Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation: Enhanced Stability and Potency as P2Y_1 Receptor Agonists

摘要

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5'-triphosphate agonists at P2Y_1, P2Y_2, P2Y_4, and P2Y_11 receptors, but not P2Y_6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y_1 or human P2Y_1 and P2Y_2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC_(50) at the hP2Y_1 receptor of 0.40 nM and was 55 fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5'-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N~6-Me analogue were also highly selective, full agonists at P2Y_1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y_1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y_1 receptor agonist with an EC_(50) of 160 nM and was selective versus hP2Y_2 and hP2Y_4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5'-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase 1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y_1 receptors.