Synthesis, Characterization, Crystal Structure and Antimicrobial Activity of Copper(II) Complexes with the Schiff Base Derived from 2-Hydroxy-4-Methoxybenzaldehyde

A novel Schiff base, ethyl 4-[(E)-(2-hydroxy-4-methoxyphenyl)methylene-amino]benzoate (HL), was prepared and structurally characterized on the basis of elemental analyses, 1H NMR, 13C NMR, UV-Vis and IR spectral data. Six new copper(II) complexes, [Cu(L)(NO3)(H2O)2] (1), [Cu(L)2] (2), [Cu(L)(OAc)] (3), [Cu2 (L)2Cl2(H2O)4] (4), [Cu(L)(ClO4)(H2O)] (5) and [Cu2(L2S)(ClO4)(H2O)]ClO4·H2O (6) have been synthesized. The characterization of the newly formed compounds was done by IR, UV-Vis, EPR, FAB mass spectroscopy, elemental and thermal analysis, magnetic susceptibility measurements and molar electric conductivity. The crystal structures of Schiff base and the complex [Cu2(L2S)(ClO4)(H2O)]ClO4·H2O (6) have been determined by single crystal X-ray diffraction studies. Both copper atoms display a distorted octahedral coordination type [O4NS]. This coordination is ensured by three phenol oxygen, two of which being related to the µ-oxo-bridge, the nitrogen atoms of the azomethine group and the sulfur atoms that come from the polydentate ligand. The in vitro antimicrobial activity against Escherichia coli ATCC 25922, Salmonella enteritidis, Staphylococcus aureus ATCC 25923, Enterococcus and Candida albicans strains was studied and compared with that of free ligand. The complexes 1, 2, 5 showed a better antimicrobial activity than the Schiff base against the tested microorganisms.


Introduction
The synthesis of new compounds that are used for the treatment of infections with less secondary effects is a biomedical problem [1,2].Recently research has focused increasingly on the synthesis of transition metal complexes with Schiff-type ligands, due to the biological properties that they present.Many compounds derived from Schiff bases exhibit antibacterial, antifungal, antitumor and anti-HIV activities [3][4][5][6][7][8][9][10][11][12][13][14].Schiff bases derived from salicylaldehyde represent an important class of ligands due to their ability to be used in various fields [15][16][17][18][19]. Having a high capacity chelating and redox potential the positive Cu 2+ ion is biologically active, and participates in many processes in the body [20][21][22].
Copper complexes are also among the most potent antiviral, antitumor and anti-inflammatory agents [23].In addition, it was found that most of benzocaine-containing compounds are biologically active.Recent research has demonstrated that, these derivatives exhibit antimicrobial activity against different species [24][25][26].Based on these observations and considering research in this area in the present study, we report the synthesis, characterization, antimicrobial studies of some copper(II) complexes containing a Schiff base ligand derived from 2-hydroxy-4-methoxybenzaldehyde and ethyl-4-aminobenzoate, with a special impetus on ligand structural investigations.The crystal structures of ligand and complex 6 were studied by X-ray diffraction.The biological properties of these compounds was tested for their antibacterial and antifungal activities against Escherichia coli ATCC 25922, Salmonella enteritidis, Staphylococcus aureus ATCC 25923 and Candida albicans strains.

Chemistry
The Schiff base HL was prepared by refluxing in ethanol an equimolar mixture of 4-aminobenzoic acid ethyl ester with 2-hydroxy-4-methoxybenzaldehyde.The structure of the formed Schiff bases was established by IR, 1 H-NMR and 13 C-NMR spectroscopies and by X-ray crystallography.All compounds were synthesized by direct reaction between ligand and the corresponding metal salts, while compound 6 was prepared by using a mixture of Cu(ClO4)2•6H2O and HL with addition of NaSCN.All complexes are microcrystalline solids.Also, the melting point values are greater than 220 °C.They are insoluble in organic solvents such as acetone and methanol, but soluble in DMF and DMSO.The molar conductivity values (8-12 ohm −1 •cm 2 •moL −1 ) of the complexes 1-5 in 10 −3 M solution DMF show that they are non-electrolytes and 6 (82 ohm −1 •cm 2 •moL −1 ) is an electrolyte [27].
The thermal decompositions of the complexes 1-4 were studied by thermogravimetry (TG).The TG and TGD curves have three or four stages of mass loss.The first weight loss step correspond of two and four water molecules respectively, per one molecule of complex (7.26% for complex 1, 8.63% for complex 4).Thermal analyses data reveal that compounds 2 and 3 not are hydratated.The final residue was analyzed by IR spectroscopy, which confirms the formation of CuO (Figure S1).
The elemental analyses data of the Schiff base and complexes (reported in Section 3) are in agreement with structure of the ligand (Figure 1) and with structures of the complexes (Figure 2).

Structural characterization of (HL)
The ligand HL structure is shown in Figure 3.The planar configuration of HL molecule is stabilized by an intra-molecular O-H⋅⋅⋅N hydrogen bond which is observed in the majority of Schiff base ligands obtained from 2-hydroxybenzaldehyde [28].
Selected bond lengths and angles for HL are presented in Tables 1 and 2. Complex 6 has a crystal structure formed from the complex binuclear cations [Cu2(L2S)2(ClO4)(H2O)] + , ClO4 − anions and H2O as solvate molecules 1:1:1 molar ratio.The structure of the complex cation is presented in Figure 6.
In the complex cation two Cu 2+ ions, separated by 3.029(1) Å, are coordinated by two mono-deprotonated HL − ligands, so that the charge balance is in agreement with the formation of species [Cu2(L2S)2ClO4H2O] + .The separate structure of the coordinated ligands along with the atomic labeling scheme is presented in Figure 7a,b.Both the ligands are identical from the chemical point of view and exhibit very close geometric parameters (Table 2).The localization of the hydrogen atoms using the difference density Fourier map has clearly shown that the non-coordinated azomethine atoms N1 and N4 are the sites of protonation associated with the hydrogen bonding towards phenolic oxygen atoms as acceptor (Figures 6 and 7).The copper atoms adopt a slightly distorted octahedral [O4NS] coordination completed by ClO4 − anion and H2O molecule as monodentate ligands for Cu1 and Cu2, respectively (Figure 8).

Infrared Spectra and Coordination Mode
The ligand and complexes have been characterized in detail by recording their IR spectra.The proposed assignments are based on previous results [28][29][30][31] and pertinent bibliography [32][33][34][35].The ʋ(C=N) band of the ligand at 1622 cm −1 is found to be shifted to lower energies (1603-1595 cm −1 ) in the spectra of the complexes, indicating coordination via the azomethine nitrogen.The phenolic ʋ(C-O) stretching vibration in the free Schiff base is observed at 1113 cm −1 , which is shifted by 10-21 cm −1 towards lower wave numbers in the complexes, thus indicating coordination of the phenolic oxygen to the Cu 2+ ion [36].
In the IR spectra of complexes 1, 4 and 5, a considerable peak observed in the 3467-3420 cm −1 range supports the presence of ʋ(H2O) in the complexes [37].The nitrate complex 1 has two bands at 1433 and 1182 cm −1 corresponding to ʋ5 and ʋ1, with a separation of 250 cm −1 , and a medium band at 930 cm −1 assigned to ʋ2 of the nitrato group, values that indicate bidentate coordination [38,39].
The acetate complex 3 has two strong bands at 1530 and 1442 cm −1 corresponding to ʋas(COO − ) and ʋs(COO − ) with a difference between frequencies of 88 cm −1 .This difference confirms the bidentate nature of the coordinated acetate [40].
The perchlorate complex 5 shows a band at 1108 cm −1 assignable to ʋ3(ClO4 − ) and a strong band at 1090 cm −1 assignable to ʋ4(ClO4 − ).The splitting of this band in two components indicates the presence of a monodentate perchlorate group [33,38].

Electronic Spectra and Magnetic Studies
The tentative assignments of the significant electronic spectral bands of ligand and of complexes are presented in Table 3.In these spectra, there is an intense band at 33,330 cm −1 which is assigned to a π→π* transition originating in the phenyl ring [41].The band at 28,570 cm −1 is attributed to an n→π* transitions originating in the -CH=N-chromophore [42].In the spectra of the complexes these bands are shifted to lower energies (Figure S2).Table 3. Electronic spectra (cm −1 ) and magnetic moments (BM) of the complexes 1-5.The electronic spectra for the Cu(II) complexes 2, 3 and 5 exhibit two bands in the region 10,980-11,100 cm −1 respectively 13,150-13,880 cm −1 , attributed to transitions d-d: 2 B2→ 2 E, 2 B2→ 2 B1( 2 A1), suggesting a distorted tetrahedral geometry [43,44].The values of the magnetic momentum (1.87, 1.92 and 1.90 BM) indicate the existence of copper complexes under monomers form.The spectrum for complex 1 exhibit three bands at 10,500, 13,420 and 19,410 cm −1 corresponding to the transitions: 2 B1g→ 2 A1g 2 , 2 B1g→ 2 B2g, 2 B1g→ 2 Eg, specific to the octahedral complexes with ground state [43].

Mass Spectra
The FAB mass spectra of Cu(II) complexes with base Schiff HL have been recorded (Table S1).The mass spectrum of ligand showing a peak at m/z = 300.12corresponds to the molecular ion [M] + (Figure S3).
[M] + peak obtained from the complexes are as follow: m/z = 386.9(1), m/z = 658.0(2), m/z = 399.1 (3), m/z = 759.0(4), m/z = 361.0(5).The results of mass spectrometry are consistent with the proposed formulas for these compounds as the peaks observed in these spectra correspond to fragments resulting from the expected fragmentations of the compounds.

EPR Spectra
The EPR spectra of the complexes in the polycrystalline state at 298 K and 77 K, were recorded at X-band, using 100 kHz field modulation.The g factors were quoted relative to the standard marker TCNE.The spectra of the compounds 1 and 4 in the polycrystalline state (293 K) show typical axial behavior with different gǁ and g ⊥ values (Table 4).The geometric parameter G which is a measure of the exchange interaction between the copper centers in the polycrystalline compound is calculated using the equation: G = (gǁ − 2.0023)/(g ⊥ − 2.0023), for axial spectra [55].In the copper(II) complexes 1 and 4, gǁ > g ⊥ > 2.0023 and G values (2.77, 3.99) are consistent with a ground state [56].The spectra of compounds 2, 3 and 5 show only one broad signal at 2.097, 2.102 and 2.127, respectively, (Figure 10) consistent with tetrahedral geometry [57].The solution of complexes were recorded in DMSO at 298 K and 77 K.They present well-resolved four hyperfine lines (Figure 11).Spectra of the complexes 1, 3 and 5 show three nitrogen superhyperfine lines in the perpendicular component (Figure 12).EPR spectral assignments of the copper (II) complexes and orbital reduction parameters are shown in Table 4.The EPR parameters gǁ, g ⊥ , Aǁ (Cu) and the energies of the d-d transition were used to evaluate the bonding parameters α 2 , β 2 and δ 2 , which may be regarded as measures of the covalency of the in-plane σ bonds, in-plane π bonds and out-of-plane π bonds [58,59].The orbital reduction factors Kǁ = α 2 β 2 and K ⊥ = α 2 δ 2 , were calculated using expressions reported elsewhere [60].Hathaway has pointed out that for pure σ bonding, Kǁ ~ K ⊥ ~ 0.77 and for in-plane π -bonding, Kǁ < K ⊥ , while for out-of-plane π-bonding, Kǁ > K ⊥ [61].

Biological Activity
The synthesized compounds and ligand were tested for their in vitro antibacterial and antifungal activity against Escherichia coli ATCC 25922, Salmonella enteritidis, Staphylococcus aureus ATCC 25923, Enterococcus and Candida albicans strains using the paper disc diffusion method [62] (for the qualitative determination) and the serial dilutions in liquid broth method [63] for determination of MIC values.Furaciline and nistatine were used as reference substances.
Data from Table 5 confirm the fact that both the ligand HL and the coordinative complexes of copper have a reduced bacteriostatic activity within the limits of concentrations 0.5-10.0mg/mL toward the gram-positive and gram-negative bacteria.A more nuanced sensitivity toward the searched complexes has been proved by the gram-positive micro-organisms.The experimental data obtained demonstrate that the bacteriostatic activity of the coordinative complexes is influenced by the number of ligand molecules HL found out in the inner sphere and ligand nature.Therefore, growing of the number of ligand molecules and replacing the ions chloro-or acetate-with the perchlorate ion leads to a growing of the antimicrobial activity.The complexes [Cu(L)2] (2), [Cu(L)(ClO4)(H2O)] ( 5) and [Cu2(L2S)(ClO4)(H2O)]ClO4•H2O (6) develop bacteriostatic activity also toward Candida albicans in the area of concentrations 0.5-0.12mg/mL, while the initial Schiff base HL which enters the inner sphere of these complexes doesn't develop antifungal activity.Table 5. Antibacterial activities of ligand HL and complexes 1-5 as MIC a /MBC b values (mg/mL).

General Information
All commercially available reagents and chemicals were of analytical-or reagent-grade purity and used as received.The chemical elemental analysis for the determination of C, H, N was done on a Carlo-Erba LA-118 microdosimeter (Lakewood, CA, USA). 1 H-NMR and 13 C-NMR spectra were recorded at room temperature on a Bruker DRX 400 spectrometer (Billerica, MA, USA) in DMSO-d6, using TMS as the internal standard.IR spectra were recorded on a Specord-M80 spectrophotometer (Leipzig, Germany) in the 4000-400 cm −1 region using KBr pellets.The complexes were studied by thermogravimetry (TG) in a static air atmosphere, with a sample heating rate of 10 °C/min using a STA 6000 Perkin Elmer (Waltham, MA, USA).Electronic spectra were recorded using the JascoV-670 spectrophotometer (Tokyo, Japan) in diffuse reflectance, using MgO dilution matrices.EPR spectra were recorded on polycrystalline powders and DMSO solutions at room temperature and 77 K with an MiniScope MS200, (Magnettech Ltd., Berlin, Germany), X-band spectrometer (9.3-9.6 GHz), connected to a PC equipped with a 100 KHz field modulation unit.High resolution mass spectra were recorded on a ThermoScientific (LTQ XLOrbitrap) spectrometer using APCI ionization technique and Orbital Ion Trap mass analyzer (Rockford, IL, USA).
The molar conductance of the complexes in N,N'-dimethylforamide solutions (10 −3 M), at room temperature, were measured using a Consort type C-533 conductivity instrument.The magnetic susceptibility measurements were done at room temperature in the polycrystalline state on a Faraday magnetic balance (home made).
Crystallographic measurements were carried out on Oxford-Diffraction XCALIBUR E CCD diffractometer (Santa Clara, CA, USA) equipped with graphite-monochromated MoKα radiation.The single crystals were positioned at 40 mm from the detector and 197 and 273 frames were measured each for 20 and 30 s over 1° scan width for HL and 6, respectively.The unit cell determination and data integration were carried out using the CrysAlis package of Oxford Diffraction [64].Both structures were solved by direct methods using Olex2 [65] software with the SHELXS structure solution program and refined by full-matrix least-squares on F² with SHELXL-97 [66].Atomic displacements for non-hydrogen atoms were refined using an anisotropic model.H atoms were placed at calculated positions and refined as riding atoms in the subsequent least-squares model refinements.The positional parameters of OH and NH hydrogen atoms were found from difference.The oxygen atoms in non-coordinated perchlorate anion were found to be severely disordered and their positional parameters were refined isotopically in combination with PART and SADI tools available in SHELXL Crystallographic data together with refinement details are summarized in Table 6.
where n is the number of reflections and p is the total number of parameters refined.

General Procedure for the Preparation of the Metal Complexes 1-6
Complexes 1-5 were prepared by direct reaction between the ligand and the corresponding metal salts.Complex 6 was obtained by stirring a mixture of ligand, Cu(ClO4)2•6H2O and NaSCN.

Antibacterial Activity
The antibacterial activity of complexes and also of their prototype furaciline has been determined under liquid nutritive environment [2% of peptone bullion (pH 7.0)] using successive dilutions method [67][68][69].Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Enterococcus stems were used as reference culture for in vitro experiment.The dissolution of studied substances in dimethylformamide, microorganisms cultivation, suspension obtaining, determination of minimal inhibition concentration (MIC) and minimal bactericide concentration (MBC) have been carried out according to the method previously reported.

Antifungal Bioassay
Antimycotic properties of the complexes were investigated in vitro on laboratory stem Candida albicans.The activity has been determined in liquid Sabouroud nutritive environment (pH 6.8).The inoculates were prepared from fungal stems which were harvested from 3-7 day-old cultures.Their concentration in suspension was (2-4) × 10 6 colony forming units per milliliter.Sowings for fungi and micelles were incubated at 37 °C during 7 and 14 days, respectively.

Figure 9 .
Figure 9.View of 2D supramolecular layer in the crystal structure 6.

Figure 11 .
Figure 11.EPR spectrum of the complex 1, in DMSO solution, registered at 77 K and (d2) second derivative spectra.

Figure 12 .
Figure12.EPR spectrum (the derived signal) for complex 5 in DMSO solution at 77.

Figure 13 .
Figure 13.EPR spectrum of the complex 4, in solution, at room temperature and 77 K.

Table 6 .
Crystallographic data, details of data collection and structure refinement parameters for HL and 6.