Discovery and Structure-Activity Relationship Studies of Novel Adenosine A₁ Receptor-Selective Agonists

A series of benzyloxy and phenoxy derivatives of the adenosine receptor agonists N⁶-cyclopentyl adenosine (CPA) and N⁶-cyclopentyl 5'-N-ethylcarboxamidoadenosine (CP-NECA) were synthesized, and their potency and selectivity were assessed. We observed that the most potent were the compounds with a halogen in the meta position on the aromatic ring of the benzyloxy- or phenoxycyclopentyl substituent. In general, the NECA-based compounds displayed greater A₁R selectivity than the adenosine-based compounds, with N⁶-2-(3-bromobenzyloxy)cyclopentyl-NECA and N⁶-2-(3-methoxyphenoxy)cyclopentyl-NECA showing ∼1500-fold improved A₁R selectivity compared to NECA. In addition, we quantified the compounds' affinity and kinetics of binding at both human and rat A₁R using a NanoBRET binding assay and found that the halogen substituent in the benzyloxy- or phenoxycyclopentyl moiety seems to confer high affinity for the A₁R. Molecular modeling studies suggested a hydrophobic subpocket as contributing to the A₁R selectivity displayed. We believe that the identified selective potent A₁R agonists are valuable tool compounds for adenosine receptor research.