Design, Synthesis, and Evaluation of Nitroxide Radical Derivatives Based on Rhein as Potential Anti-Aging Agents Targeting the Keap1-Nrf2 Pathway.

Abstract

<h4>Purpose</h4>Targeting the crucial Keap1-Nrf2-ARE antioxidant pathway, we selected Rhein - a natural anthraquinone from traditional Chinese medicine with established Keap1-Nrf2 inhibitory activity as our lead compound. Through rational structural modification by incorporating nitroxide radicals at the 3-carboxyl position, we aimed to develop enhanced Keap1-Nrf2 modulators with anti-aging potential.<h4>Patients and methods</h4>A series of rhein nitroxide derivatives were synthesized, and their free radical scavenging activity was assessed in vitro using DPPH and ABTS methods. Compound <b>4b</b>, demonstrating significant activity, was selected for further evaluation. Its effects on the survival of L02 hepatocytes under oxidative stress and the lifespan and stress tolerance of <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) were investigated. Additionally, the impacts of <b>4b</b> on antioxidant enzyme activity, malondialdehyde (MDA) levels, and reactive oxygen species (ROS) accumulation under oxidative stress were assessed. Molecular docking was conducted to analyze interactions between <b>4b</b> and the Kelch domain of Keap1.<h4>Results</h4>Compound <b>4b</b> exhibited potent free radical scavenging activity, with IC₅₀ values of 0.51 ± 0.09 mM against DPPH radicals and 0.12 ± 0.03 mM against ABTS radicals. It significantly improved the survival rate of L02 hepatocytes under oxidative stress, maintaining 95.42% viability (<i>p</i> < 0.01). In the <i>C. elegans</i> model, <b>4b</b> extended the average lifespan and enhanced stress resistance, increasing GSH activity, reducing MDA content, and decreasing ROS accumulation. Molecular docking showed that <b>4b</b> penetrates deeply into the Kelch domain of Keap1, forming stable interactions with key residues.<h4>Conclusion</h4>Compound <b>4b</b> demonstrates superior antioxidant and anti-aging effects compared to the parent compound rhein, representing a highly promising anti-aging candidate and Keap1-Nrf2 signaling pathway modulator with potential as a novel therapeutic agent for age-related diseases.