First Potent Macrocyclic A3 Adenosine Receptor Agonists Reveal G‑Protein and β‑Arrestin2 Signaling Preferences

(N)-Methanocarba adenosine derivatives (A3 adenosine receptor (AR) agonists containing bicyclo[3.1.0]­hexane replacing furanose) were chain-extended at N 6 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 12. C2-Arylethynyl-linked macrocycle 19 was more A3AR-selective than 2-ether-linked macrocycle 12 (both 5′-methylamides, human (h) A3AR affinities (K i): 22.1 and 25.8 nM, respectively), with lower mouse A3AR affinities. Functional hA3AR comparison of two sets of open/closed analogues in β-arrestin2 and Gi/o protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA 1. The potencies of 1 at all three Gαi isoforms were slightly less than its hA3AR binding affinity (K i: 1.4 nM), while the Gαi1 and Gαi2 potencies of macrocycle 12 were roughly an order of magnitude higher than its radioligand binding affinity. Gαi2-coupling was enhanced in macrocycle 12 (EC50 2.56 nM, ∼40% greater maximal efficacy than 1). Di-O-allyl precursor 18 cyclized to form 19, increasing the Gαi1 potency by 7.5-fold. The macrocycles 12 and 19 and their open precursors 11 and 18 potently stimulated β-arrestin2 recruitment, with EC50 values (nM) of 5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greater efficacy compared to 1. 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.