基于hA2AAR结构，5'-截短腺苷类似物中的疏水性C8杂芳环紧密地占据了亚口袋，将hA2AAR激动剂转化为拮抗剂，同时保持了对hA3AR的亲和力。最终的化合物，包括从d-甘露糖和d-红岛糖1,4-内酯分别合成的2,8-二取代N6-取代4'-硫代核苷或4'-酮基，通过Pd催化的定位选择性交叉偶联反应合成。所有测试化合物都完全拮抗hA2AAR，其中5d的亲和力最高（Ki,A2A = 7.7 ± 0.5 nM）。hA2AAR-5d X射线结构显示，C8杂芳环阻止了与受体激活相关的构象变化。然而，C8取代化合物仍然能够拮抗hA3AR。结构-活性关系特征和分子对接研究支持A2AAR和A3AR的不同结合模式，阐明了受体激活和选择性的药效团。有利的药代动力学研究表明，5d具有良好的口服吸收、中等半衰期和生物利用度。此外，5d显著改善了抗PD-L1的抗肿瘤效果。总体而言，本研究表明，新型的双重A2AAR/A3AR核苷类拮抗剂可能成为免疫肿瘤学领域的有希望的药物候选物。

Based on hA2AAR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA2AAR 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 hA2AAR, including 5d with the highest affinity (Ki,A2A = 7.7 ± 0.5 nM). The hA2AAR-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 A2AAR 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 A2AAR/A3AR nucleoside antagonists would be promising drug candidates for immune-oncology.