A comprehensive study of novel pregnenolone-isoxazole hybrids as selective inducers of mitochondrial apoptosis in breast cancer cells.

Heterocyclic derivatives of steroids have emerged as promising anticancer agents by concurrently targeting multiple pathways, including apoptosis induction, cell cycle arrest, and mitochondrial dysfunction. Here, we report novel pregnenolone-isoxazole hybrids as selective anticancer agents targeting mitochondrial apoptosis. Starting from 20-vinyl pregnenes, nitroisoxazole-substituted pregnenolone derivatives were synthesized and subjected to nucleophilic aromatic substitution, yielding a variety of pregnenolone-isoxazole hybrids bearing azido, phenylthio, and amino groups. In total, 33 new compounds were prepared and evaluated for their in vitro antiproliferative activity against breast cancer cells. The lead compound, 20-[3'-(5″-nitro)isoxazole]-pregna-5-ene-3β-ol-20-one, demonstrated IC50 values of 2.8 μM or lower against MCF7, MCF7/HT2, MDA-MB-231, and HCC1954 breast cancer cell lines under both normoxic and hypoxic conditions. This compound induced mitochondria-dependent apoptosis in MCF7 cells in a concentration-dependent manner. Modulation of key proliferation pathways, including the PI3K/Akt and ERK cascades, further supported the antiproliferative potential of these derivatives. Considering its high cytotoxicity and antiproliferative activity in breast cancer cells, the lead compound has been identified as a promising candidate for further development.