Synthesis and Evaluation of the First Fluorescent Antagonists of the Human P2Y₂ Receptor Based on AR-C118925

The human P2Y₂ receptor ( hP2Y₂R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y₂R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y₂R antagonists, based on the known, non-nucleotide hP2Y₂R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y₂R (p K_d = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y₂R. These properties make 98 one of the first tools for studying hP2Y₂R distribution and organization.