Functionalized congeners of 1,4-dihydropyridines as antagonist molecular probes for A(3) adenosine receptors

4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective anta gonists at human A(3) adenosine receptors, with K-i values in a radioligand binding assay vs [I-125]AB-MECA [N-6-(4-amino-3-iodobenzyl)-5'-N-methylcar bamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive ade nosine A(3) antagonists, for the purpose of synthesizing molecular probes f or this receptor subtype. Selectivity of the new analogues for cloned human A(3) adenosine receptors was determined in radioligand binding in comparis on to binding at rat brain A(1) and A(2A) receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 5-positions w ere introduced as potential sites for chain attachment. Structure-activity analysis at A(3) adenosine receptors indicated that 3,5-dibenzyl esters, bu t not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of t he 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced Ag recept or affinity resulting in a K-i value of 2.42 nM; however, a long-chain deri vative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride deriv ative appeared to bind irreversibly to the human A(3) receptor (1 h incubat ion at 100 nM resulting in the loss of 56% of the specific radioligand bind ing sites), while the binding of other potent dihydropyridines and other an tagonists was generally reversible. At the 3-position of the dihydropyridin e ring, an amine-functionalized chain attached at the 4-position of a benzy l ester provided higher A(3) receptor affinity than the corresponding 5-pos ition isomer. This amine congener was also used as an intermediate in the s ynthesis of a biotin conjugate, which bound to A(3) receptors with a K-i va lue of 0.60 mu M.