Synthesis and biological evaluation of hesperetin derivatives as agents inducing apoptosis

A flavanone, hesperetin, has been known to exert antitumor activity by inducing apoptosis. To find hesperetin derivatives showing better antitumor activity, 12 derivatives were designed and synthesized. Their antitumor activities were measured using a long-term survival clonogenic assay. Among the compounds, K-5b, hesperetin-7-butyrate, showed the half-maximal cell growth inhibitory concentration three times as low as that of hesperetin. To compare the cytotoxicity of hesperetin and K-5b, the HCT116 human colon cancer cell line was treated with various concentrations of each compound. K-5b decreased the cell viability to a larger extent than hesperetin and triggered apoptosis more efficiently than hesperetin in an apoptosis detection assay using fluorescein isothiocyanate-labeled annexin V. Immunoblotting analysis showed that K-5b promoted caspase-mediated apoptosis more efficiently than hesperetin. Because hesperetin has been reported to induce apoptosis through the activation of the c-Jun N-terminal kinase (JNK) pathway, we tested whether K-5b activates JNKs. K-5b stimulated JNK1 and JNK2 more efficiently than hesperetin as shown by western blot analysis. In conclusion, hesperetin derivatives exerting better antitumor activity than hesperetin by inducing apoptosis were found.