Discovery of a novel 10-hydroxy-12-methyl-substituted evodiamine derivative with anti-hepatocellular carcinoma activity through multi-pathway modulation involving suppression of PKA/AKT signaling cascades and moderate Topo I inhibition.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy with poor prognosis. The clinical benefits of small-molecule therapeutics represented by sorafenib remain limited, underscoring the urgent need to continuously invest in the discovery of novel anti-HCC lead compounds. In this study, structural optimization of the previously identified compound 6f with anti-HCC potential was conducted through diverse substituent modifications on the A- and E-rings, accompanied by systematic structure-activity relationship (SAR) investigations. This effort led to the identification of a new lead compound, Z4, which exhibited potent nanomolar inhibitory activity against four HCC cell lines (IC50 = 0.08 μM for HepG2, 0.16 μM for SK-Hep-1, 0.03 μM for HuH-7, and 0.03 μM for BEL-7404) as well as a doxorubicin-resistant cell line (IC50 = 0.19 μM for SMMC-7721/DOX). Cell-based phenotypic evaluations further demonstrated that Z4 more effectively inhibited proliferation and migration/invasion of BEL-7404 and HuH-7 cells than sorafenib, while inducing cell cycle arrest, mitochondrial dysfunction, reactive oxygen species (ROS) accumulation, and apoptosis. Mechanistically, Z4 exerts its anti-HCC activity primarily through synergistic inhibition of the cAMP-PKA-CREB and PI3K-AKT-mTOR pathways, with only moderate Topo I inhibition observed at high concentrations. In addition, in vivo studies indicated that Z4 possesses acceptable metabolic stability (T1/2 = 8.96 h) and exhibits significantly superior antitumor efficacy compared to sorafenib in a HuH-7 xenograft model. Collectively, this study not only further enriches the SAR of the A-ring of the evodiamine scaffold but also provides a promising lead compound Z4 for anti-HCC drug discovery.