Structure-activity relationship analysis of 5'-substituted C2-hexynyl-4'-thioadenosine analogs as dual A2A/A3 adenosine receptor agonists with tunable selectivity and anti-inflammatory activity.

Adenosine receptors (ARs), particularly the A2A and A3 receptors are complementary regulators of inflammation, motivating a dual-agonist strategy. We designed, synthesized, and evaluated 2-hexynyl-6-amino 4'-thioadenosine derivatives and established a 5'-uronamide structure-activity relationship (SAR) guided by comparative docking. Small, hydrophobic 5' substituents were favored across both A2A and A3AR, mapping a shared hydrophobic pocket and clarifying the donor/acceptor interactions between the ligands and key residues (Thr88/His250 in A2AAR; Thr94 in A3AR). Critically, steric and electronic control at the 5'-uronamide enabled the identification of A2A- and A3-selective ligands, demonstrating tunable A2A/A3 selectivity via 5'-substituent design. The lead compound 2j (SHENECA) showed high-affinity dual binding (hA2AKi = 2.9 ± 0.28 nM; hA3Ki = 0.8 ± 0.1 nM) and subnanomolar functional potency (hA2A EC50 = 0.51 ± 0.17 nM; hA3 EC50 < 0.10 nM). In LPS-stimulated bone-marrow-derived dendritic cells, 2j (and 2i) produced greater suppression of IL-6 and IL-12/IL-23p40 than selective A2A or A3 agonists under our assay conditions, findings compatible with synergistic effects of dual A2A/A3 activation. In vitro ADMET profiling indicated favorable stability in human liver microsomes and plasma and no meaningful hERG inhibition. Collectively, the study demonstrates tunable A2A/A3 selectivity via 5'-uronamide design and the successful discovery of dual agonist 2j, and supports 2j as a strong lead for further optimization toward anti-inflammatory applications.