Structure activity relationship of 2-arylalkynyl-adenine derivatives as human A₃ adenosine receptor antagonists

Recognition of nucleosides at adenosine receptors (ARs) is supported by multiple X-ray structures, but the structure of an adenine complex is unknown. We examined the selectivity of predicted A₁AR and A₃AR adenine antagonists that incorporated known agonist affinity-enhancing N⁶ and C2 substituents. Adenines with A₁AR-favoring N⁶-alkyl, cycloalkyl and arylalkyl substitutions combined with an A₃AR-favoring 2-((5-chlorothiophen-2-yl)ethynyl) group were human (h) A₃AR-selective, e.g. MRS7497 17 (∼1000-fold over A₁AR). In addition, binding selectivity over hA_2AAR and hA_2BAR and functional A₃AR antagonism were demonstrated. 17 was subjected to computational docking and molecular dynamics simulation in a hA₃AR homology model to predict interactions. The SAR of nucleoside AR agonists was not recapitulated in adenine AR antagonists, and modeling suggested an alternative, inverted binding mode with the key N250^6.55 H-bonding to the adenine N³ and N⁹, instead of N⁶ and N⁷ as in adenosine agonists.