Structure-Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A_2A/A₃ Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity

Based on hA_2AAR structures, a hydrophobic C8-heteroaromatic ring in 5′-truncated adenosine analogues occupies the subpocket tightly, converting hA_2AAR agonists into antagonists while maintaining affinity toward hA₃AR. The final compounds of 2,8-disubstituted-N⁶-substituted 4′-thionucleosides, or 4′-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA_2AAR, including 5d with the highest affinity (K_i,A2A = 7.7 ± 0.5 nM). The hA_2AAR-5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA₃AR. Structural SAR features and docking studies supported different binding modes at A_2AAR and A₃AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo. Overall, this study suggests that the novel dual A_2AAR/A₃AR nucleoside antagonists would be promising drug candidates for immune-oncology.