Methanocarba Modification of Uracil and Adenine Nucleotides:  High Potency of Northern Ring Conformation at P2Y₁, P2Y₂, P2Y₄, and P2Y₁₁ but Not P2Y₆ Receptors

Abstract

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

“…Among the more successful examples of the use of carbocyclic or sterically constrained carbocyclic substitution of the ribose moiety for P2Y receptor interactions are the "methanocarba" analogs (Nandanan et al, 2000;Kim et al, 2002). These analogs incorporate a conformationally fixed bicyclic ring system, consisting of fused cyclopentane and cyclopropane rings, in place of the ribose moiety.…”

A critical look at the function of the P2Y11 receptor

“…C-terminal eGFP-tagged human and canine P2Y 11 receptors both showed similar signalling properties to the respective non-tagged receptors [6,51]. Nearly all vectors expressing human P2Y 11 described in published studies were created from the initial P2RY11 sequence arising from the fusion transcript (AF030335) [2,34,45,47,48,52,54,56,58,59,[62][63][64][65][66][67]. The sequence difference results in a slightly altered N-terminal of the P2Y 11 protein from its rightful MAANVSGAK to MDRGAK that originates from the transgenic splicing with PPAN.…”

Agonists and Antagonists for P2 Receptors

“…fused cyclopropane and cyclopentane rings, locks the analogue in either a (N) or (S) conformation, depending on the position of the -CH 2 -bridge. These isomeric variants based on the bicyclo[3.1.0]hexane ring system produce agonists having widely differing activities at P2 receptors (Kim et al 2002). In the (N) conformation (e.g.…”

“…Dramatically, a uridine 5′-diphosphate analogue locked in the (N) envelope conformation was inactive (Kim et al 2002). Based on a prediction from docking of nucleotide derivatives to the P2Y 6 receptor model, (S)-mc-dUDP was synthesized and found to be more potent than the corresponding riboside, dUDP, indicating a preference for the South conformation .…”