Design, synthesis and biological evaluation of novel podophyllotoxin derivatives as tubulin-targeting anticancer agents.

Context: Podophyllotoxin (PPT) derivatives, used in cancer therapy, require development toward enhanced efficacy and reduced toxicity.

Objective: This study synthesizes PPT derivatives to assess their anticancer activities.

Materials and methods: Compounds E1-E16 antiproliferative activity was tested against four human cancer cell lines (H446, MCF-7, HeLa, A549) and two normal cell lines (L02, BEAS-2B) using the CCK-8 assay. The effects of compound E5 on A549 cell growth were evaluated through molecular docking, in vitro assays (flow cytometry, wound healing, Transwell, colony formation, Western blot), and in vivo tests in female BALB/c nude mice treated with E5 (2 and 4 mg/kg). E5 (4 mg/kg) significantly reduced xenograft tumor growth compared to the DMSO control group.

Results: Among the 16 PPT derivatives tested for cytotoxicity, E5 exhibited potent effects against A549 cells (IC₅₀: 0.35 ± 0.13 µM) and exceeded the reference drugs PPT and etoposide to inhibit the growth of xenograft tumours. E5-induced cell cycle arrest in the S and G2/M phases accelerated tubulin depolymerization and triggered apoptosis and mitochondrial depolarization while regulating the expression of apoptosis-related proteins and effectively inhibited cell migration and invasion, suggesting a potential to limit metastasis. Molecular docking showed binding of E5 to tubulin at the colchicine site and to Akt, with a consequent down-regulation of PI3K/Akt pathway proteins.

Discussion and conclusions: This research lays the groundwork for advancing cancer treatment through developing and using PPT derivatives. The encouraging results associated with E5 call for extended research and clinical validation, leading to novel and more effective cancer therapies.