Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: Influence of thechain at the N-8 pyrazole nitrogen

An enlarged series of pyrazolotriazolopyrimidines previously reported, in p reliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as high ly potent and selective human A(3) adenosine receptor antagonists is descri bed. The synthesized compounds showed A(3) adenosine receptor affinity in t he sub-nanomolar range and high levels of selectivity evaluated in radiolig and binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptor s. In particular, the effect of the chain at the Ns pyrazole nitrogen was a nalyzed. This study allowed us to identify the derivative with the methyl g roup at the N-8 pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N-5 position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A )/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full a ntagonists in a specific functional model where the inhibition of cAMP gene ration by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with highe r affinity at human A(3) adenosine receptors proved to be antagonists, in t he 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 th e aim to identify possible pharmacophore maps. In fact, a sterically contro lled structure-activity relationship was found for the N-8 pyrazole substit uted derivatives, showing a correlation between the calculated molecular vo lume of pyrazolo[4,3-e]1,2,4-triazolo [1,5-c]-pyrimidine derivatives and th eir experimental K-i values.