Characterization of potent ligands at human recombinant adenosine receptors

The four adenosine receptor subtypes have been stably transfected into Chinese hamster ovary (CHO) cells allowing for comparative studies in a similar cellular background, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). We are currently using the transfected CHO cells for extensive screening of nucleosides and purine derivatives of our library. Screening of a number of 2-alkynyl analogs of 5′-N-ethylcarboxamidoadenosine (NECA) indicated that introduction of particular substituents, such as the racemic 2-phenylhydroxypropynyl group, led to a highly potent, nonselective agonist at A1, A2A, and A3 subtypes (PHPNECA, Ki in the low nanomolar range at the three subtypes). In the A2B functional assay, it has been found that PHPNECA (EC50 A2B = 0.88 μM) is threefold more potent than NECA. This article is the first report in which the introduction of a bulky group in the 2-position of NECA led to a compound that is active as an agonist at the human A2B subtype. On the other hand, the presence of a phenyl ring conjugated to the triple bond as in phenylethynylNECA (PENECA) enhanced selectivity for the A3 subtype. In the purine series (potential antagonists), 8-bromo-9-ethyladenine (8-BEA) showed good affinity toward all adenosine receptor subtypes (Ki A1 = 0.28 μM, Ki A2A = 0.052 μM, Ki A2B = 0.84 μM, Ki A3 = 27.8 μM). On the other hand, the introduction of alkynyl chains in the 8-position resulted in an increased affinity at the A3 receptor (8-hexynyl-9-ethyladenine, 8-HEEA, Ki A3 = 0.62 μM and 8-phenylethynyl-9-ethyladenine, 8-PEEA, Ki A3 = 0.086 μM).