ROS-mediated antiproliferative effects of dihydrotestosterone-derived ferrocene-steroid conjugates toward human cancer cell lines of variable androgen dependence.

Ferrocene appendages often endow organic scaffolds with reactive-oxygen-species (ROS)-mediated cytotoxicity, yet ferrocene-androgen conjugates remain somewhat poorly explored, especially those linked at the steroid A-ring, known to modulate androgen receptor (AR) binding. Three C-2-substituted ferrocene-steroid conjugates derived from dihydrotestosterone (DHT) - the most potent human androgen - were synthesized; the structure of 1 was confirmed by single-crystal X-ray crystallography. Antiproliferative activity was quantified in AR-positive (LNCaP, OVCAR-3) and AR-negative (PC-3) cancer cells and in non-malignant MRC-5 fibroblasts. The balance of androgenicity and inherent cytotoxicity brought about by the presence of ferrocene proved adequate, as all conjugates acted in an antiproliferative manner toward the two hormone-responsive cancerous cell lines. Conjugate 2 (2α-ferrocenylmethyl-DHT) was the most potent analogue, inhibiting OVCAR-3 growth with an IC50 = 2.8 μM and a selectivity index of 3.0 relative to cisplatin. In OVCAR-3 cells, 2 triggered S-phase arrest, a 21-fold rise in intracellular iron, and ROS-dependent loss of viability; co-treatment with N-acetyl-l-cysteine, but not the caspase inhibitor Ac-DEVD-CHO, rescued cells. In multicellular tumor spheroids, 2 disrupted spheroid integrity (IC50 = 14 μM). These findings indicate the potential of A-ring substituted androgen-ferrocene conjugates as antiproliferative agents for hormone-dependent cancers, with 2 emerging as a promising candidate that surpasses cisplatin in potency and appears to act through a distinct mechanism.