In vitro, in silico, and DFT evaluation of antimicrobial imidazole derivatives with insights into mechanism of action

In this study, three imidazole derivatives : 2,4,5-triphenyl-1-(4-(phenyldiazenyl)phenyl)-1 H-imidazole (N1), 2-(4-chlorophenyl)-4,5-diphenyl-1-(4-(phenyldiazenyl)phenyl)-1 H-imidazole (N2), and 2-(2,4-dichlorophenyl)-4,5-diphenyl-1-(4-(phenyldiazenyl)phenyl)phenyl)-1 H-imidazole (N3), which were prepared using N-methyl-2-pyrrolidone hydrogen sulfate ionic liquid as a catalyst, were selected for detailed evaluation. The antimicrobial activities of these compounds were assessed against a panel of pathogenic microorganisms, including Gram-positive bacteria (Staphylococcus aureus, Bacillus anthracoides), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae), and the fungal strain Candida albicans. The Density Functional Theory (DFT) results showed that the N3 molecule exhibited the highest stability with the largest ΔEgap (2.9546 eV) and chemical hardness, while N1 showed the highest reactivity. Frontier molecular orbital (FMO) and electrostatic potential (ESP) analyses revealed that the HOMO orbitals are delocalized over the imidazole ring and neighboring aromatic fragments, and the LUMO orbitals are spread over the phenyldiazenyl phenyl fragments. A comparative analysis suggests that while N3 is the most chemically stable, N2 demonstrates the highest potential for biological activity, consistent with experimental antimicrobial results. Both in-vitro and in-silico results revealed that the N2 molecule exhibits superior biological activity compared to N1 and N3. Molecular docking studies of N2 were performed with its target protein to explore its binding interactions with several amino acid residues, elucidating its potential antioxidant mechanism. Docking results revealed that all ligands exhibit strong binding affinities, with N1 showing the highest MolDock score and N2 demonstrating strong specificity through key hydrogen bonding and π-interactions. Molecular dynamics simulations, including RMSD, RMSF, radius of gyration (Rg), solvent-accessible surface area (SASA), and principal component analysis (PCA), further confirmed the structural stability and dynamic behavior of the protein-ligand complexes. MMPBSA binding free energy calculations indicated that N2@1AI9 forms the most stable complex, primarily driven by favorable van der Waals and electrostatic interactions. In silico ADMET analysis suggests all compounds, particularly the lead compound N2, face challenges with high lipophilicity, low solubility, and predicted drug-drug interaction risk (CYP2C19 inhibition for all; CYP3A4 and P-gp substrate for N2), yet their lack of BBB permeation supports their further development, especially for topical or intravenous use. Overall, the results suggest that N2 is a promising candidate for further development as a potential antimicrobial anti-fungal agent targeting the ergosterol biosynthesis pathway in Candida albicans.