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

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 (Ki = 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.