Exploring C2 and N⁶ Substituent Effects on Truncated 4′-Thioadenosine Derivatives as Dual A_2A and A₃ Adenosine Receptor Ligands

Based on high binding affinity of truncated 2-hexynyl-4′-thioadenosine (3 a) at both A_2A adenosine receptor (AR) and A₃ AR, we explored structure-activity relationship (SAR) of the C2-substitution by altering chain length of the 2-hexynyl moiety, thereby evaluating the hydrophobic pocket size. A series of truncated N⁶-substituted 4′-thioadenosine derivatives with C2-alkynyl substitution were successfully synthesized from D-mannose, using a palladium-catalyzed Sonogashira coupling reaction as the key step, whose structures were confirmed by the X-ray crystal structure of 4 h. As the size of the alkynyl group at the C2-position increased, the binding affinity improved; however, when the substituted group was larger than hexynyl, the binding affinity decreased. The introduction of a bulky hydrophobic group such as 3-halobenzyl group at the free N⁶-amino group decreased the binding affinity at hA_2AAR. These results confirm our previous findings that a free amino group at N⁶-position and longer hydrophobic chain at C2-position are essential for hA_2A AR binding affinity. The introduction of a bulky hydrophobic group at free N⁶-amino group maintained the binding affinity at hA₃ AR. The binding mode of truncated 2-substituted-4′-thioadenosine derivatives to hA_2A and hA₃ AR were predicted by a molecular docking study.