Design,synthesis And Biological Activity Of 2-substituted Quinolines

Objective: MAP3K5(also known as ASK1)is a widely expressed serine tyrosine kinase,and its increased activity is related to a variety of pathologies.Some studies have shown that inhibiting MAP3K5 activity is a safe strategy for treating diseases,so the development of MAP3K5 inhibitors is of great significance.In this paper,we designed and synthesized a series of 2-substituted quinoline compounds with novel structure based on the K812 and clinical candidate drug GS-4997 as the lead compound,in order to find highly active and selective MAP3K5 inhibitors,lay a foundation for future research work and provide some new ideas.Methods: Based on the eutectic structure of GS-4997 and MAP3K5,and combined with the previously reported structure-activity relationship of K812,this paper modified the fluorobenzene ring site,pyridine ring site,and triazole ring site of GS-4997 with imidazole substitutions,synthesized a series of six membered ring compounds containing benzene and nitrogen,and preliminarily studied the structure-activity relationship of the synthesized compounds,in order to guide further structural optimization of the compounds.This paper used the ADP-GLO luminescence kinase assay to test the in vitro MAP3K5 inhibitory activity and selectivity of the synthesized compound.Finally,select a preferred compound with the best enzyme inhibitory activity,use Discovery Studio(version 2.5)for molecular docking,and study its binding mode.Subsequently,a series of in vitro experiments were conducted on the selected compounds,including the application of CCK-8detection method for cytotoxicity evaluation,flow cytometry for cell cycle arrest,and NAFLD model for testing the therapeutic effect.Results: This paper presents a fused ring design strategy for imidazole substituted fluorobenzene ring sites,and synthesizes a series of benzo hexamembered heterocyclic compounds with the aim of screening suitable benzo hexamembered heterocyclic frameworks.The test results show that the quinoline ring skeleton 2-1d(IC50=25 n M)has good inhibitory activity.Next,we synthesized and evaluated substituents at different positions on the quinoline ring,and determined the optimal substitution position for the quinoline ring.From the obtained MAP3K5 inhibitory activity data,the compound JT21-25 with bromine substitution at the 8-position of the quinoline ring exhibits the best activity.However,the activity of compound 2-2d substituted with 1-methyl-1,2,3,6-tetrahydropyridine at position 8 of the quinoline ring decreased by 500 times compared to JT21-25.In the MAP3K5 kinase selectivity experiment,compound JT21-25 showed up to 2000 times selectivity towards MAP3K5 kinase.In the molecular docking experiments of JT21-25 and MAP3K5 proteins,the results showed that compound JT21-25 formed strong hydrogen bonds with the key residues VAL575 and GLN567 of MAP3K5 protein,enhancing the binding force between the compound and the target.In vitro cytotoxicity experiments,five different concentrations of JT21-25 were measured using the CCK-8 method,and the survival rate in human normal liver cell LO2 was as high as 80%,indicating the biological safety of JT21-25.Secondly,in the flow cytometry cycle arrest experiment,JT21-25 has obvious blocking effect on G1 phase of Hep G2 cells.Finally,this paper also verified that JT21-25 has a certain therapeutic effect in the NAFLD model.Conclusion: The paper mainly focuses on the clinical drug GS-4997,designing and synthesizing a series of 2-substituted quinoline compounds,and conducting in vitro kinase activity experiments,kinase selectivity experiments,molecular docking experiments,cytotoxicity experiments,cell cycle arrest experiments,oil red O staining experiments,and CHOL,LDL,and TG content testing experiments on them.In a series of experimental studies,JT21-25,a bromine substituted compound at position 8 of quinoline ring with the best activity and selectivity,was screened out.It has a strong inhibitory effect on MAP3K5 kinase,can block the G1 phase of Hep G2 cells,has a certain NAFLD therapeutic effect,and will not cause significant damage to human normal hepatocytes.In this paper,a MAP3K5 inhibitor with high selectivity and inhibitory activity was discovered,and its cytotoxicity was relatively low in vitro evaluation,providing a prerequisite for further in vivo evaluation in the later stage.So far,there has been no market launch of MAP3K5 small molecule inhibitors.The research in this paper may provide a new research approach for the development of MAP3K5 small molecule inhibitors.