Multifaceted biological and computational assessment of aromatic and N-heteroaromatic non-substituted thiosemicarbazones

Thiosemicarbazones (TSCs) constitute a pharmacologically versatile class of compounds with documented antimicrobial, antiviral, and anticancer properties. Despite their therapeutic potential, their biological mechanisms and pharmacokinetic behaviour remain insufficiently characterized. In this study, we investigated the biological activity of the library consisting of 28 aromatic and heteroaromatic compounds against twelve cancer cell and two normal cell lines, and four parasites. Six N-heteroaromatic thiosemicarbazones based on pyridine (13, 14, and 18), quinoline (24 and 26), and indole (27) moieties show promising anticancer and/or antiparasitic activity. Density functional theory (DFT) calculations indicated that the biological activity of 13, 14, 18, 24, and 26 arises from their ability to chelate d-metals, whereas 27 operates via a distinct mechanism. The passive gastrointestinal absorption and Human serum albumin (HSA) binding ability of these six compounds were evaluated by the Parallel artificial membrane permeability (PAMPA) technique and high-performance affinity chromatography, respectively. Compound 26 emerged as the most potent and selective anticancer agent and was further examined through cell cycle profiling, caspase-3/7 activation, DNA double-strand break quantification, 3D spheroid assays, and an in ovo chorioallantoic membrane (CAM) model, supported by HSA docking and molecular dynamics simulations. Collectively, our results indicate that 26 is a selective anticancer agent that exerts cytotoxic effects preferentially in cancer cells, by inducing apoptosis and DNA double-strand breaks.