Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells

<b>Background/Objectives:</b> Breast cancer remains one of the most prevalent and life-threatening malignancies worldwide. This study describes the design and biological evaluation of a series of secosteroid-2-pyrazoline hybrids as novel antitumor agents against ERα-positive breast cancer cell lines MCF-7 and T47D. <b>Methods</b>: A simple and efficient method for synthesizing secosteroid-2-pyrazoline hybrids was developed starting from 13α-hydroxy-3-methoxy-13,17-secoestra-1,3,5(10)-triene-17-oic acid hydrazide and 1,3-diketones. The resulting secosteroid derivatives were evaluated against hormone-dependent MCF-7 and T47D breast cancer cells. Furthermore, the selectivity and effects of three lead compounds on signaling pathways in MCF-7 cells were examined. Flow cytometry was used to assess the cell-cycle distribution of MCF-7 cells treated with the lead compound. <b>Results</b>: Among the synthesized hybrids, compounds <b>3f</b>, <b>3j</b>, and <b>3k</b> exhibited potent antiproliferative activity with IC₅₀ values of 0.2-0.5 μM against breast cancer cells, while demonstrating very low cytotoxicity towards normal cells (IC₅₀ > 25 μM), indicating a favorable safety profile. The antitumor activity of lead compound <b>3j</b> was additionally investigated in combination with standard chemotherapeutics, docetaxel and doxorubicin, yielding synergistic effects. The lead compounds showed a dual mechanism of action by inhibiting S6 kinase and promoting Bcl-2 phosphorylation at 0.9 μM, without significantly affecting hormonal breast cancer targets such as ERα, GREB1, and AR. Compound <b>3j</b> induced apoptosis accompanied by a reduction of the G1/G0 phase in MCF-7 cells. <b>Conclusions</b>: These findings highlight secosteroid-2-pyrazoline hybrids as promising candidates for the development of next-generation breast cancer therapeutics targeting apoptosis and S6K signaling pathways.