Previous studies from this laboratory have revealed that two potent nonsteroidal antiestrogens, CI628 [α-(p-(2-(1-pyrrolidino)ethoxy)phenyl)4-methoxy-α′-nitrostilbene] and U23, 469 [cis-(3-(p-(l, 2, 3, 4-tetrahydro-6-methoxy-2-phenyll-naphthyl)phenoxy)1, 2-propanediol], are metabolized during their action in vivo to more polar metabolites. We have identified these metabolites as the demethylated forms of the parentantiestrogen and have synthesized them (CI628M and U23.469M). In studies reported here, we compare the potency of the metabolites and parent antiestrogens in terms of their 1) affinity for cytoplasmic estrogen receptor, 2) ability to translocate estrogen receptor to the nuclear fraction in whole uteri in organ culture in vitro and to prevent nuclear uptake of [3H]estradiol, 3) ability to prevent estradiol stimulation of induced protein synthesis in vitro, and 4) ability to inhibit estradiol stimulation of uterine weight gain and peroxidase activity in vivo. The antiestrogens compete with [3H]estradiol for binding to cytosol estrogen receptor with the following affinities: CI628M, 135%; U23.469M, 30%; CI628, 11%; and U23.469, 0.1%, where estradiol affinity is considered 100%. In whole uteri in vitro, all four compounds deplete cytoplasmic receptor and translocate estrogen receptor into the nucleus, and they prevent nuclear localization of [3H]estradiol and inhibit estradiol stimulation of induced protein synthesis in a dose-related fashion; CI628M and U23.469M are more potent, being as effective as their parent compounds at 10- to 100-fold lower doses. In 3-day in vivo assays, dose-response curves indicate that the metabolites and parent compounds are equally potent in inhibiting estradiol-stimulated uterine weight gain and peroxidase activity. These studies document that the demethylated metabolites of the antiestrogens have a higher affinity for receptor and a greater biological potency in vitro. However, in vivo, where the parent compounds are rapidly and efficiently converted to the metabolites, both forms have comparable potencies.
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