2-Substituted (N)-Methanocarba A Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds -, , , , , , , , , and , lacking a spacer, had human (h) AAR values of 2-30 nM, and others displayed lower affinity. Mouse (m) AAR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (, > , > ). However, 2-aryl-4'-truncated derivatives had greatly reduced hAAR affinity, even containing affinity-enhancing -dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical AAR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hAAR miniGα recruitment assay, (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy ( ) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, AAR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues - were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately AAR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the AAR agonist SAR for (N)-methanocarba adenosines.