The antimicrobial effects of the currently synthesized Schiff bases (6a-l) were tested against two Gram-positive bacteria (S. aureus and B. subtilis), two Gram-negative bacteria (E. coli and P. aeruginosa), and two strains of fungus (A. flavus and C. albicans). The modified disk diffusion method (Alkhaldi et al., 2019; Manso et al., 2021) was used to determine the inhibition zones (IZ, mm/mL) and the minimal inhibitory concentration (MIC, nM). Ciprofloxacin and Fluconazole was used as positive controls. Tables 1–3 display the findings.

Based on the inhibition zones (IZ) measurements in Table 1, we can conclude that five Schiff bases (6c, 6e, 6h, 6j, and 6l) had inhibition zones that were either greater than or comparable to the inhibition zones of the reference medications (ciprofloxacin and fluconazole) against pathogenic organisms. Schiff bases 6a-l were found to have strong antibacterial activity against both Gram-positive and Gram-negative bacteria, with inhibition zones (IZ) ranging from 22 to 48 mm for all pathogens tested, compared to Ciprofloxacin’s IZ of 40 mm. Moreover, Table 1 demonstrated that compounds 6h and 6l had superior efficacy compared to the reference ciprofloxacin against both gram-positive and gram-negative bacteria. In addition, most of the newly synthesized Schiff bases 6a-l had IZ values between 11 and 36 mm against A. flavus and C. albicans, whereas fluconazole, a broad spectrum antifungal medication, had a value of 40 mm. We proceeded with the investigation to ascertain the minimal inhibitory concentrations (MIC, nM) of the potent Schiff bases (6c, 6e, 6h, 6j, and 6l). The results are displayed in Tables 2, 3.

The most potent components 6c, 6e, 6h, 6j, and 6l were evaluated for antibacterial activity using a twofold serial dilution method on a 96-well microtiter plate (Wiegand et al., 2008). Table 2 showed the MICs (nM) of these compounds against the tested bacteria, with ciprofloxacin as the reference medicine. The results of this in vitro assay test are consistent with the findings of the antimicrobial sensitivity test. Compound 6h (R = Cl, X = H) was the most potent of the compounds examined, having MIC values of 19, 54, and 55 nM against S. aureus, E. coli, and P. aeruginosa. It displayed greater potency to ciprofloxacin against the investigated species but had a MIC value of 24 nM against B. subtilis, which is 2.5 times less effective than ciprofloxacin (MIC = 10 nM). Compound 6l (R = NO₂, X = H) exhibited the second highest activity. Its MIC values were comparable to those of ciprofloxacin against E. coli and P. aeruginosa, Table 2. However, it was 2.5 times less effective than ciprofloxacin against B. subtilis. Compounds 6h and 6l were 1.5 times more effective than ciprofloxacin against S. aureus, with MIC values of 19 and 21 nM, respectively, whereas ciprofloxacin had a MIC value of 30 nM. Compounds 6c (R = Br, X = Cl) and 6j (R = CH₃, X = H) exhibited significant activity against the tested species, especially against S. aureus, with MICs values of 29 and 25 nM, respectively. These values were comparable to that of ciprofloxacin (MIC = 30 nM). Finally, compound 6e (with R = OCH₃ and X = Cl) demonstrated the lowest level of efficacy. It has lower antimicrobial activity than ciprofloxacin against all pathogens tested.

The MBC differs from the MIC. The MIC test finds the lowest concentration of an antimicrobial agent that significantly inhibits growth, whereas the MBC identifies the lowest concentration that causes microbiological organisms to die. In contrast to the MBC, which does result in mortality, the MIC just inhibits, meaning that the antibacterial action does not cause death (Abdullahi et al., 2023). MBC is typically reported as MBC₅₀, indicating that the antibiotic concentration kills 50% of the initial bacterial population (Sun et al., 2019).

In general, components 6c, 6e, 6h, 6j, and 6l had strong bactericidal activity. The MBC values for Gram-positive bacteria ranged from 28 to 58 nM, whereas ciprofloxacin MBC values were 19 and 45 nM, Table 2. Compounds 6h (R = Cl, X = H) and 6l (R = NO₂, X = H), the most potent antibacterial agents, displayed bactericidal activity of 28 and 33 nM, respectively, for S. aureus, compared to ciprofloxacin’s MBC value of 45 nM, making 6h and 6l more effective as bactericidal agents. Compound 6j (R = CH₃, X = H) scored third in bactericidal activity with MBC value of 40 nM against S. aureus, which was comparable to the reference ciprofloxacin. Regrettably, compounds 6h, 6j, and 6l exhibited lower effectiveness as bactericidal agents compared to ciprofloxacin against B. subtilis. The MBC values for compounds 6h, 6j, and 6l were 38, 47, and 43 nM, respectively, while ciprofloxacin had an MBC value of 19 nM.

In the case of Gram-negative bacteria, compounds 6h, 6j, and 6l had higher MBC values than the other compounds examined. These compounds have MBC values of 74, 80, and 78 nM, making them more effective than ciprofloxacin (MBC = 90 nM) against both E. Coli and P. aeruginosa species. Again, compound 6e (with R = OCH₃ and X = Cl) was the least effective as bactericidal agents against all of the bacterial species tested.

Compounds 6c, 6e, 6h, 6j, and 6l were tested for antifungal activity with a twofold serial dilution method (Wiegand et al., 2008). Table 3 displays the MICs (µM) and MFCs (minimum fungicidal concentration, µM) of these derivatives against A. flavus and C. albicans fungus, using fluconazole as the reference medication. Overall, the investigated compounds displayed robust antifungal efficacy against the selected fungal species when compared to ciprofloxacin. Compounds 6c, 6e, 6h, 6j, and 6l exhibited MIC values ranging from 14.50 to 19.50 µM against A. flavus, while fluconazole had a MIC value of 11.50 µM. Moreover, the tested compounds demonstrated MIC values ranging from 18.20 to 22.90 µM against C. albicans, while the reference fluconazole had a MIC of 17.50 µM.

Compound 6h (R = Cl, X = H), the most potent antibacterial agent, was also the most potent antifungal agent, with MIC values of 14.50 µM against A. flavus and 18.20 µM against C. albicans, being comparable to the reference fluconazole, which had MIC values of 11.50 and 17.50 µM, respectively. Compound 6l (R = NO₂, X = H) demonstrated the second highest antifungal activity. It exhibited MIC values of 15.60 µM against A. flavus and 19.50 µM against C. albicans. Finally, Table 3 shows that all investigated compounds had fungicidal activity, with MFC/MIC ratios ranging around two.

The most potent Schiff bases 6c, 6e, 6h, 6j, and 6l were examined for their antibacterial efficacy against a panel of MDRB (multi-drug resistant bacteria), including one clinical strain S. aureus (ATCC 43300) (MRSA), two standard strain E. coli (ATCC BAA-196) and P. aeruginosa (ATCC BAA-2111). Norfloxacin, a broad-spectrum antibiotic, worked as positive control. Results are displayed in Table 4.

The MIC values for the tested compounds vary from 1.70 to 7.15 µM, while the MBC values range from 3.50 to 10.40 µM against MDRB bacteria. These values are compared to the MIC values of norfloxacin, which range from 1.20 to 3.20 µM, and the MBC values, which range from 2.80 to 4.70 µM. The majority of the investigated Schiff bases, compounds 6c, 6e, 6h, 6j, and 6l, had substantial broad-spectrum effects on both Gram-positive and Gram-negative bacteria, either by inhibiting their growth or by killing them (bactericidal effect). Notably, compounds 6h and 6l show the most potent actions compared to other derivatives. Compound 6h (R = Cl, X = H) showed a MIC value of 1.70 µM against the clinical strain S. aureus (ATCC 43300) (MRSA), making it an effective antibacterial agent. It was found to be approximately as potent as the reference drug norfloxacin, which had a MIC of 1.20 µM. Compound 6h, on the other hand, exhibited an MBC of 3.90 µM, making it around 1.2 times more potent against the MRSA strain than the reference norfloxacin. Furthermore, compound 6h demonstrated similar efficacy to norfloxacin against E. coli (ATCC BAA-196) and P. aeruginosa (ATCC BAA-2111), with MICs of 1.9 and 3.4 µM, respectively. Norfloxacin, on the other hand, showed MIC values of 1.6 and 3.2 µM. Additionally, compound 6l (R = NO₂, X = H) showed significant antibacterial activity against MDRB strains, with MIC values ranging from 2.20 to 4.50 µM and MBC values ranging from 4.50 to 6.50 µM. Based on the MBC/MIC ratio, we identified that all of the compounds tested had values less than two, indicating bactericidal activity.

The inhibitory potency of derivatives 6h, 6j, and 6l, the most potent antibacterial agents, against E. coli DNA gyrase and DHFR was determined using the E. coli DNA gyrase and DHFR assay (Durcik et al., 2018). The findings are shown as IC₅₀ values for the investigated compounds and reference drugs (Table 5). The results of this assay complement those of the antibacterial activity investigation. Compounds 6h, 6j, and 6l inhibited E. coli DNA gyrase at IC₅₀ values of 79, 117, and 87 nM, respectively, compared to the reference novobiocin (IC₅₀ = 170 nM). Compounds 6h, 6j, and 6l exhibited greater potency compared to the reference compound novobiocin, with compounds 6h and 6l being twice as potent as novobiocin against DNA gyrase.

Compounds 6h, 6j, and 6l were further evaluated against the DHFR enzyme as indicated in Table 5. Compounds 6h and 6l, the most potent E. coli gyrase inhibitors, also demonstrated promising effects on DHFR. Compounds 6h and 6l have IC₅₀ values of 3.80 µM and 4.25 µM, respectively. These values are much lower and more potent than trimethoprim’s IC₅₀ value of 5.20 µM. Based on these observations, we may infer that both compounds 6h and 6j show promise as dual-target inhibitors against DNA gyrase and DHFR, especially after optimization.

Bacterial biofilms provide health risks in hospitals, the food industry, and drinking water systems. In the current work, we investigated the antibiofilm activity of compound 6h, the most potent derivative, against S. aureus using Microtiter plate assay for biofilm quantification (Antunes et al., 2010; Niu and Gilbert, 2004). The assay was performed with three different concentrations, the first of which was equivalent to the MIC of 6h against S. aureus ATCC 43300 (Table 4), the second of which was equivalent to 1/2 MIC, and finally 1/4 MIC. The results were presented in Table 6 as Biofilm inhibition%.

The results showed that 6h has significant antibiofilm action, with biofilm inhibition percentage equal to 97 at the MIC dose. Compound 6h inhibited biofilms by 88% and 64% at ½ and ¼ MIC levels, respectively.

Blank represented absorbance of media only.

Control represented absorbance of test organism without any treatment.

This test examines the impact of compounds 6c, 6e, 6h, 6j, and 6l, which are the most potent derivatives, on normal cell lines in order to assess the safety level of these compounds. The vitality of the investigated compounds was assessed using the MCF-10A cell line, which is a normal human mammary gland epithelial cell line. Following a 4-day incubation period on MCF-10A cells with a concentration of 50 µM for each compound being studied, the viability of the cells was assessed using the MTT test (Hisham et al., 2022; Mahmoud et al., 2022). The results from Table 7 indicate that none of the compounds tested exhibited cytotoxicity, and all compounds show a cell viability of more than 89% at a concentration of 50 µM.

This investigation performed an extensive computational docking analysis to determine the binding affinities of compounds 6b, 6h, and 6l with E. coli DNA gyrase B and E. coli DHFR. Novobiocin served as the reference drug for E. coli DNA gyrase B, while trimethoprim was used as the reference for E. coli DHFR. Utilizing Discovery Studio software (Baroroh et al., 2023), the study provided an in-depth examination of the interaction mechanisms between these compounds and their respective target proteins. To ensure the accuracy and relevance of our investigation, we incorporated the crystallographic structure of the E. coli DNA gyrase B ligand complex (PDB ID: 4DUH) from the Protein Data Bank (Marinho et al., 2023). The OPLS-AA (Optimized Potentials for Liquid Simulations - All Atom) force field was utilized during the energy minimization process for the molecular systems under examination. Implementing this force field was crucial for achieving conformational stability of the molecular structures, thereby enhancing the precision and reliability of our computational analyses (Hazarika et al.; Kumar H. et al., 2023). Prior to initiating the docking procedure, the protein structure underwent comprehensive preparation to ensure its accuracy. This preparation included protonation, a critical step that significantly contributed to the robustness and reliability of the subsequent docking analysis. A comparative analysis between docking scores and in vitro activity levels of E. coli DNA gyrase B for the compounds studied revealed a direct correlation. Compound 6h, which demonstrated the highest in vitro activity against E. coli DNA gyrase B, achieved a docking score of −7.88 kcal/mol. In contrast, compound 6b, with lower in vitro antibacterial activity and a smaller inhibition zone (IZ) measurement compared to the ciprofloxacin, had a docking score of −5.78 kcal/mol. Compound 6l recorded a docking score of −7.51 kcal/mol, reflecting its relatively strong binding affinity and correlating with its moderate in vitro activity. Moreover, Novobiocin, used as a reference drug in the study, displayed a docking score of −7.26 kcal/mol, indicating its considerable binding affinity. In analyzing interactions between the tested compounds and the E. coli DNA gyrase B protein, compound 6h has exhibited notable binding characteristics. An important hydrogen bond interaction between the carbonyl oxygen of the salicylate moiety and Arg136 stabilizes 6h within the active site. This hydrogen bond is crucial as it anchors the compound, preventing its dissociation from the active site and enhancing its inhibitory efficacy (Figure 4). Moreover, the absence of a bulky atom, such as the 3-chloro group on the di-salicylate nucleus of 6h, facilitates the free rotation of the two salicylate moieties around the methylene axis. This rotational freedom is crucial as it enables the compound to adopt a conformation that effectively blocks the entrance to the active site. By fitting more snugly into the binding pocket, 6h can comprehensively obstruct substrate access, thereby enhancing its ability to inhibit E. coli gyrase activity. The isatin nucleus within 6h significantly enhances its binding affinity. The docking interactions reveal that the isatin moiety forms essential interactions with several amino acid residues within the active site. Notably, the π-π T-shaped interactions with residues such as His55 and Lys103 underscore the crucial role of the isatin nucleus in stabilizing the enzyme-inhibitor complex. The aromatic nature of isatin allows for these non-covalent interactions, which strengthen the overall binding and stability of 6h within the active site (Figure 4). Furthermore, the presence of substituents like the 5-chloro group on the isatin nucleus of 6h further amplifies its binding affinity. The 5-chloro substituent increases hydrophobic interactions with the active site residues and contributes to the electronic distribution of the molecule. The electron-withdrawing nature of the 5-chloro group enhances interactions with residues like His99. Additionally, this substituent helps position the isatin moiety in an optimal orientation for interaction with the enzyme, thereby boosting the inhibitory potential of 6h (Figure 4).

In contrast, compound 6l, which also lacks the bulky 3-chloro group on the di-salicylate nucleus like 6 h, exhibits less binding affinity due to fewer interactions with the active site. The absence of the 3-chloro group allows 6l to have a certain degree of rotational freedom, but not as effectively as 6h. This results in a less snug fit within the binding pocket, making it less efficient in blocking substrate access (Figure 5). Arg136 forms a Pi-donor hydrogen bond with the salicylate moiety, while Gly77 and Arg76 interact with the isatin moiety. Thr165 and Asp73 contribute to stabilizing the binding conformation through additional hydrogen bonds.

In addition, Novobiocin exhibited notable interactions with the E. coli DNA gyrase B protein, with its amide nitrogen functioning as a hydrogen bond acceptor in conjunction with Gly 101, as depicted in Figure 6. However, this significant interaction results in Novobiocin having a comparatively lower binding affinity to the active site than compounds 6h and 6l. This reduced affinity can be attributed to the lack of additional stabilizing interactions that compounds 6h and 6l possess, such as the π-π interactions with His55 and Lys103 in compound 6h and the hydrophobic interactions facilitated by the 5-chloro group in compound 6h (Figure 4). Moreover, the rigid structure of Novobiocin, which might limit its flexibility and ability to adopt an optimal conformation within the active site. This structural rigidity can result in fewer contacts with key residues within the binding pocket, leading to weaker overall binding interactions (Figure 6).

On the other hand, compound 6b features a bulky atom, the 3-chloro group on the di-salicylate nucleus, which significantly influences its interaction with E. coli gyrase. The presence of the 3-chloro group restricts the free rotation of the salicylate moieties around the methylene axis. This restriction limits the conformational flexibility of 6b, potentially reducing its ability to snugly fit into the binding pocket and block substrate access as effectively as 6h (Figure 7).

Comparative analysis between compounds 6h, 6l, and 6b reveals insights into their flexibility and stability. Compound 6h benefits from the absence of bulky substituents, allowing for greater conformational flexibility and a snugger fit within the active site, leading to potentially higher inhibitory efficacy. Compound 6l, while also lacking the bulky 3-chloro group on salicylate, forms fewer interactions within the active site, resulting in reduced binding affinity and inhibitory potency compared to 6h. On the other hand, compound 6b, with its bulky 3-chloro group on salicylate, faces a steric hindrance that reduces its conformational adaptability.

In our study on E. coli DHFR, we utilized the crystallographic structure of its ligand complex (PDB ID: 6CXK) from the Protein Data Bank as a foundational framework for computational modeling (Bhati, 2024). Compound 6h, which demonstrated the highest in vitro activity against E. coli DHFR, achieved a docking score of −7.42 kcal/mol. Similarly, compound 6l, with nearly comparable in vitro activity, recorded a docking score of −7.28 kcal/mol. Trimethoprim, used as the reference compound in this study, exhibited a docking score of −6.94 kcal/mol. The significance of the docking score of trimethoprim lies in its role as a benchmark, highlighting that compounds 6h and 6l exhibit stronger binding affinities, as evidenced by their lower docking scores, which correlates with their higher in vitro activities against E. coli DHFR. In the comprehensive analysis focusing on the interaction profiles between the investigated compounds and E. coli DHFR, compound 6h exhibited significant binding characteristics. The docking interactions of compound 6h with the DHFR E. coli enzyme provide detailed insights into its inhibitory potential. The carbonyl and phenolic oxygens of the salicylate moiety form two crucial hydrogen bonds with Lys32, anchoring 6h within the active site (Figure 8). Additionally, π-stacked interactions with Leu28 and Leu54 further enhance the binding affinity of 6h, while hydrogen bonds with Ile5 and Asn18 help stabilize its overall conformation within the active site. These interactions highlight the importance of specific residues in stabilizing the binding of compound 6h, allowing it to fit snugly and effectively block substrate access, thereby enhancing its inhibitory efficacy against the DHFR E. coli enzyme.

Similarly, compound 6l exhibits a binding affinity to DHFR E. coli enzyme that is comparable to 6h. The docking interactions of 6l reveal several key interactions contributing to its stability and inhibitory potential. The carbonyl oxygen of the salicylate moiety in 6l forms a crucial hydrogen bond with Lys32, mirroring the interaction seen with 6h (Figure 9). Pi-cation interactions between the aromatic ring of the salicylate moiety and Ala29 further stabilize the enzyme-inhibitor complex. Additionally, isatin-pi stacked interactions with Leu54, and Pro25 enhance the binding affinity of 6l, while hydrogen bonds with Asn23 and Ile50 contribute to the overall stability of the compound within the active site. These interactions allow 6l to occupy the DHFR active site effectively, forming stable bonds with key residues and blocking substrate access, thus inhibiting enzyme activity.

Trimethoprim exhibited notable interactions with the E. coli DHFR enzyme, with its amino nitrogen functioning as a hydrogen bond acceptor in conjunction with Asp27, as depicted in Figure 10. However, this significant interaction results in Trimethoprim having a comparatively lower binding affinity to the active site than compounds 6h and 6l. This reduced affinity can be attributed to the lack of additional stabilizing interactions that compounds 6h and 6l possess, such as the π-π stacking interactions with Ala29 and Lys32 of di-salicylate and also, the network interactions facilitated by the isatin nucleus.

This dual modeling against E. coli DNA gyrase B and DHFR enzymes has crucial implications for antibacterial drug development. Particularly, the favorable docking poses of 6h suggest its potential as dual inhibitor against E. coli DNA gyrase B and DHFR enzymes.

In conclusion, The molecular docking results provided crucial insights into the binding affinities and interaction mechanisms of the compounds with E. coli DNA gyrase B and DHFR enzymes. As summarized in Table 8, compound 6h demonstrated the strongest binding affinity due to key hydrogen bonds and π-π interactions with active site residues, making it particularly effective in blocking substrate access. Comparatively, compound 6l and reference drugs like Novobiocin and Trimethoprim exhibited fewer stabilizing interactions, resulting in relatively lower binding affinities.