Design,Synthesis And Activity Evaluation Of Novel Heat Shock Protein 90 Inhibitors

Objectives:A variety of heat shock protein 90（Hsp90）single-target inhibitors and Hsp90/histone deacetylase 6（HDAC6）dual-target inhibitors were designed and synthesized,and their anti-tumor and anti-idiopathic pulmonary fibrosis（IPF）activities were preliminarily evaluated.1、Design,synthesis and activity evaluation of novel Hsp90 inhibitors.Methods:Based on the Hsp90 inhibitor VER-50589 with strong Hsp90 inhibitory activity,three novel Hsp90 inhibitors dubbed as 12-4,12-8 and 12-9 were designed by crystal structure analysis and structural optimization.By scrutinizing the literatures,a reasonable synthetic route was designed through which three target compounds were obtained,and the inhibitory activity of three compounds against Hsp90 was tested in vitro.The cytotoxicities of three compounds against A549（non-small cell lung cancer）,95-D（human highly metastatic lung cancer cells）,MCF-7（breast cancer cells）,MDA-MB-231（breast cancer cells）,Hela（cervical cancer cells）,ES-2（human ovarian clear cell cancer cells）cell lines were determined by methyl thiazolyl tetrazolium（MTT）assay.The in vitro antiproliferative activity of the most effective compound 12-4 was further validated by colony formation assay.The cell cycle was detected by flow cytometry to explore the antiproliferation mechanism of compound 12-4.The induction of apoptosis of A549 tumor cells by 12-4 was detected by Annexin V-Fluorescein isothiocyanate/7-Aminoactinomycin D（Annexin V-FITC/7-AAD）double staining.The impact of compound 12-4 on the migratory capacity of A549 cells was tested by scratch test.The effect of compound 12-4 on the expression of Hsp90 client proteins cell cycle dependent kinase 4（CDK4）and human epidermal growth factor receptor 2（HER2）was detected by Western blot.Finally,the binding mode of compound 12-4 with Hsp90 was simulated by molecular docking.Results:Compounds 12-4 showed strong inhibitory activity against Hsp90 enzyme with half maximal inhibitory concentration(IC₅₀)value of 9 nanomole（n M）.It also showed strong cytotoxicity towards six different tumor cell lines with IC₅₀ values at n M range,outperforming two positive controls VER-50589 and geldanamycin（GA）.The colony formation assay showed that compound 12-4 could effectively repress colony formation of A549 cells,and compound 12-4 was able to induce apoptosis and arrest the cell cycle in G0/G1 phase of A549 cells,which was consistent with the observations on Hsp90 inhibitors.Compound 12-4 inhibited the migration of A549 cells.Western blot results showed that 12-4 significantly downregulated the expression of Hsp90 client proteins CDK4 and HER2.Finally,molecular docking simulation reveals that compound 12-4 fits well with the active site of Hsp90 and forms several H-bonds with Hsp90.Conclusion:Compound 12-4 is superior to the positive control drug in many biological activity experiments such as enzyme activity,in vitro antiproliferation experiment,plate colony formation experiment and scratch experiment.Overall,compound 12-4 should have a wide application as a novel Hsp90 inhibitor.2、The design,synthesis and activity evaluation of novel Hsp90 and HDAC6 dual-target inhibitors.Methods:In this study,a series of Hsp90 and HDAC6 dual-targeting inhibitors were designed and synthesized by merging the key pharmacophore of second generation Hsp90inhibitor（Cap）,hydroxamic acid（ZBG）and different kinds of linkers.The inhibitory activities of all target compounds against both Hsp90 and HDAC6 were tested in vitro.The anti-proliferative effects of the synthesized compounds on a variety of tumor cells:A549,Huh-7（human liver cancer cells）,Hep G2（human liver cancer cells）,MDA-MB-231,Hela and SKOV3（human ovarian cancer cells）and HFL-1（human embryonic lung fibroblasts）were tested by MTT assay.Results:All 17 compounds showed good inhibitory activity against both targets.The antiproliferation activity of compounds 13-1 and 13-3 was significantly better than that of the positive controls,Vorinostat（SAHA）and 30f（the reported Hsp90 inhibitor）.The IC₅₀of 13-1 against HFL-1 cells was 3.42μM,whose activity was significantly higher than that of Nintedanib(IC₅₀=30.41μM)and Pirfenidone(IC₅₀=5000μM).Conclusion:Compound 13-1 showed a strong inhibitory effect in in vitro antiproliferation experiments.