Synthesis, toxicity and antioxidant activity of phenolic benzimidazole derivatives: In vitro and in silico studies

Benzimidazole derivatives have attracted significant attention due to their diverse pharmacological activities, including antimicrobial, anticancer, and antioxidant properties. This article reports the synthesis of two benzimidazole derivatives (2-(2-benzimidazolyl)phenol ( 3a ) and 4-(1H-benzimidazol-2-yl)-2-methoxyphenol ( 3b )) via the condensation of o-phenylenediamine with aromatic aldehydes, their structural characterization using FT-IR, NMR, and HR-MS analyses, and the evaluation of their toxicity, antioxidant potential, and molecular interactions with NAD(P)H oxidase. Acute toxicity tests assessed in zebrafish reveals a concentration-dependent toxic effect. 3a lethal dose measured to kill 50 % of test fishes was 3.163 mg/L, and 1.88 mg/L for 3b Antioxidant activity was determined using the DPPH radical scavenging assay, with compound 3a demonstrating higher radical scavenging efficiency than 3b Molecular docking studies indicated that both compounds interact with NAD(P)H oxidase, with 3b showing a stronger binding affinity due to additional functional groups facilitating enhanced interactions, with H-bond interactions between 3a phenolic OH and Pro 696 and between benzimidazole NH and Asn 710, as well as H-bond interaction between 3b benzimidazole NH and Glu 482, and steric interactions between 3b O-ether and Tyr 481 and between aromatic ring and Ser 564. Molecular dynamics simulations confirmed that these interactions influenced enzyme flexibility, the 3b binding region exhibiting much greater flexibility than the ADP and 3a binding regions, what suggests a negative correlation between binding ability and rigidity, which may be associated with competitive interactions between residues within the enzyme itself and the binding of the 3b ligand. Density functional theory (DFT) calculations provided insight into the antioxidant mechanisms, suggesting that 3b predominantly follows a hydrogen atom transfer (HAT) mechanism, whereas 3a exhibits a stronger electron-donating ability. These findings contribute to the understanding of benzimidazole-based antioxidants and highlight the need for further investigations to optimize their bioactivity and minimize toxicity for potential therapeutic applications.