[³H]DU41165: A high affinity ligand and novel photoaffinity labeling reagent for the progesterone receptor

17α-Acetoxy-6-fluoro-16-methylene-(9β, 10α )pregna-4,6-dien-3,20-dione (DU41165), a retroprogestin (9β,10α) embodying a fluorine-substituted dienone system, has been prepared in high specific activity tritium-labeled form (4 Ci/mmol) and shown to be a high affinity ligand for the progesterone receptor (PgR) and a highly selective photoaffinity labeling reagent for PgR. The radiosynthesis involved conversion of DU41231 (the 17α-hydroxy analog of DU41165) to DU41165 by treatment with tritum-labeled acetic anhydride. The binding affinity of DU41165 for PgR was determined by both a competitive binding assay and a direct binding assay (Scatchard analysis) to be 1.6-2.2-times higher than that of the high affinity synthetic progestin promegestone (R5020). In unlabeled form, DU41165 demonstrates photoinactivation of PgR to the extent of 60% at 60 min. In radiolabeled form [³H]DU41165 demonstrates specific covalent attachment with an efficiency of 5-7%. SDS-polyacrylamide gel electrophoresis of photoattached [³H]DU41165 confirms that there is covalent labeling of both the B subunit (M_r = 118,000), and the A subunit (M_r = 88,000) of PgR in a molar ratio of approximately 1:3.