Schiff bases show distinct DNA binding and antibacterial activity

Researchers at a collaboration involving M.R. Aadhithya, Bhumika Joshi, Kaliappan Uma, and Murugesh Shivashankar have synthesized and characterized two novel Schiff base compounds to evaluate their biomolecular interactions and biological activities. The study, published in Results in Chemistry, focuses on a comparative analysis of N-(benzo[d]oxazol-2-yl)-1-(pyridin-2-yl) methanimine, abbreviated as ABPC, and 2-((4-bromonaphthalen-1-ylimino) methyl)-4-fluorophenol, abbreviated as FABN. By subjecting these structurally related molecules to identical experimental conditions, the team aimed to elucidate how specific structural features, such as the benzo[d]oxazole moiety and halogen substitutions, influence the compounds’ ability to interact with biological macromolecules like DNA and proteins.

The investigation began with the synthesis of both compounds through condensation reactions. ABPC was prepared by reacting benzo[d]oxazol-2-amine with pyridine-2-carbaldehyde in methanol, using acetic acid as a catalyst, while FABN was synthesized from 1-amino-4-bromonaphthalene and 5-fluorosalicylaldehyde. The successful formation of the azomethine linkage in both molecules was confirmed through comprehensive spectroscopic analysis, including 1H and 13C NMR, FT-IR, and HRMS. These characterization techniques provided unambiguous evidence of the molecular structures, with specific signals in the NMR spectra corresponding to the imine protons and carbons, and IR bands confirming the presence of the characteristic C=N stretching vibrations.

Photophysical properties were examined to understand the electronic behavior of the compounds in solution. In 1% DMSO, ABPC exhibited an intense absorption band at approximately 211 nm, assigned to π → π* transitions, while FABN showed absorption at 226 nm and a secondary band at 374 nm. The quantum yield of ABPC was notably higher at 0.61 compared to 0.01 for FABN, suggesting that ABPC is a more efficient emitter under the measurement conditions. The partition coefficients indicated that both compounds possess moderate lipophilicity, with FABN being slightly more lipophilic, a factor that may influence their interaction with biological membranes and targets.

The interaction of both Schiff bases with Calf Thymus DNA (ct-DNA) was evaluated using UV–Vis absorption titration. The researchers observed a hyperchromic effect, where the absorbance increased as the DNA concentration was raised, which is typically indicative of groove binding or electrostatic interactions rather than classical intercalation. The binding constant (Kb) for ABPC was calculated to be 1.97 × 10^6 M−1, whereas FABN demonstrated a higher affinity with a Kb of 4.93 × 10^6 M−1. This difference in binding strength suggests that the structural features of FABN, including its bromine and fluorine substituents, may facilitate more favorable interactions with the DNA grooves.

To further investigate the specificity of these interactions, the binding of the compounds to guanine, a key nucleobase in DNA, was studied. Both ABPC and FABN showed significant hyperchromic effects upon the addition of guanine, with binding constants of 6.80 × 10^6 M−1 and 7.66 × 10^6 M−1, respectively. These values were significantly higher than their overall DNA binding constants, indicating a preferential binding to guanine-rich sequences. This selective recognition is a characteristic feature of many clinically relevant anticancer agents and suggests that these compounds may target specific regions of the DNA molecule.

Complementary evidence for the binding mode was obtained through ethidium bromide (EtBr) displacement assays. EtBr is a planar dye that intercalates into DNA, and its fluorescence is quenched when displaced by other intercalators. The modest quenching constants observed for both ABPC and FABN suggest that they do not compete strongly with EtBr for intercalation sites. Instead, the data supports a non-intercalative binding mode, consistent with the hyperchromic shifts observed in the UV–Vis titrations, pointing towards groove binding and electrostatic interactions with the DNA phosphate backbone.

The interaction of the compounds with human serum albumin (HSA), the principal carrier protein in blood plasma, was investigated using fluorescence quenching measurements. The fluorescence intensity of HSA decreased systematically with increasing concentrations of both ABPC and FABN, without any significant shift in the emission maximum. The calculated bimolecular quenching rate constants were much higher than the diffusion-controlled limit, indicating a static quenching mechanism involving the formation of ground-state complexes. FABN exhibited a higher binding affinity to HSA compared to ABPC, with a binding constant of 1.53 × 10^5 M−1 versus 0.19 × 10^5 M−1 for ABPC.

Synchronous fluorescence spectroscopy provided insights into the local microenvironment of the HSA residues. The quenching effect was more pronounced at the tryptophan (Trp-214) emission wavelength compared to the tyrosine (Tyr) wavelength, suggesting that both compounds interact primarily with the hydrophobic subdomain IIA of HSA. The lack of significant peak shifts indicates that the binding does not induce large conformational changes in the protein backbone. Stability studies in Tris-HCl buffer, water, and glutathione solution over 72 hours confirmed that both compounds remained spectroscopically stable under biologically relevant conditions, with no evidence of degradation or reaction with the thiol group of glutathione.

The biological activities of the synthesized Schiff bases were evaluated through antibacterial and anti-inflammatory assays. Against Staphylococcus aureus and Escherichia coli, both compounds exhibited dose-dependent antibacterial activity, with FABN showing slightly higher inhibition zones than ABPC at the highest tested concentrations. The presence of bromine and fluorine in FABN may enhance its lipophilicity and electronic properties, facilitating better interaction with bacterial targets. In the in vitro anti-inflammatory assay, which measures the inhibition of albumin denaturation, both compounds showed moderate activity, with IC50 values of 190.50 μg/mL for ABPC and 182.65 μg/mL for FABN, comparable to the standard aspirin.

This comparative study provides a detailed understanding of how structural modifications in Schiff bases affect their biomolecular interactions and biological profiles. The findings highlight the potential of benzo[d]oxazole and halogen-substituted Schiff bases as candidates for further development in medicinal chemistry. The data on DNA and protein binding, stability, and biological activity serve as a foundation for the design of new bioactive molecules. However, the in vitro nature of the biological assays means that further cellular and in vivo studies are required to fully assess the therapeutic potential of these compounds.

The experimental design prioritized direct comparison by maintaining identical conditions for both ABPC and FABN throughout the spectroscopic and biological assays. This approach allows for the isolation of structural variables, specifically the benzo[d]oxazole-pyridine framework of ABPC versus the brominated naphthyl-fluorophenol framework of FABN. The synthesis of these compounds involved condensation reactions in methanol with acetic acid as a catalyst, yielding milky-white ABPC and yellow FABN solids. The structural integrity of the azomethine linkages was verified through the disappearance of aldehyde proton signals in NMR spectra and the presence of characteristic imine stretching bands in FT-IR data, confirming the successful conversion of carbonyl groups to azomethine bonds.

Photophysical characterization revealed distinct electronic behaviors between the two molecules. While both compounds exhibited π → π* transitions, FABN displayed an additional absorption band centered around 374 nm, a feature attributed to its phenolic hydroxyl and fluorine substituents. These structural elements modify the electronic distribution, influencing how the molecules interact with light and potentially with biological targets. The significant difference in quantum yield, with ABPC at 0.61 and FABN at 0.01, suggests that the halogenated structure of FABN alters excited-state behavior, possibly through non-radiative decay pathways associated with its specific substituent arrangement.

The stability of these Schiff bases under biologically relevant conditions was a critical aspect of the evaluation. Both compounds were incubated in Tris-HCl buffer at pH 7.4, aqueous solution, and glutathione-containing medium for 72 hours. The spectroscopic data indicated that the position and intensity of absorption peaks remained consistent over this period, demonstrating that the compounds do not undergo significant degradation or nucleophilic attack by the thiol group of glutathione. This stability is essential for any potential biological application, as it ensures that the measured interactions with DNA and proteins are not artifacts of compound decomposition in physiological environments.

The antibacterial assays utilized agar-well diffusion tests against clinical isolates of Staphylococcus aureus and Escherichia coli, with amoxicillin serving as the positive control. The results showed that both compounds produced dose-dependent zones of inhibition, with FABN generally exhibiting slightly larger zones than ABPC at the highest concentrations tested. For instance, against S. aureus, FABN produced a 14 mm zone at 0.08 mM, compared to 11 mm for ABPC at 0.13 mM. This enhanced activity is linked to the bromine and fluorine atoms in FABN, which can increase membrane permeability and facilitate interactions with essential bacterial enzymes, a mechanism previously observed in halogenated Schiff base derivatives.

The anti-inflammatory evaluation employed an in vitro albumin denaturation inhibition assay, a standard method for assessing NSAID-like activity. The denaturation of proteins such as albumin is a hallmark of inflammation, and inhibiting this process suggests potential anti-inflammatory properties. Both ABPC and FABN demonstrated concentration-dependent inhibition, with IC50 values of 190.50 μg/mL and 182.65 μg/mL, respectively. These values are comparable to aspirin, which had an IC50 of 169.24 μg/mL. The slightly lower IC50 for FABN may be attributed to the additional hydrogen-bonding capability of its phenolic hydroxyl group and the electronic effects of the fluorine substituent, which enhance interactions with serum albumin.

Despite the promising in vitro results, the study remains limited to biochemical and cellular-free assays. The interactions with DNA and HSA provide mechanistic insights into how these molecules might function in a biological system, but they do not confirm efficacy in living organisms. The groove-binding mode of DNA interaction, rather than intercalation, suggests a different mechanism of action compared to many traditional anticancer agents. Further research involving cellular models and in vivo studies is necessary to determine whether the observed biomolecular interactions translate into therapeutic benefits, particularly given the moderate lipophilicity and stability profiles of these compounds.

Subject of Research: Biomolecular interactions and biological activities of benzoxazole and halogen-substituted Schiff bases

Article Title: Structure-related biomolecular interactions and biological activities of benzoxazole and bromo/fluoro-substituted Schiff bases

Article References: Aadhithya, M. R., Joshi, B., Uma, K., & Shivashankar, M. (2026). Structure-related biomolecular interactions and biological activities of benzoxazole and bromo/fluoro-substituted Schiff bases. Results in Chemistry, 32, Article 104037. https://doi.org/10.1016/j.rechem.2026.104037

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DOI: 10.1016/j.rechem.2026.104037

Keywords: Schiff bases, DNA binding, Human serum albumin, Antibacterial activity, Anti-inflammatory activity, Fluorescence spectroscopy, Benzoxazole, Halogen substitution, Structure-related, biomolecular, interactions, biological