Discovery of IMHDPA derivatives with selective cytotoxicity and autophagy-modulating properties: Structure-activity relationships and mechanistic insights.

Phenotypic drug discovery enables the identification of compounds with novel mechanisms of action and context-dependent biological activities that may not emerge from target-based approaches. Through high-content phenotypic screening, we identified the (E)-6-(2-((1H-indol-3-yl)methylene)hydrazineyl)-N,N-diethylpyrimidin-4-amine (IMHDPA) scaffold as a potent and highly selective inhibitor of HeLa cell proliferation. To further explore this scaffold, a library of 70 analogues was synthesized, and structure-activity relationship studies revealed stringent structural requirements for maintaining cellular potency and selectivity. Among them, compound 39 emerged as the most active derivative, exhibiting a GI50 value of 2.96 nM against HeLa cells and an exceptional selectivity index exceeding 16,000-fold across a panel of 15 cell lines. Compound 39 suppressed colony formation, migration, invasion, and spheroid growth, while inducing pronounced morphological alterations in HeLa cells. Mechanistic investigations indicated that its antiproliferative activity was associated with autophagy activation rather than apoptosis, necrosis, ferroptosis, or reactive oxygen species accumulation. Integrated transcriptomic and proteomic analyses implicated perturbation of cholesterol metabolism and inhibition of mTORC1 signaling as potential upstream events linked to autophagy induction. Collectively, these findings establish IMHDPA derivatives as promising chemical probes for investigating the molecular basis of context-dependent autophagy-associated cytotoxicity and support future efforts aimed at elucidating the molecular determinants underlying this selective phenotype.