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.

Ribose-based nucleoside 5′-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5′-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y 2 receptor but were inactive or weakly active at P2Y 4 receptors. Uridine 5′-(diphospho)phosphonate was approximately as potent at the P2Y 2 receptor as at the UDP-activated P2Y 6 receptor. These results suggest that removal of the 5′-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y 2 receptor. Uridine 5′-(phospho)phosphonate as well as the 5′-methylphosphonate equivalent of UMP were inactive at the P2Y 4 receptor and exhibited maximal effects at the P2Y 2 receptor that were ≤50% of that of UTP suggesting novel action of these analogues.The P2Y receptors are a family of eight G protein-coupled receptors (GPCRs) of class A that respond to diverse nucleotides. 1 The human P2Y 1 , P2Y 12 , and P2Y 13 receptors are activated preferentially by ADP, while the human P2Y 11 receptor is activated preferentially by ATP. The human P2Y 4 , P2Y 6 , and P2Y 14 subtypes respond exclusively to various uracil nucleotides, and the human P2Y 2 receptor responds to both UTP and ATP. Depending on the subtype, the effector coupling of these receptors is typically through G q or G i proteins, to stimulate phospholipase or to inhibit of adenylate cyclase, respectively. P2Y receptors are targets for therapeutic approaches, some of which are currently being explored at an early stage. 2 Several clinical applications of P2Y receptor ligands are more advanced, such as P2Y 12 receptor antagonists as antithrombotic agents 3 and P2Y 2 receptor agonists as drugs for cystic fibrosis and dry eye disease. 4 To aid in the design of P2Y receptor ligands, we have reported rhodopsin-based molecular models of all known subtypes of P2Y receptors. 5,6 Various amino acid residues have been Correspondence to: Kenneth A. Jacobson. Supporting material: Procedure for biological assays, the synthetic route for compounds 5 and 6, and detailed procedures for compounds 3 -7 are provided.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In the present study, we have explored the removal of the 5′-oxygen or its replacement by carbon moieties, resulting in 5′-phosphonate derivatives. The activity of various phosphonate derivatives was compared with the native ribosides at various P2Y receptor subtypes. Potency was best preserved at the P2Y 2 receptor following this modification. However, the apparent efficacy of activa...

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