In this study, a series of novel oxazol-5-one derivatives containing a chiral trifluoromethyl and isoxazole moiety were synthesized and evaluated for cytotoxic activities. Among them, 5t was the most effective compound against HepG2 liver cancer cells with an IC₅₀ of 1.8 μM. 5t inhibited cell proliferation, migration, invasion, and induced cell cycle arrest and apoptosis in vitro. Nevertheless, the potential anti-hepatocellular carcinoma (HCC) target and mechanism of 5t were unclear. This work aimed to seek the molecular target of 5t against HCC and investigate its mechanism. Liquid chromatography tandem-mass spectrometry was used to identify peroxiredoxin 1(PRDX1) as a possible target of 5t. Cellular thermal shift assay, drug affinity responsive target stability, and molecular docking provided conclusive evidence that 5t targeted PRDX1 and inhibited its enzymatic activity. 5t augmented the level of reactive oxygen species (ROS) and led to ROS-dependent DNA damage, endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis in HepG2 cells. Silencing PRDX1 also resulted in ROS-mediated apoptosis in HepG2 cells. In vivo, 5t inhibited mouse tumor growth by increasing oxidative stress. Briefly, our studies revealed that compound 5t targeted PRDX1 through a ROS-dependent mechanism, highlighting the future development of compound 5t as a novel therapeutic drug for HCC.

在这项研究中，合成了一系列含有手性三氟甲基和异恶唑部分的新型恶唑-5-酮衍生物，并评估了细胞毒活性。其中，5t是对HepG2肝癌细胞最有效的化合物，IC ₅₀为1.8 μM。5t在体外抑制细胞增殖、迁移、侵袭，并诱导细胞周期停滞和凋亡。然而， 5t的潜在抗肝细胞癌 (HCC) 靶点和机制尚不清楚。本工作旨在寻找5t抗HCC的分子靶点并探讨其作用机制。采用液相色谱-串联质谱法鉴定过氧化物酶 1(PRDX1) 作为5t的可能靶标. 细胞热转移测定、药物亲和反应靶点稳定性和分子对接提供了5t靶向 PRDX1 并抑制其酶活性的确凿证据。5t增加活性氧 (ROS) 的水平并导致 ROS 依赖性 DNA 损伤、内质网应激、线粒体功能障碍和 HepG2 细胞凋亡。沉默 PRDX1 还导致 HepG2 细胞中 ROS 介导的细胞凋亡。在体内，5t通过增加氧化应激来抑制小鼠肿瘤的生长。简而言之，我们的研究揭示了化合物5t通过 ROS 依赖性机制靶向 PRDX1，突出了化合物5t作为 HCC 新型治疗药物的未来发展。