Hexestrol diazirine photoaffinity labeling reagent for the estrogen receptor.

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. Kd: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at > 315 nm. The tritium-labeled diazirine [3H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M(r) 65,000, the same species that is labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.

Oxohexestrol derivatives labeled with fluorine-18. Synthesis, receptor binding and in vivo distribution of two non-steroidal estrogens as potential breast tumor imaging agents.

We have prepared two non-steroidal estrogens in the 2-oxohexestrol series labeled with the positron-emitting radionuclide fluorine-18, 1-fluoro-5-oxohexestrol (4) and 1-fluoro-2-oxohexesterol (5). We anticipated that the polar ketone function at the interior of these ligands would reduce their level of non-specific binding, which might increase the selectivity of their uptake in vivo. The two compounds were prepared by total synthesis: compound 4 was prepared in fluorine-18 labeled form by [18F]fluorine ion displacement on a suitably protected methanesulfonate precursor followed by deprotection under acidic hydrogenolytic conditions; the isomer 5 was prepared from a protected alpha-keto trifluoromethanesulfonate precursor with deprotection under basic conditions as the final step. The binding affinity of these hexestrol derivatives for the estrogen receptor was determined by competitive radiometric binding assays at 0 and 25 degrees C, and their lipophilicity (as octanol-water partition coefficients, log P values) and non-specific binding were estimated. The log P values determined by a reversed phase HPLC method were higher, relative to estradiol, than those calculated by the fragment method of Rekker. In tissue distribution studies in immature (50 g) rats, both of these compounds showed selective uptake in estrogen target tissues. At 1 h, activity in the uterus reached the level of 2.5-3.0% of the injected dose per gram tissue, with uterus-to-blood and uterus-to-muscle ratios of 14-20 and 8-14, respectively. The uptake efficiency and selectivity of these fluoro-oxohexestrols in principal estrogen target tissues is less than that of fluorine-18 labeled steroidal estrogens we have prepared previously, but their receptor-mediated uptake in certain secondary target tissues is substantial. The specific and non-specific components of target tissue uptake of these two compounds appear to be directly related to their non-specific binding and their binding selectivity.