Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.

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.