Diethylstilbestrol metabolites and analogs. Stereochemical probes for the estrogen receptor binding site

Indenestrol A (IA) and indenestrol B (IB) are analogs and metabolites of diethylstilbestrol (DES). These compounds have high binding affinity with the estrogen receptor (ER) but possess weak uterotropic activity. Due to their chemical structures, IA and IB exist as mixtures of enantiomers. We investigated whether the poor biological activity of these compounds was due to differential activity of the enantiomers. We also utilized these compounds as probes to determine the extent of stereochemical sensitivity in the ER ligand binding site. The IA and IB enantiomers were separated to >98% purity using a chiral high pressure liquid chromatography column. Their enantiomeric nature was confirmed by mass spectrometry and NMR. The purified IA enantiomer peak 1 was derivatized with 4-bromobenzoyl chloride. The resulting di(4-bromobenzoate) IA was analyzed by x-ray crystallography and the absolute enantiomeric conformation assigned as C(3)-R. The IA enantiomers designated IA-R and IA-S were assayed by competitive binding to cytosolic ER. The competitive binding index was estradiol, 100; DES. 286; IA-Rac (racemic mixture of IA), 143; IA-R, 13; and IA-S, 285; the index showed that ER demonstrates a stereochemical chiral preference. The IB enantiomers did not show a binding preference: IB, 145; IB-1, 100; and IB-2, 143. The differences in the IA enantiomer binding were shown to be due to competitive interactions by Lineweaver-Burk analysis of saturation binding of estradiol to ER in the presence of 1-, 5-, and 10-fold molar excess of competitor. Differences in binding affinity of the enantiomers could be partially explained by differences in the association rate constant (k₊₁) determined by association rate inhibition studies in which IA-S was 15 times more active than IA-R. Nuclear estrogen receptor levels were measured 1 h after in vivo treatment with doses of 5-20 μg/kg. The IA-Rac produced only 60% of the levels as compared with DES. Nuclear ER levels were checked every 30 min up to 2 h with no apparent difference, indicating that the low early levels were not due to a delayed estrogen receptor retention. When the enantiomers were tested individually only a dose of 10 μg/kg IA-S translocated ER to a level comparable to DES, while IA-R showed low levels at several doses. These results suggest that the poor biological activity of IA may be related to the differential ER interaction of its enantiomers. The use of these compounds has shown that the ER ligand binding site demonstrates specific significant high affinity stereochemistry for non-steroidal compounds which influences its nuclear form. These findings may be relevant in explaining som of the agonist/antagonist properties of other types of non-steroidal estrogens such as triphenylethylenes.