Synthesis and evaluation of vanillin Schiff bases as potential antimicrobial agents against ESBL-producing bacteria: towards novel interventions in antimicrobial stewardship

Abstract

The escalating challenge of antimicrobial resistance necessitates the development of novel antibacterial agents. In this study, a series of five vanillin Schiff bases (SB-1 to SB-5) were synthesized from vanillin and various aromatic amines. The chemical structures of these compounds were characterized using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR), proton nuclear magnetic resonance ( $_{}^1\text{H}$ -NMR), carbon-13 NMR ( $_{}^{13}\text{C}$ -NMR), and mass spectrometry techniques. Antibacterial efficacy was evaluated against strains of bacteria producing extended-spectrum beta-lactamases (ESBL), including Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae using the disc diffusion method. Cytotoxic effects were assessed through haemocompatibility and brine shrimp lethality assays. The Schiff bases demonstrated notable antibacterial activities, with SB-1, SB-2, SB-4, and SB-5 exhibiting zones of inhibition up to 16.0, 16.5, 16.6, and 15.5 mm against ESBL E. coli, respectively. SB-3 showed a maximum inhibition zone of 15.0 mm against ESBL K. pneumoniae. In cytotoxicity assays, the compounds exhibited IC $_{50}^{}$ values against red blood cells (RBCs) greater than 200 μg/mL and ranging from 45.7 to 50.5 μg/mL for the brine shrimp assay. While demonstrating potent antibacterial properties, the toxicity towards human RBCs suggests that further toxicity evaluations and structural modifications are essential for developing safer therapeutic agents based on vanillin Schiff bases.

Keywords: 1 H -NMR; 13 C -NMR; Antibacterial activity; Cytotoxicity; Drug resistance; ESBL-producing bacteria; Vanillin Schiff bases.

Fig. 1

General procedure for the synthesis of vanillin Schiff bases.

Fig. 2

Structures of synthesized compounds with variable R groups, illustrating the effect of different substituents on the molecular structure. The groups are as follows: R = -CH$_3^{}$ (Methyl), R = -Cl (Chloro), and R = -OH (Hydroxy). The representations are derived from their respective SMILES notation.

Fig. 3

Chemical structure of 2-Methoxy-4-((4-nitrophenylimino)methyl)phenol (SB-1). The nitro-substituted aromatic ring enhances its electron-withdrawing properties, potentially influencing its bioactivity.

Fig. 4

Chemical structure of 4-((2-Bromophenylimino)methyl)-2-methoxyphenol (SB-2). The presence of a bromine substituent may influence its reactivity and potential as an antimicrobial agent.

Fig. 5

Chemical structure of 4-((2-Chloro-4-methylphenylimino)methyl)-2-methoxyphenol (SB-3).

Fig. 6

Chemical structure of 4-((3-Hydroxyphenylimino)methyl)-2-methoxyphenol (SB-4).

Fig. 7

Chemical structure of 4-((2,4-Dichlorophenylimino)methyl)-2-methoxyphenol (SB-5).

Fig. 8

ESBL-positive E. coli showing clear zones of inhibition indicative of beta-lactamase activity.

Fig. 9

ESBL-positive P. aeruginosa displaying resistance patterns against beta-lactam antibiotics.

Fig. 10

ESBL-positive K. pneumoniae with enhanced expression of beta-lactamase, as observed in the synergy test.

Fig. 11

Antibacterial activity of Schiff base-1 against Escherichia coli.

Fig. 12

Antibacterial activity of Schiff base-2 against Escherichia coli.

Fig. 13

Antibacterial activity of Schiff base-3 against Klebsiella pneumoniae.

Fig. 14

Antibacterial activity of Schiff base-4 against Pseudomonas aeruginosa.

Fig. 15

Antibacterial activity of Schiff base-4 against Escherichia coli.

Fig. 16

Antibacterial activity of Schiff base-5 against Klebsiella pneumoniae.

Fig. 17

Hemocompatibility of SB1-2.

Fig. 18

Hemocompatibility of SB3-5.

Fig. 19

Brine shrimp cytotoxicity of SB1-2.

Fig. 20

Brine shrimp cytotoxicity of SB3-5.