The activation of the human A(3) adenosine receptor (AR) by a wide range of N(6)-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N(6)-alkyl substitutions, small N(6)-alkyl groups were associated with selectivity for human A(3)ARs vs. rat A(3)ARs, and multiple points of branching were associated with decreased hA(3)AR efficacy. N(6)-Cycloalkyl-substituted adenosines were full (/=6 carbons) hA(3)AR agonists. N(6)-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A(1)ARs. Numerous N(6)-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A(1) and A(3) vs. A(2A)ARs (with variable degrees of partial to full A(3)AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A(3)AR affinity and efficacy of N(6)-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N(6)-(R-1-phenylethyl)adenosine vs. N(6)-(S-1-phenylethyl)adenosine, as well as for the N(6)-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A(3)AR. Interestingly, DPMA, a potent agonist for the A(2A)AR (K(i)=4nM), was demonstrated to be a moderately potent antagonist for the human A(3)AR (K(i)=106nM). N(6)-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (K(i)=0.63nM) than rat A(3)ARs. Dual acting A(1)/A(3) agonists (N(6)-3-chlorobenzyl- 29, N(6)-(S-1-phenylethyl)- 39, and 2-chloro-N(6)-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.

We examined the effects on allosteric modulation and ligand binding of the mutation of amino acid residues of the human A 3 adenosine receptor (A 3 AR) that are hypothesized to be near one of three loci: the putative sodium binding site, the putative ligand binding site, and the DRY motif in transmembrane helical domain 3. The effects of three heterocyclic allosteric modulators [the imidazoquinoline 2-cyclopentyl-4-phenylamino-1H-imidazo [4,5-c]

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.