The investigational anti-breast cancer agent 1,1-dichloro-cis2.3- diphenylcycloprane (Analog II, 1), has antiestrogenic and anti-tumor activity in rodents, but appears to be exerting its effects in a manner different from classical antiestrogen. The precise mechanism(s) by which 1 is effecting its activity are into known. Compound 1 is a highly lipophilic agent with little affinity for the high affinity-lo capacity estrogen receptor. It is known to be active in both hormone-dependent and independent breast cancer study systems (rodents and human cell lines). It is also known to be metabolized in rodent in vivo systems to electrophilic intermediates which bind to macromolecule. In rodent in vitro metabolizing systems it is also known to be converted to compounds which closely resemble known antimitotic agents and nuclear type II binding sites antiprofilerative agents. The actions and metabolic profile of this compound in human breast cancer cell lines with differing hormone dependence have yet to be completely characterized. The subcellular fractions affected by 1, both in vitro and in vivo, are also unknown. The overall goal of this focused project is to begin to define the molecular mechanism of action of 1 at the structural level so that potent anti-breast cancer agents can be rationally designed, synthesized and evaluated. Preliminary results generated in this laboratory demonstrate that 1 is metabolized to an active metabolite, which has antiproliferative and cytotoxic activity above that seen with the patent compound in hormone dependent and independent human breast cancer cell lines. Therefore, specific aims are: 1. To further characterize the growth inhibitory action of 1 in estrogen receptor (ER) positive-estradiol (E2) responsive, ER negative-E2 unresponsive, and ER positive-E2 unresponsive human breast cancer cell lines (MCF-7, MDA-MB-231 and MCF-7/LY-2) by cytostasis, cytotoxicity, and nuclear type II site binding assays. 2. To synthesize both radiolabeled and heavy atom labeled analogues of 1,1-dichloro-2,3-cis-diphenylcyclopropane to probe its metabolism and mechanism of antitumor action. 3. To determine the structures of the metabolites formed in vitro by metabolic activation of 1 by mouse and human liver microsomal systems, as well as those formed in the human breast cancer cell lines. 4. To determine if covalent adducts of macromolecule are formed in vitro by metabolic activation of 1 as a means to elucidate its molecular mechanism of action. 5. To determine the distribution of metabolites and macromolecular adducts formed in vivo in targets tissues, with emphasis on hormone- sensitive tissues such as uterus, ovary, adrenal, liver, brain testes and mammary gland of mice treated with 1.
