2-aminothienopyridazines as novel adenosine A1 receptor allosteric modulators and antagonists

A pharmacophore-based screen identified 32 compounds including ethyl 5-amino-3-(4-tert-butylphenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxylate (8) as a new allosteric modulator of the adenosine A(1) receptor (A(1)AR). On the basis of this lead, various derivatives were prepared and evaluated for activity at the human A(1)AR. A number of the test compounds allosterically stabilized agonist-receptor-G protein ternary complexes in dissociation kinetic assays, but were found to be more potent as antagonists in subsequent functional assays of ERK1/2 phosphorylation. Additional experiments on the most potent antagonist, 13b, investigating A(1)AR-mediated [S-35]GTP gamma S binding and [H-3]CCPA equilibrium binding confirmed its antagonistic mode of action and also identified inverse agonism. This study has thus identified a new class of A(1)AR antagonists that can also recognize the receptor's allosteric site with lower potency.