Design, Synthesis, and Evaluation of o‑(Biphenyl-3-ylmethoxy)nitrophenyl Derivatives as PD-1/PD-L1 Inhibitors with Potent Anticancer Efficacy In Vivo

Two series of novel o-(biphenyl-3-ylmethoxy)­nitrophenyl compounds (A1–31 and B1–17) were designed as programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) inhibitors. All compounds showed significant inhibitory activity with IC50 values ranging from 2.7 to 87.4 nM except compound A17, and compound B2 displayed the best activity. Further experiments showed that B2 bound to the PD-L1 protein without obvious toxicity in Lewis lung carcinoma (LLC) cells. Furthermore, B2 significantly promoted interferon-gamma secretion in a dose-dependent manner in vitro and in vivo. Especially, B2 exhibited potent in vivo anticancer efficacy in an LLC-bearing allograft mouse model at a low dose of 5 mg/kg, which was more active than BMS-1018 (tumor growth inhibition rate: 48.5% vs 17.8%). A panel of immunohistochemistry and flow cytometry assays demonstrated that B2 effectively counteracted PD-1-induced immunosuppression in the tumor microenvironment, thereby triggering antitumor immunity. These results indicate that B2 is a promising PD-1/PD-L1 inhibitor worthy of further development.本研究设计了两系列新型邻联苯-3-基甲氧基硝基苯基类化合物（A1–31与B1–17），作为程序性细胞死亡蛋白1（programmed cell death protein 1，PD-1）/PD配体1（PD-ligand 1，PD-L1）抑制剂。除化合物A17外，所有化合物均展现出显著的抑制活性，其半最大抑制浓度（IC50）范围为2.7~87.4 nM，其中化合物B2的活性最优。进一步实验结果显示，B2可特异性结合PD-L1蛋白，且在路易斯肺癌（Lewis lung carcinoma，LLC）细胞中无明显毒性。此外，B2在体外与体内实验中均能以剂量依赖方式显著促进γ干扰素的分泌。尤为突出的是，在携带LLC异体移植瘤的小鼠模型中，仅5 mg/kg的低剂量B2即展现出强效的体内抗肿瘤活性，其活性优于阳性对照化合物BMS-1018（肿瘤生长抑制率：48.5% vs 17.8%）。通过一系列免疫组织化学与流式细胞术检测证实，B2可有效逆转PD-1介导的肿瘤微环境免疫抑制，进而激活抗肿瘤免疫应答。上述研究结果表明，B2是一款极具开发潜力的PD-1/PD-L1抑制剂，值得开展进一步的深入研究。