The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]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 A 1 and/or A 2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA 2B and hA 3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (K i = 150 nM) and the best selectivity for the hA 2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA 2A (K i = 123 nM) and A 1 AR affinity (K i = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (K i = 11 nM) and good selectivity for the hA 1 AR. The 5-(N 4 -substituted-piperazin-1-yl) derivatives 15-24 bind the hA 1 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.