A facile and novel synthesis of N2-, C6-substituted pyrazolo[3,4-d]pyrimidine-4 carboxylate derivatives as adenosine receptor antagonists

• Synthesis of N2-, C6-substituted pyrazolo[3,4-d]pyrimidine-4 carboxylate.
• Bicyclic analogues of PTP retained some crucial interactions.
• Improved selectivity towards human A3 adenosine receptor (hA3AR) was obtained.
• Docking evaluations on both homology-based hA3AR and crystallographic hA2AAR.
• Docking studies were able to rationalize the pharmacological data.

An efficient synthetic procedure was adopted to synthesize a series of new molecules containing the pyrazolo[3,4-d]pyrimidine (PP) scaffold, which have been evaluated as promising human adenosine receptor (AR) antagonists. The effect of substitutions at the N2, C4 and C6 positions of PPs on the affinity and selectivity towards the adenosine receptors were explored. Most of the pyrazolo[3,4-d]pyrimidine-4-carboxylates displayed from moderate to good affinity at the human A3AR (hA3AR), as indicated by the low micromolar range of Ki values (Ki hA3AR = 0.7–34 μM). In particular, compounds 60 and 62 displayed good affinity at the hA3AR (60, Ki hA3AR = 2.2 μM and 62, Ki hA3AR = 2.9 μM) and selectivity towards the other AR subtypes (60, >46-fold selective and 62, >34-fold selective, respectively). In view of these results, these novel PP analogues were docked both in the crystallographic structure of the hA2AAR and in a homology model of the hA3AR in order to support the structure activity relationship (SAR) analysis. These preliminary results demonstrated that pyrazolo[3,4-d]pyrimidine can be considered a promising scaffold to obtain new molecules with potent hA3AR antagonist activity.

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