ER alpha selective chromone, isoxazolylchromones, induces ROS-mediated cell death without autophagy

Chromones are recognized as privileged structures and useful templates for the design of novel compounds with promising pharmacological activity. Several reports implicate chromone scaffold as an antitumor agent. The present study highlights synthesis, docking, and potential activity of isoxazolylchromones, 3(a-f), a new class of compounds as potential agents exhibiting ERα antagonism and ERβ agonism. Molecular docking studies determined the binding site of compounds 3(a-f) in ERα and ERβ. All the analogues synthesized showed preferential cytotoxicity in ERα+ cell line (MCF-7) compared to ERα- cell line (MDA-MB-231). Among the analogues synthesized, analogue 3d exhibited increased cytotoxicity. ERα silencing experiments confirmed the ERα selective nature of ligands. Transactivation assay on compound 3d indicated the down-regulation of ERα luciferase reporter gene expression and induction of ERβ GFP in the treated cells. Cell cycle analysis revealed an increase in sub-G0/G1 population on treatment with analogue 3d as compared to control. Similar to tamoxifen, 3d-induced cell death is mediated through an increase in ROS as evidenced by change in roGFP ratio. Interestingly, the compound 3d induced mitochondrial trans-membrane potential loss and caspase activation without indication of autophagy compared to tamoxifen that induced autophagy in the treated cells. Lack of significant autophagy and induction of ERβ signaling by the new compound place them as a better ERα antagonist.