Structure-Activity Relationship of Truncated 2,8-Disubstituted-Adenosine Derivatives as Dual A 2A /A 3 Adenosine Receptor Antagonists and Their Cancer Immunotherapeutic Activity.

Based on hA2A AR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA2A AR agonists into antagonists while maintaining affinity toward hA3 AR. The final compounds of 2,8-disubstituted- N 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 hA2A AR, including 5d with the highest affinity ( K i,A2A = 7.7 ± 0.5 nM). The hA2A AR- 5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA3 AR. Structural SAR features and docking studies supported different binding modes at A2A AR and A3 AR, 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 A2A AR/A3 AR nucleoside antagonists would be promising drug candidates for immune-oncology.