We have previously shown that conjugated ferrocenyl p-phenols show strong cytotoxic effects against both the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell lines, possibly via metabolic quinone methide (QM) formation. To further evaluate this proposed mechanism, we have created a series of ferrocenyl prodrugs containing methyl and acetyl-protected thio- and oxo-phenols: 2-ferrocenyl-1,1-bis(4-acetoxyphenyl)-but-1-ene (5), 2-ferrocenyl-1,1-bis(4-thioacetylphenyl)-but-1-ene (6), 2-ferrocenyl-1,1-bis(4-methoxyphenyl)-but-1-ene (7), and 2-ferrocenyl-1,1-bis(4-thiomethylphenyl)-but-1-ene (8), which might be activated by hydrolysis enzymes in situ. Only the acetoxy 5 displayed antiproliferative effects (IC50 on MDA-MB-231 of 0.5 mu M) while 6-8 act as pure estrogens (proliferative on MCF-7 and little to no effect on MDA-MB-231). The behaviour of 5 is similar to that previously found for the free phenol 2-ferrocenyl-1,1-di(4-hydroxyphenyl)-but-1-ene (2), indicating that 5 is metabolized in situ to 2, which could then undergo oxidative QM formation. The observation that the thioacetyl 6 is not cytotoxic suggests that the in situ oxidative chemistry of the putative ferrocenyl thiophenol is different from that of 2. Because p-thioquinone methides are practically unknown, the negative results for 6 further implicate the bioformation of the QM in the case of 2 and related compounds. The lack of cytotoxicity of 7 and 8 can be attributed to lack of efficient hydrolysis in situ. Estrogen receptor binding affinity studies for the compounds and the X-ray structure of 8 are also reported. (C) 2008 Elsevier B.V. All rights reserved.