The adenosine A1 receptor (A1AR) is a G-protein-coupled
receptor (GPCR) that provides important therapeutic opportunities
for a number of conditions including congestive heart failure, tachycardia,
and neuropathic pain. The development of A1AR-selective
fluorescent ligands will enhance our understanding of the subcellular
mechanisms underlying A1AR pharmacology facilitating the
development of more efficacious and selective therapies. Herein, we
report the design, synthesis, and application of a novel series of
A1AR-selective fluorescent probes based on 8-functionalized
bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl)
xanthine scaffolds. These fluorescent conjugates allowed quantification
of kinetic and equilibrium ligand binding parameters using NanoBRET
and visualization of specific receptor distribution patterns in living
cells by confocal imaging and total internal reflection fluorescence
(TIRF) microscopy. As such, the novel A1AR-selective fluorescent
antagonists described herein can be applied in conjunction with a
series of fluorescence-based techniques to foster understanding of
A1AR molecular pharmacology and signaling in living cells.