Tuning the Triazolotriazine Scaffold Toward Selective A₃ Adenosine Receptor Antagonists: From Molecular Recognition...

A series of 2,5,7-trisubstituted [1,2,4]triazolo[1,5-a][1,3,5]triazines (TTs) was developed to identify selective A₃ AR antagonists. Guided by previous structure-activity relationship studies, the C2-furyl substituent was replaced with phenyl or 4-methoxyphenyl moieties to improve A₃ AR affinity and selectivity. C2 phenyl substitution, together with appropriate C5 and C7 amino substituents, critically modulates receptor recognition. In particular, benzylamino substitution at C5 markedly enhanced A₃ AR affinity, yielding compound 15 as the most potent antagonist of the series (K_i = 0.485 nM), whereas incorporation of a 4-methoxyphenyl group at C2 substantially improved subtype selectivity, exemplified by compound 30 (>2800-fold selective for A₃ AR). Molecular docking at the recently determined antagonist-bound structure of the A₃ AR rationalized these trends and identified key structural determinants of A₃ AR preference. The biological relevance of A₃ AR blockade was investigated in rat hippocampal slices subjected to oxygen-glucose deprivation. Compound 15 delayed anoxic depolarization and, when administered during ischemia, prevented its onset in most slices and promoted near-complete recovery of synaptic transmission. These findings identify TT derivatives as promising and highly selective A₃ AR antagonists with potential neuroprotective applications in cerebral ischemia.