Design, synthesis, in vitro and in silico evaluation of novel 5-(1-benzyl-1H-indol-3-yl)-3-phenyl-4,5-dihydroisoxazole derivatives against ER-positive breast cancer

Estrogen receptor alpha (ERα) overexpression is the primary dominant factor responsible for breast Cancer (BC) development. Despite survival success, clinical use of anti-estrogenic agents is hindered by endometrial risk, thromboembolic complications, and rising drug resistance. Therefore, novel, more potent ERα-targeted agents are essential to reduce the BC burden. A series of 21 5-(1-benzyl-1H-indol-3-yl)-3-phenyl-4,5-dihydroisoxazole hybrids were designed, synthesized, and evaluated in vitro on ERα-predominant and triple-negative cells. The structural and biological novelty of the designed backbone was confirmed through searches of the SciFinder and PubChem databases. Hybrids 19 (MCF-7, IC₅₀ = 13.1 ± 0.0711 μM), 20 (T-47D, IC₅₀ = 9.666 ± 0.059 μM), and 21 (T-47D, IC₅₀ = 13.19 ± 0.0416 μM) were identified as promising candidates against ER+ BC. Compounds 19 and 21 underscored significant human ERα inhibition in contrast to tamoxifen and bazedoxifene. In the cytotoxicity study, compound 21 exhibited a selectivity index higher than bazedoxifene, 12.4-fold in T-47D, and 4.4-fold in MCF-7, indicating low healthy cell toxicity. Compounds 19, 20, and 21 demonstrated cell cycle arrest predominantly at the G0/G1 checkpoint to induce Apoptosis. Compound 21 was observed with the highest early and total apoptotic effect. In addition, MD simulations confirmed stable ERα binding for compounds 19 and 21 with low average RMSD and RMSF values, while compound 20 formed the most compact complex. Moreover, pharmacokinetics and DFT analysis ensured drug-likeness and bio-feasibility of promising molecules. Finally, based on interaction-analysis, bio-assay findings, and 3D-QSAR mapping, an overall structure-activity correlation was established for future-optimization and lead-development of indole - 4,5-dihydroisoxazole hybrids against ER+ BC.