Recently, the drug-resistant bacteria represented a distinctive hindrance, especially those having the capability to proliferate in the presence of several antibiotic groups (Matin et al., 2019). The resistance of bacterial pathogens toward at least one agent in three or more antibacterial families was characterized as MDR. Moreover, the bacterial pathogen was called XDR when it’s non-susceptible to at least one agent in all but two or fewer antimicrobial categories. In this regard, our vision is aimed at investigating the ability of the targeted compounds to cause bactericidal effects against the clinically important bacterial pathogens. In which, these models were isolated from different urinary tract infected samples, which had a significant resistance toward the classical antibacterial agents as mentioned in the antibiotic profile for each pathogen (Table 1). The new coumarin-based derivatives conjugated with the thiazole scaffold were examined as a potential drug against these pathogens due to the potential antibacterial activity of coumarin and thiazole derivatives.

The target compounds 6a-i were prepared, and their antibacterial activities were investigated against XDR and MDR pathogens. As can be seen in Supplementary Figure S1, the sensitivity of the XDR pathogens (C. jejuni and S. fonticola) towards the tested compounds was found to be negligible, as no inhibition zone appeared around all tested molecules. High resistance demonstrated by these pathogens emphasized the ineffective action of the tested compounds; hence, those were excluded from our examination. Interestingly, the efficiency of the targeted compounds was notably performed toward the MDR pathogens (A. xylosoxidans) and XDR (E. faecalis) (Table 1). A significant activity was clearly indicated in the case of most tested compounds against both pathogens except for compound 6g. On the other hand, the activity of some tested compounds was relatively preferred when compared to the standard antibacterial agents. Since some compounds of this group obtained an inhibition zone higher than that carried out by the potent antibacterial standards. Surprisingly, compounds 6a-e had higher sensitivity towards E. faecalis than that obtained by ciprofloxacin (4 mm). In addition, the ability of these compounds to inhibit A. xylosoxidans was observed in the equal efficacy of the reference drug (5 mm) as occurred in compounds 6b and 6c. Our results clearly showed that compounds 6a-f and h were very effective against A. xylosoxidans and E. faecalis. Therefore, further studies using potent compounds were implemented against A. xylosoxidans and E. faecalis to establish more details about their activity.

The ability of the potent compounds to inhibit the targeted bacterial pathogens at the lowest concentration (MIC) could be determining the optimal dosage that maximizes the killing of bacterial colony-forming units (CFUs). Accordingly, stock solutions of each potent compound were prepared at different concentrations and incubated with each bacterial pathogen. Bacterial growth was measured using turbidimetric methods, and the percentage of survival inhibition was calculated based on the reduction of CFUs. The activity of the potent compounds 6a and 6b was clearly observed at significant MIC values, which were sometimes close to the values of the standard antibiotic Ciprofloxacin (see Table 2). Comparatively, the potency of the most active compounds was significantly greater against E. faecalis than against A. xylosoxidans, indicating the specificity of these compounds in combating E. faecalis. Notably, compound 6c demonstrated the highest MIC values, with 12.5 μg/mL against E. faecalis and 25 μg/mL against A. xylosoxidans. Overall, the superiority of compounds 6a and 6c, with MIC values not exceeding 50 μg/mL, positions them as promising candidates for application in the pharmaceutical industry.

Bacterial biofilms are slimy structures created by the aggregation of bacteria that can protect bacterial cells from outside influences and enhance resistance to antimicrobial agents (Yang et al., 2022). Commonly, microbial pathogens utilize this mechanism to increase their virulence and evade adverse conditions. The assessment of biofilm formation by the treated bacterial pathogens was performed using both qualitative and quantitative methodologies.

Preliminarily, the evaluation of bacterial biofilm formation was conducted at a fixed concentration of 50 μg/mL (which considered an effective concentration for the potent molecules) for all potent molecules using a Congo red plate assay. After incubating each bacterial pathogen with the targeted molecule, the resulting bacterial suspension was cultivated on the Congo red plate and incubated for 48 h. The colonial growth was then observed. Notably, the black pigment of the growth colonies was clearly visible in the untreated samples after 24 h Supplementary Figure S2. Variable antibacterial activity was observed in all treated samples, indicating the efficacy of the tested compounds at this concentration, particularly compounds 6a and 6c. The bacterial cells appeared stressed and exhibited difficulty in growth. Furthermore, the black pigment did not develop in any of the treated samples, with only a slight appearance noted in the cases of P25 and P100.

Indeed, the qualitative assay demonstrated that compounds 6c and 6a have the potential to act as strong antagonists against bacterial biofilm formation, particularly against E. faecalis (Supplementary Figure S2).

Quantitative assay methodologies were utilized to assess the effectiveness of the potent compounds 6a and 6c in inhibiting biofilm formation by specific bacterial pathogens. Initially, the assessment of microbial biofilm formation was conducted at a fixed concentration of 50 μg/mL. The target compound 6c demonstrated greater effectiveness against both bacterial pathogens, as shown in Table 3. Both compounds yielded significant results regarding the inability of bacterial pathogens to produce biofilm while maintaining cell proliferation without negative consequences. The assessment of biofilm formation demonstrated a significantly enhanced ability to suppress biofilm formation in both Gram-positive and Gram-negative bacteria compared to the standard drug. The percentage of bacterial biofilm inhibition exceeded 50% for E. faecalis, indicating that it is a promising antibiofilm agent. Conversely, it is noteworthy that 6a exhibited moderate antibiofilm efficacy against A. xylosoxidans, with inhibition levels below 40%. The observed differences in biofilm production among the tested pathogens in the presence of these potent molecules may be attributed to variations in the composition of the embedded biofilm.

An assay to evaluate the inhibition of E. coli DNA gyrase was conducted for the two most potent compounds, 6a and 6c, with Ciprofloxacin serving as a reference control. The findings are presented in Table 6, indicating the IC₅₀ values in μg/mL. Compound 6a exhibited notable inhibitory activity against DNA gyrase, with an IC₅₀ value of 23.75 μg/mL, relative to the Ciprofloxacin control, which recorded an IC₅₀ of 15.95 μg/mL. Conversely, compound 6c demonstrated a comparatively lower level of inhibition, being approximately 2.5 times less effective than the control.

The suggested binding mode of compound 6a demonstrated a binding energy of −8.35 kcal/mol in relation to DNA Gyrase B (PDB ID: 4duh). A total of fifteen interactions were identified, including hydrophobic π-π, π-cation, π-anion, π-alkyl, and π-sigma interactions with Val120, Ile94, Lys103, Ile78, Gly77, Glu50, Pro50, and Arg76 amino acids sidechain. Moreover, compound 6a formed five hydrogen bonds with Ala100, Gly101, Asp73, Thr165, and Arg136 with a distance of 3.29, 1.76, 1.82, 2.37, and 2.42 Å, Figure 2. The binding mode of compound 6c showed a binding energy that was not constrained (∆G) equal of −7.14 kcal/mol. fourteen hydrophobic π-π, π-anion, π-cation, and π-alkyl interactions were conducted with Pro79, Arg76, Glu50, Lys103, Ile94, Val120, Val43, Val167, Ile78, and Asn46. Additionally, the interaction of Compound 6c was stabilized by the formation of three hydrogen bonds with Arg76, Gly77, and Asp73 with distances of 2.59, 2.50, and 2.05 Å (Figure 3). Furthermore, The co-crystalized ligand complexed with DNA Gyrase B exhibited an affinity score of −8.12 kcal/mol with RMSD value equal 1.12 Å, the co-crystalized ligand formed twelve hydrophobic π-alkyl, π-cation, and π-π interactions with Val167, Val43, Val71, Asn46, Gly77, Ile78, Lys103, Ile94, Pro79, and Arg76, additionally, the interaction was supported by three hydrogen bonds with Asp73, Arg136, and Gly101 with distances of 1.87, 2.19 and 1.81 Å (Figure 4).

Molecular dynamics (MD) simulations were performed over duration of 100 nanoseconds to investigate the molecular stability of the ligand 6a, which exhibited the most favorable docking results, in relation to the DNA Gyrase B target pocket. The resulting root mean square deviation (RMSD) values for both the complex and the ligand, in comparison to their initial positions within the active site, were documented and subjected to analysis. Additionally, the interactions of the lead compound 6a were thoroughly examined and assessed (Table 7).

A reaction was performed utilizing a combination of thiosemicarbazones 4 (0.001 mol) and their corresponding hydrazonoyl chloride derivatives 5a-i (0.001 mol) (These derivatives were synthesized using established methods that involve the reaction of diazonium chloride with appropriately activated halogenated methylene groups) in 15 mL of dioxane, with triethylamine (TEA) employed as a catalytic agent. The reaction mixture underwent reflux for a period of 8 h. Following the cooling phase, the resulting precipitate was filtered and subsequently recrystallized from ethanol, leading to the synthesis of the desired compounds 6a-i.

To assess the antibacterial effectiveness of the synthesized coumarin derivatives, various extensively drug-resistant (XDR) bacterial pathogens were employed. In this regard, two Gram-positive (Campylobacter jejuni and Enterococcus faecalis) and two Gram-negative (Serratia fonticola and Achromobacter xylosoxidans) bacteria were supplemented by faculty of medicine (Boys), Al-Azhar University. Antibiogram sensitivity profile demonstrated highly resistance of these pathogens towards at least three groups of classical antibiotic references. Activation of each of pathogen prior to screening of the targeted compounds was implemented Cultivating in Mueller Hinton broth for 24 h at 37 °C with shaking. The rationale for the inoculum size for each pathogen was determined through the use of the McFarland scale and precisely quantified in terms of colony forming units (CFU/mL) to ensure a constant bacterial concentration across all experimental assays. Thus, The agar-well diffusion method was developed in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) (Yassin et al., 2025). Subsequently, screening of each of targeted molecule was performed at fixed concentration (50 μg/mL). Incubation of each tested pathogen was carried out and the observed inhibition zone diameter (mm) were conducted in triplicate and subsequently compared to established standard antibacterial agents (Ahmed et al., 2024).

The most-active compounds that efficiently inhibited the tested bacterial pathogens were subjected to determine the MIC using broth microdilution procedure according to CLSI protocol. Briefly, a predetermined quantity, 10 mg of each compound and standard drug was solubilized in 1 mL of dimethyl sulphoxide (DMSO) to create a stock solution. Ten successive dilutions were prepared with a concentration 10 times greater than the final solution of the targeted compounds. The dilutions were further diluted to 1:5 in Mueller Hinton broth medium and 100 µL aliquots were sequentially dispensed into the microdilution plates to obtain the desired concentrations ranging from 20 to 250 μg/mL. In comparison, the standard antibacterial drug was also prepared as same as the targeted compounds and determined its MIC value. After incubation period, 80 µL of each microbial cell was swapped onto nutrient agar plates. The determination of the minimum inhibitory concentration (MIC) for each targeted compound is defined as the lowest concentration of each sample that results in a minimum number of colony-forming units (CFUs) when compared to the untreated samples (Mohamed et al., 2023).

Briefly, 10 mg of each selected compound and reference drugs were dissolved in 1 mL of dimethyl sulphoxide (DMSO) to prepare the stock solution. Ten successive dilutions were prepared with a concentration 10 times greater than the final solution of the synthesized compounds. The dilutions were further diluted to 1:5 in PDB and 100 µL aliquots were sequentially dispensed into the microdilution plates to get the desired concentrations ranging from10–400 μg/mL. Each tested compound was incubated with the tested fungal strain for 48 h at 28 °C. The PDB mixed with DMSO was served as the control growth samples. After that, inoculated of each treated sample with 50 µL to Potato dextrose agar plate and incubated for 72 h at 28 ^oC

Reactive Oxygen Species is one of the assumed mechanisms that may responsible for the antibacterial activity of the Sulfa drugs. ROS are known as a group of highly reactive molecules like molecular oxygen (O₂), superoxide anion (O₂ ^•), hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^•) having an ability to induced an oxidative stress onto the bacterial cells. Therefore, the susceptibility of the potent compounds to induced ROS toward the microbial pathogens was assessed intracellularly. In this regard, ROS could be quantifying using a fluorescent prop 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, the treated microbial samples were subjected to permeabilization process by ultrasonciation of cells and the resultant supernatant was harvested and reacted with 10 μM DCFH-DA for 30 min at 37 °C (Farid et al., 2024). At the same time, the microbial cells were treated with hydrogen peroxide H₂O₂ at 155 μM as a positive control. Immediately, the DCF fluorescence intensity was measured by Spectrofluorometric apparatus (JASCO FP-6500, light source Xenon arc lamp, Japan). The fluorescence intensity for each sample was measured with an excitation and emission wavelength at 485 nm and 530 nm, respectively.

A treated bacterial cell using the most potent molecules was subjected to investigate the LPO process. Utilizing the LPO colorimetric kit assay, a particular reagent called thiobarbituric acid (TBA) was utilized to indicate the fatty acid peroxidation byproduct, malondialdehyde (MDA), producing a pink color complex known as MDA-TBA (Mallique Qader et al., 2021). In this study, a consistent concentration of each potent molecule (20 μg/mL) was employed to evaluate the level of oxidation occurring in the lipids of the bacterial cell membrane. To assess lipid peroxidation (LPO), 1 mL of each treated bacterial pathogen was combined with 300 μL of malondialdehyde (MDA) lysis buffer at 4 °C. Subsequently, 3 μL of Butylated Hydroxytoluene (BHT) was added to mitigate interference from pigments generated by the decomposition of lipophilic peroxides. Each sample was then subjected to centrifugation at 8,000 rpm for 10 min, after which the insoluble materials were discarded. A volume of 200 μL of the clear supernatant was mixed with 600 μL of thiobarbituric acid (TBA) solution and incubated at 95 °C for 60 min. Following incubation, the samples were cooled to 25 °C, and the resultant pink color was quantified using a spectrophotometer (Agilent Cary 100, Germany) at a wavelength of 532 nm. Treatment of both bacterial pathogens using 5% hydrogen peroxide for 20 min as a positive control was also involved. The increase in the malondialdehyde concentration in the tested sample indicated the increase of the lipid peroxidation activity of SL towards bacterial cell membrane, malondialdehyde concentration was calculated based on the following equation: Malondialdehyde nmol / mL = A  sample / A  standard × 10 …

Where A sample is the lipid peroxidation absorbance in the treated bacterial cells and A standard is the absorbance of the standard lipid peroxidation sample.

The in vitro assessment of enzyme inhibition for the most active derivative 6a and 6c was performed against E. coli DNA gyrase B. The anticipated assay was done using E. coli DNA gyrase microplate assay kit (Inspiralis) according to the optimized protocol of the manufacturer. Ciprofloxacin was used as a reference drug for E. coli DNA gyrase B according to the previously reported methods (Fayed et al., 2022). The obtained data are tabulated as IC₅₀ values in Table 6.

Molecular docking studies were conducted to evaluate the potential binding affinity of the compounds 6a and 6c towards DNA Gyrase B. the tested compounds were docked against DNA Gyrase B obtained from Protein Data Bank (PDB ID: 4duh) (Brvar et al., 2012).

Initially, water molecules and extraneous components were eliminated from the protein complex. Subsequently, crystallographic disorders and unoccupied valence atoms were rectified. The energy of the protein structure was minimized and subsequently saved in a PDBQT file format. The two-dimensional structures of each compound were constructed utilizing Chem-Bio Draw Ultra 16.0, saved as SDF files, and subsequently converted into three-dimensional structures. Protonation and energy minimization were performed, with the results also saved as PDBQT files. The docking processes were executed using AutoDock Vina version 1.5.7, employing a rigid docking technique in which the receptor remained fixed while the ligands were permitted to adopt flexible conformations. Furthermore, each molecule was allowed to generate twenty distinct poses. The docking scores, representing the affinity energy of the most favorable poses in relation to the target protein, were documented, and both three-dimensional and two-dimensional representations were produced using Discovery Studio 2024 for visualization purposes (El-Demerdash et al., 2024).

The Desmond simulation package developed by Schrödinger LLC was employed to perform molecular dynamics (MD) simulations (Kumar et al., 2022). All simulations were conducted using the NPT ensemble, maintaining a temperature of 300 K and a pressure of 1 bar throughout the experimental runs. Each simulation was executed for a duration of 100 ns, with a relaxation time of 1 ps allocated for the ligands under investigation. The OPLS_2005 force field parameters were utilized for all simulations. Long-range electrostatic interactions were calculated using the particle mesh Ewald method, with a cutoff radius of 9.0 Å designated for Coulomb interactions (Ivanova et al., 2018).

Water molecules were represented explicitly through the simple point charge model. Pressure regulation was achieved using the Martyna–Tuckerman–Klein chain coupling scheme, characterized by a coupling constant of 2.0 ps, while temperature regulation was conducted via the Nosé–Hoover chain coupling scheme. Nonbonded interactions were computed utilizing the r-RESPA integrator, wherein short-range forces were updated at every time step and long-range forces were updated every three-time steps. Trajectories were recorded at intervals of 4.8 ps for further analysis.

The interactions and behavior of ligands and proteins were analyzed using the Simulation Interaction Diagram tool available in the Desmond molecular dynamics (MD) package. The stability of the MD simulations was evaluated by tracking the root mean square deviation (RMSD) of the positions of ligand and protein atoms over time (Case et al., 2008).