Synthesis and Biological Evaluation of a Caffeic Acid Phenethyl Ester Derivatives as Anti-Hepatocellular Carcinoma Agents via Inhibition of Mitochondrial Respiration and Disruption of Cellular Metabolism

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Background: In this study, 28 caffeic acid phenethyl ester (CAPE) derivatives were designed and synthesized, and their anti-proliferative activities were evaluated against two representative human hepatocellular carcinoma (HCC) cell lines. The half-maximal inhibitory concentration (IC50) was used as the activity metric. Among these derivatives, compound WX006 displayed the most potent anti-proliferative effect, with IC50 values of 3.332 μM and 3.764 μM after 48 h of treatment, significantly lower than those of the parent compound CAPE. Consequently, WX006 was selected for further investigation into its antitumor efficacy and underlying mechanisms. Methods: To investigate the pharmacological mechanism of WX006, we employed a combination of high-throughput transcriptomics, metabolomics, and mitochondrial function analysis to elucidate its intracellular mechanisms of action. Results: WX006 disrupts cytoplasmic-mitochondrial metal ion homeostasis, triggering ferroptosis and cuproptosis through iron-copper dysregulation. Computational modeling revealed that WX006 selectively inhibits mitochondrial NDUFS2 subunit of respiratory chain complex I, which may induce NAD+ exhaustion and consequent energy metabolism collapse in tumor cells. These “metabolism & metal homeostasis” dual mechanisms collectively underpin its robust anti-tumor effects. Therapeutic efficacy of WX006 was further validated in murine H22 ectopic xenograft and Hepa1-6-Luc orthotopic xenograft models, where WX006 exhibited superior tumor suppression compared to sorafenib, alongside favorable safety profiles. Conclusions: Our findings establish a foundational rationale for further pharmaceutical development of CAPE derivates as a promising therapeutic candidate for hepatocellular carcinoma.