C-4 analogs of podophyllotoxin as tubulin inhibitors: Synthesis, biological evaluation, and structure-activity relationship

The rich diversity of lignin small molecules derived from podophyllotoxin, a non-covalent tubulin inhibitor isolated from the Podophyllum family, has led to the discovery and clinical development of several anticancer agents including etoposide and teniposide, which received FDA approval for the treatment of various cancers through DNA topoisomerase inhibition. While these compounds share the same tetracyclic core, two subtle structural changes that differentiate 1 from its DNA topoisomerase-binding analogs—the presence of 4’ methylation on the aromatic ring and stereospecific glycosylation at the C-4 hydroxyl—yield two independent mechanisms. Given the immense pharmacological importance of these two features, we sought to establish a structure-activity relationship regarding modification at C-4 on the potency, specificity, and chemical properties of podophyllotoxin. Here, we synthesized and evaluated a systematic library of close-in diversified esters at the C-4 position of podophyllotoxin to evaluate the effect of alkyl bulk at C-4 on the potency of 4-acyl podophyllotoxin analogs as tubulin inhibitors. Additionally, we evaluate their biological target and activity through cell viability assays, tubulin polymerization assays, and cell cycle analysis. Furthermore, we rationalize our results by analyzing the interactions between each ester and the binding site of tubulin through computer docking models. Altogether, we show that increasing steric hindrance at C-4 leads to a loss in potency against human cancer cells but has an insignificant impact on the ex-vitro kinetics of tubulin inhibition. This suggests that the biological activity of our compounds may be attributed to cytosolic and membrane distribution rather than binding interactions within the colchicine site.