Discovery of Novel Benzo[4,5]imidazo[1,2‑<i>a</i>]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-<i>a</i>]­pyrazin-1-amine derivatives bearing an amide substitution at 3-position to obtain potent antitumor antagonist <i>in vivo</i>. The structure–activity relationship studies were performed by molecular modeling and radioactive assay. The <i>in vitro</i> anticancer activities were evaluated by 3′,5′-cyclic adenosine monophosphate (cAMP) functional and T cell activation assay. The most potent compound <b>12o·2HCl</b> showed much higher affinity toward A_2AAR (<i>K</i>_i = 0.08 nM) and exhibited more significant <i>in vitro</i> immunostimulatory anticancer activity than clinical antagonist AZD4635. More importantly, <b>12o·2HCl</b> 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 <b>12o·2HCl</b> a promising immunotherapy anticancer drug candidate.