Regioselective Synthesis, Characterization, and Antimicrobial Evaluation of Novel Pyrazole, Hydrazone, and Azo-Benzoyl Acetone Derivatives under Catalyst-Free Conditions

Abstract

This study presents a catalyst-free, regioselective synthesis of a series of heterocyclic and azo-linked compounds derived from benzoyl acetone. The initial phase investigated the condensation of benzoyl acetone with hydrazine hydrate and ethylenediamine under varied molar ratios (1:1, 2:1, and excess) at room temperature. For the hydrazine series, reactions at 1:1 and 2:1 stoichiometries both yielded the same pyrazole product (I), while an excess of hydrazine produced a distinct hydrazone derivative (II). In contrast, the reaction with ethylenediamine consistently afforded a single product (VI) regardless of the molar ratio employed. The intermediate (II) was further functionalized via condensation with various aldehydes (1:1) to generate a new series of schiff bases (III, IV, V). In the final phase, benzoyl acetone was subjected to azo-coupling with a diazonium salt derived from 2,4-dichloro aniline to yield an azo-benzoyl acetone derivative. Subsequent reaction of this azo intermediate with hydrazine hydrate under different molar ratios surprisingly led to the same product (VII) in all cases, indicating a highly regioselective transformation. All synthesized compounds were characterized by melting point determination, TLC, and comprehensive spectroscopic techniques, including ¹H-NMR (with D₂O exchange), ¹³C-NMR, and APT analyses. The antimicrobial activity of selected compounds (I, II, V, VI, and VII) was evaluated against three Gram-negative bacterial strains: Escherichia coli, Klebsiella pneumoniae, and Salmonella spp., and compared with standard antibiotics. Compound (I) demonstrated the highest antibacterial potency across all tested bacterial strains. At a 5% concentration, compound (VII) exhibited notable activity specifically against Salmonella spp. However, compounds (II), (V), and (VI) showed no inhibitory effect against Salmonella spp., while compounds (II), (V), and (VII) were found to be inactive against K. pneumoniae. The results highlight the influence of structural modification on antimicrobial selectivity and position compound (I) as a promising lead for further development. To examine the binding interactions and possible binding modes of the synthesized compounds, molecular docking studies were conducted against dihydrofolate reductase (DHFR) from Klebsiella pneumoniae (PDB ID: 4OR7). With a docking score of −7.167 kcal/mol, compound VI had the best binding affinity among the tested compounds, while compound I had the lowest, with a score of −5.166 kcal/mol. Additionally, the electronic characteristics and global reactivity of the synthesized compounds were examined through Density Functional Theory (DFT) calculations. Compound VII had the smallest HOMO–LUMO energy gap (3.7239 eV), indicating relatively higher chemical reactivity, according to the computed frontier molecular orbital energies and global reactivity descriptors, which showed variations in the electronic characteristics and reactivity of the examined compounds.