# 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

## TL;DR

This study develops a new anti-cancer compound, WX006, derived from caffeic acid phenethyl ester, which effectively targets liver cancer by disrupting mitochondrial function and metal ion balance.

## Contribution

The study introduces WX006, a novel CAPE derivative, as a potent anti-HCC agent with a unique dual mechanism targeting mitochondrial respiration and metal homeostasis.

## Key findings

- WX006 selectively inhibits NDUFS2 in complex I, leading to NAD+ depletion and energy metabolism collapse in HCC cells.
- The compound disrupts metal ion homeostasis, inducing ferroptosis and cuproptosis in tumor cells.
- WX006 shows superior tumor suppression in mouse models compared to sorafenib with a favorable safety profile.

## Abstract

Hepatocellular carcinoma (HCC) is characterized by high incidence and mortality rates, yet effective therapeutic drugs remain scarce, creating an urgent need for viable treatment strategies. This study aimed to develop novel small-molecule anti-HCC agents based on the naturally derived antitumor compound caffeic acid phenethyl ester (CAPE), evaluate their efficacy against HCC, and investigate their underlying pharmacological mechanisms. A total of 28 structurally analogous small-molecule derivatives were synthesized, from which the most potent compound, designated WX006, was selected. Using an integrated approach encompassing molecular biology, cell biology, computational biology, and multi-omics analysis, we demonstrated that WX006 effectively depletes Nicotinamide adenine dinucleotide (NAD+) in HCC cells by binding to NADH: Ubiquinone Oxidoreductase Core Subunit S2 (NDUFS2), thereby disrupting mitochondrial function and intracellular metal ion homeostasis. This study provides valuable and independent insights into the development of derivatives based on the natural small molecule CAPE and establishes a further foundation for the advancement of anti-HCC therapies.

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.