[H-3]MRS 1754, a selective antagonist radioligand for A(2B) adenosine receptors

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl -1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A(2B ) adenosine receptors. This is the least well-defined adenosine receptor su btype, and A(2B) antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was trit iated on the propyl groups in a two-step reaction using a p-carboxamido pre cursor, which was dehydrated to the cyano species using trifluoroacetic anh ydride. [H-3]MRS 1754 (150 Ci/mmol) bound to recombinant human A(2B) adenos ine receptors in membranes of stably transfected HEK-293 cells. Specific bi nding was saturable, competitive, and followed a one-site model, with a K-D value of 1.13 +/- 0.12 nM and a B-max value of 10.9 +/- 0.6 pmol/mg protei n. Specific binding utilizing 0.7 nM [H-3]MRS 1754 was > 70% of total bindi ng. The affinity calculated from association and dissociation binding const ants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A(1 ) and A(3), adenosine receptors was not significant, and binding in membran es of HEK-293 cells expressing human A(2A) receptors was of low affinity (K -D > 50 nM). The effects of cations and chelators were explored. Specific b inding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [H-3 ]MRS 1754 was consistent with the structure-activity relationship for agoni sts and antagonists at A(2B) receptors. The K-i values of XAC (xanthine ami ne congener) and CPX (8-cyclopentyl-1,3-diproplylxanthine) were 16 and 55 n M, respectively. NECA (5'-N-ethylcarboxamidoadenosine) competed for [H-3]MR S 1754 binding with a K-i of 570 nM, similar to its potency in functional a ssays. Thus, [H-3]MRS 1754 is suitable as a selective, high-affinity radiol igand for A(2B) receptors. (C) 2001 Elsevier Science Inc. All rights reserv ed.