Functionalized congeners of 1,4-dihydropyridines as antagonist molecular probes for A3 adenosine receptors

4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists 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-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A(3) antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A(3) adenosine receptors was determined in radioligand binding in comparison 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 were introduced as potential sites for chain attachment. Structure-activity analysis at A(3) adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced Ag receptor affinity resulting in a K-i value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A(3) receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A(3) receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A(3) receptors with a K-i value of 0.60 mu M.