Irreversible Antagonists for the Adenosine A(2B) Receptor †

Blockade of the adenosine A(2B) receptor (A(2B)AR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A(2B)AR antagonists based on an 8-p-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα(15) 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(2B)AR antagonist of the present series with an apparent K(i) value of 10.6 nM at the human A(2B)AR 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 A(1)- and A(2A)ARs. 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(2B)AR, indicating that 6a and related irreversible A(2B)AR antagonists do not interact with K269(7.32). The new irreversible A(2B)AR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs.