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_2AAR binding modes were predicted computationally. The optimal n-butyl spacer was incorporated into the following A_2AAR-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_2AAR-expressing HEK-293 cells displayed saturable binding (low nonspecific) and inhibition by known A_2AAR antagonists. Water-soluble sulfonate 13 was a highly potent (K_i = 6.2 nM) and selective A_2AAR 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_2AAR. 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_2AAR drug discovery and characterization.