Discovery of Novel Benzo[4,5]imidazo[1,2‑a]pyrazin-1-amine-3-amide-one Derivatives as Anticancer Human A_2A Adenosine Receptor Antagonists

The blockade of A_2A adenosine receptor (A_2AAR) activates immunostimulatory response through regulating signaling in tumor microenvironment. Thus, A_2AAR has been proposed as a promising target for cancer immunotherapy. In this work, we designed a new series of benzo­[4,5]­imidazo­[1,2-a]­pyrazin-1-amine derivatives bearing an amide substitution at 3-position to obtain potent antitumor antagonist in vivo. The structure–activity relationship studies were performed by molecular modeling and radioactive assay. The in vitro anticancer activities were evaluated by 3′,5′-cyclic adenosine monophosphate (cAMP) functional and T cell activation assay. The most potent compound 12o·2HCl showed much higher affinity toward A_2AAR (K_i = 0.08 nM) and exhibited more significant in vitro immunostimulatory anticancer activity than clinical antagonist AZD4635. More importantly, 12o·2HCl significantly inhibited the growth of triple-negative breast cancer by reversing immunosuppressive tumor microenvironment in the xenograft mouse model without severe toxicity at the testing dose. These results make 12o·2HCl a promising immunotherapy anticancer drug candidate.