Click chemistry-based synthesis of fluorescent digitoxin derivatives with anticancer activity and cellular imaging...

Cardiac glycosides are secondary plant metabolites with considerable potential as therapeutic agents in cancer treatment. However, digitoxin (Dg), one of the most studied members of this class, is reported to exhibit dose-limiting systemic toxicity and cannot be directly tracked in cancer cells. To address these limitations, we investigated whether Dg derivatization and conjugation with fluorescent moieties could modulate its cytotoxic profile in vitro while enabling cellular imaging. Such dual-function compounds may provide useful tools for studying the intracellular behavior of Dg derivatives while retaining anticancer activity. In this study, we designed and synthesized fluorescently labelled Dg derivatives via conjugation with BODIPY, Nile red, fluorescein, fluorescein isothiocyanate (F9), coumarin, rhodamine, and tetramethylrhodamine (F10) using copper-catalyzed click chemistry. To determine whether these derivatives retained interaction with Na⁺/K⁺-ATPase, molecular docking and molecular dynamics simulations were performed, confirming stable complexes for selected lead compounds. We then evaluated 72-h cytotoxicity of the new Dg derivatives in lung adenocarcinoma (A549), noncancerous lung fibroblasts (MRC-5), and several additional cancer cell lines. Overall cytotoxicity decreased 5-49-fold relative to Dg, with FD10 and FD9 retaining the highest activity. Fluorescence microscopy revealed intracellular localization of the derivatives in the endoplasmic reticulum, lysosomes, and cytoplasmic membrane in both A549 and MRC-5 cells. To further assess their effects in a more advanced model, Dg derivatives were evaluated in A549 cancer cell spheroids. Morphological changes were observed, with FD10 and FD9 inducing the greatest reduction in spheroid compactness after 3 and 17 days. Additionally, apoptosis was identified as the predominant mode of cell death in A549 cells treated with FD9 and FD10 after 48 h. Overall, these findings suggest that fluorescent Dg derivatives represent promising candidates for further development as anticancer agents with integrated imaging capability as tools for studying the cellular distribution of Dg-based tumor compounds.