The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y 2 , P2Y 4 , and P2Y 6 receptors. The 2-thio modification, found previously to enhance P2Y 2 receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y 2 receptor, in the form of Up 4 -sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include: 9, α,β-methylene-UDP, a P2Y 6 receptor agonist; 30, Up 4 -phenyl ester and 34, Up 4 -[1]glucose, selective P2Y 2 receptor agonists; 43, the 2-thio analogue of INS37217 (P 1 -(uridine 5′)-P 4 -(2′-deoxycytidine 5′) tetraphosphate), a potent and selective P2Y 2 receptor agonist.

Abstract"Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Binding affinity at the human A 1 , A 2A , and A 3 ARs (adenosine receptors) and relative efficacy at the A 3 AR were determined. Some triazol-1-yl analogues showed A 3 AR affinity in the low nanomolar range, a high ratio of A 3 /A 2A selectivity, and a moderate-to-high A 3 /A 1 ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A 3 AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A 3 AR efficacy. Thus, several 5′-OH derivatives appeared to be selective A 3 AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A 1 AR and displaying a characteristic docking mode in an A 3 AR model. The corresponding 5′-ethyluronamide analogues generally showed increased A 3 AR affinity and behaved as full agonists, i.e., 17, with 910-fold A 3 /A 1 selectivity. Thus, N 6 -substituted 2-(1,2,3-triazolyl)-adenosine analogues constitute a novel class of highly potent and selective nucleosidebased A 3 AR antagonists, partial agonists, and agonists.

A rhodopsin-based homology model of the nucleotide-activated human P2Y2 receptor, including loops, termini, and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine. Docked uridine 5'-triphosphate (UTP) formed a nucleobase pi-pi complex with conserved Phe3.32. Selectivity-enhancing 2'-amino-2'-deoxy substitution interacted through pi-hydrogen-bonding with aromatic Phe6.51 and Tyr3.33. A "sequential ligand composition" approach for docking the flexible dinucleotide agonist Up4U demonstrated a shift of conserved cationic Arg3.29 from the UTP gamma position to the delta position of Up4U and Up4 ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2'-Amino and 2-thio modifications were synergized to enhance potency and selectivity; compound 8 (EC50 = 8 nM) was 300-fold P2Y2-selective versus P2Y4. 2'-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance P2Y2 potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-favored Northern conformation of ribose. This detailed view of P2Y2 receptor recognition suggests mutations for model validation.