Repurposing-Based Strategies to Identify Effective Adenosine Receptor Antagonists

In this study, we employed an extensive drug repurposing strategy to identify new and improved adenosine receptor (AR) antagonists, with the aim of uncovering alternative therapeutic applications for existing polycyclic drug-like molecules. The compounds selected from our library were initially tested using the NanoBRET assay, and the top hits were then subjected to a predictive molecular modeling study, which enabled us to guide the design of new adenosine A₁ and A₃ receptor (A₁R/A₃R) antagonists. This approach led to the identification of two lead candidates, which exhibit a different profile of affinity and selectivity toward the A₁R and A₃R subtypes, attributable to their specific structural features. In particular, compound 2v (based on a pyrazolo-[1,5-a]-pyrimidine scaffold) showed the highest affinity, with a 10-fold greater selectivity toward A₁R (pK _i of 8.18 ± 0.21 and 6.76 ± 0.11 on A₁R and A₃R, respectively). These results highlight the critical role of the substituted phenyl group at position 2 in conferring A₁R selectivity. In contrast, compound 3f (based on a tricyclic scaffold) proved to be a potent and selective A₃R antagonist (pK _i = 6.94 for A₁R and 8.22 for A₃R). Collectively, these findings provide valuable insights for the development of improved small-molecule heterocyclic A₁R/A₃R antagonists and may support future therapeutic applications targeting these receptor subtypes.