Exploring novel A2AAR antagonists: Design, synthesis, and evaluation of 2,6,9-trisubstituted purine derivatives as promising antifibrotic agents.

A series of 2,6,9-trisubstituted purine derivatives were designed and synthesized with diverse chemical moieties. Through a comprehensive biological evaluation, we identified 4-(6-(methylamino)-2-(phenylethynyl)-9H-purin-9-yl)phenol (6a) as a promising A2AAR antagonist with potent antifibrotic properties. Compound 6a demonstrated significant efficacy in inhibiting CRE promoter activity and in reducing the expression of fibrogenic marker proteins and downstream effectors of A2AAR activation, surpassing the A2AAR antagonist ZM241385 and initial screening hits, 9-benzyl-N-methyl-2-(phenylethynyl)-9H-purin-6-amine (5a) and 9-((benzyloxy)methyl)-N-methyl-2-(phenylethynyl)-9H-purin-6-amine (5j). Further validation revealed that compound 6a effectively inhibited fibrogenic marker proteins induced by A2AAR overexpression or TGF-β1 treatment in hepatic stellate cells, alongside reducing PKA and CREB phosphorylation. These findings suggest that compound 6a exerts its antifibrotic action by modulating the cAMP/PKA/CREB pathway through A2AAR inhibition. Overall, our study provides valuable insights for the development of novel therapeutics that target hepatic fibrosis through A2AAR antagonism.