Synthesis, Physicochemical Characterization, and Antimicrobial Evaluation of Halogen-Substituted Non-Metal Pyridine Schiff Bases

Abstract

Four synthetic Schiff bases (PSB1 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4,6-dibromophenol], PSB2 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4,6-diiodophenol], PSB3 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4-iodophenol], and PSB4 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4-chloro-6-iodophenol]) were fully characterized. These compounds exhibit an intramolecular hydrogen bond between the hydroxyl group of the phenolic ring and the nitrogen of the azomethine group, contributing to their stability. Their antimicrobial activity was evaluated against various Gram-negative and Gram-positive bacteria, and it was found that the synthetic pyridine Schiff bases, as well as their precursors, showed no discernible antimicrobial effect on Gram-negative bacteria, including Salmonella Typhi (and mutant derivatives), Salmonella Typhimurium, Escherichia coli, and Morganella morganii. In contrast, a more pronounced biocidal effect against Gram-positive bacteria was found, including Bacillus subtilis, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus haemolyticus. Among the tested compounds, PSB1 and PSB2 were identified as the most effective against Gram-positive bacteria, with PSB2 showing the most potent biocidal effects. Although the presence of reactive oxygen species (ROS) was noted after treatment with PSB2, the primary mode of action for PSB2 does not appear to involve ROS generation. This conclusion is supported by the observation that antioxidant treatment with vitamin C only partially mitigated bacterial inhibition, indicating an alternative biocidal mechanism.

Keywords: ROS; antimicrobial activity; intramolecular hydrogen bond; pyridine Schiff bases.

Figure 1

Chemical structures of (E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4,6-dibromophenol (PSB1), (E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4,6-diiodophenol (PSB2), (E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4-iodophenol (PSB3), and (E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4-chloro-6-iodophenol (PSB4).

Figure 2

FTIR (ATR) spectrum of PSB1, PSB2, PSB3, and PSB4.

Figure 3

¹H-NMR spectrum of the corresponding PSB1 to PSB4 at 400 MHz and 25 °C, with samples dissolved in deuterated DMSO.

Figure 4

¹³C-NMR spectrum of the corresponding PSB1 to PSB4 at 100 MHz and 25 °C, with samples dissolved in deuterated DMSO.

Figure 5

UV-VIS absorption spectra of PSBs under study in dichloromethane (A) or DMSO (B) at room temperature.

Figure 6

The intracellular ROS (reactive oxygen species) production was assessed in Gram-negative (A,B) and Gram-positive (C,D) bacteria following treatment with two concentrations of PSB2 (3.1 and 6.3 µM). For comparison, 1.5 mM of H₂O₂ was used as a positive control. We obtained similar results to those shown in (C) with Staphylococcus aureus strains 2 and 7 and Staphylococcus haemolyticus. Conversely, similar outcomes were observed with Streptococcus agalactiae and Enterococcus faecalis, as shown in (D) (Figure 7). With Bacillus subtilis, we observed intermediate results compared to (C,D). The bars represent the standard error of the mean (SEM) for n = 3. ROS was determined using the H₂DCFDA probe.

Figure 7

Effect of vitamin C (VitC) on quenching ROS (reactive oxygen species) action in Streptococcus pyogenes (A), Streptococcus agalactiae (B), or Enterococcus faecalis (C). Bacteria were treated with PSB2 (6.3 µM). For comparison, 1.5 mM of H₂O₂ was used as a positive control. The bars represent the standard error of the mean (SEM) for n = 3. ROS was determined using the H₂DCFDA probe.

Figure 8

Vitamin C (VitC) affects bacterial growth in the presence of PSB2 for Streptococcus pyogenes (A), Streptococcus agalactiae (B), or Enterococcus faecalis (C). Bacteria were treated with the vehicle alone (DMSO), PSB2, or PSB2 + vitamin C (0.1 mM VitC). Bacterial growth was estimated according to OD₆₀₀. 1 × MIC corresponds to the minimum inhibitory concentration of PSB2 (refer to Table 2). Furthermore, 0.5 × MIC corresponds to half of the MIC, while 0.25 × MIC corresponds to a quarter. Bars represent the standard error (SEM), n = 3. One-way ANOVA was performed as the statistical test. * p < 0.05, ** p < 0.01; NS = not significant.