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

Abstract

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-<i>N</i>⁶-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 <b>5d</b> with the highest affinity (<i>K</i>_{i,A_2A} = 7.7 ± 0.5 nM). The hA_2AAR-<b>5d</b> 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 <b>5d</b> displayed high oral absorption, moderate half-life, and bioavailability. Also, <b>5d</b> significantly improved the antitumor effect of anti-PD-L1 <i>in vivo</i>. Overall, this study suggests that the novel dual A_2AAR/A₃AR nucleoside antagonists would be promising drug candidates for immune-oncology.