Bromodomain containing protein 4 (BRD4) has been demonstrated to play critical roles in cellular proliferation and cell cycle progression. In this study, using the BRD4 inhibitor Fragment 9 as a lead compound, a series of imidazolopyridone derivatives were designed and tested for their inhibitory activity against BRD4 protein in vitro. Among them, HB100-A7 showed excellent BRD4(1) inhibitory activities with an IC₅₀ value of 0.035 μM in amplified luminescent proximity homogeneous assay (Alphascreen). The result of MTT assay showed that HB100-A7 could suppress the proliferation of pancreatic cancer cells. In addition, flow cytometry further illustrated that HB100-A7 treatment resulted in G0/G1 phase arrest and promoted apoptosis of BxPc3 cells. Furthermore, the in vivo study found that HB100-A7 displayed significant tumor growth inhibition in a pancreatic mouse tumor model (Panc-02). Moreover, IHC staining suggested that HB100-A7 induce cell apoptosis in pancreatic cancer tumor tissue. Together, this study revealed, for the first time, HB100-A7 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the BRD4 protein.

已证明含溴结构域蛋白 4 (BRD4) 在细胞增殖和细胞周期进程中发挥关键作用。本研究以BRD4抑制剂片段9为先导化合物，设计并测试了一系列咪唑并吡啶酮衍生物对BRD4蛋白的体外抑制活性。其中，HB100-A7显示出优异的 BRD4(1) 抑制活性，IC ₅₀值为 0.035 μM，在放大发光邻近均相测定 (Alphascreen) 中。MTT实验结果表明HB100-A7能抑制胰腺癌细胞的增殖。此外，流式细胞术进一步说明HB100-A7治疗导致 G0/G1 期停滞并促进 BxPc3 细胞的凋亡。此外，体内研究发现HB100-A7在胰腺小鼠肿瘤模型 (Panc-02) 中显示出显着的肿瘤生长抑制作用。此外，IHC 染色表明HB100-A7诱导胰腺癌肿瘤组织中的细胞凋亡。总之，这项研究首次表明，HB100-A7是一种很有前景的先导化合物，可进一步开发为靶向 BRD4 蛋白的新一代小分子抑制剂。