Lead Discovery Of Rho Kinase Inhibitors: Design,biological Evaluation And Molecular Mechanism Study Of 4-aminopyridine Derivatives

Multiple sclerosis(MS)is a kind of autoimmune disease that occurs in the human central nervous system,such as the demyelination of the spinal cord and the damage of the optic nerve.With the deterioration of MS,patient will become disabled in action and finally paralytic.Th1 T cells and Th17 T cells are the most important pathogenic effector cells of MS.IFN-? cytokines are mainly secreted by Th1 T while IL-17 cytokines are mainly secreted by Th17 T.Meanwhile,IL-10 cytokines that secreted by Th2 T cell have the function of immunomodulatory,they can regulate the biological activity of Th1 T and Th17 T cells.In other words,IL-10 cytokine plays a important role in anti-inflammation and protection of myelin.One of the Rho kinase inhibitors,Fasudil,has a good effect on the treatment of degenerative neurological disorders,especially MS.Another Rho kinase inhibitor,Y27632,also has a well effect of neuroprotection0 and its analogue,WAR5,has been reported to have considerable potential for neuroprotection.Our study could be mainly divided into three parts:First part,the design of a new synthetic route of WAR5 that 4 aminopyridine and phenyl chloroformate were considered as initial materials.And then,the product was performed substitution reaction with N-boc-piperazine to take off phenol.Finally,the boc-functional group was taken off and product transform into salt,which was the target molecule named N-pyridine-4-ylpiperazine-1-carboxamide dihydrochloride.Compared with previous synthetic routes,this new route has many advantages such as mild conditions,the product is easy to be separated and purified.Meanwhile,the most significant contributes are the yield has been greatly improved(the total yield of 84%)and can easily achieve kilo-class zoom produce.Second,a series of in vivo and cell experiments about pharmaceutical evaluation of WAR-5 and Fasudil were performed through animal model of EAE mice.The results indicate that WAR5 has very ideal effect on the treatment of spinal cord Sheath,inflammatory cell infiltration,regulation of release of lymphocyte inflammatory,NO and so on.Also,the safety window of WAR-5 is superior to Fasudil.Finally,in order to explore the role of WAR5 in the treatment of human multiple sclerosis,we use a listed Rho kinase inhibitor drug Fasudil as a positive control for EAE lesions mice to do a series of vivo experiments and cell experiments.It was concluded that WAR5 had a very good effect on remission of demyelinating symptoms in EAE lesion mice and alleviating the release of inflammatory factors of lymphocytes,and the safety window of WAR5 was superior to Fasudil.In order to explore the potential interaction between WAR5 and Rho kinase,docking and molecular dynamics simulations were performed by AutoDock 4.0 and AMBER 11.The inhibitory effect of WAR5 against Rho kinase was contributed to high binding affinity generated by hydrogen bond,hydrophobic effect,and aromatic effect.