Effects of changing ions on the crystal design, non-covalent interactions, antimicrobial activity, and molecular docking of Cu(II) complexes with a pyridoxal-hydrazone ligand

The present work reports the influence of the presence of different ions (Cl−, Br−, NO3 −, or SO4 2−) on the formation and proprieties of Cu(II) complexes with pyridoxal-benzoylhydrazone (PLBHZ). Four new complexes were successfully synthesized, [CuCl2(PLBHZ)] (1), [CuBr2(PLBHZ)] (2), [CuCl(PLBHZ)H2O]⋅NO3⋅H2O (3), and [CuSO4(PLBHZ)H2O]⋅3H2O (4), and characterized by spectroscopic and physicochemical methods. A single-crystal X-ray study reveals the Schiff base coordinated to the metal center tridentate by the ONS-donor system, resulting in distorted square pyramidal coordination geometries. Noncovalent interactions were investigated by 3D Hirshfeld surface analysis by the d norm function, 2D fingerprint plots, and full interaction maps. The ion exchange is important in forming three-dimensional networks with π⋅⋅⋅π stacking interactions and intermolecular hydrogen bonds. The in vitro biological activity of the free ligand and metal complexes was evaluated against Gram-positive and Gram-negative bacterial strains and the free pyridoxal-hydrazone ligand showed higher activity than their Cu(II) complexes. Molecular docking was used to predict the inhibitory activity of the ligand and complexes against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria.


Introduction
B vitamins are essential for human metabolism and their lack can result in a wide range of health problems.Pyridoxal is among the six water-soluble vitamins of vitamin B 6 that can be used for cell or bioimaging and anticancer applications (Gupta, 2022;Karthick et al., 2023).Pyridoxal and their metal complexes have been found useful in catalytic reactions and biological activities (Askarian et al., 2021;Ma et al., 2022;Dherbassy et al., 2023).The aldehyde group of pyridoxal can form through condensation reaction a variety of Schiff bases (Casas et al., 2012).These Schiff bases can offer diverse coordination possibilities with several transition metals (Casas et al., 2012;Askarian et al., 2021;Murašková et al., 2021;Poladian et al., 2021;Gamov et al., 2023).Their effectiveness is increased when coordinated with specific metals since the charges of the molecules are stabilized due to the binding of the metal ion with the ligand (Gatto et al., 2019;Gan et al., 2024).Pyridoxal-based ligands also exhibit antitumor activity, particularly those resulting from the reaction with semicarbazide or thiosemicarbazide (Qi et al., 2022;Jevtovic et al., 2023).
Hydrazones belong to a class of organic compounds that possess an azomethine-NHN=CR2 group showing wide application and biological properties due to their structural versatility and chelating capability (Sharma et al., 2020;Yan et al., 2024).Their wide popularity also is due to their straightforward synthesis from the condensation of hydrazides or hydrazines with aldehydes and ketones.Moreover, the attractiveness of hydrazone-based ligands is their stability and flexibility of coordination, evidenced in medicinal chemistry and enriched with pharmacological potential-including antibacterial, antifungal, antioxidant, and antitumor activity (Casas et al., 2012;Liu et al., 2022;Arora et al., 2023;Yuan et al., 2023).Pyridoxal-hydrazone compounds have many possible applications, such as promising candidates for the future development of antitubercular drugs (Shtyrlin et al., 2021), chemosensors for aluminum detection (Kejík et al., 2016), and iron-chelating agents for anticancer therapy (Sarno et al., 2018).Additionally, metal complexes with Schiff bases, particularly Cu(II) complexes, are important in the development of new compounds with applications such as coordination chemistry, industries, medicine, and biological activities (Soroceanu and Bargan, 2022;Boulechfar et al., 2023;Li et al., 2024).Studies have reported these complexesas having great antibacterial activity (Salehi et al., 2016;Fekri et al., 2019;Xu et al., 2020;Omidinia et al., 2022).
The antibiotic drug resistance generated in the treatment of bacterial infections in hospitals is critical in the cure of diseases caused by microorganisms (Marrero et al., 2006;Ziervogel and Roux, 2013).One of these resistances is the Gram-positive methicillin-resistant Staphylococcus aureus strain, which presents strong resistance to most beta-lactam antibiotics.This profile is determined for the biosynthesis of the penicillin-binding protein that molecularly decreases the affinity for beta-lactam drugs.The extracellular antibiotic-sensor domain of S. aureus (MecR1) is a serine protease-like enzyme responsible for the signal-transduction system related to antibiotic resistance.Once this enzymatic machinery is inhibited, the resistance of S. aureus to known antibiotic drugs is reduced (Marrero et al., 2006).
Another important pathway in antibiotic resistance for Gramnegative Escherichia coli and Salmonella typhimurium is the reduction of the permeability of their outer membrane (Ziervogel and Roux, 2013).This membrane has a barrier function that selects outside harmful agents, molecules, and compounds.E. coli produce porin channels that are responsible for cell diffusion (OmpF).When this porin channel develops mutations in key residues, the biological response is the modification of the cellular antibiotic levels which reduces their activity and inactivity.The development of new molecules for antibiotic therapy strategies is urgent and is vital for solving the problem of bacteria resistance to known antibiotic drugs (Ziervogel and Roux, 2013).
Due to our structural and biological interests in the study of new metal complexes based on hydrazone (Santiago et al., 2020;Santiago et al., 2022), we here study the reactions and resulting crystal structures between pyridoxal-benzoylhydrazone ligand and different Cu(II) salts.The compounds were analyzed by singlecrystal X-ray diffraction, mass spectrometry, FT-IR, UV-Vis, and molecular docking.The influence of ion exchange on the structure and noncovalent interactions was investigated by 3D Hirshfeld surface analysis using the d norm function, 2D fingerprint plots, and full interaction maps.Furthermore, biological activity was evaluated against Gram-positive S. aureus and Gram-negative E. coli bacterial strains.The results of this study of antibacterial activity show an interesting comparison between the free ligand and the Cu(II) complexes with the changing ions.

Materials, methods, and instruments
All reagents and solvents used were obtained from commercial sources (Merck) and used as received.Elemental analyses were performed with a Perkin Elmer/Series II 2400 analyzer.Melting point was determined using the Micro Chemistry MQAPF-302 digital instrument.For comparison purposes, a flame atomic absorption spectrometer (AAS), Model PSD 120, operating with an acetylene/air flame, was used to determine the Cu(II) content in each complex.The lamp used was of the multi-element type Co/Cr/Cu/Fe/Mn/Ni, and each complex sample was made at a concentration of 3 mg L −1 .Infrared spectra in ATR were recorded from KBr pellets (4,000-400 cm −1 ) using FT-IR Varian 640.A UV-Vis-NIR Varian Cary 5000 spectrophotometer was used, and the concentration used for all analyses was 2 × 10 −5 M in methanol (MeOH) and N, N-dimethylformamide (DMF). 1 H NMR and 13 C NMR analyses were conducted using an NMR YH 600 Oxford.The ESI-MS and ESI-MS/MS spectra were obtained from AB Sciex TripleTOF 5600+ spectrometer equipment in positive mode, 5,500 V, and 200 °C.Electrospray ionization mass spectrometry (ESI-MS) analysis was performed on an AB Sciex Triple TOF 5600+ mass spectrometer in positive mode, with a voltage of 5500 V and source temperature of 200 °C.

Synthesis of pyridoxalbenzoylhydrazone hydrochloride monohydrated (PLBHZ)
The synthesis of PLBHZ was based on the literature (Back et al., 2009).The mixture of 3 mmol of pyridoxal hydrochloride and 3 mmol of benzoylhydrazide with 15 mL of ethanol was refluxed and heated for 2 h.A yellow solid product was obtained and filtered off the mother solution.Yield: 89.10%.Melting point: 197

Crystal structure determinations
X-ray diffraction data were collected at room temperature (296 K) on a Bruker CCD SMART APEX II single crystal diffractometer with Mo Kα radiation (0.71073 Å).The structures were solved using the Olex2 program (Dolomanov et al., 2009) directly with SHELXS (Sheldrick, 2008); subsequent Fourier-difference map analyses yielded the positions of the nonhydrogen atoms.The refinement was performed using SHELXL (Sheldrick, 2015).The empirical absorption corrections were applied by the SADABS program (Sheldrick, 1997).All refinements were made by full-matrix least-squares on F 2 with anisotropic displacement parameters for all non-hydrogen atoms.Hydrogen atoms were included in the refinement in calculated positions, but those performing special bonds were located in the Fourier map.Molecular graphics were generated via Olex2 and MERCURY software (Dolomanov et al., 2009;Macrae et al., 2020).Crystal data, experimental details, and refinement results are summarized in Table 1.

Computational details
CrystalExplorer 21.5 software (Spackman et al., 2021) was used to calculate the Hirshfeld surfaces (HS) and related 2D fingerprint plots, using the crystallographic information files (CIFs) obtained by single-crystal X-ray.A fixed color scale of −0.4740 (red) to 2.6654 (blue) was used to map the 3D d norm surfaces (normalized contact distances) for the ligands and complexes.A 2D fingerprint plot of d i versus d e was used to identify and quantify in percentages the intermolecular interactions of the compounds.The full interaction maps were produced using MERCURY software (Macrae et al., 2020).The interaction maps are indicative of hydrogen bond acceptors (red), donors (blue), and hydrophobic interactions (brown), respectively.

Biological activity
Antibacterial tests were performed by the microdilution broth method in 96-well microplates, in triplicate, using bacterial strains obtained from the American Type Culture Collection (ATCC): Gram-positive S. aureus ATCC 29213 and Gram-negative E. coli ATCC 25922 bacterial.Both strains were grown in Müller-Hinton Broth (MHB) at 37 °C overnight, resulting in approximately 5 × 10 8 colony forming units (CFU) per mL according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Patel et al., 2015).The positive control was oxacillin for S. aureus at concentration 1 μg mL −1 and ampicillin at concentration 6 μg mL −1 for E. coli.Minimal inhibitory concentrations (MICs) of 0.1 and 99.9 were determined by the broth microdilution method, according to CLSI (Patel et al., 2015).

Molecular docking
Molecular docking is a computational chemistry tool used on a large-scale system to theoretically predict the most favorable orientation of the ligand when interacting with a given target protein.Molecular docking empirically determines the interaction energy and predicts the inhibition constant of the receptor-ligand complex (R-L).Knowing the energetically most favorable orientation of a ligand is an important step in predicting the binding affinity of the R-L system.The score functions are applied to determine the energies involved in the activation or inhibition process of a given biochemical target (Morris et al., 2009;Nascimento et al., 2017).
In this work, two rigid-flexible docking studies (receptor-ligand) were carried out to elucidate a possible interaction path between enzymatic bioreceptors of Gram-positive (S. aureus) and Gramnegative (E.coli) bacteria and the PLBHZ molecules, their metal complexes (1-4), and the classic inhibitors ampicillin (to E. coli) and oxacillin (to S. aureus).For the docking study, the AutoDockTools 4 package (Morris et al., 2009) was used with the AutoDock Vina docking algorithm (Trott and Olson, 2010).The enzymatic systems used to evaluate the effectiveness of inhibition of the PLBHZ ligand The crystallographic space group is a set of geometrical symmetry operations that take a three-dimensional periodic object (i.e., the crystal) into itself according to the International Table of Crystallography.
and their Cu(II) complexes were (a) the methicillin resistance protein MecR1, which is an extracellular penicillin-sensor domain of S. aureus, whose crystallographic structure is deposited in the Protein Data Bank under the code 2IWD (Marrero et al., 2006), complexed with oxacilloyl and with a resolution of 2.40 Å; (b) the outer membrane protein porin of E. coli (OmpF/A), with crystallographic structure deposited in the PDB under the code 4GCP, complexed with ampicillin at 1.98 Å resolution.
For the molecular docking protocol, the systems were prepared as follows: i) 3D molecular structures: The bioreceptors were obtained by searching crystallographic data deposited in the PDB.The protein structures were extracted directly from the PDB under the codes 2IWD and 4GCP.Sequentially, only the residues of the protein structure were maintained, and the amino acid residues with the best crystallographic resolution were selected.The geometric structures of the classical inhibitors were extracted directly from the PDBs: oxacilloyl (PDB: 2IWD) and ampicillin (PDB: 4GCP).
The crystallographic structures of the PLBHZ ligand and their Cu(II) complexes were obtained directly from the crystallographic data carried out in this work.ii) Hydrogen atoms: Hydrogen atoms were added to proteins and small molecules using the standard AutoDock protocol.iii) Addition of charges: For proteins, the Kollman-type charges were addressed; for ligands, Gasteiger charges were used; for both, the standard protocol of AutoDockTools was followed.iv) Binding site setup: A grid box was built around the center of mass of each complexed classical inhibitor with the bioreceptor target.To study the structure of MecR1 (PDB: 2IWD), the dimensions of the grid box were 18.0 Å × 18.0 Å × 15.0 Å, with a spacing of 0.375 (points spacing).
The box was centered at the point (20.622,12.347, and 79.033).For the Ompf/A study (PDB: 4GCP), the dimensions of the grid box were 17.25 Å × 18.0 Å × 17.25 Å, with a spacing of 0.375 (points spacing) centered on the point (39.044,−26.679, and −18.162).These dimensions ensured that all ligands had an area of probable interactions suitable for their volume and the active site residues of the enzymes.v) Docking run: To perform this study, the AutoDock Vina algorithm was applied to search for a maximum of 1,000 solutions at an exhaustiveness level of 32.vi) The score obtained through the score function of the program relates the negative score value directly to the binding energy, which is given in kcal.mol−1 .This value represents the highest interaction affinity between a given ligand, in the most appropriate conformation and the proteomic target of interest.vii) For both enzymatic targets, redocking studies were performed to validate the proposed docking protocols above.For MecR1 and its oxacilloyl ligand, the value of the RMSD was 0.86 Å, and the binding energy (score) was −6.90 kcal mol −1 .In the case of OmpF/A and its ampicillin ligand, the RMSD value was 0.36 Å and the binding energy (score) was −6.90 kcal mol −1 .The values of the RMSD, under 2.0 Å for both redocking studies, validate our docking protocol properly.viii) Hydrogen and van der Waals interactions are fundamental for carrying out biochemical processes.In these studies, the hydrogen interactions were considered with the maximum distance between a hydrogen atom and a carbon atom or heteroatoms of 3.4 Å.For medium-and long-range (van der Waals) interactions, such as π•••π stacking, π-sigma, π-alkyl, and π-sulfur interactions, interactions with a maximum distance of 6.0 Å between the ligand were taken into account.To visualize the results and the main distances between the ligand atoms and the amino acid, residue of the active site Discovery Studio Visualizer software was used (Biovia and Release, 2020).

Results and discussion
Pyridoxal-benzohylhydrazone ligand (PLBHZ) was synthesized by the condensation reaction of pyridoxal hydrochloride with benzohylhydrazine.The reactions were carried out in ethanol with a catalytic amount of acetic acid to ensure the formation of the azomethine and the separation of the product from the mixture.Their Cu(II) complexes were obtained from the reactions between the PLBHZ ligand and different Cu(II) salts (Figure 1).The compounds were characterized by physicochemical and spectroscopic methods.The crystal structures of the complexes were established by single-crystal X-ray diffraction.

Structural analyses
The single-crystal structural analysis revealed isoelectronic structures of the Cu(II) complexes (1) and (2) (Figure 2).The metal centers are found in distorted square pyramidal environments and coordinated by the phenolic oxygen atom, the nitrogen atom of the azomethine group, and the oxygen atom of the ketone group of ligand molecules.Two chloride or bromide ions from the starting metal salt fulfill the coordination sphere.The bond length Cu-Cl of 2.238 (2) and 2.585 (2) Å in (1) and Cu-Br of 2.332 (2) and 2.699 (2) Å in (2) are in agreement with the range found for Cu(II) complexes (Vidovic et al., 2011;Gatto et al., 2019).The apical bond copper-halogen is longer than the axial bond due to the repulsion along the z-axis with the presence of two electrons in the dz (Gupta, 2022) orbitals.The data show that the ligand was coordinated to the Cu(II) atom by the isomer E and keto tautomer, with bond lengths C9-O3 of 1.259 (4) Å and 1.235 (9) Å and C9-N3 of 1.340 (4) Å and 1.351 (9) Å, respective of (1) and (2).
The Cu(II) atoms in both complexes (3) and ( 4) show square pyramidal distorted geometry, with a water molecule and sulfate ion in the apical position of the pyramid, and Addison's parameters of 0.262 and 0.045, respectively (Addison et al., 1984).The coordination mode of these complexes is similar to the two previous examples by the ONO-donor system.In the case of complex (3), there is, in addition, the coordination of a chloride ion and a water molecule, and the asymmetric unit shows a nitrate ion and a water molecule.In the case of complex (4), coordination of a sulfate ion and a water molecule occurs, and there are three water molecules in the asymmetric unit.Figure 3 shows the ORTEP representations of the structures of complexes (3) and ( 4).
Additionally, it is important to note that the substitution of the Cu(II) salt produces similar crystal structures with the same ligand coordination mode and with the metal center presenting a square pyramidal geometry.In complexes (1), (2), and (3), the Cu(II) ion is coordinated with Cl − , Br − , and SO 4 − , respectively, from the starting material.However, it was observed when Cu(NO 3 ) 2 was used that the NO 3 − ion is not coordinated with the metal atom and there is a chloride ion coordinated with the free ligand.
It is noteworthy that, in all complexes, the ligand is in zwitterionic form with the protonation of the N1 nitrogen pyridine ring and deprotonation of the O2 atom, as reported in the literature for other similar metal complexes with pyridoxal ligands (Jevtovic et al., 2023;Jevtovic et al., 2022).The value found between 123.7 (7) and 125.3 (2) °for the bond angle C3-N1-C4 indicates the presence of a proton on the N1 atom, as this angle has been observed for deprotonated forms of similar ligands with the value of about 118 ° (Jevtovic et al., 2022).Comparing the free ligand PLBHZ (Back et al., 2009) with complexes (1-4), the bond distances found N2-N3 between 1.370  Molecular structure of (1) and ( 2) with crystallographic labeling (50% probability displacement).
The abundant non-covalent interactions observed, such as π•••π stacking interactions and hydrogen bonds, are vital to connecting adjacent molecules to form supramolecular structures (Man et al., 2023).The different ions present in the unit cell play an important role in the formation of the intermolecular interactions and are essential for the formation and assembly of the complex.X-ray analysis data of all complexes show the presence of a π•••π stacking interaction between the pyridine rings and benzene groups with distances between the centroids of 3.685 and 3.825 (5) Å (Supplementary Figures S1-S4).In all complexes, due to the presence of protonated nitrogen atoms N1 and N3, water molecules, and different ions, it is possible to find many intra-and intermolecular interactions.Figure 4 shows the π•••π stacking interactions and hydrogen bonds in complex (1).Supplementary Table S1 lists the non-covalent interactions found in complexes (1-4).

Spectroscopy data
In order to certify the coordination modes of PLBHZ ligand in complexes (1-4), the vibrational infrared spectra of the free hydrazone and complexes were collected (Supplementary Figures S7-S11).The infrared spectrum of pyridoxal-benzohylhydrazone shows the ν(C=O) and ν(C=N) bands at 1,589 cm −1 , and after the coordination of Cu(II) atom with the hydrazone, they are shifted to lower wavelength values and decrease the strength of the connection.The bands of ν(O-H) at 3,349 cm -1 , ν(N-N) at 1053 cm −1 , and ν(N-H) at 2936 cm −1 are observed for the free ligand.The ν(N-H) vibration was observed as broadband between 2830 and 2760 cm −1 in the spectrum of complexes (1-4), characteristic of the vibrational mode ν(N-H) in pyridine groups, suggesting that the PLBHZ acts as a neutral ligand (Sharma et al., 2020).The band of ν(C=O) shows in the ligand at 1669 cm −1 ; a shift of this band to lower and weak wave number by 70-60 cm −1 suggests Molecular structure of ( 3) and ( 4) with crystallographic labeling (50% probability displacement).

Bond length (Å)
(1) (2) (3) (4) the coordination of the carbonyl group to the metal ion (Jevtovic et al., 2022).The same behavior is observed for the strong band of ν(C=N) at 1589 cm −1 ; a shift of this band to lower wave number by 50-40 cm −1 suggests the coordination of the hydrazone to the metal ion through the azomethine nitrogen (Jevtovic et al., 2023).It is possible to find the band of ν(C-N) in the spectra of the complexes between 1,100 and 1,000 cm −1 .For the complexes, bands in the region of 3,600 cm −1 are associated with the asymmetric and symmetric OH stretching vibrations of water molecules.In (3), the strong absorption at 1,380 cm −1 is relative to the nitrate in ionic form, according to previous data (Burgos-Lopez et al., 2019).The presence of ν(SO 4 2− ) in (4) at 1000 cm −1 confirms the monodentate coordination with the Cu(II) atom.Weak bands around 500 and 400 cm −1 that are not present in the spectra of the free ligand are assigned to ν(N1-Cu-O1), ν(Cu-N1), and ν(Cu-O2) modes in the complexes.The infrared analysis is according to similar complexes described previously and supports the crystal data analysis (Back et al., 2009;Gatto et al., 2019;Santiago et al., 2020).
It is possible to observe the absorption spectrum of free ligand bands corresponding to the π→π* transition of the azomethine group at around 285 nm in MeOH.The bands at 306 and 341 nm are attributed as being π→π* transitions of aromatic groups and n→π* of the hydrazone moiety, respectively, and underwent shifts upon complexation.The hypsochromic displacement of the π→π* band corresponding to the π→π* transition of the azomethine can be observed, which decreases to 326 nm in MeOH and 331 nm in DMF; this is indicative of the coordination of the group to the Cu(II) atom.The ligand-metal charge transition (LMCT) can be attributed in the range of 406-411 nm in MeOH and 413-415 nm in DMF as an indication of the coordination of the hydrazone to the Cu(II) atom (Santiago et al., 2022).In addition, to observe d-d transitions of the metal center, the electronic spectra of the complexes were determined at a higher concentration (2 mM) in methanol (Supplementary Figure S15 and Supplementary Table S3).The absorption bands of the d-d transitions were observed in the range of 672-680 nm, with values of log ε between 1.54 and 1.85 being more evidence of the complexation of the copper ion with the ligand (Ramírez-Contreras et al., 2022).
ESI(+)-MS(/MS) electrospray mass spectra, with a concentration of 250 μM (methanol) in an acid medium (0.1% acetic acid), were obtained to evaluate the real species in the solution of the ligand and complexes.The spectra containing the fragmentations are shown in Figure 5 and Supplementary Figures S16-S20.In the ESI(+)-MS spectrum of the free hydrazone, it is possible to observe the isotopic distribution for the [M+H] + ion of m/z = 286.1192and a base peak at m/z=150.0551 for the substituted pyridine group; this can be explained by a rearrangement of the hydroxyl followed by an alpha cleavage of the azomethine group.Two other peaks can be observed at m/z=165.0663 and m/z=119.0612due to the breaking of the azomethine group via a concerted mechanism.
In the case of complexes (1-4), it is possible to observe that some fragmentations are common to the ligand due to the similarity in their structures.The first peak corresponds to the molecular ion [M+H] + after the loss of the coordinated ions and water molecules of m/z=347.0330 in (1), m/z=347.0326 in (2), m/z=347.0334 in (3), and m/z=347.0323 in (4).Other characteristic peaks observed are m/ z=269.9929 in (1), m/z=269.9930 in (2), and m/z=269.9935 in (3) and ( 4) after the loss of the benzene group via alpha cleavage.The peaks m/z=251.9928 in (1), m/z=251.9825 in (2), m/z=251.9829 in (3), and m/z=251.9929 in (4) are observed due to the loss of water molecules by rearrangement of the hydroxyl group and the hydrogen attached to the azomethine group.

Hirshfeld Surface
The Hirshfeld surface (HS) allowed the evaluation of the electronic density distributions at the crystal structures to predict Frontiers in Chemistry frontiersin.org08 the intermolecular interactions between neighboring molecules of the complexes (McKinnon et al., 2004;Spackman and Jayatilaka, 2009).HS was obtained by the combination of normalized distances (d norm ) between atoms inside (d i ) and outside (d e ) the surface.The contacts shorter than the sum of the van der Waals radii show a red color, close to the sum (white) and longer than the sum of the radii (blue).4) with an internuclear distance between 2.00 and 2.43 Å. Non-classical interactions are verified in the HS of the four compounds, such as C-H•••O interactions (Man et al., 2023;Yilmaz et al., 2024).The shape index map allows a clearer observation of the π•••π interactions, which show a pattern of red and blue triangles.The shape index for complexes (1-4) is shown in Supplementary Figure S21, where it is possible to observe the presence of π•••π stacking interactions in all complexes, agreeing with the observed single-crystal X-ray diffraction.
The 2D-fingerprint plots (FP) associated with the Hirshfeld surface of the Cu(II) complexes (Supplementary Figures S22-S25) were obtained to verify the contribution of each contact and to help better comprehend the interactions occurring within the crystal (Man et al., 2023;Yilmaz et al., 2024)

Full interaction maps
The full interaction maps (FIMs) were used to predict the most likely locations for a variety of acceptor and donor groups in the crystal structures and evaluate the preferred interactions of the molecules (Wood et al., 2013).The interaction maps highlight the red regions that indicate the acceptor positions and the blue regions where the donor atoms are expected.Figure 7 shows the FIMs of complexes (1-4) where the intense red regions near the NH or OH are indicative of hydrogen bond acceptors, and the intense blue regions near oxygen atoms are indicative of hydrogen bond donors.Large and intense red and blue regions are observed in complexes (3) and (4) due to the presence of solvent water molecules, nitrate, and sulfate ions.The hydrophobic regions in brown can be found above and below the phenyl rings of the ligands.The green color found for complex (1) in the FIM is indicative of noncovalent interactions with the chloride ions.

Biological activity analysis
To investigate the antibacterial activity of the synthesized compounds, the free ligands and the Cu(II) complexes were tested on Gram-positive S. aureus and Gram-negative E. coli bacteria strains.The MICs of the compounds were determined, corresponding to the lowest concentration of the tested compound necessary to inhibit the growth of a microorganism, and are compared in Figure 8. Table 3 shows the tested different formulations for antimicrobial activity and the MIC values determined for PLBHZ and their metal complexes (1-4).
The compounds were generally more effective on Gram-positive than Gram-negative bacteria.The results show that the free ligand and complexes (1-3) presented a better MIC value than the other complexes for Gram-positive bacteria, and that complex (3) Full interaction maps for complexes (1-4) showing the acceptor and donor likelihood regions, and hydrophobic groups.
Frontiers in Chemistry frontiersin.org10 presented a lower MIC value for Gram-negative bacteria.The free ligand and complexes (1-3) presented IC 50 values lower than the positive control oxacillin used for S. aureus with an IC 50 of 0.05 μg mL −1 .However, PLBHZ and complexes 2 and 3 presented IC 50 values much lower than the positive control, ampicillin with IC 50 of 1.78 μg mL −1 , for E. coli.The results of the biological activity also show various compounds with significant antibacterial activity against S. aureus (MIC < 1 μg mL −1 ) (Gibbons, 2004).Comparing the obtained values, it is possible to verify the high antimicrobial activity of the hydrazone before coordination with the Cu(II) ion.In contrast, Gram-negative bacteria have an asymmetrical functional lipid bilayer outer membrane that protects them from harmful chemicals present in their environment.This outer membrane is composed mainly of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer layer.Due to the polar nature of the LPS, the outer membrane is charged and creates a significant barrier against lipophilic molecules, thus protecting the bacteria from various detergents (e.g., bile salts), dyes (e.g., methylene blue), and hydrophobic antibiotics (Neidhardt and Roy, 1996).In addition to containing lipoproteins, the outer membrane features porin proteins, with their conserved β-barrel fold that encloses a central aqueous channel that regulates the passage of hydrophilic molecules (Koebnik et al., 2000).

Molecular docking simulation
The molecular modeling of the complexes formed by the MERC1 and OpmF/A enzymes and the small molecules was performed using a molecular docking simulation with AutoDock Vina.The protocols for the docking simulations were validated through previous redocking studies with crystallographic structures of the complexes and their ligands molecules, oxacilloyl (S. aureus) and ampicillin (E.coli).In both cases, the superposition of the docked pose and crystallographic pose agree well (see Supplementary Figure S26).The RMSD was 0.86 Å for MecR1oxacilloyl and 1.36 for the OmpF/A-ampicillin system.After establishing the docking protocol, the molecular modeling of both systems proceeded.The docking simulation revealed good agreement in the tendency of the inhibitory power with the Percentage of inhibition for minimal inhibitory concentration (μg/mL) of the synthesized compounds.experimental IC 50 values for both enzymatic systems and their ligands.The same trend of inhibition was observed comparing the antibacterial activity in the experimental essay (Table 3) and the theoretical energy of binding (score values in Table 4).
The relative trend of inhibition to quantitatively evaluate the correlation and assertiveness between the experimental values of IC 50 versus the in silico energy of binding (score) is shown in Figure 9.For the S. aureus proteomic system (Figure 9A), the order of power of inhibition of the studied molecules is PLBHZ > (3) > (1) > (2) > oxacillin > (4); the trend of the power of inhibition for the E. coli proteomic system (Figure 9B) is PLBHZ > (3) > (2) > ampicillin > (1) > (4).The PLBHZ and compound (3) are the most powerful inhibitors for both enzymes in the set of ligands studied, and compound (4) presents for both bacteria a low power of inhibition.
In general, the compounds show a promising inhibitory power against Gram-positive and Gram-negative bacteria, although the experimental and theoretical studies pointed to a better affinity between the Gram-positive bacteria system-S.aureus.

Molecular docking simulation of MecR1 S. aureus
The crystallographic structure of the MecR1 (PDB 2IWD) bioreceptor representing the proteomic of S. aureus is originally complexed with the reacted oxacillin, forming a covalent bond with the catalytic residue Ser391, thus forming the oxacilloyil adduct (Figure 10A).The docking study for oxacilloyl and oxacillin molecules was performed and showed good agreement in superposition with the original crystallographic structure.
As presented in Figure 10B, the active site of the MecR1 enzyme is compound of catalytic Ser391 residue and Pro389, Asn390, Thr392, Lys394, Ser439, Phe423, Trp426, Asn441, Thr529, Thr531, and Ile533 residues.To block the activity of the MecR1, and consequently interrupt the machinery of resistance in the S. aureus, a covalent interaction (set as a, see Figure 10C) between the antibacterial inhibitor and the Ser391 active site residue is necessary.In addition, some important interactions were observed by Marrero et al. (2006) for effective inhibition.
Important hydrogen bonds should be established between the Thr529 (2.50 Å), Thr531 (2.70 Å), and Asn441 (2.80 Å) residues (set as b, c, and f in Figure 10C) to perform efficient inhibition.Furthermore, medium range pi-alkyl, pi-sulfur interaction types between the adduct specie and the residues Phe423 (set as d, Figure 10C) and Trp426 (set as e, Figure 10C) residues are very important for stabilizing the adduct in the active site of MecR1.Table 5 presents the main interactions performed for the ligand PLBHZ, complexes (1-4), oxacilloyl, and oxacillin during the molecular docking study with the MecR1 enzyme, with distances (Marrero et al., 2006).
The inhibitory power of the ligands is related to the strong, short-length hydrogen bond interactions between the compounds and the residues Thr529, Thr531, and Asn441.The pi-alkyl interaction with the Phe423 significantly stabilizes the ligand in position to react with the catalytic Ser391 residue.(1) −7.5 (1) −6.8 (2) −6.9 (2) −7.0 (3) −7.7 (3) −7.4 (4) −6.7 (4) −6.6 Frontiers in Chemistry frontiersin.org The best-ranked compound as a good inhibitor for MecR1 was the ligand PLBHZ, followed by ( 3) and (1).The measured distances shown in Table 5 reveal that PLBHZ is the molecule that interacts with all active site residues with short distances for c, d, and e distances, and also mimics the profiles of interaction of the oxacillin.The shorter hydrogen interaction with the residues Thr531 (c) and Asn411 (f), observed with PBLHZ, and oxacillin suggests the importance of this range length during the inhibition process.In addition, the closer range length of the interactions (d) and (e) shows the same importance in the inhibitory process.

Molecular docking simulation of OmpF/ A E. coli
The crystallographic structure of the OmpF (PDB 4GCP) bioreceptor from proteomic of the S. aureus bacteria complexed with ampicillin is shown in Figure 11A.
The crystallographic 3D structure of Ompf/a reveals that ampicillin is oriented perpendicular to the axis of the channel.This orientation promotes proper electrostatic interactions between the negatively charged carboxylic group of ampicillin and the positive NH 3 + groups of the residues Arg167 (b) and Arg168 (c), Figure 11B.These interactions stabilized the complex.Other important interactions are the hydrogen bond type, observed between the Ser125 (a) and the hydroxyl group of the side chain of ampicillin, and the non-conventional hydrogen bond between the Asp121 (d) residue and the hydrogen of the C1 atom of ampicillin.The phenyl ring of ampicillin is located in the hydrophobic pocket composed of several aromatic residues, especially the residues Tyr22, Tyr32, and Phe118.The phenyl ring of ampicillin makes a π•••π T-shaped interaction with the phenyl ring of Phe118 residue (e), Table 6.This interaction is responsible for stabilizing the ampicillin in the active site of OmpF/a without inducing a large conformational change in the protein active site (Ziervogel and Roux, 2013).The PLBHZ ligand presents stronger hydrogen bond interaction with the residue Ser125 (a: 2.80 Å) than other ligands, including ampicillin.The non-conventional hydrogen-bond interaction between the Asp121 (d) residue and the ligands plays an important role in the inhibitory process, comparing the size of the distance for this interaction and the inhibitory trend obtained through the experimental essay and in silico approach.[PLBHZ > (3) > (2) > ampicillin > (1) > (4)] is the correlation between the length of this interaction and the binding energy of the compounds.
analysis revealed that the different ions in the crystal structures have no significant influence on the spectral properties of the complexes.
In addition, non-covalent interactions were detected in all compounds, showing the relevance of pyridoxal, water molecules, and different ions in establishing hydrogen bonds.Hirshfeld surface analysis combined with 2D fingerprint plots showed that the different anions in the Cu(II) complexes, together with their noncovalent interactions, are essential in determining the final crystal structure networks.The results of the biological evaluation and molecular docking of the new Cu(II) complexes with pyridoxalbenzoylhydrazone can allow the development of innovative new strategies for developing potential new drugs.

FIGURE 1
FIGURE 1Summary of products obtained from the reactions between pyridoxal-benzoylhydrazone ligand (PLBHZ) and different Cu(II) salts.

FIGURE 4
FIGURE 4Projection view of (1) showing an intramolecular hydrogen bond and the π•••π stacking interactions (as a dashed line).
. The H•••H contact contributes most to the molecular surface of the complexes with values 32.3% -38%.The Cl•••H, Br•••H, and O•••H interactions appear as long and narrow spikes in the FP, demonstrating that these interactions are strong and intense.The C•••C contacts have contribution values between 5% and 7% due to the presence of π•••π stacking interactions in all compounds.

FIGURE 9
FIGURE 9Relative trend of inhibition and the correlation to IC 50 versus in silico energy of binding (score).(A) S. aureus-MecR1, (B) E. coli-OmpF/A.

TABLE 3
Antibacterial activity was determined for PLBHZ and complexes (1-4), expressed as MIC and MBC (μg•mL −1 ) for the different formulations against S. aureus and E. coli strains.

TABLE 4
Theoretical binding energy (score) of docking studies with the complexes systems formed between Gram-positive and Gram-negative bacteria and the small molecules PLBHZ, complexes (1-4), and control molecules oxacillin and ampicillin.