Shikonin Derivatives:Design,Synthesis And Anticancer Activity

Shikonin has a wide range of pharmacological effects,such as anti-inflammatory,anti-viral and anti-oxidative,especially good anti-cancer effects,and these effects have attracted wide attention.However,its potential utility is limited because of its relative side effects and toxicity.In order to develop high efficiency and low toxicity molecules,Twenty one shikonin derivatives targeting PI3K were designed and synthesized.All compounds synthesized have been verified by ¹H-NMR,¹³C-NMR and HR-MS.First,The activity of the compounds 4a⁴u against human gastric cancer cells?SGC-7901?,human gastric cancer cells?MGC-803?,human hepatocyte cancer cells?SMMC-7721?,human glioma cells?U-87?and human normal liver cells?L02?was evaluated in vitro by MTT assay.We found that compound 4s has more research significance on SGC-7901 cells.Therefore,Compound 4s was chosen to explore the action mechanism.We further investigated the effects of compound 4s on cell proliferation by colony formation assay and cell cycle assay;AnnexinV/PI staining and western blot assay were used to detect the effect of compound 4s on apoptosis;Cell autophagy was investigated by acridine orange staining,GFP-LC3 transfection,immunofluorescence and western blot assay;And AnnexinV/PI staining and western blot assay were used to investigate the role of autophagy in the apoptosis induced by the compound 4s;Finally,western blotanalysiswasusedtoassessetheeffectsofcompound4son phosphoinositide3-kinase/protein kinase B and mammalian target of rapamycin?PI3K/AKT/mTOR?pathway,Further exploring whether the compound has an effect on the PI3K protein through molecular docking.The results showed that the colony formation rate decreased with the increase concentration of compound 4s,it indicated that cell proliferation was inhibited by compound 4s;The ratio of compound 4s arrested in G1 phase increased in a concentration-dependent manner,It mean that inhibition of cell proliferation was related to retardarce in G1 phase;The apoptotic rate of compound4s on SGC-7901 increased with increasing concentration?1,2,4?M?,the levels of apoptosis-related proteins Bax and cl-PARP were up-regulated,and Bcl-2 and PARP were down-regulated,indicating that apoptosis was induced after cells were blocked in G1 phase by compound 4s;An increase in autophagic lysosomes and LC3 showed the occurrence of autophagy;When the compound 4s was incubated with the autophagy inhibitor 3-methyladenine?3-MA?,the apoptosis rate was inhibited,indicating that autophagy promoted the cell apoptosis induced by compound 4s;Compound 4s negatively regulated autophagy by PI3K/AKT/mTOR pathway.Preliminary mechanism suggested that compound 4s mediates apoptosis in an autophagy-dependent manner by inhibiting PI3K/AKT/mTOR pathway.