Imidazo[1,2- a ]pyrazine Derivatives as Dual A2A/A2B Adenosine Receptor Antagonists Enhancing Anti-PD-1 Antibody Therapy in Colorectal Cancer

Abstract Adenosine signaling through A2A and A2B adenosine receptors (ARs) suppresses antitumor immunity and promotes tumor progression, limiting immune checkpoint blockade in microsatellite-stable colorectal cancer (CRC). Here, we identified imidazo[1,2-a]pyrazine-urea derivatives as a new chemotype of dual hA2A/hA2B AR antagonists. SAR studies revealed that 3-iodination of the core scaffold enhanced receptor affinity and functional antagonism, with 5p emerging as a representative lead. MD simulations showed that 5p can be accommodated in the orthosteric pockets of both AR subtypes through distinct binding modes, with the 3-iodo substituent occupying adjacent subpockets. Representative compounds inhibited adenosine-induced CREB phosphorylation, restored Th1 cytokine production, and increased CD4+ and CD8+ T cell infiltration. In CD73-high CRC models, 5p suppressed tumor growth and potentiated anti-PD-1 efficacy. These findings support dual A2A/A2B AR antagonism as a strategy to reprogram the adenosine-rich tumor microenvironment and improve CRC immunotherapy.