Molecular probes for the A₂A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A₂A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, Kᵢ 15nM). The potent and A₂AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A₂AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A₂AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A₂AAR.