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 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.腺苷A2A受体（A2A adenosine receptor, A2AAR）的阻断可通过调控肿瘤微环境中的信号通路激活免疫刺激性应答，因此A2AAR已被提出作为癌症免疫治疗的极具潜力的靶点。本研究设计了一系列全新的苯并[4,5]咪唑并[1,2-a]吡嗪-1-胺类衍生物，在3位引入酰胺取代基，以获取体内活性强效的抗肿瘤拮抗剂。通过分子建模与放射性结合实验开展了构效关系研究。采用3',5'-环磷酸腺苷（3′,5′-cyclic adenosine monophosphate, cAMP）功能实验与T细胞活化实验评估了体外抗癌活性。活性最优的化合物12o·2HCl对A2AAR展现出更高的亲和力（Ki=0.08 nM），其体外免疫刺激性抗癌活性优于临床拮抗剂AZD4635。更为重要的是，12o·2HCl可通过逆转异种移植小鼠模型中的免疫抑制性肿瘤微环境，显著抑制三阴性乳腺癌的生长，且在受试剂量下未出现严重毒性。上述结果表明，12o·2HCl是一款极具潜力的免疫治疗类抗癌候选药物。