Estrogen receptor stereochemistry: ligand binding orientation and influence on biological activity

Racemic (Rac) 4'- and 5-deoxyindenestrol A (4'-DIA and 5-DIA), monohydroxyl analogs of the diethylstilbestrol (DES) oxidative metabolite indenestrol A (IA), were synthesized, and their enantiomers were resolved and isolated. Each compound was then tested for estrogen receptor (ER) binding affinity, uterotropic activity, and nuclear ER levels, to further define the stereochemical preference of the ER and to structually evaluate the function of each IA hydroxyl group for binding and biological activity. Competitive binding to cytosolic ER determined the relative binding affinity of racemic mixtures of 4'- and 5-DIA as 1.3 and 3.7, respectively, compared with that of DES, 286. The ER exhibited a binding preference for the S-enantiomer of both compounds, with relative binding affinities of 4'-DIA-R, 0.2; 4'-DIA-S, 1.8; 5-DIA-R, 0.9; and 5-DIA S, 5.6. 4'-DIA-Rac produced 3 times the in vivo stimulation of 5-DIA-Rac in the uterotropic bioassay (with mouse uterine doubling doses of 302.4 and 800 micrograms/kg, respectively). Nuclear ER levels measured 1 hr after in vivo treatment with either 160 micrograms/kg 4'-DIA or 80 micrograms/kg 5-DIA showed a maximum binding level of 2 (4'-DIA) and 1.5 (5-DIA) times saline control, with these doses producing levels nearly equal to that caused by a 10 micrograms/kg dose of IA. Metabolic studies were carried out by treating mice with [3H]4'- and [3H]5-DIA-Rac, to determine the differential binding affinity and biological stimulation of 4'-DIA and 5-DIA. The in vivo metabolism of the [3H]DIA compounds showed formation of [3H]IA-Rac in urine extracts, as analyzed by chiral high performance liquid chromatography. Furthermore, in vitro incubation of unlabeled 4'- and 5-DIA-Rac with mouse liver microsomes showed stereospecific metabolism, with IA-S primarily formed from 4'-DIA-Rac and IA-R from 5-DIA-Rac. Metabolism of 4'-DIA-Rac to the more active IA S-enantiomer and of 5-DIA-Rac to the less active IA R-enantiomer contributes to the different biological activities, because the ER exhibits a chiral preference for these compounds. The higher binding affinity of 5-DIA indicates that the phenyl ring hydroxyl group is required for high affinity binding; however, both hydroxyl groups are needed for subsequent biological activity. These data further suggest that the ER demonstrates stereochemical ligand binding and that IA binds in an orientation relative to 17 beta-estradiol in which the IA phenyl ring corresponds to the estradiol A-ring.