Agonist derived molecular probes for A 2 adenosine receptors

The adenosine agonist 2‐(4‐(2‐carboxyethyl)phenylethylamino)‐5′‐ N ‐ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A 2 adenosine receptor subtype, which mediates its hypotensive action. To investigate structure/activity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p ‐hydroxyphenylpropionic, 2‐thiophenylacetic, and p ‐aminophenylacetic acids were prepared. The latter derivative (PAPA‐APEC) was iodinated electrophilically using [ 125 I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A 2 adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350‐fold selective for A 2 receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl‐1‐piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta ‐ and para ‐phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A 2 receptors is relatively insensitive to distal structural changes at the 2‐position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity lebelling methodology.