Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

Efficient, large-scale synthesis and preclinical studies of MRS5698, a highly selective A3 adenosine receptor agonist that protects against chronic neuropathic pain.

We reported that 2-(3,4-difluorophenylethynyl)-N (6)-3-chlorobenzyl (N)-methanocarba adenosine derivative 1 (MRS5698) binds selectively to human and mouse A3 adenosine receptors (A3ARs, K i 3 nM). It is becoming an important pharmacological tool for defining A3AR effects and is orally active in a chronic neuropathic pain model. Here, we introduce a new synthetic route for MRS5698 from D-ribose, suitable for a scale-up on a multi-gram scale, and we measure in vitro and in vivo ADME-Tox parameters. MRS5698 was very stable in vitro, failed to inhibit CYPs at <10 µM, and was largely bound to plasma proteins. It was well tolerated in the rat at doses of ≤200 mg/kg i.p. A 1 mg/kg i.p. dose in the mouse displayed t 1/2 of 1.09 h and plasma Cmax of 204 nM at 1 h with an AUC of 213 ng × h/mL. CACO-2 bidirectional transport studies suggested intestinal efflux of MRS5698 (efflux ratio 86). Although the oral %F is only 5 %, the beneficial effect to reverse pain lasted for at least 2 h in the CCI model in rats, using the same vehicle for oral administration of a high dose. The stability, low toxicity, lack of CYP interaction, pharmacokinetic half-life, and in vivo efficacy suggest that MRS5698 is a preferred compound for further consideration as a treatment for neuropathic pain.