Discovery of Novel Benzo[4,5]imidazo[1,2‑a]pyrazin-1-amine-3-amide-one Derivatives as Anticancer Human A2A Adenosine Receptor Antagonists

The blockade of A2A adenosine receptor (A2AAR) activates immunostimulatory response through regulating signaling in tumor microenvironment. Thus, A2AAR 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 A2AAR (Ki = 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.