Design,Synthesis And Bioactivity Studies Of Specific Small Molecule Inhibitors Targeting RIPK1、ROCK And SARS-CoV-2 M^pro

Protein kinases are a key class of factors that regulate cell signal transduction pathways.Their abnormal regulation is closely related to the occurrence and development of tumors,inflammation,immunity and other diseases,and has become a key target for the treatment of these diseases.As of the end of 2020,63 kinase inhibitors have been approved by the Food and Drug Administration（FDA）.However,most of the clinical indications of these inhibitors are tumors,and there are few kinase inhibitors for nononcologic diseases such as inflammation and autoimmunity.One of the important reasons is that most of nononcologic diseases are chronic diseases and require long-term medication,so the requirement of inhibitors selectivity is relatively high.Due to the high homology of kinases,however,it is a challenge to develop highly selective inhibitors for specified kinase targets.During the period of my Ph.D.degree study,I mainly focused on the design,synthesis and structure-activity relationship of specific inhibitors for two kinases,namely RIPK1 and ROCK.For those compounds with the best bioactivity and selectivity,pharmacokinetic properties and safety evaluation studies have been carried out,and the pharmacodynamics of them in inflammatory disease models and ocular disease models have been further explored.In addition,the Coronavirus（COVID-19）pandemic broke out and quickly developed into a global pandemic at the beginning of 2020.In response to the national call to fight the COVID-19 epidemic,I also participated in the design,synthesis and structure-activity relationship study of novel inhibitors of SARS-CoV-2 main protease(M^pro)from 2020 to 2021,carried out anti-SARS-CoV-2 bioactivity in vitro and in vivo,and evaluated the preliminary draggability.PartⅠ.Design,synthesis and structure-activity relationship of specific RIPK1inhibitors and their pharmacodynamics in inflammatory disease modelsRIPK1（Receptor-interacting protein kinase 1）,a serine/threonine protein kinase,is a key mediator of necroptosis and inflammation.In addition to being closely related to the occurrence and development of tumors,RIPK1 is also involved in the regulation of various nononcologic diseases such as inflammation,neurodegenerative diseases and autoimmune diseases.Although many RIPK1inhibitors have been reported,most of the kinases show the poor selectivity.Therefore,the development of a new type of high specificity and good draggability of RIPK1 inhibitors would be of great worth for the treatment of nononcologic diseases.computer aidedIn this study,we firstly discovered a hit compound（5-（2-（cyclopropanecarboxamido）benzo[d]thiazol-6-yl）-2-methoxy-N-（4methoxybenzyl）nicotinamide,Hit-1）by the computer-aided virtual high-throughput screening combined with programmed necrosis cell models and kinase activity testing methods.Compound Hit-1 showed the inhibition activity against RIPK1with an IC50 value of 191 n M,and the cell protection effect in the TSZ-induced HT29 cell necroptosis model with an EC50 value of 1.009μM.Subsequently,the docking model between Hit-1 with RIPK1 was carried out,and the research strategy of determining the dominant nucleus through the scaffold hopping was established,and then the structure-activity relationship of other regions was successively optimized.Firstly,we locked the 5-substituted 1H-indole-3-carboxamideas as the dominant essential scaffold by the performing ring closure and scaffold hopping to the methoxypyridine moiety of Hit-1.Secondly,the substitution of4-methoxybenzylamine was investigated in detail,and finally it was found that the（S）-1-（3-fluorophenyl）ethylamine group can significantly help to improve the bioactivity.Finally,the benzothiazole moiety was optimized,but no more ideal substituent groups were found.A total of 80 compounds were synthesized,and the compound 18an showed good bioactivity（RIPK1,IC50=19 n M;HT29,EC50=0.117 n M）.Screening of the full kinase profile showed that 18an has the kinase selectivity.18an could efficiently protect cells from necroptosis in vitro.It also showed a good anti-inflammatory effect in the systemic inflammatory response syndrome（SIRS）model.Further in-depth research found that although 18an has good efficacy and safety,its oral pharmacokinetic properties are very poor.To this end,we conducted a more detailed study of the structure-activity relationship starting from 18an.We gradually blocked the NH of indole,replaced the benzothiazole moiety with a variety of heterocycles,and found the[1,2,4]triazolo[1,5-a]pyridin is an ideal choice.A total of 60 compounds were synthesized.After comprehensive analysis of bioactivity and pharmacokinetic properties,we selected the best one,compound 45b（RIPK1,IC50=15 n M;HT29,EC50=0.012 n M）.Compound 45b has good pharmacokinetic properties,and has very ideal RIPK1 selectivity.Subsequently,we analyzed the complex crystal structure of 45b and RIPK1,and clarified the inherent reason why 45b has high potency and selectivity.We also found that 45b has a significant protective effect against necroptosis in vitro.In the SIRS and inflammatory bowel disease（IBD）mouse models,Furthermore,45b showed significant anti-inflammatory effects,and its activity was significantly better than the positive control GSK3145095.In addition,the safety of 45b is very well.In summary,45b is a new class of RIPK1 inhibitor with high potency,selectivity,and good draggability,and has the potential value of further development as a clinical drug candidate.PartⅡ.Design,synthesis and structure-activity relationship of specific ROCK inhibitors and their pharmacodynamics in animal models of ophthalmic diseasesROCK（Rho-related protein kinase）is a serine/threonine protein kinase.ROCK is involved in mediating various cellular functions such as actin cytoskeleton regulation and actin fiber dynamics,and is closely related to smooth muscle contraction,cell adhesion,proliferation and apoptosis.The research has shown that ROCK is closely related to the course of diseases such as glaucoma,age-related macular degeneration,organ fibrosis,and myocardial infarction,and is considered as a key therapeutic target for these diseases.Although there are a variety of ROCK inhibitors currently reported,but still very lack of specific ROCK inhibitor.In this study,we discovered a seed compound ROCK-1 through a ligand-based pharmacophore,and then successively carried out structural optimization and structure-activity relationship studies for its 3 regions（RegionⅠ/Ⅱ/Ⅲ）.Structure–activity relationship analyses led to the identification of the most active compound,4-（dimethylamino）-N-（3-[2-[（4-oxo-4H-chromen-7-yl）oxy]acetamido]ph enyl）（12j）.This compound showed excellent kinase selectivity for ROCK I and ROCK II against 387 other kinases.In retinal explants,compound 12j protected retinal neurons from high glucose-induced oxidative stress and apoptosis-mediated cell death.Furthermore,12j administration suppressed the improper proliferation of Müller cells and promoted the regression of vascular vessels in retinal explants cultured in a high glucose microenvironment.Collectively,our data suggest that 12j could be a potential lead compound for the treatment of DR,hence deserving further in-depth studies.Related studies have been published in J.Med.Chem.（2019）.We have further learned that ROCKⅡselective inhibitors for a single subtype can significantly improve the safety of the compound and reduce toxic and side effects.To this end,we started from another hit compound ROCK-2,through structure-based drug design and two-step scaffold hopping.We gradually transformedtheessentialscaffoldfromhittolead（3,4-dihydrobenzo[f][1,4]oxazepin-5（2H）-one→1,2,3,4-tetrahydro-5H-benzo[e][1,4]diazepin-5-one→3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione）.The ROCKⅡselective inhibitors 69e was found,has 36 n M inhibitory activity against ROCKⅡ,but has no obvious inhibitory activity against ROCKⅠ(IC₅₀>10μM).Most of all,the subtype selectivity is more than 277 times.Further studies have shown that 69e showed less cell toxicity and can effectively protect neuronal cell damage in the oxygen-glucose deprivation（OGD）model.In addition,69e can significantly reduce the intraocular pressure of animals.Compared with the positive control Netarsudil,69e can obviously reduce the eye irritation.69e also has good oral pharmacokinetic properties as evidenced by oral bioavailability of 41.43%.In conclusion,the present study found a novel highly ROCKⅡselective inhibitor with good draggability,which can be used as lead compound for the depth study of various indications against ROCKⅡ.PartⅢ.Design and synthesis of a new class of SARS-CoV-2 M^pro inhibitors,which Showed Potent anti-SARS-CoV-2 pharmacodynamics in vivo and in vitroThe COVID-19 pandemic caused by SARS-CoV-2 poses a serious threat to global public health security.Unfortunately,there is currently no specific anti-SARS-CoV-2 drug.M^pro,also known as main protein or 3CL^pro,is a key enzyme that mediates viral RNA replication and transcription,and there is no known human protease that has the same enzyme cleavage site.Therefore,M^pro is considered an ideal target against SARS-CoV-2.In this study,we carried out the research on small molecule drugs against SARS-CoV-2 M^pro.Firstly,we established a screening system based on fluorescence resonance energy transfer（FRET）.Through screening the library of listed compounds,we found that the anti-HCV drugs,Telaprevi and Boceprevir,have certain inhibitory activity.The co-crystal structures of SARS-CoV-2 M^pro in complex with Telaprevir and Boceprevir is parsed respectively.In order to obtain highly active molecules in a short period,we performed the rational drug design based on the splicing of dominant fragments,and synthesized 32 novel potent compounds,and the inhibitory bioactivity was between 7.6-748.5 n M.The co-crystal structure analysis of the representative compound 18i and M^pro is completely consistent with our previous drug design.Subsequently,we accomplished cell-level tests on 20 compounds with an enzyme inhibitory activity of less than 50 n M,and then evaluated the rat pharmacokinetics of 6 compounds with antiviral activity of less than 1μM in vitro.After the step-by-step screening and comprehensive analysis,two compounds（11i and 18p）were selected to evaluate the antiviral efficiency in the SARS-CoV-2 infection transgenic mouse model.Oral or intraperitoneal treatment with 11i or 18p significantly reduced lung viral loads and lung lesions.Both also displayed good safety in rats.In summary,this study has discovered a new class of high-efficiency and low-toxicity SARS-CoV-2 M^pro inhibitors,which have good antiviral activity in vivo and in vitro,and their pharmacokinetic properties and safety evaluation have confirmed that they have the potency of being developed as anti-SARS-CoV-2 drug candidate.This has laid a good foundation for the research and development of innovative anti-SARS-CoV-2 drugs.Related studies have been published in Science（2021）.In conclusion,this graduation thesis mainly focused on the design,synthesis and bioactivity of specific small molecule inhibitors for RIPK1,ROCK and SARS-CoV-2 M^pro.The main research results are as follows:（1）A novel highly potent and specific RIPK1 inhibitor 45b was obtained.This compound shows good protective effect against necroptosis in vivo and in vitro,and has significant anti-inflammatory effects in animal models;（2）A highly effective pan-ROCK inhibitor 12j and a highly potent specific ROCKⅡinhibitor 69e were discovered.Both compounds showed good efficacy in ocular disease models;（3）We obtained two novel safe and highly effective SARS-CoV-2 M^pro inhibitors,11i and18p,which have significant anti-SARS-CoV-2 effects in vivo.In addition,the research of this thesis also pays great attention to the draggability of the compounds,and the obtained inhibitors have undergone preliminary evaluation about pharmacokinetics and safety.In short,this study has established a key foundation for the research and development of innovative drugs for anti-inflammatory diseases,anti-eye disorders and anti-SARS-CoV-2.