The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-<i>d</i>]pyrimidine core in affecting adenosine A₁ and A_2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-<i>d</i>]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A₁ and/or A_2A receptor subtypes. On the whole, the novel derivatives <b>1</b>–<b>24</b> shared scarce or no affinities for the off-target hA_2B and hA₃ ARs. The 5-(4-hydroxyphenethylamino)- derivative <b>12</b> showed both good affinity (<i>K_i</i> = 150 nM) and the best selectivity for the hA_2A AR while the 5-benzylamino-substituted <b>5</b> displayed the best combined hA_2A (<i>K_i</i> = 123 nM) and A₁ AR affinity (<i>K_i</i> = 25 nM). The 5-phenethylamino moiety (compound <b>6</b>) achieved nanomolar affinity (<i>K_i</i> = 11 nM) and good selectivity for the hA₁ AR. The 5-(N⁴-substituted-piperazin-1-yl) derivatives <b>15</b>–<b>24</b> bind the hA₁ AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.