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 hA(2A)AR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA(2A)AR agonists into antagonists while maintaining affinity toward hA(3)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 hA(2A)AR, including 5d with the highest affinity (K(i,A(2A)) = 7.7 ± 0.5 nM). The hA(2A)AR-5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA(3)AR. Structural SAR features and docking studies supported different binding modes at A(2A)AR and A(3)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 A(2A)AR/A(3)AR nucleoside antagonists would be promising drug candidates for immune-oncology.