Repositioning of 3-(isoxazol-3-yl)thiazolidin-4-one-based compounds via Inverse Virtual Screening: discovery of new ERα inhibitors with in vitro activity in breast cancer models

Herein, we report the design, synthesis and biological evaluation of a novel series of 23 racemic thiazolidin-4-one/isoxazole derivatives. The compounds were initially designed as putative BRD9 ligands; however, biophysical assays revealed weak BRD9 binding, prompting the search for alternative molecular targets through an Inverse Virtual Screening approach. This strategy identified Estrogen Receptor alpha (ERα) as a prioritized target, which was subsequently validated by TR-FRET assays, revealing five compounds as promising ERα ligands. The evaluation of their effects against Breast Cancer cell lines (MCF-7 and MDA-MB-231) showed antiproliferative activities in the low micromolar range and good selectivity over non-cancerous cells. Moreover, mechanistic studies demonstrated that the most active compounds, 4 and 14, significantly reduced ERα expression levels in MCF-7 cells, and, notably, downregulation occurs through ubiquitin-proteasome-mediated degradation. Based on molecular modelling predictions, enantiomerically pure compounds were isolated and characterized. Biological evaluation confirmed that R enantiomers display superior ERα affinity and enhanced antiproliferative activity compared with their S counterparts, while maintaining favorable target selectivity and more suitable pharmacokinetic properties. Therefore, this new class of chiral thiazolidin-4-one/isoxazole-based compounds, endowed with a satisfactory in vitro safety profile, was identified as promising candidate for further investigation in Breast Cancer therapy.