Targeting Acetylcholinesterase: Design, Synthesis and Multimodal Evaluation of Ferrocenyl Acetylaniline Derivatives as Potential AntiAlzheimer Agents

Abstract

This thesis presents the design, synthesis, physicochemical characterisation, and comprehensive in silico evaluation of a series of ferrocenyl acetylaniline derivatives (FcMe2Ac, FcMe3Ac, and FcMe4Ac), with particular emphasis on elucidating the relationships between molecular structure, electronic properties, and interaction behaviour toward acetylcholinesterase (AChE), a key enzymatic target in Alzheimer’s disease. The target compounds were synthesised via a nucleophilic substitution strategy, yielding the desired regioisomers in moderate to high yields (48–80%) with good reproducibility. Structural confirmation was achieved through complementary spectroscopic techniques, including FT-IR, UV–Visible spectroscopy, and one- and two-dimensional NMR analyses. FTIR spectra displayed characteristic bands corresponding to N–H stretching (~3290–3445 cm⁻¹) and carbonyl functionalities (~1647–1651 cm⁻¹), while UV–Visible spectra revealed absorption maxima in the range of 259–432 nm, attributed to π→π* transitions within the aromatic system and metal-centred d–d transitions. NMR data confirmed the expected molecular architecture, with distinctive signals corresponding to the ferrocenyl unit (~4.1–4.3 ppm) and aromatic protons (~6.6–7.8 ppm). Electrochemical characterisation by cyclic voltammetry demonstrated a quasi-reversible Fe(II)/Fe(III) redox process for all derivatives, with formal potentials ranging from 43.9 to 57.5 mV. The observed peak separations (ΔEp = 87.8–115 mV) and near-unity current ratios confirmed quasi-reversible behaviour. Diffusion coefficients (1.11 × 10⁻⁷–5.74 × 10⁻⁷ cm²·s⁻¹) and heterogeneous electron-transfer rate constants (0.14 × 10⁻³–0.62 × 10⁻³ cm·s⁻¹) indicated that both transport and kinetic properties are strongly influenced by substitution pattern. Density functional theory calculations provided detailed insight into the electronic structure, revealing HOMO energies between −5.83 and −5.68 eV and HOMO–LUMO gaps of 3.99–4.29 eV, indicative of moderate chemical stability and controlled reactivity. Global reactivity descriptors confirmed this behaviour, with hardness values ranging from 1.995 to 2.143 eV and electrophilicity indices up to 3.519 eV. The progressive increase in dipole moment (3.82–5.12 D) reflects enhanced molecular polarity across the series. Molecular docking studies targeting acetylcholinesterase demonstrated favourable binding affinities (−7.34 to −8.58 kcal·mol⁻¹), with FcMe4Ac exhibiting the strongest interaction. Binding mode analysis revealed stabilisation within the catalytic gorge through hydrophobic contacts, π–π stacking, and hydrogen bonding interactions involving key residues. Molecular dynamics simulations over 100 ns confirmed the stability of the ligand–enzyme complexes, with RMSD values stabilising within ~0.10–0.25 nm and reduced residue fluctuations, indicating a stable and persistent binding mode. In silico ADMET predictions revealed favourable pharmacokinetic and safety profiles, including high gastrointestinal absorption, balanced lipophilicity (LogP = 3.12–3.45), and absence of predicted mutagenic or hepatotoxic effects. However, the limited predicted blood– brain barrier permeability suggests that further optimisation may be required to enhance central nervous system availability. Overall, the results demonstrate that structural modulation of ferrocenyl derivatives through acetylaniline substitution significantly influences their electronic properties, electrochemical behaviour, and interaction profiles with acetylcholinesterase. Among the investigated compounds, FcMe4Ac emerges as the most promising candidate, combining optimal redox characteristics, strong binding affinity, high dynamic stability, and favourable pharmacokinetic properties, thereby representing a valuable scaffold for the development of novel anti-Alzheimer agents.