Characterization of potent ligands at human recombinant adenosine receptors

The four adenosine receptor subtypes have been stably transfected into Chin ese hamster ovary (CHO) cells allowing for comparative studies in a similar cellular background, using radioligand binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity assays (A(2B)). We are currently using the tr ansfected CHO cells for extensive screening of nucleosides and purine deriv atives of our library. Screening of a number of 2-alkynyl analogs of 5'-N-e thylcarboxamidoadenosine (NECA) indicated that introduction of particular s ubstituents, such as the racemic 2-phenylhydroxypropynyl group, led to a hi ghly potent, nonselective agonist at A(1), A(2A), and A(3) subtypes (PHPNEC A, K-i in the low nanomolar range at the three subtypes). In the A(2B) func tional assay, it has been found that PHPNECA (EC50 A(2B) = 0.88 mu M) is th reefold more potent than NECA. This article is the first report in wh ich t he introduction of a bulky group in the 2-position of NECA led to a compoun d that is active as an agonist at the human A(2B) subtype. On the other han d, the presence of a phenyl ring conjugated to the triple bond as in phenyl ethynylNECA (PENECA) enhanced selectivity for the A(3) subtype. In the puri ne series (potential antagonists), 8-bromo-9-ethyladenine (8-BEA) showed go od affinity toward all adenosine receptor subtypes (K-i A(1) = 0.28 mu M, K -i A(2A) = 0.052 mu M, K-i A(2B) = 0.84 mu M, K-i A(3) = 27.8 mu M). On the other hand, the introduction of alkynyl chains in the 8-position resulted in an increased affinity at the A(3) receptor (8-hexynyl-9-ethyladenine, 8- HEEA, K-i A(3) = 0.62 mu M and 8-phenylethynyl-9-ethyladenine, 8-PEEA, K-i A(3) = 0.086 mu M). Drug Dev. Res. 45:176-181, 1998. (C) 1998 Wiley-Liss, I nc.