| Summary: | Blockade of the adenosine A<sub>2B</sub> receptor (A<sub>2B</sub>AR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A<sub>2B</sub>AR antagonists based on an 8-<i>p</i>-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα<sub>15</sub> protein activation assays by performing ligand wash-out and kinetic experiments. <i>p</i>-(1-Propylxanthin-8-yl)benzene sulfonyl fluoride (<b>6a</b>, PSB-21500) was the most potent and selective irreversible A<sub>2B</sub>AR antagonist of the present series with an apparent K<sub>i</sub> value of 10.6 nM at the human A<sub>2B</sub>AR and >38-fold selectivity versus the other AR subtypes. The corresponding 3-cyclopropyl-substituted xanthine derivative <b>6c</b> (PSB-21502) was similarly potent, but was non-selective versus A<sub>1</sub>- and A<sub>2A</sub>ARs. Attachment of a reactive sulfonyl fluoride group to an elongated xanthine 8-substituent (<b>12</b>, K<sub>i</sub> 7.37 nM) resulted in a potent, selective, reversibly binding antagonist. Based on previous docking studies, the lysine residue K269<sup>7.32</sup> was proposed to react with the covalent antagonists. However, the mutant K269L behaved similarly to the wildtype A<sub>2B</sub>AR, indicating that <b>6a</b> and related irreversible A<sub>2B</sub>AR antagonists do not interact with K269<sup>7.32</sup>. The new irreversible A<sub>2B</sub>AR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs.
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