Cu(II), Ni(II), and Zn(II) Complexes of Salan-Type Ligand Containing Ester Groups: Synthesis, Characterization, Electrochemical Properties, and In Vitro Biological Activities

A salen ligand on reduction and N-alkylation affords a novel [N2O2] chelating ligand containing ester groups [L = diethyl-2,2′-(propane-1,3-diylbis((2-hydroxy-3-methoxy benzyl)azanediyl))diacetate]. The purity of the ligand was confirmed by NMR and HPLC chromatograms. Its Cu(II), Ni(II), and Zn(II) complexes were synthesized and characterized by a combination of elemental analysis, IR, NMR, UV-Vis, and mass spectral data, and thermogravimetric analysis (TG/DTA). The magnetic moments, UV-Vis, and EPR spectral studies support square planar geometry around the Cu(II) and Ni(II) ions. A tetrahedral geometry is observed in four-coordinate zinc with bulky N-alkylated salan ligand. The redox properties of the copper complex were examined in DMSO by cyclic voltammetry. The voltammograms show quasireversible process. The interaction of metal complexes with CT DNA was investigated by UV-Vis absorption titration, ethidium bromide displacement assay, cyclic voltammetry methods, and agarose gel electrophoresis. The apparent binding constant values suggest moderate intercalative binding modes between the complexes and DNA. The in vitro antioxidant and antimicrobial potentials of the synthesized compounds were also determined.


Introduction
Salen metal complexes are the interest of many workers because of their applications in food industry, in the treatment of cancer [1], as antibactericide agents [2,3], as antivirus agents [4], as fungicide agents [5], and for other biological properties [6]. The antitumor activity of salen complex arises due to its DNA binding properties. The salen complexes are conformationally flexible and adopt a variety of geometries. Also, salen metal complexes have a unique flat electron-rich aromatic surface that may facilitate their interactions with nucleic acids. Hydroxyl groups in the salen complexes act as a quinone system which would cooperate to facilitate the formation of free radicals responsible for DNA cleavage [7]. The biological properties of salen complexes are enhanced by functionalization with a variety of substituents [8][9][10][11]. When salen compounds are reduced at the imine function, the more flexible, reduced salen derivatives (salan) are obtained.
Considerable attention has been devoted to the preparation and structural characterization of metal complexes containing salen-type ligands. However, little attention has been paid to systems in which functionalized salan is used as ligands. In the present investigation N-alkylated salan complexes are used for DNA binding and antimicrobial and antioxidant properties. In continuation of our earlier works on salen-type ligands [12][13][14], the present investigation reports on the synthesis and spectral characterization of Cu(II), Ni(II), and Zn(II) complexes with N-alkylated salan ligand. The interaction of the metal complexes with calf thymus (CT) DNA was studied by UV-Vis and fluorescence spectroscopy and cyclic voltammetric method. The DNA cleaving nature of the compounds was tested against pUC19 DNA in the absence and presence of hydrogen peroxide. The in vitro antimicrobial activity of the compounds was assessed against various microorganisms. The antioxidant activity of the metal complexes was investigated systematically.

Materials and Methods.
All chemicals employed for the synthesis were of analytical reagent grade and of highest purity available. o-Vanillin and 1,3-diaminopropane were purchased from Sigma Aldrich and used as received. Solvents used for spectroscopic and electrochemical studies were purified and dried by standard procedures [15]. Metal acetates were purchased from Merck. CT DNA and pUC19 DNA were purchased from GeNei, Bangalore, and used without purification. Tris-(hydroxylmethyl)-aminomethane-HCl (Tris-HCl) and ethidium bromide (EB) were obtained from HiMedia. Tris-HCl-NaCl buffer solution was prepared with doubledistilled water. Tetrabutylammonium perchlorate (TBAP) was used as a supporting electrolyte for recording cyclic voltammograms.

Physical Measurements.
Elemental analyses were recorded on a Thermo Finnigan Flash EA 1112 elemental analyzer. Molar conductance values of the complexes in DMSO were obtained on a Systronics Model 611 digital conductivity meter. Magnetic susceptibility measurements on powder samples were carried out on a Gouy balance at room temperature using mercuric tetra(thiocyanato)cobaltate (II) as the calibrant. The infrared (IR), ultraviolet-visible (UV-Vis), and emission spectra were recorded on Shimadzu 8400 S, Shimadzu UV-2450, and Shimadzu RF-5301 PC spectrophotometers, respectively. The 1 H and 13 C NMR of ligand in CDCl 3 and zinc complex in DMSO-d 6 were recorded on Bruker AV 300 MHz spectrophotometer. Electrospray ionization (ESI) mass spectral measurements were recorded on Micromass Quattro II mass spectrometer. Electron paramagnetic resonance (EPR) spectrum of the Cu complex in DMSO solution was recorded on JES-FA200 spectrometer at 300 K and at 77 K using tetracyanoethylene (TCNE, = 2.00277) as the marker. The thermogravimetric analysis/differential thermal analysis (TGA/DTA) was carried out in dynamic nitrogen atmosphere with a heating rate of 20 ∘ C/min using NETZSCH STA 449F3 thermal analyzer. Cyclic voltammograms were recorded on a CHI 603 C electrochemical analyzer with a three-electrode compartment.

Synthesis of the Ligand (L).
The synthetic procedure of the ligand was reported in our earlier work [14]

DNA Binding Experiments
2.5.1. UV-Vis Spectroscopic Studies. The DNA binding experiments were performed at room temperature. A solution of CT DNA in the buffer (5 mM Tris-HCl and 50 mM NaCl) gave a ratio of UV absorbance at 260 and 280 nm of about 1.8-1.9 : 1, indicating that the CT DNA was sufficiently free from protein [16]. The concentration of DNA was measured using its extinction coefficient at 260 nm (6600 mol L −1 cm −1 ) [17]. Concentrated stock solutions of the compounds in DMSO were prepared and diluted suitably with the buffer to the required concentrations for all the experiments. The absorption titrations of the compounds in buffer were performed using a fixed concentration (10 M) to which increments of the DNA stock solution were added (R = [DNA]/[complex] = 0, 2, 4, 6, 8, and 10). Compound DNA solutions were allowed to incubate for 30 min before the spectra were recorded. From the absorption data, the intrinsic binding constant, , was determined using the following [18]: where , , and are the apparent, free, and bound compound extinction coefficients, respectively. In the plots

Fluorescence Studies.
The interaction of the synthesized compounds with DNA was further studied by ethidium bromide (EB) displacement method. The excitation wavelength was fixed at 530 nm, and the emission range was adjusted before measurements. The changes in the fluorescence intensities at 595 nm of EB-bound CT DNA in Tris-HCl buffer (pH 7.2) were measured with respect to different concentrations of the compounds (0-120 M). The magnitude of the binding strength of the compounds with CT DNA can be calculated using linear Stern-Volmer equation [19]:

Electrochemical Studies.
The cyclic voltammetric studies of the copper complex were performed with a threeelectrode system of glassy carbon as working electrode, Pt wire as auxiliary electrode, and Ag/AgCl as reference electrode. The supporting electrolyte is 0.05 M TBAP in DMSO solution. The cell was maintained oxygen-free by passing dry nitrogen through the solution. The interaction of the copper complex with CT DNA has been investigated by monitoring the changes observed in the cyclic voltammogram of CuL in buffer (5 mM Tris-HCl/50 mM NaCl) with increasing amount of DNA.
2.6. DNA Cleavage Experiment. The DNA cleavage experiment was conducted by gel electrophoresis on pUC19 DNA. The reaction mixture was prepared as follows: 1 L of pUC19 DNA, 5 L of the compound in DMSO, and 1 L of H 2 O 2 followed by dilution with buffer (50 mM Tris-HCl and 50 mM NaCl) to a total volume of 25 L. The reaction mixture was incubated at 37 ∘ C for 1 h. The 1% agarose gel was prepared and stained using ethidium bromide. The samples were then loaded on gel after mixing with 3 L of loading dye (0.25% bromophenol and 40% sucrose). The gel was electrophoresed at 100 V using Tris-boric acid-EDTA buffer (pH = 8.0) until the bromophenol blue reached one-third of the gel.
The bands were visualized and photographed under a UV transilluminator. The experiment was also carried out in the absence of H 2 O 2 .

Antioxidant
Property. 2,2 -diphenyl-1-picrylhydrazyl (DPPH•) scavenging capacity (antioxidant activity) was measured according to the following procedure [21,22]. The concentration of DPPH• used for antioxidant activity was 50 M. Different concentration of the ligand and metal complexes in methanol was added to DPPH• in methanol solution and kept at room temperature for 30 min in dark. The reduction of the DPPH• was monitored by observing the decrease in absorbance at 517 nm using UV-Vis spectrophotometer. The radical scavenging capacity of the antioxidant was expressed in terms of % inhibition and IC 50 . The capability to scavenge the DPPH• was calculated using the following [23]: where 0 is the absorbance of DPPH• in methanol solution without an antioxidant and sample is the absorbance of DPPH• in the presence of an antioxidant. The IC 50 value is the concentration of the antioxidant required to scavenge 50% DPPH• and is calculated from the inhibition curve.

Antimicrobial
Activity. All the synthesized compounds were screened for their antibacterial activity against grampositive bacteria: Streptococcus pyogenes and Staphylococcus aureus, and gram-negative bacteria: Escherichia coli, Klebsiella mobilis, Aeromonas aquariorum, and Serratia marcescens, by well diffusion method [24]. Standard antibiotics, ampicillin and amoxicillin, were used as controls. Stock solutions of tested compounds were prepared in DMSO to a final concentration of 10 mg mL −1 . 20 mL of sterilized agar media was poured into each presterilized Petri dish and allowed to solidify by placing it in an incubator at 37 ∘ C for an hour. 24 h culture suspension was poured and neatly swabbed with the presterilized cotton swabs. Then holes of 5 mm diameter were punched carefully using a sterile cork borer, and these wells were completely filled with the prepared L or the metal complex solutions (50 L). These dishes were transferred to an incubator maintained at 37 ∘ C for 24 h. During this period, the test solution diffused and the growth of the inoculated microorganism was affected. The inhibition zone was developed and measured at the end of the incubation period. Experiments were performed in triplicate, and standard deviation was calculated.

Results and Discussion
The ligand was synthesized by three steps. In the first step Schiff 's base was obtained by the condensation of o-vanillin with 1,3-diaminopropane. The Schiff base was reduced using sodium borohydride in the second step. Finally ester compound is obtained by N-alkylation reaction using 2-bromoethylacetate in the presence of potassium carbonate. The ester compound obtained as yellow oily substance on complexation with metal ions forms powdered metal complexes.

Molar Conductance.
The molar conductance of the synthesized metal complexes was measured in DMSO at 10 −3 M solution. The values were found to be in the range of 12.72-16.56 mho cm 2 mol −1 suggesting the nonelectrolytic nature of the complexes.

NMR Spectra.
Formation of nickel and zinc complex is confirmed by comparing the 1 H NMR of ligand and its metal complex (Table S1). The N-methylene protons (H8) of ester part of L give the singlet at 3.35 . The methylene protons (H6) to amino part of L show the signal at 2.63 . The sharp singlet at 3.86 corresponds to methylene protons (H5) to phenyl ring ( Figure S2). These methylene proton signals undergo higher deshielding up to 0.1 to 0.5 . This demonstrates that the tertiary amine nitrogen is involved in coordination with metal ion. The ligand shows that multiple signals at 4.16 and 1.43 are assignable to methylene and methyl protons of ester groups. These signals are not altered in the metal complex (Table S1). This suggests that the ester group of L is free from coordination with the metal ions.
The aromatic protons of L show multiple signals in the region 6.58-6.87 . The sharp singlet at 3.86 corresponds to methoxy protons. These protons undergo smaller deshielding up to 0.06 . The 13 C NMR spectral data of L is compared with its ZnL complex ( Figure S3 and Figure 1). The 13 C NMR signal for ester group of L (C-9) is not altered in the zinc complex (Table S2). This suggests that the ester group of L is free from coordination with the metal ion. The signals for carbon atoms Bioinorganic Chemistry and Applications 5 adjacent to nitrogen (C-7 and C-13) are observed at 54.20 and 45.37 , respectively. These signals are shifted to lower value in the metal complexes. Similarly, the carbon atom adjacent to phenolic oxygen (C-1) of L is shifted to higher value in the ZnL complex. The shifts in the positions of the carbon atoms adjacent to nitrogen and phenolic oxygen clearly demonstrate the bonding of the two nitrogen atoms of tertiary amine and two oxygen atoms of phenol to the Zn(II) ion forming tetrahedral geometry.

Electronic Absorption Spectra.
The UV-Vis absorption spectra of L and its metal complexes in DMSO were recorded at room temperature ( Figure S4). The absorption spectrum of L shows bands at 267 and 335 nm, which are due to → * transitions of phenyl ring and H-bonding induced changes of OH proton-donor aromatic molecules and amine NH (intraligand charge transfer band), respectively. The spectrum of CuL ( Figure S5) displaying the band at 527 nm is assigned to 2 B 1 → 2 A 1 transition confirming the square planar geometry of the CuL ( Table 1). The magnetic moment value for CuL (1.85 BM) is consistent with the square planar Cu(II) system. The NiL complex showed absorption at 553 nm ascribed to d-d transition ( 1 A 1 → 1 A 2 ) which supports the square planar geometry around Ni(II) ion [25].

Mass Spectra.
ESI mass spectra of all the metal complexes support the proposed structure of the complexes. The copper complex shows main peak at m/z 580 corresponding to the molecular weight of the complex ( Figure S6). The fragmentation peaks of copper complex are observed at m/z 379 and 491. The molecular ion and fragmentation peaks have half intensity peaks due to isotopic distributions of copper ( 63 Cu and 65 Cu) [26,27]. The spectral result shows that metal complexes are monomeric in nature and the metal to ligand ratio is 1 : 1. Nickel and zinc complexes are the same as copper, supported by analytical and spectral analysis.
3.6. EPR Spectra. The X-band EPR spectrum of the copper complex was recorded at 300 K and at 77 K using TCNE as the marker ( Figure S7). The absence of a half-field signal at 1600 G due to the = ±2 transitions ruling out any Cu-Cu interaction suggests the monomeric nature of the CuL complex. The observed values are II (2.29) > ⊥ (2.11) > (2.0027), suggesting the unpaired electron is in the x2−y2 orbital ( Table 2). The II / II value calculated for CuL (138 cm) lies between 90 and 140 cm indicating a square planar structure around the Cu (II) ion [28][29][30]. The II value of 2.29 for the CuL complex indicates the covalent nature of the metal-ligand bond. The values are related to exchange interaction coupling constant ( ) by the expression = ( II − 2.0027)/( ⊥ − 2.0027). If < 4, the ligand forming the copper complex is regarded as a strong-field ligand. For the present square planar complex, = 2.67 indicates that the ligand is strong field and the metal-ligand bonding in the complex is covalent [31].
The bonding parameters 2 , 2 , and 2 which may be regarded as covalency of the in-plane bond, in-plane bond, and out-of-plane bond, respectively, were evaluated from the following expressions [ where = −828 cm −1 for Cu(II) d 9 system and is the electronic transition energy. The 2 value of 0.83 for CuL demonstrates that the complex has covalent character in the ligand environment. The observed 2 and 2 values indicate that there is an interaction in the in-plane bonding between the metal ion and ligand. This is also confirmed by orbital reduction factors, II and ⊥ : II = 2 2 and ⊥ = 2 2 . In the present investigation the trend II < ⊥ for the copper complex implies a considerable in-plane bonding is between the metal ion and the ligand [33].

Thermal Studies.
The metal complexes show gradual loss in weight due to the decomposition with increasing temperature ( Figure S8). The thermogram shows four decomposition steps within the temperature range of 25-1000 ∘ C. In the first step upto 100 ∘ C, the mass loss (3-4.2%) corresponds to loss of lattice water molecule. In the second step (100-275 ∘ C), the mass loss of 22-25% corresponds to removal of the ester and methoxy groups with evolution of CO 2 gas. The third step of decomposition is noticed in the temperature range 275-450 ∘ C with loss of 28-30% due to the removal of the amino part of the ligand in the complexes. The fourth stage of decomposition occurs in the range 450-1000 ∘ C, which corresponds to the removal of the remaining part of the ligand leaving metal oxide as a residue.

Absorption Spectroscopic Studies.
All the metal complexes show intraligand ( → * ) transition in the region 270-280 nm. On addition of DNA, this band of the complexes was affected resulting in the tendency of hypochromism lying in the range 20-32% and a slight bathochromic shift in the range of 1.6-1.7 nm (Figure 2). These phenomena indicate that the complexes probably interact with CT DNA by intercalation binding mode. The extent of hypochromism is commonly consistent with the strength of intercalative interaction [34]. In order to study the binding ability of the compounds with CT DNA, the binding constant, , was determined. The values of the ligand (1.23 × 10 5 M −1 ) and its copper complex (1.00 × 10 5 M −1 ) are comparable. But the values are found to be lower than those reported for typical intercalators (for ethidium bromide and [Ru(Phen) 2 (dppz)] 2+ ; the binding constants have been found to be of the order 1.4 × 10 6 and >10 6 M −1 ) [35].   ligand (L) and CuL with DNA is stronger than that of salen, salan [14], NiL, and ZnL (Table 1).

Fluorescence Spectral Studies.
The EB fluorescence displacement experiment has been widely used to investigate the interaction of metal complexes with DNA. The EB shows weak fluorescence in buffer solution. The fluorescence intensity of EB in presence of DNA can be greatly enhanced due to intercalation with DNA [36]. On addition of metal complexes (0-120 M) to DNA-EB mixture, the metal complex competes with EB to bind with DNA. This leads to a decrease in the binding sites of DNA available for EB, and hence quenching of fluorescence intensity of EB-DNA mixture occurs (Figure 3). The quenching plot illustrates that the quenching of ethidium bromide bound to DNA by metal complexes is in agreement with the linear Stern-Volmer equation. The value of sv for CuL, NiL, and ZnL is found to be 8.44 × 10 3 , 6.58 × 10 3 , and 7.89 × 10 3 , respectively.
The apparent binding constant ( app ) values obtained for the CuL, NiL, and ZnL compounds are found to be 6.25 × 10 5 , 5 × 10 5 , and 4 × 10 5 , respectively (Table 1). Furthermore, the quenching constants and binding constants calculated for the complexes suggest that the interaction of all the compounds with DNA occurs through intercalation. The DNA binding abilities of the complexes follow the order Cu(II) > Ni(II) > Zn(II), which is in conformity with the trend in DNA binding affinities obtained from absorption spectral studies.
Binding Analysis. The equilibrium binding constant and the number of binding sites can be analyzed according to the Scatchard equation [37,38]: where bin is the binding constant of complex with DNA and is the number of binding sites. From the plot of log   Table 1). The values of sv and bin suggest that the complexes interact strongly with DNA.  (Table S3). The limiting peak to peak separation (ΔEp) for Cu(III)/Cu(II) process is greater than 59 mV which revealed that the couple is quasireversible. The ratio of anodic to cathodic peak current value is 1.1 demonstrating the simple one-electron process. Further, the anodic peak is shifted towards positive potential value, and the cathodic peak is shifted towards negative potential with a function of scan rate 25 to 125 mVs −1 which supports quasireversible process.
The cyclic voltammetric technique provides information about interaction between the metal complexes and DNA. DNA is denatured in DMSO medium, so we recorded the CV of copper complex in Tris-HCl buffer containing 10% DMSO. In Buffer medium, the copper complex shows a Cu(II)/Cu(I) couple with Ep at −0.438 V and Ep at 0.137 V ( Figure S9). The separations of anodic and cathodic peaks (ΔEp) are found to be 0.575 V indicating quasireversible one-electron redox process (Table S4). In the presence of CT DNA with R = 10 (R = [DNA]/[complex]) both the anodic and cathodic peak currents are decreased with shifting of potential values indicating that there exist interactions between copper complex and CT DNA. The drop of the voltammetric current in the presence of CT DNA is due to slow diffusion of the copper complex bound to CT DNA. The formal potential, 1/2 , taken as the average of Ep and Ep shifts slightly towards the positive side on binding to DNA which suggests that copper complex binds intercalatively to CT DNA [39]. 3.10. Antioxidant Property. The antioxidant activity of the ligand and the metal complexes was measured in terms of their hydrogen donating or radical scavenging capability by DPPH assay method. Upon addition of metal complexes, the reduction of DPPH radical is monitored by the decrease of the absorbance of its radical at 517 nm ( Figure 6). The absorbance decreases as a result of color changes from purple to yellow as the radical is scavenged by antioxidants. The 50% inhibitory concentration (IC 50 ) values of L, CuL, NiL, and ZnL are 103, 1019, 771, and 1429 M, respectively. The higher free radical scavenging activity of the ligand may be due to the presence of free phenolic -OH groups. The IC 50 values of synthesized compounds are much higher than the positive control like ascorbic acid, 11.55 M [40].
3.11. Antibacterial Activity. The in vitro antibacterial activities of the synthesized compounds were tested against six human pathogenic microorganisms (gram-positive bacteria: Streptococcus pyogenes and Staphylococcus aureus; gram-negative bacteria: Escherichia Coli, Klebsiella mobilis, Aeromonas aquariorum, and Serratia marcescens) by well diffusion method using ampicillin and amoxicillin as standards. The susceptibility of the strains of bacteria towards the present compounds was judged by measuring the size of inhibition diameter ( Figure S11). The comparison of the antimicrobial activity of the synthesized compounds and the known antibiotics showed that the metal complexes were more effective than the ligand or metal salts but less active than the controls against all the bacteria tested. The bulky N-alkylated ligand on chelation to the metal cation reduces the polarity of the metal ion due to the ligand orbital overlap with the metal orbitals, resulting in a delocalization of positive charge. This increases the lipophilic character of the metal chelate and favors its permeation through the lipoid layer of the bacterial membranes. Cu(II) complex has higher antibacterial activity against Streptococcus pyogenes and Escherichia Coli than the other metal complexes. Zn(II) complex has higher activity against Klebsiella mobilis, Aeromonas aquariorum. Ni(II) complex is found to have moderate activity towards the bacteria tested.

Conclusion
Salan-type ligand containing ester groups was synthesized. The N-alkylated salan was used to prepare Cu(II), Ni(II), and Zn(II) complexes. The synthesized compounds were characterized by spectral and analytical techniques. Spectral studies reveal that the ligand coordinates to the metal ion through the phenolic -O and tertiary -N atoms. The presence of ester groups in tertiary -N leads to distortion from the regular square planar geometry of the complexes. The cyclic voltammogram of copper complex reveals that the complex exhibits well-defined quasireversible Cu(III)/Cu(II) couple along with Cu(II) irreversible process. The synthesized compounds bind to CT DNA through intercalation mode. Copper complex has been found to promote cleavage of pUC19 DNA from the super coiled form to nicked form in presence of H 2 O 2 . The metal complexes show higher bacterial activity than the ligand. The ligand shows more effective free radical scavenger activity than the metal complexes.