Design,Synthesis And Activity Of Novel RIPK1 Inhibitors

In recent years,small molecule kinase inhibitors have become one of the hot spots in clinical drug development.RIPK1 is a member of the serine/threonine kinase family whose kinase activity plays an important role in inflammation and cell death(apoptosis and necroptosis).Chemical inhibition or genetic ablation of RIPK1 kinase activity can ameliorate necroptosis-associated pathologies in various diseases models in mice,including inflammatory and neurodegenerative diseases.Therefore,selective RIPK1 small molecule inhibitors have become a research hotspot of many international pharmaceutical companies and scientific research institutions.Nec-1 is the first reported RIPK1 inhibitor,but Nec series compounds have been limited to preclinical due to the narrow SAR window that can be optimized.At present,there is no selective RIPK1 inhibitor on the market,but companies such as GSK,Denali,Rigel,and Sironax have successively launched selective RIPK1 inhibitors that have entered clinical trials,and their indications cover many diseases such as inflammation and the central nervous system.The main research object of this thesis is to develop a novel RIPK1 inhibitor with a novel structural framework and drug developability.We first screened the activity of our group’s compound library by using the necroptosis model and obtained the compound PK6 with a pyridyl-benzothiazole skeleton.We performed preliminary structural optimization of PK6 to obtain PK68,which has good anti-necroptosis activity(HT29:22 nM,L929:14 nM)Subsequently,we carried out a target identifications and kinase selectivity study on PK68,and the results show that PK68 is a selective RIPK1 inhibitor.Molecular docking simulation experiments show that PK68 exhibits a type Ⅱ binding conformation with RIPK1,which is significantly different from most reported RIPK1 inhibitors(type Ⅲ).Despite its good activity,PK68 exhibits extremely poor metabolic stability in liver microsomes.In order to solve the metabolic problem and further improve the activity,we carried out a systematic structure-activity relationship study on PK68.First,under the guidance of the results of molecular docking simulation experiments,we immobilized some key groups with hydrogen bonding and performed heteroatom migration in the pyridyl-benzothiazole skeleton of PK68,but these compounds show a significant decrease in potency.Then,we adopted a cyclization strategy to obtain a series of RIPK1 inhibitors with pyridopiperidine,pyridopiperazine,and pyridopyrrolidine structural backbones.Among them,the activity of pyridopyrrolidine compounds is significantly improved(about twenty times).While optimizing the activity,we reduced the clogP of the compound by introducing heteroatoms to improve the metabolic stability.Finally,through the structural reorganization of the advantageous fragments in different regions,we obtained four compounds with good activity and liver microsome stability(30,40,44,52).Finally,through pharmacokinetic screening in rats,we identified compound 44 as a candidate compound for further study.Compound 44 has the extremely high anti-necroptosis activity in both human and murine cells(HT29:6.5 nM,L929:5.9 nM).In the competitive binding assay and kinase activity inhibition assay,44 is proved to have a strong binding affinity(Kd=8.6 nM)and kinase inhibitory activity(IC50=10 nM)with RIPK1.Subsequent kinase selectivity experiments show that 44 has excellent selectivity among 468 kinases at 10 μM.In vitro hERG and Ames experiments show that 44 exhibits no risk of cardiotoxicity and mutagenesis.In CYP inhibition test,44 also does not show significant inhibition in five major metabolic enzymes,suggesting a lower risk of possible drug-drug interactions.In addition,compared with PK68,the stability of liver microsomes of 44 has been greatly improved.The favorable in vitro activity and druggability results motivated us to conduct in vivo safety,pharmacokinetic and pharmacodynamic studies of 44.Under the gavage of mice for 14 consecutive days,even at a high dose of 100 mg/kg,the mice survived normally,and no body weight loss and important tissue and organ damage were found,indicating that 44 has a good safety profile in mice.In pharmacokinetic studies of mice and rats,44 exhibits low clearance and good bioavailability,indicating good oral druggability.The in vivo efficacy of 44 was validated in the SIRS model.In SIRS model,at a very low dose(0.1 mg/kg),compound 44 can 100%rescue TNFα-induced shock death in mice.We believe that 44 is a preclinical candidate for RIPK1 inhibitor with great druggable potential.In the next step,we will conduct more research on the safety and efficacy of 44,hoping to advance 44 to clinical research and benefit clinical patients.