Synthesis and cytotoxicity of novel cyanochalcones: induction of cell cycle arrest, apoptosis, and autophagy in HEP2 and MCF-7 cells

Developing new chemotherapeutic agents is essential for improving cancer treatment and overcoming resistance. In this study, four novel synthetic cyanochalcone derivatives were designed, synthesized, and structurally characterized using various spectroscopic techniques. Their anticancer potential was evaluated via an MTT cytotoxicity assay against three human cancer cell lines and one normal cell line. Among the tested compounds, 5b exhibited the highest cytotoxic activity against laryngeal carcinoma HEP2 cells (IC₅₀ = 23.93 ± 1.3 µM), whereas 5c demonstrated potent cytotoxicity against breast cancer MCF-7 cells (IC₅₀ = 39.08 ± 3.4 µM). Mechanistically, treatment of HEP2 cells with compound 5b significantly downregulated cyclin-dependent kinase (CDK)4 and CDK6, key regulators of the cell cycle, while modulating ferroptosis-associated genes by suppressing transferrin (TF) and upregulating heme oxygenase-1 (HO-1). In contrast, compound 5c induced apoptosis in MCF-7 cells by upregulating TF while downregulating autophagy-related 3 (ATG3). Flow cytometric analysis indicated that both compounds promoted ATG3-independent autophagy and cell cycle arrest at G0/G1 (in HEP2) and G2/M (in MCF-7). Furthermore, these chalcone derivatives effectively induced P53-independent apoptosis, as evidenced by a significant increase in both early and late apoptotic phases. Molecular docking studies demonstrated a strong binding affinity of compound 5b toward CDK4 and CDK6, supporting its role in cell cycle inhibition. Overall, these findings suggest that the synthetic chalcone derivatives 5b and 5c possess promising anticancer activity, modulating key pathways involved in cell cycle regulation, apoptosis, ferroptosis, and autophagy, and emerge as strong candidates for further investigation in the development of new therapeutic agents.