not affect the interaction of TNFR1 with RIPK1, TRAF2, and TRADD proteins. Figure 8 (C). Since both RIPK1 and TRADD proteins are present in both complex I and complex II, the applicant further verified the effect of Cpd-8 on complex I and complex II using three rounds of coIP stimulated with TSZ for 2.5 h. Figure 8As shown in Figure D, in the first round of TNFR1 IP, Cpd-8 (10 μM) did not affect the interaction between TNFR1 and RIPK1, TRAF2, and TRADD proteins in complex I. The applicant performed a second round of TNFR1 IP in the lysate after removing the first round of coIP beads. At this point, the TNFR1 protein in the cell lysate was significantly reduced, and RIPK1, TRAF2, and TRADD proteins in complex I were undetectable, indicating that the first two rounds of TNFR1 removed RIPK1 and TRADD proteins from complex I. After removing the second round of coIP beads, the applicant performed a third round of TRADD IP to enrich the proteins in complex II. The results showed that Cpd-8 could inhibit the interaction between TRADD and RIPK1 in complex II. Given that apoptosis requires the interaction of FADD with caspase 8 (also a component of the necroptosis complex II) to activate caspase 8, and that Cpd-8 can simultaneously inhibit both apoptosis and necroptosis. Therefore, Cpd-8 may exert its protective effect by inhibiting the formation of necroptosis complex II and blocking death signal transduction.

[0059] Example 9: Cpd-8 protects mice from TNF-α / D-GalN-induced acute liver injury

[0060] To investigate whether Cpd-8 can inhibit necrotizing apoptosis-related inflammation in vivo, the applicant constructed an animal model of acute liver injury induced by TNF-α / D-GalN (D-galactosamine). Eight-week-old C57BL / 6J mice were divided into three groups: a drug toxicity group (Cpd-8, n=7), a model group (TNF-α / D-GalN, n=8), and an experimental group (TNF-α / D-GalN + Cpd-8, n=7). One hour after intraperitoneal administration of Cpd-8 (15 mg / kg) or an equivalent volume of DMSO, mice were intraperitoneally injected with D-GalN (600 mg / kg), followed by a tail vein injection of TNF-α (15 μg / kg) 15 minutes later. The drug toxicity group received direct intraperitoneal administration of Cpd-8 (15 mg / kg). Mouse body temperature was measured every two hours, and mortality was recorded. Figure 9 As shown in Figure A, the body temperature of mice in the model group decreased significantly from 4 to 8 hours, while the body temperature of mice in the experimental group only began to decrease after 12 hours. The body temperature of mice in the drug toxicity group showed no significant change (body temperature measurements were stopped after mouse death). Survival curve results indicate that Cpd-8 can significantly prolong the survival time of mice, and long-term monitoring shows that Cpd-8 has no obvious toxic side effects on mice. Figure 9 (B)

[0061] Subsequently, the applicant tested liver injury markers in mice using the same animal model, setting up a control group, a model group, and an experimental group (n=5). The concentrations of TNF-α / D-GalN and Cpd-8 were consistent with the former. Four hours after modeling, blood was collected from the eyeballs of mice in each group, followed by euthanasia by cervical dislocation, and the livers were rapidly dissected. Figure 9 As shown in Figure C, the livers of the model group exhibited liver function impairment due to acute hepatitis, resulting in increased pressure in the hepatic veins and hepatic congestion. Figure 9 As shown in Figure D, HE staining of liver tissue further indicated that Cpd-8 in the experimental group, compared to the model group, could inhibit tissue vacuolation, hepatocyte enlargement, and hematopoiesis caused by liver inflammation, while the overall histological morphology was similar to that of the control group. TUNEL staining results showed that Cpd-8 could reduce TNF-α / D-GalN-induced hepatocyte apoptosis (…). Figure 9 (E, F). For example Figure 9 As shown in Figures G and H, the applicant used aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assay kits to detect liver injury markers in serum samples from each group of mice. Cpd-8 significantly inhibited the increase in AST and ALT concentrations in the blood caused by TNF-α / D-GalN. In conclusion, Cpd-8 can inhibit TNF-α / D-GalN-induced acute liver injury in vivo, alleviate inflammation, and thus prolong the survival of mice.

[0062] The above experimental results show that the compound Cpd-8 of this invention has excellent anti-TNF-induced cell death activity and can be used as a novel anti-cell death inhibitor to resist TNF-α / D-GalN-induced acute liver injury. It can be used to prepare drugs for cardiovascular diseases (such as atherosclerosis and ischemic heart disease), ischemia-reperfusion injury (such as myocardial infarction and stroke), nervous system diseases (such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, etc.), acute organ injury (such as acute lung injury, acute kidney injury, acute liver injury, etc.), intestinal diseases (such as ulcerative colitis, inflammatory bowel disease, Crohn's disease, etc.), liver diseases (such as non-alcoholic fatty liver disease and chronic hepatitis, etc.), and inflammatory disorders (such as systemic inflammatory response syndrome and sepsis, etc.).

[0063] In summary, the compounds of this invention and their pharmaceutically acceptable salts can be used to prepare novel small molecule inhibitors against TNF-induced cell death.

[0064] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. The application of compound Cpd-8 in the preparation of drugs for treating TNF-induced cell death-related diseases, characterized in that, TNF-induced cell death-related diseases include acute liver injury; The structural formula of compound Cpd-8 is shown in formula (Ⅰ): （Ⅰ）。

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