Using commercially available ethyl 4-chlorobenzoate (A) as the starting material and referring to literature methods (Liu et al., 2025; Tan et al., 2022), a series of N′-substituted methylene-4-chlorobenzohydrazide derivatives (C1–C16) were successfully designed and synthesized. The general synthetic route is illustrated in Scheme 1. Among the synthesized compounds, C1 and C16 are novel structures that have not been previously reported in the literature. The remaining compounds (C2–C15) are known and have been reported previously (Abdel-Aziz et al., 2021; Al-Wahaibi et al., 2024; Khan et al., 2018). The synthesis of the key intermediate, 4-chlorobenzohydrazide (B), was achieved via an efficient ester aminolysis reaction. Specifically, compound A was heated under reflux with an excess of hydrazine monohydrate in ethanol. The reaction progress was monitored dynamically by thin-layer chromatography (TLC). Upon completion, the mixture was cooled to room temperature, resulting in the quantitative precipitation of intermediate B as a high-purity white solid. Simple filtration, washing with cold ethanol, and vacuum drying afforded pure product suitable for the subsequent step without the need for tedious column chromatography. This method is straightforward, provides high yield (86%), and demonstrates practicality and cost-effectiveness for large-scale preparation. After obtaining key intermediate B, the characteristic bioactive pharmacophore–the hydrazone bond (-NHN = CH-) – of the target molecules was successfully constructed via condensation reactions with 16 structurally diverse aldehydes. This step was performed under reflux in anhydrous ethanol. Leveraging the inherent strong nucleophilicity of the hydrazide, the reactions proceeded efficiently and reached completion within 8 h, as confirmed by TLC. After cooling the reaction mixtures, most target compounds (C1–C16) precipitated directly and were obtained as high-purity samples meeting analytical requirements through recrystallization (ethanol/water system). The chemical structures of all final compounds were unambiguously confirmed using modern spectroscopic techniques, including ¹H NMR, ¹³C NMR, and High-Resolution Mass Spectrometry (HRMS). In the ¹H NMR spectra of all target compounds, the characteristic imine proton signal adjacent to the hydrazone bond was observed as a sharp singlet or broad peak in the range of δ 7.60–8.40 ppm, providing definitive evidence for the formation of the hydrazone structure. Concurrently, the -NH- proton signal adjacent to the carbonyl group in the hydrazide fragment was also clearly identifiable, typically appearing in the downfield region of δ 10.20–11.20 ppm. Furthermore, HRMS analysis provided precise molecular weight evidence for all target molecules. The detected molecular ion peaks [M + H]⁺ matched the theoretically calculated values, offering indisputable confirmation of the correct molecular formulas at the mass spectrometry level.

According to literature reports, certain Schiff base derivatives exhibit promising in vitro antibacterial activity (Chung., 2022; Sindelo et al., 2023). Consequently, this study initially determined the in vitro antibacterial activity (Minimum Inhibitory Concentration, MIC) of all synthesized compounds against the following strains via the broth microdilution method: Gram-positive bacteria: Staphylococcus aureus ATCC 29213, S. aureus ATCC 43300, Methicillin-resistant S. aureus MRSA2; Gram-negative bacteria: Escherichia coli ATCC 25922 and Salmonella enterica subsp. enterica SM012, with the results summarized in Table 1. Among the tested compounds, all were ineffective against the Gram-negative strains; notably, compound C12 (MIC = 8 μg/mL) exhibited inhibitory activity against all tested S. aureus strains, while other salicylaldehyde derivatives (C13-C15) also demonstrated anti-Gram-positive activity capable of inhibiting all tested S. aureus strains, whereas the remaining compounds showed weaker antibacterial activity, potentially attributable to their overall poor solubility. The distinct activity profile observed among the synthesized derivatives reveals a clear structure-activity relationship. The emergence of antibacterial activity specifically in the salicylaldehyde-derived compounds (C12–C15) strongly indicates that the ortho-hydroxy substituent on the aromatic aldehyde moiety is a critical pharmacophore for anti-staphylococcal activity. This functional group is known to enhance biological activity through mechanisms such as facilitating intramolecular hydrogen bonding (improving stability) or enabling metal chelation. The superior potency of C12, compared to C13-C15, suggests that its unique substituent (R group) optimally balances molecular properties like lipophilicity and steric bulk, allowing for more effective interaction with the bacterial target. Conversely, the inactivity of compounds C1-C11 and C16 against all tested strains implies that their respective aldehyde components lack the essential structural features required for effective antibacterial action, which may be linked to inadequate target binding or poor cellular penetration. Furthermore, the consistent inactivity against Gram-negative bacteria across the entire series underscores a common limitation, likely attributable to the impermeable outer membrane of these bacteria, which prevents the compounds from reaching their intracellular target site. We subsequently evaluated C12 against additional Gram-positive strains, including clinical isolates LN38 and LN51, as well as Bacillus subtilis ATCC 6633, Bacillus cereus CMCC 63303, Listeria monocytogenes CICC 21662, and Enterococcus faecalis ATCC 29212; as shown in Table 2, C12 maintained good antibacterial activity against these diverse strains.

To systematically evaluate the biosafety of the lead compound, this study first conducted a comprehensive assessment of the hemolytic activity of compound C12, which exhibited significant in vitro antibacterial potency (Greco., 2020; Kindrachuk et al., 2011). In the standard hemolysis assay, 1% Triton X-100 solution and sterile PBS buffer were used as positive and negative controls, respectively. As shown in Figure 2A, compound C12 did not induce any visible hemolysis across a broad concentration range (13–832 μM), indicating no significant disruptive effect on the membrane integrity of rabbit red blood cells. Notably, its effective antibacterial concentration (≤104 μM) was substantially lower than any concentration observed to initiate hemolysis, demonstrating excellent antibacterial specificity and favorable hemocompatibility. Building upon these findings, and to further investigate the potential toxicity of C12 towards mammalian cells, this study employed African green monkey kidney epithelial cells (VERO) as a normal cell model. The impact of the compound on cellular metabolic activity was determined using the CCK-8 assay. Results presented in Figure 2B showed that even after 24-h treatment at a high concentration of 832 μM, cells in the C12-treated group maintained normal metabolic levels, with no statistically significant metabolic inhibition compared to the negative control group. In conclusion, while demonstrating specific antibacterial activity, compound C12 exhibited no significant toxicity towards mammalian cells or blood components. These results preliminarily confirm its favorable biosafety profile and development potential as an antibacterial candidate, providing crucial toxicological evidence for subsequent in vivo pharmacodynamic evaluation and structural optimization.

To systematically evaluate the antibacterial properties of compound C12, this study focused on its bactericidal kinetics against Methicillin-resistant S. aureus MRSA2 and the potential risk of inducing resistance (Bhattacharya et al., 2022). As a common pathogen prone to developing resistance, evaluating the bactericidal efficacy and resistance control of S. aureus is crucial for developing novel antimicrobial agents. In the time-kill kinetics assay, bacterial colony counts were determined at various time points to assess the immediate bactericidal effect of C12, using dimethyl sulfoxide (DMSO) as a negative control. As shown in Figure 3A, C12 at 4 × MIC (4 × the minimum inhibitory concentration) completely inhibited the growth of MRSA2 after 16 h of incubation, while at a higher concentration of 8 × MIC, it achieved complete growth inhibition within 6 h. Furthermore, given that Schiff base compounds typically exhibit multi-target mechanisms of action and membrane-disrupting effects, such structures are generally considered less likely to induce bacterial resistance. To further evaluate its capacity to induce resistance, we conducted a serial passage experiment for 21 generations. The results (Figure 3B) demonstrated that the MIC of C12 against MRSA2 increased by no more than 8-fold, accompanied by a low spontaneous resistance frequency, suggesting a potentially high resistance barrier for this compound in vivo. In summary, C12 exhibits rapid and potent antibacterial activity at effective concentrations while significantly reducing the probability of resistant mutant development. These findings indicate that this Schiff base derivative, leveraging its multi-mechanistic action, holds considerable promise for development against drug-resistant Gram-positive bacterial infections.

Over 80% of human chronic bacterial infections are associated with biofilm formation. Biofilms are structured bacterial communities encased in a protective extracellular polymeric matrix, which significantly enhances their tolerance to antimicrobials and host immune defenses. These resilient infections, often associated with medical devices, chronic wounds, and cystic fibrosis lungs, are persistent and notoriously difficult to eradicate. This study evaluated the effect of compound C12 against biofilm formation in S. aureus ATCC 43300 and MRSA2 (Table 3). Quantitative analysis using a crystal violet assay demonstrated that C12 inhibited biofilm formation at 4 × MIC for each strain: 52 µM for S. aureus ATCC 43300 and 104 µM for MRSA2. These results confirm the potential of C12 in effectively preventing staphylococcal biofilm formation.

To systematically evaluate the drug development potential of the synthesized Schiff base derivative C12, key pharmaceutical properties were assessed following the demonstration of its significant anti-Gram-positive activity, low systemic toxicity, unique membrane-targeting mechanism, and anti-biofilm capability. As summarized in Table 4, C12 exhibits a high human plasma protein binding (PPB) rate of 88.5%, a logD_7.4 value of 3.47 ± 0.15 indicating moderate lipophilicity, and intermediate metabolic stability in liver microsomes (T₁/₂ = 50.72 min; CL = 4.87 μL/min/mg). These properties suggest favorable membrane penetration and acceptable in vivo residence time, counterbalanced by potentially limited free drug concentration and aqueous solubility, thus providing clear guidance for subsequent structural optimization and formulation strategies.

To explore the potential molecular target of compound C12, we performed molecular docking studies. The PBP2a (PDB ID: 1VQQ), which is closely associated with bacterial membrane integrity, was selected as the receptor (Lang et al., 2025). The results showed that C12 could stably bind within the active pocket of this protein, with a calculated binding free energy (ΔG) of −5.8 kcal/mol. Specifically, the hydroxyl group of C12 forms a hydrogen bond with residue His291, while its hydrophobic backbone is accommodated within a hydrophobic pocket (Figure 7). This suggests that C12 may interfere with the normal function of the protein through competitive inhibition or allosteric effects, thereby contributing to the observed membrane damage, which is consistent with our phenotypic results.

During the 200 ns production phase, the protein backbone RMSD entered a stable plateau after an initial relaxation period, exhibiting only minor overall fluctuations (Figure 8A). By the end of the simulation (200 ns), the backbone RMSD was maintained within a range of 0.25–0.37 nm (approximately 0.25 nm at the terminus), indicating that the main chain conformation remained stable throughout the production run. Residue-wise RMSF analysis revealed low fluctuations for most residues, with higher fluctuations concentrated in a few flexible regions (Figure 8B), consistent with the expected local flexibility profile of the protein. Concurrently, the radius of gyration (Rg) of the protein showed no sustained drift during the entire simulation, fluctuating only slightly around its mean value (Figure 8C). The solvent accessible surface area (SASA) also displayed stable oscillations (Figure 8D). The consistent results from RMSD, RMSF, Rg, and SASA collectively support that the overall protein fold and compactness remained stable over the 200 ns timescale, with no signs of significant abnormal collapse or unfolding.

To more directly assess whether ligand “dissociation/diffusion” occurred, we further analyzed geometric and contact-based evidence. The minimum distance between the ligand and protein remained within a low range throughout the simulation, stabilizing at 0.09–0.13 nm (around 0.11 nm) by the end (Figure 8E), indicating the ligand persistently resided in the vicinity of the binding pocket. Consistent with this, the number of ligand-protein contacts within a 0.35 nm threshold remained non-zero for the duration of the simulation (Figure 8F), suggesting the ligand maintained close contact with pocket residues rather than diffusing away.

Hydrogen bond analysis showed that 1–2 hydrogen bond events were observable during the early phase of the simulation. However, this number gradually decreased and approached zero in the later stages (Figure 8G). Notably, while the hydrogen bond count decreased, the corresponding minimum distance and contact number did not show a synchronous decline (Figures 8E,F). This suggests that the system likely underwent a dynamic rearrangement of the binding mode: the initial recognition, primarily driven by hydrogen bonding, gradually evolved into a stable binding state dominated by hydrophobic/van der Waals contacts (and possibly other non-bonded interactions such as aromatic stacking). The ligand RMSD showed considerable variation over time (reaching 1.8–2.1 nm at the terminus; Figure 8H). This metric is sensitive to the choice of reference conformation and fitting procedures and can increase significantly during “in-pocket reorientation/conformational rearrangement.” Therefore, it must be interpreted in conjunction with the minimum distance and contact data. Synthesizing the direct evidence from Figures 8E,F, the ligand maintained stable binding over the 200 ns timescale, accompanied by an evolution of its interaction network from hydrogen-bond-dominated to non-polar-contact-dominated.

All commercially available chemicals and reagents were purchased from Adamas Biochemical Co., Ltd. (Shanghai, China) and were used as received without further purification. Solvents were of analytical grade and were employed directly or dried over activated 4Å molecular sieves when anhydrous conditions were required. Thin-layer chromatography (TLC) analyses were performed on silica gel GF254 pre-coated plates (Yantai Jiangyou Silicone Development Co., Ltd.) to monitor reaction progress. Visualization was achieved under ultraviolet light (254 nm). Nuclear Magnetic Resonance (NMR) spectra, including ¹H (400 MHz) and ¹³C (100 MHz), were acquired on a Bruker Avance 400 spectrometer at ambient temperature. Chemical shifts (δ) are reported in parts per million (ppm) and are referenced to the residual solvent signals of DMSO-d6 (δH 2.50 ppm, δC 39.5 ppm). High-Resolution Mass Spectrometry (HRMS) data were obtained using an AB Sciex TripleTOF 5,600+ mass spectrometer equipped with an electrospray ionization (ESI) source.

For detailed procedures, refer to the Supplementary Material (Chung et al., 2021; Sindelo et al., 2023).

For detailed procedures, refer to the Supplementary Material (Kong et al., 2023).

For detailed procedures, refer to the Supplementary Material (Kong et al., 2023).

For detailed procedures, refer to the Supplementary Material (Tian et al., 2025).

For detailed procedures, refer to the Supplementary Material (Tian et al., 2025).

For detailed procedures, refer to the Supplementary Material (Liu et al., 2025).

For detailed procedures, refer to the Supplementary Material (Kong et al., 2023; Xu et al., 2025).

For detailed procedures, refer to the Supplementary Material (Xu et al., 2025; Tian et al., 2025).

For detailed procedures, refer to the Supplementary Material (Xu et al., 2025; Tian et al., 2025).

For detailed procedures, refer to the Supplementary Material (Xu et al., 2025; Tian et al., 2025).

For detailed procedures, refer to the Supplementary Material (Ueda et al., 2025; Wu et al., 2012).

For detailed procedures, refer to the Supplementary Material (Andrés et al., 2015; Slavik et al., 2015).

For detailed procedures, refer to the Supplementary Material (Knights et al., 2016; Liu et al., 2020).

For detailed procedures, refer to the Supplementary Material (Lang et al., 2025).

For detailed procedures, refer to the Supplementary Material (Fang et al., 2020).

Data are presented as the mean ± SEM from at least three independent experiments. Statistical significance was assessed by one-way analysis of variance (ANOVA) using SPSS software (version 21.0).