5-(Thiophen-2-yl)isoxazoles as novel anti-breast cancer agents targeting ERα: synthesis, <i>in vitro</i> biological evaluation, <i>in silico</i> studies, and molecular dynamics simulation

Herein, we report the design and synthesis of novel 5-(thiophen-2-yl)-4-(trifluoromethyl)isoxazoles (<b>TTI</b>), and <i>in vitro</i> evaluation of their anti-cancer activities. Based on the molecular structure of our previously developed isoxazole-based anti-breast cancer lead molecule, 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)-4-(trifluoromethyl)isoxazole (TTI-4), we designed a set of 14 new analogues of TTI-4. The <b>TTIs</b> are a synthetically challenging class of molecules, and we synthesized them with high purity by utilizing our in-house developed novel synthetic strategy, <i>i.e.</i>, metal-free, cascade regio- and stereoselective trifluoromethyloximation, cyclization, and elimination strategy, with readily available α,β-unsaturated ketones by using commercially available and cheap reagents such as CF₃SO₂Na and ^<i>t</i> BuONO (cost-effective and sustainable synthesis). Subsequently, the anti-cancer activities of the newly synthesized molecules were evaluated against various cancer cell lines such as MCF-7, 4T1, and PC-3, and the molecules showed potential and more selective cytotoxicity against the human breast cancer cell line, MCF-7, among others. The <i>in vitro</i> screening revealed a new molecule, <i>i.e.</i>, 5-(thiophen-2-yl)-4-(trifluoromethyl)-3-(3,4,5-trimethoxyphenyl)isoxazole (TTI-6), possessing an IC₅₀ value of 1.91 μM against MCF-7, is superior to the previous lead molecule (TTI-4) and also the best anti-cancer agent among all. The structure-activity relationship (SAR) studies revealed the importance of an unsubstituted thiophene ring in the 5th position, a -CF₃ functional group in the 4th position, and a highly electron-rich benzene ring bearing three -OCH₃ functional groups in the 3rd position of the isoxazole core to have superior activity. Further studies with TTI-6, such as apoptosis induction, cell cycle analysis, and nuclear staining, revealed an apoptotic cell death mechanism. The <i>in silico</i> molecular docking, induced fit analysis, and ADMET studies further supported the effects of various functional groups of <b>TTIs</b> on their anti-breast cancer activity by inhibiting the estrogen receptor alpha (ERα), a crucial nuclear hormone receptor involved in gene regulation that plays an important role in several human cancers.