Synthesis, Crystal Structure, Antioxidant, Antimicrobial, and Mutagenic Activities and DNA Interaction Studies of Ni(II) Schiff Base 4-Methoxy-3-benzyloxybenzaldehyde Thiosemicarbazide Complexes

Three new Ni(II) square planar complexes of 4-methoxy-3-benzyloxybenzaldehyde thiosemicarbazide(4m3BTSC) having polypyridyl bases of general formulation [ML 2 ] (1) and [MLB] (2, 3), where L = 4m3BTSC and B is N,N-donor heterocyclic bases, namely, 1,10-phenanthroline (phen, 2), 2,2-bipyridine (bpy, 3), are synthesized and characterized.The free radical scavenging assay results showed that complex 1 possesses significant activity when compared to complexes 2 and 3. The biological studies showed that the ligand and its complexes exhibited significant and different biological activities and also the prepared compounds are nonmutagenic. They may be potential commercial antioxidants because of their nonmutagenic and nontoxic nature. The DNA interaction of the new complexes is evaluated by absorption, emission, andmelting temperature methods, and the results suggested that the binding affinity of the complexes increases with the presence of planar ligand in the molecule. The nickel (II) complexes with planar phenanthroline bases show moderate DNA binding and cleavage ability.


Introduction
Schiff bases have great importance in coordination chemistry due to their ability to form a range of complexes with applications in different fields [1].Thiosemicarbazones have variable bonding modes, ability to form stable chelates with metal ions, and structural diversity [2].The sulfur, oxygen, and nitrogen may be involved in coordination providing a useful model for bioinorganic processes [3].These thiosemicarbazide ligands have good complexing ability, and their activity increases on complexation with transition metal ions [4].These compounds are also of interest due to their biological activities including enzyme inhibition [5] and antifungal and pharmacological applications [6,7].The toxicological importance of the -N-C=S moiety has been well established in antifungal, antibacterial agents and pesticidal activities [8,9].It has been suggested that the azomethine linkage in Schiff bases is responsible for the biological activities such as antitumor, antibacterial, antifungal, and herbicidal activities [10].
The biological activities of thiosemicarbazones often showed a high dependence on their substituents.Minor modifications in the structure of thiosemicarbazones can lead to widely different biological activities.The biological properties of thiosemicarbazones are often modulated by metal coordination.In some cases, the highest biological activity is associated with a metal, and some side effects may decrease upon complexation [11].
Nickel(II) complexes have found several potential applications in medicine, and very recently they have been screened for inhibition of cancer cell proliferation [12,13].In the last 20 years transition metal complexes have become increasingly important as drugs and artificial nucleases.One goal of designing Ni(II) complexes is developing more reactive chemical systems that can efficiently cleave DNA under physiological conditions [14][15][16].
Substantial progress has been made during the past few decades to develop metal-based small molecules as DNA foot-printing as well as the therapeutic agents that are capable of binding and cleaving DNA under physiological condition [17,18].Among the metal complexes so far investigated, those of polypyridyl phenanthroline bases have attracted great attention by virtue of its binding propensity to nucleic acid under the physiological condition [19,20].
In view of the varying ligation behavior and interesting biological activity shown by these nitrogen-sulfur ligands and their complexes, we are also interested to prepare the thiosemicarbazide Schiff base and its metal complexes.Designing of these metal complexes has been undertaken, and their biological activities have been studied.The study of biological activities of benzyloxy benzaldehyde-Schiff bases is less explored.Herein, we present the syntheses, structure, antioxidant, antimicrobial, and mutagenic activities and DNA interactions of nickel(II) complexes of general formulae 3) where L is ligand 4m3BTSC (4-methoxy-3-benzyloxybenzaldehyde thiosemicarbazide) and B is N,N-donor heterocyclic base, namely, 1,10-phenanthroline (phen), 2,2  -bipyridine (bpy).

Materials and Instrumentation.
All reagents and chemicals were of AR grade and used as purchased.The 4-methoxy-3-benzyloxy benzaldehyde, thiosemicarbazide, and various metal salts were Merck products and used as supplied.The agarose (molecular biology grade) and ethidium bromide (EB) were obtained from Sigma.Calf thymus (CT) DNA and Supercoiled (SC) pUC19 DNA (cesium chloride purified) were purchased from Bangalore Genie (India).The tris(hydroxymethyl)-aminomethane-HCl (tris-HCl) buffer was prepared by doubly distilled H 2 O.
Bacterial Strains.The four Salmonella typhimurium mutant strains (histidine-dependant) TA98, TA100, TA1535, and TA1538 were procured from IMTECH, Chandigarh, India.For mutagenicity assay, each bacterial culture was inoculated in 10 mL of fresh nutrient broth and incubated at 37 ∘ C for 14 h.All chemicals and bacterial media were purchased from Himedia.
The elemental analyses were carried out by using variomicro CHNS15106062 analyzer. 1H NMR and ESI-MS data of the compounds were recorded at the Sophisticated Analytical and Instrument Facility, IISc, Bangalore.The IR spectra of the samples were recorded on a Shimadzu spectrophotometer from 4000 to 400 cm −1 using KBr pellets.The UV-Vis spectra were recorded on a Shimadzu UV-3101PC spectrophotometer using DMF as solvent.The molar conductances of the complexes were measured using Equiptronics digital conductivity meter number EQ-660A, and the melting points were checked by melting point apparatus used in laboratories.Magnetic susceptibility data at 300 K for the polycrystalline samples of the complexes were obtained by VSM, IIT Madras.

Synthesis.
The Schiff base ligand 4m3BTSC was synthesized by reported procedure [21].
phen, bpy) Complexes.The complexes were prepared by following a modified reported [22] synthetic procedure in which a 10 mL methanolic solution of Ni(CH 3 COO) 2 ⋅4H 2 O (0.24 g, 1 mmol) was reacted with 4m3BTSC (0.315 g, 1 mmol) in acetonitrile (10 mL) under magnetic stirring at room temperature.The resulting precipitate was filtered off, washed with methanol, and air-dried.The obtained precipitate was dissolved in dimethyl formamide and leaving it to stand for a week on slow evaporation of the solution at room temperature yielded a crystalline material (1).Crystals of complexes were suitable for X-ray diffraction studies.After 30 min, a 20 mL methanolic solution of the heterocyclic base [phen (0.198 g, 1 mmol) bpy (0.156 g, 1 mmol)] was added to the solution, and the resulting mixture was stirred for 2 h at room temperature.On cooling the solution to an ambient temperature, it was filtered and the filtrate on slow concentration yielded a crystalline solid of the product (2, 3).The solid was isolated and washed with cold methanol and finally dried over P 4 O 10 .The FT-IR, 1H NMR and ESI-MS spectras for all the complexes are given in supplementary material (Figures S1-S9).

X-Ray Crystallographic
Procedures.Single crystal of the complex 1 was grown by slow evaporation of DMF solution.Darkgrey single crystal was mounted on a glass fiber with epoxy cement.The X-ray diffraction data were measured in frames with increasing  (width of 0.3 ∘ per frame) and with a scan speed at 15 s/frame on a Bruker SMART APEX CCD diffractometer, equipped with a fine focus 1.75 kW sealed tube X-ray source.Empirical absorption corrections were carried out using multiscan program [23].The structure was solved by the heavy atom method and refined by full matrix leastsquares using SHELX system of programs [24].

Antioxidant Activity
The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm which can be induced by antioxidants [25].Antioxidant activity of ligand and metal complexes was determined by the method described by Brand-Williams et al. [26].The complex solutions (100 L, 1 mmol) were mixed with 0.8 mL of tris-HCl buffer (pH 7.4) to which 1 mL of DPPH • (500 M in ethanol) was added.The mixture was shaken vigorously and left to stand for 30 min.Absorbance of the resulting solution was measured at 517 nm in a Uv-Vis Spectrophotometer.The radical scavenging activity was measured by decrease in the absorbance of DPPH.Lower absorbance of the reaction mixture indicated higher free radical scavenging activity.

Antimicrobial Activity.
The antibacterial activity was tested against clinical isolates like Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa.The test organisms were maintained on nutrient agar slants.In vitro antibacterial activity was determined by the agar well-diffusion method as described by Mukherjee et al. [27].The overnight bacterial culture was centrifuged at 8000 rpm for 10 min.The bacterial cells were suspended in saline to make a suspension of 10 5 CFU/mL and used for the assay.Plating was carried out by transferring the bacterial suspension to a sterile Petri plate and mixed with molten nutrient agar medium, allowing the mixture to solidify.About 75 L of the sample (2 mg/mL) was placed in the wells.Plates were incubated at 37 ∘ C, and activity was determined by measuring the diameter of the inhibition zones.The assay was carried out in triplicate.

Mutagenicity Assay. Mutagenicity of compounds was
studied by the preincubation assay as described by Maron and Ames [28].100 L of fresh culture of Salmonella strains was treated with test compounds dissolved in DMSO with concentrations of 2, 5, and 10 mg/plate at 37 ∘ C. For the mutagenicity assay, the controls and compound-treated cells were mixed with 2 mL of sterile top agar and poured onto minimal glucose agar plate.The plates were then inverted and incubated at 37 ∘ C for 48 hrs.Revertant colonies of bacteria were counted manually after 48 h.Each experiment consisted of three replicates for each concentration.

DNA Binding Experiments.
The UV absorbance at 260 and 280 nm of the CT-DNA solution in 5 mM tris-HCl buffer (pH 7.2) gave a ratio of 1.9, indicating that the DNA was free of protein [29].The concentration of CT DNA was measured from the band intensity at 260 nm with a known  value (6600 M −1 cm −1 ) [30].Absorption titration measurements were done by varying the concentration of CT DNA, keeping the metal complex concentration constant in 5 mM tris-HCl/5 mM NaCl buffer (pH 7.2).Samples were kept for equilibrium before recording each spectrum.The intrinsic binding constant (  ) for the interaction of the complexes with CT-DNA was determined from a plot of [DNA]/(  −  ) versus [DNA] using absorption spectral titration data and the following equation: where [DNA] is the concentration of DNA in base pairs.The apparent absorption coefficients   ,   , and   correspond to  obsd./[Ni], the extinction coefficient for the free nickel(II) complex, and extinction coefficient for the nickel(II) complex in the fully bound form, respectively [31].  is given by the ratio of the slope to the intercept.The apparent binding constant ( app ) of the complexes was determined by fluorescence spectral technique using ethidium bromide (EB) bound CT DNA solution in tris-HCl/NaCl buffer (pH 7.2).The fluorescence intensities of EB at 600 nm (546 nm excitation) with an increasing amount of the ternary complex concentration were recorded.Ethidium bromide was nonemissive in tris-buffer medium due to fluorescence quenching of the free EB by the solvent molecules [32,33].In the presence of DNA, EB showed enhanced emission intensity due to its intercalative binding to DNA.A competitive binding of the copper complexes to CT DNA could result in the displacement of EB or quenching of the bound EB by the paramagnetic nickel(II) species decreasing its emission intensity.
DNA-melting experiments were carried out by monitoring the absorbance of CT-DNA (200 M NP) at 260 nm with varying temperature in the absence and presence of the complexes in 2 : 1 ratio of DNA to complex with a ramp rate of 0.5 ∘ C min −1 in phosphate buffer (pH 6.85) using a Peltier system attached to the UV-Vis spectrophotometer.
2.8.DNA Cleavage.The extent of cleavage of supercoiled (SC) DNA in the presence of the complex and oxidizing agent H 2 O 2 was determined by agarose gel electrophoresis.In a typical reaction, supercoiled pUC19 DNA (0.2 g), taken in 50 mM tris-HCl buffer (pH 7.2) containing 50 mM NaCl, was treated with the complex.The extent of cleavage was measured from the intensities of the bands using UVITEC Gel Documentation System.For mechanistic investigations, inhibition reactions were done on adding the reagents prior to the addition of the complex.The solutions were incubated  The proposed structures of complexes 2 and 3 are given in Figure 1.The molar conductance values of 1 mM solutions of the metal complexes in DMF at room temperature are in the range of 8-12 Ω −1 cm 2 mol −1 , indicating their nonelectrolytic nature [35].They are characterized from the analytical and physicochemical data (Table 1).The complexes 2 and 3 display an intense charge transfer (CT) band in the range 200-310 nm, which can be attributed to the  →  * transition of the coordinated N,N-donor heterocyclic base.The band around 330 and 340 nm in all the complexes is due to the imine (-HC=N-) and C=S groups, and the occurrence of two d-d bands in the regions 527-538 and 430-438 nm ( 1 A 1 g→ 1 B 2 g and 1 A 1 g→ 1 B 1 g transitions) is consistent with a square planar structure for all the all complexes.

Results and Discussion
3.2.Crystal Structure of 1.The Ni(L) 2 (DMF) complex is structurally characterized using single-crystal X-ray diffraction technique.The ORTEP diagram and numbering scheme for Ni(L) 2 are shown in Figure 2. Crystallographic data for complex 1 is presented in Table 2. X-ray studies reveal that the complex has square planar geometry as is evident from their bond parameters and bond angles (Table 3).Complex 1 crystallizes in the centrosymmetric P2 1 /n space group of the monoclinic crystal system with cell parameters,  = 15.669(5)Å,  = 9.127(5) Å,  = 16.227(5)Å, and

IR Spectra.
In principle, the ligand can exhibit thionethiol tautomerism since it contains a thioamide -NH-C=S functional group.The ](S-H) band at 2565 cm −1 is absent in the IR spectrum of ligand, but ](N-H) band at ∼3265 cm −1 is present, indicating that in the solid state the ligand remains as the thione tautomer.The position of ](C=N) band of the thiosemicarbazone appeared at 1616 cm −1 is shifted towards lower wave number in the complexes indicating coordination via the azomethine nitrogen.This is also confirmed by the appearance of band in complexes in the range of 450-455 cm −1 , which has been assigned to the ](M-N) [36].A medium band found at 1014 cm −1 is due to the ](N-N) group of the thiosemicarbazone.The position of this band is shifted towards higher wave number in the spectra of complexes.
It is due to the increase in the bond strength, which again confirms the coordination via the azomethine nitrogen.The band appearing at ∼830 cm −1 corresponding to ](C=S) in the IR spectrum of ligand is shifted towards lower wave number.It indicates that thione sulfur coordinates to the metal ion [37].Thus, it may be concluded that the ligand behaves as bidentate chelating agent coordinating through azomethine nitrogen and thiolate sulfur.The C-H stretching and bending vibrations appear at 2780-2890 cm −1 and 1460-1420 cm −1 , respectively.There is no appreciable change in the other signals of the complexes (Table 4).

DPPH Radical Scavenging Activity.
Various researchers have used scavenging effect of a chemical on DPPH radical as a quick and reliable parameter to assess the in vitro antioxidant activity.The efficient concentration of complex required to scavenge 50% of DPPH, EC 50 , is shown in Table 5.The lower the EC 50 is, the better its radical scavenging activity is.
It is evident from results that free radical scavenging activities of these compounds were concentration dependent.Among the examined compounds, the complex 1 showed a strong interactive ability with DPPH, expressed as EC 50 value of 40 M concentration, which was close to that of ascorbic acid (30 M).Maximum free radical scavenging activity (56.9%) was found in complex 1 followed by complex 3 (52.41%)and complex 2 (51.8%), while least activity (32.88%) was observed for free ligand 4m3BTSC.The comparative time-dependent antioxidant activity of compounds and standards were shown by graph in Figure 4. Antiradical efficiency (AE) involves the potency (1/EC 50 ) and the reaction time ( EC 50 ).The AE of all the complexes are in the range of 2.5-3.5 × 10 −3 , the value near to standards and the complexes are good antioxidants [38].

Antibacterial Activity.
The in vitro antibacterial activity of the Schiff bases and their Ni(II) complexes were evaluated against three Gram positive, S. aureus, B. subtilis, and M. luteus, and three Gram negative bacteria, P. aeruginosa, K. pneumoniae, and E. coli.Table 6 illustrates the antimicrobial activity of the synthesized compounds.In general, the synthesized Schiff bases have more antibacterial activity compared to their metal complexes; this may be due to the greater liphophilic nature of the Schiff bases than their metal complexes.The results showed that the complex 2 exhibited antibacterial activity against all the tested organisms, namely, E. coli, P. aeruginosa, K. pneumoniae, B. subtilis, M. luteus, and S. aureus.The result also indicated that the complex 1 exhibited antibacterial activity against E. coli and B. subtilis and that complex 3 exhibited antibacterial activities against K. pneumonia and B. subtilis, respectively.The factors that govern antimicrobial activities are strongly dependent on the central metal ion and coordination numbers and are also due to the presence of nitrogen and sulfur donor groups [39,40].

Mutagenic Assay.
The mutagenic activity was carried out by Ames test.The results indicate that all the complexes are nonmutagenic in nature under assay condition.According to the Ames test, the tested ligand and its complexes did not show mutagenicity with bacterial strain Salmonella typhimurium types TA 98, TA 100, TA 1535, and TA 1538 in the assayed range.
3.8.DNA Binding Properties.It was reported that DNA binding mode and affinity are affected by a number of factors, such as planarity of ligands [41], the coordination geometry, the ligand donor atom type [42], the metal ion type, and its flexible valence [43].The UV binding of the complexes 1-3 to the calf thymus (CT) DNA has been studied by electronic absorption spectral technique.The intrinsic binding constant (  ) value was 3.99(±0.04)× 10 6 M −1 for complex 2 (Figure 5).The complexes 1 and 3 did not show binding affinity to CT-DNA due to lack of planarity.The experimental values of   revealed that complex 2 binds to DNA via intercalative mode, and this may be due to the presence of the phenanthroline ring in the structure.
The emission spectral method is used to study the relative binding of the complexes to CT-DNA.The emission intensity of ethidium bromide (EB) is used as a spectral probe.EB shows reduced emission intensity in buffer solution because of solvent quenching and an enhancement of the emission intensity when intercalatively bound to DNA.The binding of the complexes to DNA decreases the emission intensity of EB (Figures 6(a), 6(b), and 6(c)).The relative binding propensity of the complexes to DNA is measured from the extent of reduction in the emission intensity (Figure 7).The apparent binding constant ( app ) values for 1, 2, and 3 are given in Table 7.The phen complex showed significantly higher  app values than those of the bpy and bis ligand complexes.The bis ligand complex is a poor binder to DNA.The nature of the binding of the complexes to CT-DNA was further investigated by DNA melting experiment.Thermal behavior of the DNA in the presence metal   complexes can give insight into their conformational changes and information about the interaction strength with DNA.DNA melting is observed when ds DNA molecules are heated and separated into two single strands; it occurs due to a disruption of the intermolecular forces, such as  stacking and hydrogen bonding interactions, between DNA base pairs.A classical intercalator such as EB stabilizes the duplex DNA, causing the DNA to melt at a higher temperature [44].The melting temperature of CT-DNA in the absence of any added complex was found to be 65.5 ∘ C in our experimental conditions.Under the same set of conditions, in the presence of all the complexes, they gave Δ  values ranging from 0.4 to +0.8 ∘ C indicating primarily DNA groove binding propensity of the complexes.The phen complex 2 with a relatively high Δ  value of +0.8 ∘ C could have a partial intercalative mode of DNA binding with the planar ring of 1,10-phen intercalating to the DNA base pairs.8.The phen complex 2 (60 M) shows moderate "chemical nuclease" activity, and 35% conversion of the SC (form I) to its nicked-circular form (NC, form II) of DNA is observed.Control experiments with H 2 O 2 or the complexes alone did not show any apparent conversion of SC to its nicked-circular (NC) form (Figure 8).The bpy complex 3 and bis ligand complex 1 are inactive due to their poor binding ability to DNA.The pathways involved in the DNA cleavage reactions are believed to be analogous to those proposed by Sigman and coworkers for the chemical nuclease activity of bis(phen)copper species (Scheme 1) [45,46].Based on the mechanistic studies it is observed that chemical nuclease activity is high in oxidizing condition compared to reducing condition.

Conclusions
Three new Ni(II) complexes having N,S-donor ligand and N,N-donor heterocyclic bases are prepared and characterized.The complex 1 was structurally characterized by X-ray crystallography, and the structure shows the square planar geometry.The free radical scavenging assay results showed that complex 1 posseses significant activity when compared to complexes 2 and 3.The biological studies showed that the ligand and its complexes exhibited significant and different biological activities and also the prepared compounds are nonmutagenic.They may be potential commercial antioxidants because of their nonmutagenic and nontoxic nature.
The DNA interaction of the new complexes was evaluated by absorption, emission, and melting temperature methods, and the results suggested that the binding affinity of the complexes increases with the presence of planar ligand in the molecule.The nickel(II) complexes with planar phenanthroline bases show moderate DNA binding and cleavage ability.

Figure 3 :
Figure 3: (a) Molecular packing for the complex NiL 2 , viewed down the c-axis; (b) dashed lines indicate hydrogen bonds.

Figure 6 :
Figure 6: The emission spectral changes on addition of complexes 1 (a), 2 (b), and 3 (c) to the CT-DNA bound to ethidium bromide (shown by arrow).

Figure 7 :
Figure 7: The effect of addition of metal complexes 1, 2, and 3 to the emission intensity CT-DNA bound ethidium bromide in a 5 mM tris-HCl (pH = 7.2) at 25 ∘ C.

3. 9 .
DNA Cleavage Studies.The oxidative DNA cleavage activity of the complexes in the presence of oxidizing agent hydrogen peroxide (H 2 O 2 , 50 mM) is investigated by agarose gel electrophoresis using supercoiled (SC) plasmid pUC19 DNA (0.2 g, 33.3 M NP) in 50 mM tris-HCl/50 mM NaCl buffer (pH 7.2), and the selected DNA cleavage data are given in Table

Scheme 1 :Figure 8 :
Scheme 1: Proposed mechanistic pathway for the chemical nuclease activity of complexes in tris-buffer medium.

Table 1 :
[34]ytical and physical data of the ligand and its complexes.Melting point, b KBr Phase, c in DMF medium, d in DMF medium at 25 ∘ C, and e  eff for solid at 300 K.for 1 h in a dark chamber at 37 ∘ C followed by addition to the loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol (2 L), and the solution was finally loaded on 0.8% agarose gel containing 1.0 g/mL ethidium bromide (EB).Electrophoresis was carried out for 2 h at 60 V in tris-acetate-EDTA (TAE) buffer.Bands were visualized by UV light and photographed for analysis.Due corrections were made to the observed intensities for the low level of NC form present in the original sample of SC DNA and for the low affinity of EB binding to SC in comparison to nicked-circular (NC) and linear forms of DNA[34]. a
1H NMR Spectra.The 1 H NMR spectra of ligand and its nickel complexes were recorded in CDCl 3 .The 1 H NMR spectrum of 4m3BTSC shows signal at 8.30  indicating the presence of azomethine (HC=N) proton.The singlet peaks between 9.3-10.1  and ∼6.8  are assigned for the presence of NH and NH 2 protons; however the multiplet in the region 7.10-7.6 indicates the presence of the phenolic protons.No signals are observed in the range 11.3-11.4 which indicates the absence of thiol (-SH) group.The other important bands were observed at ∼3.9  assigned for methyl (O-CH 3 ) protons and 5.16  assigned for methylene (O-CH 2 ) protons.The azomethine proton in the Ni(II) complexes is shifted downfield compared to free ligand, suggesting deshielding of the azomethine group due to coordination with nickel.

Table 6 :
The values of zone inhibition (mm) of microorganisms for the 4m3BTSC and its complexes 1, 2, and 3.

Table 7 :
Intrinsic binding, apparent DNA binding, and melting temperature studies.
a Intrinsic binding constant from absorption spectral method.b Apparent binding constant from competitive binding assay by emission method.c Changes in melting temperature of CT-DNA.

Table 8 :
Selected cleavage data of SC pUC19 by ligand and its complexes 1, 2, and 3.
a SC: supercoiled DNA.b NC: nicked circular DNA.