Development of Covalent, Clickable Probes for Adenosine A₁ and A₃ Receptors

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A₁ receptor (A₁R) and adenosine A₃ receptor (A₃R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure–activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A₁R and A₃R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A_2A and A_2B adenosine receptors. Once bound to the receptor, ligands were successfully “clicked” with a cyanine-5 fluorophore containing the complementary “click” partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A₁R and A₃R localization and trafficking in native cells and living systems.