Bioorganometallic Safety Assessment of Ferrocene-Based Candidates: Rat Cytotoxicity Benchmarked Against Computational Toxicology

Abstract

The growing pharmaceutical interest in ferrocene derivatives is attributed to their unique physicochemical and redox properties, which may enhance biological activity and therapeutic potential. However, toxicological safety assessment remains essential prior to their biomedical application. Therefore, the present study aimed to evaluate the acute toxicological profile and biological safety of three ferrocene derivatives: N-Ferrocenylmethylaniline (FMA), NFerrocenylmethyl-N-acetylaniline (FCMphA), and N-Ferrocenylmethyl-N-benzoylaniline (FCMphB), using complementary in vivo and in silico approaches. The in vivo study was performed on adult Wistar rats of both sexes exposed to graded doses (0.1, 1, 10, and 100 mg/kg). Toxicological evaluation was based on clinical observations, body weight evolution, food and water intake, relative organ weights, hematological and biochemical analyses, and histopathological examination of major organs, including the liver, kidneys, and brain, in order to determine dose-dependent toxic effects and organ-specific alterations. In parallel, the in silico investigation involved ADMET prediction and molecular docking analysis to evaluate pharmacokinetic properties, drug-likeness, absorption profile, biological distribution, and toxicity prediction of the tested compounds. Molecular docking was additionally conducted to investigate ligand–protein interactions with selected target proteins, including CYP3A4 and KEAP1, to better understand the relationship between molecular structure and biological behavior. The experimental findings revealed the absence of mortality at all administered doses, indicating relatively low acute toxicity of the tested compounds. Nevertheless, several doserelated clinical, physiological, biochemical, and histopathological alterations were observed. Among the investigated derivatives, FCMphA demonstrated the highest toxicological effect, showing more pronounced organ alterations and biological disturbances. In contrast, FCMphB exhibited the best biological tolerance and a comparatively safer toxicological profile, while FMA induced moderate and mostly reversible effects. Furthermore, the computational analyses supported the experimental observations, revealing differences in absorption, blood–brain barrier permeability, and protein-binding affinity among the compounds. Docking results showed strong molecular interactions, particularly for FCMphB, suggesting a favorable pharmacological profile. In conclusion, the tested ferrocene derivatives demonstrated promising pharmaceutical potential with relatively low acute toxicity; however, structural modifications significantly influenced their toxicological and biological behavior. The integration of experimental and computational findings highlights the importance of comprehensive toxicological evaluation to support the safe development of ferrocene-based therapeutic candidates.