Molecular modelling study of 2-phenylethynyladenosine (PEAdo) derivatives as highly selective A(3) adenosine receptor ligands

A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N(6)- and 4′position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity assay (A(2B)). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A(3) receptors, resulting in compounds endowed with high affinity and selectivity for the A(3) subtype. Additional substitution of the N(6)- and 4′position increases both A(3) affinity and selectivity. The results showed that the new compounds have a good affinity for the A(3) receptor and in particular, the N(6)-methoxy-2-phenylethynyl-5′N-methylcarboxamidoadenosine, with a K(i) at A(3) of 1.9 nM and a selectivity A(1)/A(3) and A(2A)/A(3) of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A(3) adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A(3) receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A(3) receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data.