N-Adamantyl-1-Pentyl-4-Oxo-1,4-Dihydroquinoline-3-Carboxamide and N-(Adamantan-1-Yl)-2-(Alkyloxy)benzamide Derivatives as Fluorescent Probes for Selective CB2R Targeting

Background/Objectives: The cannabinoid receptor subtype 2 (CB2R) is an increasingly important therapeutic and diagnostic target because of its involvement in inflammation-associated disorders, including cancer and neurodegenerative diseases. CB2R biology has traditionally been investigated using radioligands, whose broader application is limited by safety, regulatory, operational, and environmental constraints. Fluorescent ligands provide a complementary approach for real-time, high-resolution analysis of receptor expression and dynamics in living systems. This study aimed to develop structurally diverse fluorescent CB2R ligands as molecular tools for investigating receptor localization, trafficking, and signaling across imaging and cell-based platforms. Methods: Probe design was based on two complementary CB2R chemotypes: the 4-oxoquinolinone scaffold of the previously reported fluorescent ligands SM15 and VM7 and the salicylamide scaffold of the reference ligand CC48. These pharmacophores were conjugated to Cyanine 5 (Cy5), Cyanine 7 (Cy7), BODIPY-TR, 7-nitrobenzo-2-oxa-1,3-diazole (NBD), or 7-nitrobenzo-2-selena-1,3-diazole (Se-NBD), generating ligands with emission profiles spanning the green to near-infrared spectral regions. Compound 2, which showed the highest CB2R affinity among the new ligands (Ki = 143.4 nM) and negligible CB1R displacement at 1 µM, was characterized photophysically in organic and aqueous media and evaluated by flow cytometry in MCF7 and PANC-1 cancer cells, which display low and high CB2R expression, respectively. Results: Compound 2 retained nanomolar CB2R affinity and exhibited detectable fluorescence in organic solvents and aqueous buffer down to nanomolar concentrations. In flow cytometry experiments, its concentration-dependent fluorescence differentiated MCF7 from PANC-1 cells. Partial displacement by the CB2R reference ligand 13, particularly in PANC-1 cells, supported a receptor-specific component of the observed labeling. Conclusions: Compound 2 combines CB2R selectivity with robust fluorescence across chemically distinct environments and can distinguish cellular models with different receptor-expression levels. These findings establish compound 2 as a promising tool for CB2R detection and provide a rational basis for developing optimized fluorescent ligands for receptor imaging, target validation, and related translational applications.