Ferroptosis-Driven Anticancer Activity of 1,3,4-Oxadiazole-Benzimidazole/Acetamide Hybrids: Design, Synthesis, and Biological Profiling.

A series of 1,3,4-oxadiazole-benzimidazole/acetamide derivatives (7a-r) was designed, synthesized, and evaluated for their anticancer activity. NCI-screening results at 10 µM against the 60 human tumor cell line panel revealed a broad-spectrum of antiproliferative effects of the tested compounds. Compounds 7b, 7e, 7f, 7g, 7h, 7k, 7l, and 7r were further tested in a five-dose assay, and they exhibited half-maximal growth inhibitory (GI50) values ranging from 0.90 to 43.50 µM. Focused studies in MDA-MB-231 triple-negative breast cancer (TNBC) cells demonstrated that compounds 7b, 7g, 7h, 7k, and 7l possessed potent antiproliferative activity, with half-maximal inhibitory concentration (IC50) values between 1.92 and 3.69 µM. Mechanistic investigations using cell death pathway inhibitors indicated that compounds 7b, 7k, and 7l did not induce necrosis, apoptosis, or autophagy. Instead, these compounds significantly increased intracellular ferrous iron (Fe2+) and malondialdehyde (MDA) levels and induced lipid peroxidation in MDA-MB-231 cells. In parallel, GPX4 expression was markedly reduced at both the mRNA and protein levels, supporting ferroptosis as the primary mechanism of action. In addition, molecular docking studies and molecular dynamics simulations were performed for derivatives 7b, 7k, and 7l in order to confirm the mechanistic study. Collectively, these findings suggest that the 1,3,4-oxadiazole-benzimidazole/acetamide scaffold represents a promising chemotype for developing ferroptosis-inducing therapeutics targeting TNBC.