Antitumor activity of clomiphene analogs in vitro: relationship to affinity for the estrogen receptor and another high affinity antiestrogen-binding site.

The antitumor activity of three enclomiphene (trans-1- (p-beta-diethylaminoethoxyphenyl)1,2-diphenyl-2-chloroethylene) analogs and the cis isomer zuclomiphene was investigated in MCF 7 human mammary carcinoma cells in culture. Relative antitumor activity was compared with relative binding affinities (RBAs) for the nuclear estrogen receptor (RE; estradiol = 100%) and a microsomal antiestrogen-binding site (AEBS; tamoxifen = 100%) measured in cell-free extracts from MCF 7 cells. Modifications in the structure of the diethylaminoethoxy side chain influenced affinity of both RE and AEBS. Deethylation of the side chain (9599) reduced affinity for both sites by 65-70%, while a diethylaminoproproxy side chain (6866) resulted in a 3-fold increase in affinity for RE (enclomiphene = 2%; 6866 = 6%), but reduced the RBA for AEBS by 66% (enclomiphene = 140%; 6866 = 45%). Conversion of the ether linkage to an amine (10222) increased affinity for RE (10222 = 5%), but markedly reduced affinity for the AEBS (10222 = 15%). All five compounds inhibited the growth of MCF 7 cells in culture, but with markedly different potencies. At low doses (0.25-1.0 microM), where the growth-inhibitory effects were reversed by estradiol (except in the case of zuclomiphene), the relative antitumor activity (6866 greater than 10222 greater than enclomiphene greater than 9599 greater than zuclomiphene) was in the same order as RBA for RE. At higher doses (greater than 2.5 microM), where the growth-inhibitory effects were unaffected or partially reversed by estradiol, the relative potencies of these compounds as antitumor agents changed. Zuclomiphene became the most active, while the potency of enclomiphene increased relative to 6866 and 10222 (zuclomiphene greater than enclomiphene greater than 6866 greater than 10222 greater than 9599). At these doses, estrogen-irreversible antitumor activity was unrelated to affinity for RE, but showed some correlation with affinity for AEBS. It is concluded that the major effect of these compounds on mammary carcinoma growth in vitro is an estrogen-reversible growth-inhibitory effect, the magnitude of which is correlated with affinity for RE. However, studies with zuclomiphene and estrogen-irreversible doses of enclomiphene and 6866 indicate that these antiestrogens also influence cell proliferation in vitro by mechanisms that probably do not involve RE.