2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors

A number of 2-substituted5'-N-ethylcarboxamidoadenosine(NECA) derivatives was investigated for their affinity and selectivity at human A(3) adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. Tn binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A(3) adenosine receptors with a K-i value of 0.4 nM. As opposed to the limited A(3) selectivity of PHPNECA, a 100-fold selectivity compared to both A(1) and A(2A) receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; K-i 6 nM). The EC50 values for activation of adenylyl cyclase via A(2A) adenosine receptors were in good agreement with the respective K-i values from binding experiments. Tn contrast, IC50, values for A(1) and A(3) receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A(1) adenosine receptor in previous studies. Therefore, the same A(3) selectivity of PENECA compared to A(1) receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A(2A) receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A(3)-selective high-affinity ligands.