We synthesized phenyl ring-substituted analogues of N 6 -(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A 3 AR with a K i value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA 3 AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A 3 AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A 3 AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A 3 AR binding. Other related N 6 -substituted adenosine derivatives were included for comparison. Although the N 6 -(2-phenyl-1-cyclopropyl) derivatives were full A 3 AR agonists, several other derivatives had greatly reduced efficacy. N 6 -Cyclopropyladenosine was an A 3 AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N 6 -(2,2-Diphenylethyl)adenosine was an A 3 AR antagonist, and either adding a bond between the two phenyl rings (N 6 -9-fluorenylmethyl) or shortening the ethyl moiety (N 6 -diphenylmethyl) restored efficacy. A QSAR study of the N 6 region provided a model that was complementary to the putative A 3 AR binding site in a rhodopsin-based homology model. Thus, a new series of highaffinity A 3 AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N 6 -substitutions known to enhance human A 3 AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A 1 , A 2A , A 2B , and A 3 ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA 3 AR affinity and efficacy in the cases of N 6 -(3-iodobenzyl) and N 6 -(trans-2-phenyl-1-cyclopropyl), for which a full A 3 AR agonist was converted into a selective antagonist; the 2-cyano-N 6 -methyl analogue was a full A 3 AR agonist. The combination of N 6 -benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A 1 AR. The environment surrounding the 2-position within the putative A 3 AR binding site was explored using rhodopsin-based homology modeling and ligand docking.

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