Design,Synthesis And Anti-hepatocellular Carcinoma Activity Evaluation Of Novel Artesunate Derivatives

Objective:Artesunate can inhibit the growth of various tumor cells and has practical value in the potential development of anticancer drugs.However,artesunate has the problem that it also has an inhibitory effect on the growth of normal cells.The potency and efficacy of the anti-tumor effect need to be improved.Therefore,it is of great significance to use medicinal chemistry to modify its structure to obtain derivatives with better efficacy and less toxicity.Artesunate is hybridized with the pharmacophore of anti-tumor drug tyrosine kinase inhibitors such as gefitinib,regorafenib,and sorafenib by molecular hybridization technology.It is desirable to obtain novel artesunate derivatives with higher biological activity and lower toxicity.Methods:In this paper,by means of organic chemistry,11 compounds of three types,characterized by amide and benzene-substituted pyridine,were designed and synthesized through amide condensation,Suzuki coupling reaction,esterification reaction,and other processes.The purity of these compounds was preliminarily identified by thin-layer chromatography and HPLC chromatography.The structure and purity of this series of compounds were confirmed by mass spectrometry,¹H NMR,and ¹³C NMR.Their antitumor activity against three hepatoma cell lines,Hep3B,Hep G2,and Huh7,and the cytotoxicity of human normal hepatocyte LO2 were detected by MTT（Methyl Thiazolyl Tetrazolium）assay.Then,6c stimulated the production of reactive oxygen species（ROS）in Huh7 and Hep3B cells by DCFH-DA（2,7-Dichlorodi-hydrofluorescein diacetate）probe detection,and NAC（N-Acetyl-L-cysteine）intervention experiments reversed cell death induced by elevated ROS levels.The ferroptosis inhibitor（Liproxstatin-1）was used to study the effect of compound 6c in inducing ferroptosis in hepatoma cells.Western blot was used to observe the effect of 6c on the expression of apoptosis-related proteins in Huh7 cells.The ability of 6c to induce apoptosis of Huh7 cells was detected by Annexin V-FITC/PI double staining.The morphological changes of Huh7 cells apoptosis induced by 6c were observed by hoechst33342 staining experiment.The effect of 6c in Huh7 cells cycle was detected by cell cycle detection kit.Molecular simulations of several hybrids were performed using the crystal structure of VRGFR-2（PDB code:3IAI）using MOE molecular docking software.Results:All compounds were confirmed by ¹H NMR,¹³C NMR,and mass spectrometry.The results of MTT assay showed that the anticancer activities of the 11 final products obtained were improved to varying degrees compared with the lead compound,artesunate（AS）.Among all evaluated compounds,compounds 6a,6b,6c,and 12c showed robust inhibitory activity comparable to the positive drug sorafenib,and 6c showed excellent selectivity for normal hepatocytes and hepatoma cells.In view of the high efficiency and excellent selectivity of 6c,we chose 6c for following research.Compound 6c inhibited the proliferation of hepatoma cells in a time-and dose-dependent manner.6c increased the level of intracellular ROS,regulated the expression of Bax and Bcl-2 through ROS,activated the mitochondrial apoptosis pathway,and induced apoptosis and ferroptosis in hepatoma cells.At the same time,6c caused G2/M phase arrest to prevent the cell cycle progression of hepatoma cells.The molecular simulation results of 6c on VEGFR-2（PDB code:3IAI）showed that the artesunate moiety of 6c was in good agreement with the hydrophobic entrance of VEGFR-2,and both the artesunate moiety and 4-（4-substituted phenoxy）pyridine can be inserted into the active pocket.Conclusion:In this work,11 derivatives with artesunate as the parent nucleus were synthesized,among which 6c showed the advantages of high anti-hepatoma cell activity and low toxicity to normal cells.Compound 6c exerts its anti-tumor effect by increasing the level of intracellular ROS to induce ferroptosis and apoptosis of HCC,and induce G2/M phase arrest of HCC.