Novel α-Cyano indolylchalcone derivatives as potential candidates for hepatocellular carcinoma treatment (HepG2 cell line)

Four novel α-cyano-indolylchalcone derivatives ( 3a, 3b, 5a, and 5b ), incorporating biologically active indole, pyrazole, and chalcone pharmacophores within a single molecular framework, were successfully synthesized and structurally characterized using FTIR, NMR ( 1 H and 1 3 C), and mass spectrometry. The cytotoxic potential of the synthesized compounds was evaluated against breast cancer (MCF7), hepatocellular carcinoma (HepG2), and colorectal cancer (HCT116) cell lines using the MTT assay. Doxorubicin was used as a reference drug and exhibited an IC₅₀ value of 4.4 µg/mL against HepG2 cells. Among the synthesized derivatives, compounds 5a and 5b showed the highest cytotoxic activity, particularly against HepG2 cells, with IC₅₀ values of 49.01 and 21.89 µg/mL, respectively.To elucidate their mechanism of action, the apoptotic effects of compounds 5a and 5b were investigated using gene expression analysis, cell-cycle assessment, caspase activation assays, and flow cytometry. Treatment with both compounds resulted in significant upregulation of the pro-apoptotic genes Bax and p53 , downregulation of the anti-apoptotic gene Bcl-2 , activation of caspase-3 and caspase-9, and induction of G1-phase cell-cycle arrest. Flow cytometric analysis further confirmed enhanced apoptotic cell death in treated HepG2 cells.These findings demonstrate that α-cyano-indolylchalcone derivatives, particularly compound 5b, exert potent anticancer activity through activation of the intrinsic apoptotic pathway and inhibition of cell proliferation. The study highlights the therapeutic potential of integrating indole, pyrazole, and chalcone scaffolds into a single molecular architecture and identifies compounds 5a and 5b as promising lead candidates for the development of novel treatments for HepG2.