First Potent Macrocyclic A Adenosine Receptor Agonists Reveal G-Protein and β-Arrestin2 Signaling Preferences.

()-Methanocarba adenosine derivatives (A adenosine receptor (AR) agonists containing bicyclo[3.1.0]hexane replacing furanose) were chain-extended at and C2 positions with terminal alkenes for ring closure. The resulting macrocycles of 17-20 atoms retained affinity, indicating a spatially proximal orientation of these receptor-bound chains, consistent with molecular modeling of . C2-Arylethynyl-linked macrocycle was more AAR-selective than 2-ether-linked macrocycle (both 5'-methylamides, human (h) AAR affinities (): 22.1 and 25.8 nM, respectively), with lower mouse AAR affinities. Functional hAAR comparison of two sets of open/closed analogues in β-arrestin2 and G protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA . The potencies of at all three Gα isoforms were slightly less than its hAAR binding affinity (: 1.4 nM), while the Gα and Gα potencies of macrocycle were roughly an order of magnitude higher than its radioligand binding affinity. Gα-coupling was enhanced in macrocycle (EC 2.56 nM, ∼40% greater maximal efficacy than ). Di-O-allyl precursor cyclized to form , increasing the Gα potency by 7.5-fold. The macrocycles and and their open precursors and potently stimulated β-arrestin2 recruitment, with EC values (nM) of 5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greater efficacy compared to . This example of macrocyclization altering the coupling pathways of small-molecule (nonpeptide) GPCR agonists is the first for potent and selective macrocyclic AR agonists. These initial macrocyclic derivatives can serve as a guide for the future design of macrocyclic AR agonists displaying unanticipated pharmacology.