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 N⁶ 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 G_i/o protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA 1. The potencies of 1 at all three Gα_i isoforms were slightly less than its hA₃AR binding affinity (K_i: 1.4 nM), while the Gα_i1 and Gα_i2 potencies of macrocycle 12 were roughly an order of magnitude higher than its radioligand binding affinity. Gα_i2-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 Gα_i1 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.