Design And Synthesis Of Selective RIP1-Targeting Necroptosis Inhibitors Based On TAK-632 And Investigation On The SAR And Anti-Ulcerative Colitis Efficacy

Ulcerative colitis（UC）is a chronic inflammatory disease with a gradually increasing morbidity worldwide.The dysregulated or excessive cell death,including necroptosis in the intestinal epithelium,is sufficient to induce and/or amplify inflammation,which may contribute to intestinal barrier dysfunction resulting in UC.Recent evidence has supported that active receptor-interacting protein kinase 1（RIP1）can actuate inflammation through directly regulating necroptosis.Necroptosis is a caspase-independent cell death form mediated by death receptors,and is regulated mainly by receptor-interacting protein kinase 1（RIP1）,receptor-interacting protein kinase 3（RIP3）and mixed lineage kinase domain like protein（MLKL）.Therefore,discovery of necroptosis inhibitors is of great significance to the clinical treatment of UC.This thesis mainly includes the structure optimization,structure-activity relationship study and anti-ulcerative colitis research of necroptosis inhibitor.1.Design and synthesis of selective RIP1-targeting necroptosis inhibitors based on TAK-632 and investigation on the SAROur research used a ligand-based substituent-anchoring design strategy,taking dual target necroptosis inhibitor TAK-632 as the lead compound,26 target compounds were obtained through four rounds of structural optimization.Among them,15 target compounds showed nanomolar protective activity,and a class of benzothiazole and monosubstituted compounds has high selectivity to RIP1.Compound 25（RIP1,K_d=15 n M,RIP3,K_d>5000 n M）has a protein binding activity of RIP1 which is 333 times of RIP3,and can protect human HT-29 cells from necroptosis,with an EC₅₀ of 8±1 n M.It can also protect mice-derived cells（L929）from necroptosis.The mechanism study of the compound found that compound 25 can inhibit the phosphorylation of RIP1,RIP3 and MLKL,and can directly block the formation of RIP1 and RIP3 complex.In a mice systemic inflammatory response syndrome（SIRS）model induced by TNF-α,when compound 25 was administered intragastrically at a dose of 10 mg/kg,the survival rate of mice was 60%,at 20 mg/kg,the survival rate increased to 100%.Compound 25 was well tolerated at a dose of 5 g/kg,indicating that the compound has low toxicity.Compared with TAK-632 and SZM-594,the survival rate of mice is significantly improved.The study found a class of high activity,high selectivity and low toxicity RIP1 small molecule inhibitors,which provided important lead compound for the development of new drugs against necroptosis.2.Investigation on anti-UC efficacy of selective RIPK1 necroptosis inhibitor.The selective RIP1 inhibitor compound 25 was selected as an anti-ulcerative colitis therapeutic drug,and its therapeutic effect in dextran sulfate sodium（DSS）-induced UC mice was observed.The compound 25 was administered by gavage.The results showed that the weight of the mice recovered,and the disease activity index（DAI）scores decreased significantly.The length of the colons also recovered compared with the model group.HE staining showed ulcer tissue in the colon tissues of treatment groups mice were repaired,and the histological scores showed that the high-dose and low-dose treatment groups were different from the model group.The level of fluorescein isothiocyanate（FITC）in the serum of mice showed that the level of FITC in the treatment groups were significantly reduced compared with the model group,and the macromolecular substance markers were significantly reduced,the intestinal permeability of the mice in the treatment groups were improved in the colon section tissues.The leakage of FITC macromolecules was significantly reduced.TUNEL experiment found that the number of colonic cell deaths in the treatment group was decreased.Western Blotting experiment of mice colon tissues observed that the phosphorylation levels of RIP1,RIP3 and MLKL in the necroptosis pathway were inhibited to a certain extent.PCR revealed that the inflammatory factors IL-1β,IL-6,CCR-2 and CCR-5 were significantly inhibited.The above results all showed statistical differences.In addition,the pharmacokinetics of the compound showed its bioavailability was9.7%.In summary,we obtained a small molecule inhibitor candidate for the treatment of UC through a series of studies,which provides new ideas and strategies for the clinical treatment of UC.