3-aryl[1,2,4]triazino[4,3-alpha]benzimidazol-4(10H)-ones: A new class of selective A(1) adenosine receptor antagonists

Radioligand binding assays using bovine cortical membrane preparations and biochemical in vitro studies revealed that various 3-aryl[1,2,4]triazino[4, 3-a]benzimidazol-4(10H)-one (ATBI) derivatives, previously reported by us a s ligands of the central benzodiazepine receptor (BzR) (Primofiore, G.; et al. J. Med. Chem. 2000, 43, 96-102), behaved as antagonists at the Al adeno sine receptor (A(1)AR). Alkylation of the nitrogen at position 10 of the tr iazinobenzimidazole nucleus conferred selectivity for the A(1)AR vs the BzR . The most potent ligand of the ATBI series (10-methyl-3-phenyl[1,2,4]triaz ino[4,3-a]benzimidazol-4(10H)-one 12) displayed a K-i value of 63 nM at the A(1)AR without binding appreciably to the adenosine A(2A) and A(3) nor to the benzodiazepine receptor. Pharmacophore-based modeling studies in which 12 was compared against a set of well-established A(1)AR antagonists sugges ted that three hydrogen bonding sites (HB1 acceptor, HB2 and HB3 donors) an d three lipophilic pockets (L1, L2, and L3) might be available to antagonis ts within the A1AR binding cleft. According to the proposed pharmacophore s cheme, the lead compound 12 engages interactions with the HB2 site (via the N2 nitrogen) as well as with the L2 and L3 sites (through the pendant and the fused benzene rings). The results of these studies prompted the replace ment of the methyl with more lipophilic groups at the 10-position (to fill the putative L1 lipophilic pocket) as a strategy to improve A1AR affinity. Among the new compounds synthesized and tested, the 3,10-diphenyl[1,2,4]tri azino[4,3-a]benzimidazol-4( 10H)-one (23) was characterized by a K-i value of 18 nM which represents a 3.5-fold gain of A(1)AR affinity compared with the lead 12. A rhodopsin-based model of the bovine adenosine A(1)AR was bui lt to highlight the binding mode of 23 and two well-known A(1)AR antagonist s (III and VII) and to guide future lead optimization projects. In our dock ing simulations, 23 receives a hydrogen bond (via the N1 nitrogen) from the side chain of Asn247 (corresponding to the HB1 and HB2 sites) and fills th e L1, L2, and L3 lipophilic pockets with the 10-phenyl, 3-phenyl, and fused benzene rings, respectively.