Synthesis of Formononetin Derivatives and Their Protective Effects Against Oxidative Stress

Abstract— Objective: With the molecular formula C₁₆H₁₂O₄, formononetin (FMN) is an appealing synthon exhibiting a wide range of biological activities, including antioxidant, anti-infective, and anti-apoptotic effects. Due to increasing interest in the mechanisms of action of antioxidants, our group aimed to identify the cellular target sites of these compounds. Methods: Cell Counting Kit-8 (CCK-8), enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM), TdT-mediated dUTP nick end labeling (TUNEL) staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot were used to analyze the effects of oxidative stress on PC12 cell survival, antioxidant enzyme activity, mitochondrial morphology, apoptosis, key intracellular proteins, and signaling pathways. The potential roles of candidate targets were further investigated using proteomics, molecular docking, kinetic simulation, and cellular thermal shift assay (CETSA). Results and Discussion: One of the compounds, (VI) (5-(8-(((1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl)amino)methyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)-2-methoxybenzenesulfonic acid), was highly active. It enhanced PC12 cell proliferation, reduced lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels, and increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, effectively inhibiting apoptosis and ameliorating mitochondrial damage. Meanwhile, (VI) upregulated the expression of PI3K/Akt and Nrf2/HO-1 proteins. The study also demonstrated that (VI) binds significantly to the oxidative stress-related protein HO-1. Conclusions: Compound (VI) effectively protects PC12 cells against H₂O₂-induced oxidative damage. Its protective mechanism may involve activation of the PI3K/Akt and Nrf2/HO-1 signaling pathways, with HO-1 serving as a potential key target of its action.