Estradiol (E2) plays an important role in cell proliferation and certain brain functions. To reveal its mechanism of action, its detectability is essential. Only a few fluorescent-labeled hormonally active E2s exist in the literature, and their mechanism of action usually remains unclear. It would be of particular interest to develop novel labeled estradiol derivatives with retained biological activity and improved optical properties. Due to their superior optical characteristics, aza-BODIPY dyes are frequently used labeling agents in biomedical applications. E2 was labeled with the aza-BODIPY dye at its phenolic hydroxy function via an alkyl linker and a triazole coupling moiety. The estrogenic activity of the newly synthesized fluorescent conjugate was evaluated via transcriptional luciferase assay. Docking calculations were performed for the classical and alternative binding sites (CBS and ABS) of human estrogen receptor α. The terminal alkyne function was introduced into the tetraphenyl aza-BODIPY core via selective formylation, oxidation, and subsequent amidation with propargyl amine. The conjugation was achieved via Cu(I)-catalyzed azide–alkyne click reaction of the aza-BODIPY-alkyne with the 3-O-(4-azidobut-1-yl) derivative of E2. The labeled estrogen induced a dose-dependent transcriptional activity of human estrogen receptor α with a submicromolar EC₅₀ value. Docking calculations revealed that the steroid part has a perfect overlap with E2 in ABS. In CBS, however, a head-tail binding deviation was observed. A facile, fluorescent labeling methodology has been elaborated for the development of a novel red-emitting E2 conjugate with substantial estrogenic activity. Docking experiments uncovered the binding mode of the conjugate in both ABS and CBS.

Background/Objectives: Aldo–keto reductase isoforms AKR1C1–3 and 17β-hydroxysteroid dehydrogenase 1 and 2 (17β-HSD1 and 17β-HSD2) are key enzymes in steroid metabolism and validated targets in hormone-dependent cancers. Methods: In this study, Δ¹⁵- and 15β-substituted estrone derivatives were evaluated as inhibitors of AKR1C1–3 and 17β-HSD1 using enzymatic assays, cell viability assaysand computational modeling. Cellular uptake of the fluorescent estrone-based inhibitor was investigated using confocal microscopy. Results: The Δ¹⁵-estrone derivative showed potent and selective inhibition of 17β-HSD1 in the low nanomolar range, while 15β-O-propargyl and 15β-azide derivatives exhibited dual inhibitory activity against 17β-HSD1 and AKR1C2. The Δ¹⁵- and 15β-azide derivatives reduced cell viability in hormone-dependent breast, endometrial, and ovarian cancer cell lines in the sub- to low-micromolar range. A BODIPY-labeled 15β-O-propargyl analogue retained submicromolar inhibitory potency toward 17β-HSD1, representing the first fluorescent estrane-based inhibitor with preserved biological activity. Confocal microscopy confirmed efficient cellular uptake and predominant cytosolic localization in MCF-7 cells. Conclusions: These findings identify Δ¹⁵- and 15β-modified estrone derivatives as promising single- and dual-target inhibitors and introduce a fluorescent probe suitable for investigating intracellular steroid metabolism in hormone-dependent malignancies.