2-Substituted (N)-Methanocarba A 3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A 3 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 9 - 11 , 13 , 14 , 19 , 22 , 23 , 27 , 29 , 31 , and 34 , lacking a spacer, had human (h) A 3 AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A 3 AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues ( 7 , 8 > 3c , 3d > 3b ). However, 2-aryl-4'-truncated derivatives had greatly reduced hA 3 AR affinity, even containing affinity-enhancing N 6 -dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A 3 AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA 3 AR miniGα i recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy ( E max ) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A 3 AR 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 3a - 3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A 3 AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A 3 AR agonist SAR for (N)-methanocarba adenosines.