Antimicrobial potential of iron complexes of benzothiazole-based Schiff bases against bacterial and phytopathogenic fungal species

Introduction

In present, one of the major problem in the context of infectious diseases is the persistent rise and spread of Antimicrobial Resistance (AMR) to currently available antimicrobial agents. As existing antimicrobial drugs are becoming less effective in controlling the bacterial/fungal infections due to emergence of resistant microorganisms, this highlights the urgent and continuous need for new, safe and effective antimicrobial drugs (Singh et al., 2025) [18] . In 1864, a German chemist named Hugo Schiff first synthesized the compound known as Schiff base by condensation reaction of primary amine with the carbonyl compounds aldehydes or ketones (Dinku et al., 2024; Ashraf et al., 2011; Al-Saadawy et al., 2016) [6,2,3] . These can be classified as aldimine or ketimine based on their structure (Sanjurani et al., 2024) [17] . Imine or azomethine functional group is the characteristic feature of these compound. The sp² hybridized nitrogen atom of azomethine group having a lone pair of electrons is of considerable biological and chemical importance (Isiyaku et al., 2021; Uddin et al., 2020) [11,20] . Various studies have reported that these compounds exhibit diverse biological properties such as antidepressant, antidiabetic, anti-inflammatory, antiviral, antifungal, cytotoxic, anticancer, antioxidant, and antibacterial activities (Sahib et al., 2021; Etaiw et al., 2011; Ceramella et al., 2022) [16,7,5] . Schiff bases have been known to form complexes with nearly all the transition metals (Hameed et al., 2017; Tawfiq et al., 2025) [9,19] . Due to their property of being easy to synthesize, synthetic flexibility, and presence of the C=N group, these serve as an important class of ligands in coordination chemistry and can coordinate with a wide range of metal ions in different coordination geometries and oxidation states. Heterocyclic Schiff base compounds are the most bioactive class of organic compounds (Kumar et al., 2023; Mesbah et al., 2018; Ismail & Lateef, 2022) [12,13,10] . Metal complexes of Schiff bases derived from heterocyclic compounds having nitrogen, sulfur, and oxygen as ligand atoms have been reported for their enhanced biological activities. Because of their wide range of pharmacological actions and structural resemblance to naturally occurring biological molecules, Schiff bases synthesized from 2aminobenzothiazole have garnered significant interest in medicinal chemistry. These have been shown to have antidepressant, antidiabetic, anti-inflammatory, antiviral, antifungal, cytotoxic, anticancer, antioxidant, and antibacterial properties (Akyol et al., 2025; Farhan et al., 2025; Abrol et al., 2019) [4,8,1] .

Materials and Methods

All chemicals were of analytical reagent grade and used without further purification. 2-amino-benzothiazole, 4methoxy-2-aminobenzothiazole and 4-chloro-2aminobenzothiazole were purchased from Sigma Aldrich chemical company. 2-Hydroxy-1-Napthaldehyde, Ferric chloride (FeCl 3 ), Ferrous chloride (FeCl 2 ), and Methanol (CH 3 OH) were purchased from CDH. The melting point of the synthesized compounds was determined using a digital melting point apparatus (LT-108) from the Department of Chemistry, COBS&H, CCS HAU Hisar. Infrared spectra of the Schiff base ligands and their metal complexes synthesized during research were recorded in KBr pellets in the region 4000-400 cm -1 on a PerkinElmer FTIR spectrometer from the Department of Physics, COBS&H, CCS HAU Hisar. UV-Visible spectra of the synthesized compounds were recorded in range of 200-800 nm on Model SHIMADZU (UV 3600 PLUS) UV-Visible spectrophotometer from Department of Chemistry, COBS&H, CCS HAU Hisar. Proton NMR spectra of the synthesized compounds were recorded in DMSO-d 6 using TMS as an internal reference on an NMR cryo-magnet Spectrometer 400 MHz (Bruker) NMR spectrometer at GJUS&T, Hisar.

Synthesis of Schiff base ligands and metal complexes

The Schiff base ligands (L 1 -L 3 ) were prepared by refluxing in equimolar quantities (5 mmol each) of 4-methoxy-2aminobenzothiazole, 4-chloro-2-aminobenzothiazole and 2aminobenzothiazole with 2-hydroxy-1-naphthaldehyde in methanol for 5 hours. The reaction mixtures were cooled to room temperature, and the precipitated products were filtered, washed with methanol, and dried over anhydrous CaCl₂. The iron(II) and iron(III) complexes of the synthesized Schiff base ligands were prepared by reacting equimolar amounts (5 mmol) of the respective ligand (L 1 -L 3 ) and iron salts (FeCl₂ or FeCl₃) in methanol. The reaction mixtures were refluxed for 5 hours (Ndiolene et al., 2025) [14] . After completion of the reaction, the mixtures were cooled to room temperature, and the resulting precipitates were collected by filtration, washed thoroughly with methanol, and dried over anhydrous CaCl₂ in a desiccator (Scheme 1).

Results

Physiochemical properties

The physicochemical properties of the synthesized Schiff base ligands (L 1 -L 3 ) and their corresponding iron(II) and iron(III) complexes are summarized in Table 1. All the synthesized compounds were obtained as colored, crystalline solids that were stable at room temperature and non-hygroscopic in nature. The compounds were readily soluble in polar organic solvents such as dimethyl sulfoxide (DMSO), methanol, ethanol, acetone, and chloroform. The synthesized compounds were obtained in good to excellent yields ranging from 52 to 91% and molar conductance measurements were carried out in methanol at room temperature. The observed molar conductance values ranged from 13.92 to 42.00 S cm² mol⁻¹, indicating the nonelectrolytic behavior of ligands and complexes in solution.

Spectral studies FTIR analysis

The spectra of free ligands L 1 , L 2 , and L 3 displayed bands at 1621, 1642, and 1631 cm -1 , which are due to the stretching frequency of ν(C=N), and bands at 1252, 1311, and 1273 cm -1 are due to ν(C-O). In the spectra of their metal complexes, a slight shift in bands was observed for ν(C=N) and ν(C-O), confirming the coordination of azomethine nitrogen and oxygen atom (Table 2). The shift in these bands and the presence of bands in the regions 481-499 cm -1 and 740-799 cm -1 are due to ν(M-O) and ν(M-N) also indicate complex formation. Based on IR results, it was confirmed that the ligands L 1 , L 2 , and L 3 act as neutral bidentate (N, O) ligands bonded with the metal ion through the azomethine-nitrogen and oxygen atoms (Figure 1-3).

UV-visible analysis

The UV-Visible spectra of Schiff base ligands and their corresponding complexes are presented in Figures 456. The intense bands observed at 235-273 nm were assigned to π→π* transitions of the aromatic system, while the bands appearing at 297-302 nm were attributed to n→π* transitions associated with the azomethine (C=N) group. In the spectra of the corresponding metal complexes, the ligand-centered π→π* and n→π* bands show slight bathochromic shifts, indicating coordination of the ligand through the azomethine nitrogen atom. Furthermore, broad absorption bands at 328 nm and 357 nm in Fe 2+ and Fe 3+ complexes of ligand L 2 are attributed to Ligand-to-Metal Charge Transfer (LMCT) transitions, confirming interaction between ligand and metal ions (Table 3). ppm were assigned to the phenolic -OH proton. The aromatic protons of the ligands appeared as multiplet signals in the region δ ~6.60-8.60 ppm, which is consistent with the presence of aromatic and heteroaromatic rings in the ligand structure. In the spectra of some metal complexes, a slight shifting of the azomethine proton signal was observed, indicating coordination of the ligand to the metal ion through the azomethine nitrogen atom. However, the detailed interpretation of certain complex spectra was limited by poor solubility and insufficient spectral resolution. The combined spectroscopic results provide evidence for the successful synthesis of the Schiff base ligands and their corresponding iron complexes. The presence of the azomethine ν(C=N) stretching bands and the characteristic azomethine proton signals confirmed the formation of the Schiff base ligands. The shift of the azomethine ν(C=N) band to lower wavenumbers in the FTIR spectra and bathochromic shift of ligand-centered transitions indicate the coordination of the ligand to the metal. Thus, from the above spectral studies, it was concluded that all the Schiff bases (L 1 , L 2 , and L 3 ) act as neutral bidentate (N, O) donor ligands and spectral data support the proposed structures of the synthesized complexes.

Antibacterial activity

The antibacterial activity of the synthesized compounds was evaluated against Gram-positive strains, i.e. Staphylococcus aureus VTCCBAA20 and Bacillus cereus VTCCBAA443 and Gram-negative strains, i.e. Xanthomonas spp. and Pseudomonas aeruginosa VTCCBAA2 using the agar well diffusion method. Muller Hinton agar medium was used to culture the bacteria. The standard antibiotic chloramphenicol was used as a positive control for the study. The experimental details and Zone of Inhibition (ZOI) in respect antibacterial activity of synthesized compounds against pathogenic bacterial strains which are given in Table 5-6. Among the synthesized compounds, ligand L 1 exhibited the highest antibacterial activity, producing inhibition zones of 17 mm against S. aureus, 13 mm against B. cereus, and 15 mm against Xanthomonas spp. at 1500 µg mL⁻¹. Ligand L 2 and its complexes demonstrated moderate antibacterial activity, with L 22 showing slightly improved inhibition against B. cereus compared with the parent ligand. Similarly, the unsubstituted ligand L 3 exhibited moderate activity, whereas its iron complexes (L 31 and L 32 ) generally showed comparable or slightly reduced antibacterial effects. Overall, the synthesized compounds were more effective against Gram-positive bacteria than Gram-negative bacteria, which may be attributed to the simpler cell wall structure of Gram-positive organisms, allowing easier penetration of antimicrobial agents.

Antifungal activity

The antifungal activity of the synthesized compounds was evaluated against fungal strains Fusarium oxysporum, Alternaria spp., and Rhizopus spp. using the poison plate technique at different concentrations (10-1500 µg mL -1 ) and the percentage growth inhibition was calculated. The standard antibiotic cycloheximide was used as a positive control for the study. All synthesized compounds exhibited concentration-dependent antifungal activity, with inhibitory effects generally increasing as the concentration increased.

Among the tested compounds, the chloro-substituted ligand L 2 consistently demonstrated the highest antifungal activity against all three fungal pathogens, showing complete inhibition against Fusarium oxysporum at all tested concentrations and showed nearly complete inhibition of Alternaria spp. and Rhizopus spp. over a broad concentration range. The corresponding Fe(II) complex (L 21 and L 22 ) also exhibited excellent antifungal activity, maintaining complete or near-complete inhibition at moderate to high concentrations. L 2 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 6.

L 21 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 80.49±0.17 7.

L 22 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 75.60±0.21 70.73±0.12 56.10±0.09 29.27±0.30 0.00±0.00 8.

L 3 100.00±0.00 100.00±0.00 100.00±0.00 100.00±0.00 75.60±0.22 43.90±0.81 24.39±1.12 0.00±0.00 0.00±0.00 0.00±0.00 9.

Discussion

Among all tested compounds Ligand L 1 with an electrondonating group showed the highest antibacterial activity among the series, followed by unsubstituted Ligand L 3 and then Ligand L 2 with an electron-withdrawing group. As biological activity of complex depends on many factor like chelate effect of the ligand, type of donor atoms involved, type of metal ions, overall charge of the complex, the nature of counter ion, and geometry of the complex (Ramadan et al., 2025) [15] . Ligand L 2 with chloro group and its complexes exhibited the highest antifungal activity among tested compounds against all the tested fungal strains, which may be attributed to the presence of an electronwithdrawing group. Ligand L 1 with an methoxy group also exhibited good antifungal activity, achieving 100% inhibition at higher concentrations, its activity decreased progressively at lower concentrations. As Schiff bases containing sulfur-bearing heterocyclic moieties such as thiazole and benzothiazole derivatives, have been widely reported to exhibit significant antifungal properties, and presence of halogen, methoxy and naphthyl like group on these ligands further enhances their antifungal potential (Uddin et al. 2020) [20] . The unsubstituted ligand L 3 showed moderate antifungal activity, with inhibition values around 85% at higher concentrations, decreasing significantly at lower concentrations. Its complexes (L 31 and L 32 ) exhibited moderate to low activity, particularly at lower concentrations where inhibition was minimal or absent.

Conclusion

Three Schiff base ligands (L 1 -L 3 ) derived from substituted 2-aminobenzothiazole with 2-Hydroxynapthaldehyde and their corresponding iron (II) and iron (III) complexes were successfully synthesized and characterized by various spectroscopic technique. Synthesized compounds were evaluated for antibacterial and antifungal activities. Among the ligand series, the methoxy-substituted ligand (L 1 ) exhibited superior antibacterial activity, whereas the chlorosubstituted ligand (L 2 ) showed comparatively lower activity and the unsubstituted ligand (L 3 ) displayed intermediate behavior. The enhanced activity of L 1 may be associated with the electron-donating methoxy group, which can increase electron density within the conjugated system and favor interactions with bacterial targets. Synthesized compounds, particularly ligand L 2 and its complexes L 21 and L 22 ) matched or exceeded the activity of the standard across a wider concentration range, highlighting their potential as effective antifungal agents. These findings demonstrate that the nature of the substituent on the benzothiazole ring significantly influences antibacterial and antifungal activity.

Acknowledgement

AcknowledgementThe authors gratefully acknowledge the Department of Chemistry, Chaudhary Charan Singh Haryana Agricultural University, Hisar, for providing the research facilities and infrastructure essential for this work. The laboratory support provided by the Department of Microbiology for the antimicrobial studies is sincerely appreciated. The authors are also thankful to Chaudhary Charan Singh Haryana Agricultural University, Hisar, for the fellowship support received during the course of this research.

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