Irreversible Antagonists for the Adenosine A2B Receptor

Blockade of the adenosine A_2B receptor (A_2BAR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A_2BAR antagonists based on an 8-p-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα₁₅ protein activation assays by performing ligand wash-out and kinetic experiments. p-(1-Propylxanthin-8-yl)benzene sulfonyl fluoride (6a, PSB-21500) was the most potent and selective irreversible A_2BAR antagonist of the present series with an apparent K_i value of 10.6 nM at the human A_2BAR and >38-fold selectivity versus the other AR subtypes. The corresponding 3-cyclopropyl-substituted xanthine derivative 6c (PSB-21502) was similarly potent, but was non-selective versus A₁- and A_2AARs. Attachment of a reactive sulfonyl fluoride group to an elongated xanthine 8-substituent (12, K_i 7.37 nM) resulted in a potent, selective, reversibly binding antagonist. Based on previous docking studies, the lysine residue K269^7.32 was proposed to react with the covalent antagonists. However, the mutant K269L behaved similarly to the wildtype A_2BAR, indicating that 6a and related irreversible A_2BAR antagonists do not interact with K269^7.32. The new irreversible A_2BAR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs.