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

(N)-Methanocarba adenosine derivatives(A(3) adenosine receptor (AR) agonists containing bicyclo[3.1.0]hexanereplacing 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-boundchains, consistent with molecular modeling of 12. C2-Arylethynyl-linkedmacrocycle 19 was more A(3)AR-selective than2-ether-linked macrocycle 12 (both 5 & PRIME;-methylamides,human (h) A(3)AR affinities (K (i)): 22.1 and 25.8 nM, respectively), with lower mouse A(3)AR affinities. Functional hA(3)AR comparison of two setsof open/closed analogues in & beta;-arrestin2 and G(i/o) proteinassays showed certain signaling preferences divergent from referenceagonist Cl-IB-MECA 1. The potencies of 1 at all three G & alpha;(i) isoforms were slightly less thanits hA(3)AR binding affinity (K (i): 1.4 nM), while the G & alpha;(i1) and G & alpha;(i2) potencies of macrocycle 12 were roughly an order ofmagnitude higher than its radioligand binding affinity. G & alpha;(i2)-coupling was enhanced in macrocycle 12 (EC50 2.56 nM, & SIM;40% greater maximal efficacy than 1). Di-O-allyl precursor 18 cyclized to form 19, increasing the G & alpha;(i1) potency by 7.5-fold.The macrocycles 12 and 19 and their openprecursors 11 and 18 potently stimulated & beta;-arrestin2 recruitment, with EC50 values (nM) of5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greaterefficacy compared to 1. This example of macrocyclizationaltering the coupling pathways of small-molecule (nonpeptide) GPCRagonists is the first for potent and selective macrocyclic AR agonists.These initial macrocyclic derivatives can serve as a guide for thefuture design of macrocyclic AR agonists displaying unanticipatedpharmacology.