Bitopic fluorescent antagonists of the A(2A) adenosine receptor based on pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine functionalized congeners

A pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine antagonist of the A(2A) adenosine receptor (AR) was functionalized as amine congeners, fluorescent conjugates and a sulfonate, and the A(2A)AR binding modes were predicted computationally. The optimal n-butyl spacer was incorporated into the following A(2A)AR-selective (K(i), nM) conjugates: BODIPY630/650 derivative 11 (MRS7396, 24.6) and AlexaFluor488 derivative 12 (MRS7416, 30.3). Flow cytometry of 12 in hA(2A)AR-expressing HEK-293 cells displayed saturable binding (low nonspecific) and inhibition by known A(2A)AR antagonists. Water-soluble sulfonate 13 was a highly potent (K(i) = 6.2 nM) and selective A(2A)AR antagonist based on binding and functional assays. Docking and molecular dynamics simulations predicted the regions of interaction of the distal portions of these chain-extended ligands with the A(2A)AR. The BODIPY630/650 fluorophore of 11 was buried in a hydrophobic interhelical (TM1/TM7) region, while AlexaFluor488 of 12 associated with the hydrophilic extracellular loops. In conclusion, we have identified novel high affinity antagonist probes for A(2A)AR drug discovery and characterization.