SCH-76050

The M₂ muscarinic acetylcholine receptor (M₂R) is a G protein-coupled receptor involved in regulating cardiovascular functions and mediation of central muscarinic effects, such as movement, temperature control, and antinociceptive responses. Molecular probes targeting this receptor are therefore important in exploring its pathophysiological role at a molecular level. Herein, we report the design, synthesis, and evaluation of a new fluorescent probe for M₂R based on an anthranilamide ligand. In radioligand binding experiments, the presented Oregon Green 488-labeled conjugate (33) exhibited high M₂R affinity (K _i = 2.4 nM), a moderate preference for the M₂R over the M₄ receptor, and excellent to pronounced M₂R selectivity compared to the M₁, M₃, and M₅ receptors. The utility of the probe was demonstrated in confocal, two-photon, and stimulated emission depletion nanoscopy (STED) imaging to specifically label the receptors in human embryonic kidney (HEK) 293T cells. These properties suggest that our probe may be utilized in advanced microscopy to study the pharmacology of the M₂R.