Synthesis,Structural Characterization,Molecular Docking And Bioactivity Studies Of Novel MAP3K5 Inhibitors

Mitogen-activated protein kinase kinase 5（MAP3K5,also known as ASK1）is an important potential drug target that plays an important role in the treatment of non-alcoholic steatohepatitis（NASH）,pulmonary arterial hypertension（PAH）,diabetic nephropathy（DKD）and neurological disorders.This study was designed to identify a group of highly potent and selective MAP3K5 inhibitors using the clinical candidate Selonsertib as the lead compound and the principles of thickened cyclization and bioelectronic equipartition as the guiding strategy.A preliminary summary of the conformational relationships of selonsertib was performed by docking using,Discovery Studio software.Based on the structure of selonsertib and the molecular docking model,the lead compound selonsertib was modified by chemical means such as closed-loop and bioisostere.The compounds were synthesized by reduction,substitution,ring formation,Suzuki coupling and amide formation.All compounds were structurally confirmed by ¹H NMR and high-resolution mass spectrometry,while the structures of key intermediates in the synthesis were further verified by ¹³C NMR.MAP3K5inhibitory activity was measured using the ADP-GLO luminescence kinase assay.Its MAP4K1 and TAK1 kinase inhibitory activities were also assayed to test inhibitor selectivity.The compound was further tested for cytotoxicity using the CCK-8 assay at a later stage.Flow cytometry analysis of cell cycle distribution and application of oil red O staining to evaluate lipid droplet formation in LO2 cells.Combined with the structure-activity relationship analysis of slonsente on MAP3K5protein,selonsertib was used as the lead compound for the pharmacochemical modification strategies such as bioelectronic isosteric substitution and closed-loop cyclization.The compounds were designed by three different pathways and synthesized,isolated and purified to obtain 27 novel backbone MAP3K5 inhibitors that have not been reported in the literature.These include indoline series 2-4,2-5;indole series 2-7a,2-7b,2-8;benzimidazole series13;1,2,3,4-tetrahydroquinoxaline series 2-17,2-18;benzo[b][1,4]oxazine series 2-23a,2-23b,2-24;1,2,3,4-tetrahydroquinoline series 2-29a,2-29b;phenol series compounds 2-31a～2-31h;nitrobenzoid series compounds 2-35a～2-35d;pyridine series compounds2-38a,2-38b.MAP3K5 kinase inhibitory activity was tested and compounds 2-38a and 2-38b showed significant inhibition with IC₅₀values of 0.15μM n M and 0.31μM,respectively.2-38a showed great similarity to the lead compound,selon certis,by analysis of the eutectic structure of the 2-38a-MAP3K5 complex,which showed hydrogen bonding with MAP3K5protein.In the selectivity assay,compounds 2-38a and 2-38b were 2.1 and 20-fold selective（MAP4K1/MAP3K5）;compounds 2-38a and 2-38b were 3.3 and 4.5-fold selective（MAP4K1/TAK1）.CCK-8 assays were performed to detect LO2 cytotoxicity and cell survival was>75%for both compounds 2-38a and 2-38b at concentrations ranging from 0.05to 1u M.Compounds 2-38a and 2-38b showed a G1 phase cycle arrest effect on Hep G2 cells,while compound 2-38a also significantly reduced the fat droplets in LO2 cell models.Three series of novel skeleton compounds were designed and synthesized,and screened for MAP3K5 inhibitory activity.It was verified that the structure of the pyridine ring of selonsertib plays an important role in its inhibitory activity and that modification of the pyridine ring may cause a drastic reduction in the MAP3K5 inhibitory activity of the compounds.At the same time,this study obtained compounds 2-38a and 2-38b with good in vitro activity by structural modification of the solvent exposed area of Slonserti.In kinase selectivity experiments,compound 2-38a did not show better selectivity for MAP4K1 kinase,but compound 2-38b showed up to 20-fold selectivity（MAP4K1/MAP3K5）.This study designed and synthesized MAP3K5 inhibitors 2-38a and 2-38b,and conducted pharmacological and pharmacological experiments on them,providing new research ideas for obtaining efficient and highly selective MAP3K5 inhibitors.