An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A3 adenosine receptor antagonists is described. The synthesized compounds showed A3 adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A1, A(2A), A(2B), and A3 adenosine receptors. In particular, the effect of the chain at the N8 pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N8 pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N5 position as the compound with the best binding profile in terms of both affinity and selectivity (hA3 = 0.2 nM, hA1/hA3 = 5485, hA2A/hA3 = 6950, hA(2B)/hA3 = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A3 receptor. The new compounds are among the most potent and selective A3 antagonists so far described. The derivatives with higher affinity at human A3 adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC50 values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N8 pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.
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