Design, synthesis, in silico and in vitro anti-breast cancer evaluation of novel indole-isoxazole/isoxazoline hybrids as ERα inhibitors.

Abstract

Breast cancer (BC) remains the foremost cause of cancer mortality in women, with ER + subtypes accounting for over 70% of cases. Disease progression is driven by aberrant receptor signaling, marked by elevated ERα and diminished ERβ expression, underscoring the urgency of potent and targeted ERα inhibitors. Although selective estrogen receptor modulators like tamoxifen have significantly improved patient survival, their clinical utility is constrained by adverse effects like endometrial carcinoma, blood clot formation, deep vein thrombosis, hot flashes and VTE, alongside emergence of drug resistance. To overcome current limitations in ERα targeted therapy, we designed and synthesized a novel series of indole-conjugated isoxazole/ isoxazoline hybrids through a streamlined five-step synthetic route, affording moderate to high yields (70-90%). These hybrids were systematically evaluated against ERα-predominant and triple-negative BC cell lines, with promising candidates subjected to healthy cell cytotoxicity, receptor inhibition assays and comprehensive in silico validation. Among the synthesized hybrids, C04 and C06 emerged as lead candidates, demonstrating markedly superior anti-proliferative activity on ERα dominant BC cells compare to tamoxifen and bazedoxifene. Cell cycle arrest and apoptotic studies further confirmed capability of both compounds in apoptosis induction primarily through G0/G1 cell-cycle arrest. ERα inhibition assays highlighted the exceptional potency of C06, exceeding bazedoxifene by 3.3-fold and tamoxifen by 7.9-fold. C04 was identified with broad safety profile in HEK cell cytotoxicity studies. Molecular docking and dynamic simulations further validated their robust target engagement. Collectively, these findings established C04 and C06 as optimistic ERα targeted leads with superior efficacy and safety profiles than standard drug candidates.