Synthesis, Electrochemical, Spectroscopic, Antimicrobial, and Superoxide Dismutase Activity of Nickel (II) Complexes with Bidentate Schiff Bases

Five new nickel (II) complexes, namely, [Ni(L 1 ) 2 ](ClO 4 ) 2 ( 1 ); [Ni(L 2 ) 2 ](ClO 4 ) 2 ( 2 ); [Ni(L 3 ) 2 ](ClO 4 ) 2 ( 3 ); [Ni(L 4 ) 2 ](ClO 4 ) 2 ( 4 ); [Ni(L 5 ) 2 ](ClO 4 ) 2 ( 5 ), where L 1 = benzoylhydrazide; L 2 = N-[(1)-1-(2-methylphenyl)ethylidene]benzohydrazide; L 3 =N-[(1)-1-(4-methylphenyl)ethylidene]benzohydrazide; L 4 =N-[(1)-1-(2-methoxyphenyl)ethylidene]benzohydrazide; L 5 = N-[(1)-1-(4-methoxy-phenyl)ethylidene]benzohydrazide, have been synthesized and characterized by various physicochemical and spectroscopic techniques. The synthesized complexes are stable powders, insoluble in common organic solvents such as ethanol, benzene, carbon tetrachloride, chloroform, and diethyl ether, and are nonelectrolytes. The magnetic and spectroscopic data indicate a distorted square planar geometry for all complexes. The superoxide dismutase activity of these complexes has been measured and discussed. Antibacterial and antifungal properties of these complexes were also tested.


Introduction
The synthesis of low molecular weight nickel (II) complexes mimicking superoxide dismutase (SOD) activity has been challenging for bioinorganic chemists and recently some complexes with high catalytic activity have been reported [1][2][3]. Nickel-containing superoxide dismutase (Ni-SOD) has been isolated from several Streptomyces species [4]. The enzymatic activity of Ni-SOD [5] is as high as that of Cu-Zn SOD at about 10 9 M −1 S −1 per metal center. Oberley and Buettner [6] have reported that cancer cells had less superoxide dismutase (SOD) activity than normal cells. Superoxide ion is toxic to cells; a defense mechanism must have been initiated by nature. All organisms, which use dioxygen and many that have to survive an oxygenated environment, contain at least one SOD. The superoxide radical (O 2 − ) is an inevitable byproduct of aerobic metabolism which if not eliminated may cause significant cellular damage and has been implicated in numerous medical disorders [7]. To avoid such harmful consequences, all oxygen metabolizing organisms possess metalloenzymes known as superoxide dismutases (SODs). These SODs disproportionate the toxic O 2 − radical to molecular oxygen and hydrogen peroxide [8,9]. All SODs employ the two-step Ping-Pong mechanism shown in where M is a redox active metal center capable of both oxidizing and reducing superoxide. Metal complexes of bidentate Schiff bases have been extensively studied [10,11], because such ligands can bind with one, two, or more metal centers involving various coordination modes and allow synthesis of homo-and heteronuclear metal complexes with interesting stereochemistry [12,13]. A number of papers [14,15] highlight the flexible nature of bidentate ligands, their analytical, and biological properties. Recently, we have studied [16][17][18] various Schiff bases and their metal (II) complexes. This paper describes synthesis, electrochemical, spectroscopic, antimicrobial, 2 International Journal of Inorganic Chemistry and superoxide dismutase (SOD) activity of nickel (II) complexes namely [Ni( (4), and [Ni(L 5 ) 2 ] (ClO 4 ) 2 (5). All the Schiff bases (L 2 , L 3 , L 4 , and L 5 ) are bidentate ligand having donor site nitrogen and oxygen atom (Scheme 1). Superoxide dismutase activity of all these complexes has been revealed to catalyze the dismutation of superoxide (O 2 − ), and IC 50 values were evaluated and discussed.

Experimental
2.1. Physical Measurements. Nickel (II) chloride hexahydrate was purchased from S. D. Fine-Chem Limited, India. All other chemicals used were of synthetic grade and used without further purification. Elemental analyses were performed on an Elementar Vario EL III Carlo Erba 1108 analyzer. FAB mass spectra were recorded on a JEOL SX 102/DA 6000 mass spectrometer data system using xenon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was 10 kV and the spectra were recorded at room temperature. UV-Vis spectra were recorded at 25 ∘ C on a Shimadzu UV-Vis recording spectrophotometer UV-1601 in quartz cells. IR spectra were recorded in KBr medium on a Perkin-Elmer 783 spectrophotometer in the 4000-600 cm −1 region. Cyclic voltammetry was carried out with a BAS-100 Epsilon electrochemical analyzer having an electrochemical cell with a three-electrode system. Ag/AgCl was used as a reference electrode, glassy carbon as working electrode, and platinum wire as an auxiliary electrode. 0.1 M NaClO 4 was used as supporting electrolyte and DMSO as solvent. Molar conductivities of freshly prepared 2 × 10 −3 M of acetonitrile solutions were measured on a Systronics conductivity TDS meter 308. The in vitro SOD activity was measured using alkaline DMSO as a source of superoxide radical (O 2 − ) and nitroblue tetrazolium chloride (NBT) as O 2 − scavenger [19]. In general, 400 L sample to be assayed was added to a solution containing 2.1 mL of 0.2 mol L −1 potassium phosphate buffer (pH 8.6) and 1 mL of 56 mol L −1 alkaline DMSO solution prepared under similar condition in DMSO (except NaOH). A unit of SOD activity is the concentration of complex, which causes 50% inhibition of alkaline DMSO-mediated reduction of NBT. The in vitro antimicrobial (antibacterial) activities of these complexes were tested using paper disc diffusion method [20]; the chosen strains were Streptococcus aureus and Escherichia coli. The liquid medium containing the bacterial subcultures was autoclaved for 20 min at 121 ∘ C and at 15 lb pressure before inoculation. The bacteria were then cultured for 24 h at 36 ∘ C in an incubator. The antifungal activity of the present complexes has been evaluated against Aspergillus sp. and Penicillium sp. by the Radial Growth Method [21] using Czapek's agar medium. The compounds were added directly with the medium in 5, 10 and 15 mM concentrations.

2.2.
Synthesis of L 2 , L 3 , L 4 , and L 5 . The Schiff bases L 2 , L 3 , L 4 , and L 5 were prepared by general condensation reaction and recrystallized from ethanol or methanol. A methanol

Spectroscopic Study.
The room temperature ligand field spectra (electronic spectra) of these complexes have been recorded in 100% DMSO solution at 25 ∘ C. In present Ni (II) complexes, the highest energy d-d energy transition from the lower lying fully occupied 3d 2 − 2 orbital to the upper empty 3d orbital ("B ← A" transition) at 405 ± 5 nm is obscured by the MLCT transition. The other three lower energy d-d transitions from the occupied 3d 2 , 3d , and 3d orbitals to the empty 3d orbital ( 1 B ← 1 A, 1 B ← 1 A, and 1 A ← 1 A transition, resp.) are less intense, appear as a broad envelope around at the range 615 ± 5 nm region, and are typical of square planar monoligated nickel (II) complexes [24]. In addition, MLCT transition is located at the range 420 ± 5 nm. The azomethine (-HC=N-) characteristic band in the IR for the free ligand was observed at ∼1650 ± 5 cm −1 . The IR-spectra of the complexes show coordination C=N bonds [25] in the range of 1602-1628 and of 460-490 cm −1 . A strong band, observed at ca. 1205-1329 cm −1 is assigned to coordination through phenolic oxygen [26]. In addition, these complexes show strong bands at 1053 cm −1 and 1083 cm −1 indicating the presence of ClO 4 groups [27] in agreement with their noncoordinating character. Vibrations at ∼460 weak (and expected below 400 cm −1 , out of our measuring limit) can be attributed to M-O and M-N vibrations [28].

Electrochemical Studies.
Electroactivity of the complexes was studied in DMSO with 0.1 M NaClO 4 as supporting electrolyte using cyclic voltammetry at a platinum working electrode. "Supplementary material" and redox potential values are given in Table 1. The redox processes assigned as Ni (II)/Ni (I) couples are fully irreversible [29]. The voltammograms of these complexes consist of two well-separated peaks, one cathodic potential ( pc ) and one anodic potential ( pa ). In these complexes reduction waves are observed at more negative potentials. In these cases, the peak potential differences increase as the scan rate increases. Constancy of 0 shows that in all the cases both peaks are complementary to each other. The peak current ratio pa / pc is less than unity showing that the electron transfer reaction is followed by a chemical reaction (EC mechanism) [30].

Superoxide Dismutase Activity.
The SOD activities for the complexes were measured. Superoxide was enzymatically supplied from alkaline DMSO and SOD activity was evaluated by the NBT assay [31] following the reduction of NBT to MF + kinetically at 560 nm. These complexes exhibit significant catalytic activity towards the dismutation of superoxide anions. The concentration causing 50% inhibition of NBT reduction is IC 50 . The SOD activity of [Ni(L 1 ) 2 ](ClO 4 ) 2 (1) was shown in Figure 1. The observed IC 50 values of the nickel (II) complexes (50 for 1, 40 for 2, 49 for 3, 41 for 4, and 48 for 5 mol dm −3 ) are higher than the value exhibited by the native enzyme (IC 50 = 0.04 mol dm −3 ) on a molar base (note that the smaller the IC 50 value, the higher the SOD activity). The observed IC 50 values of the present complexes are comparable to reported values [32] for nickel (II) ( Table 2). The catalytic activity of NiSOD [6], however,   [Ni(L 1 ) 2 ](ClO 4 ) 2 (1) 5 0 Th i s w o r k (8) [Ni(L 2 ) 2 ](ClO 4 ) 2 (2) 4 0 Th i s w o r k (9) [Ni(L 3 ) 2 ](ClO 4 ) 2 (3) 4 9 Th i s w o r k (10) [Ni(L 4 ) 2 ](ClO 4 ) 2 (4) 4 1 Th i s w o r k (11) [ 3.5. Antimicrobial Activity. The in vitro antimicrobial (antibacterial) activities of these complexes were tested using paper disc diffusion method; the chosen strains were G (+) Streptococcus aureus and Escherichia coli. Three concentrations of the present complexes were taken, that is, 5 mM, 10 mM, and 15 mM. Paper disc were prepared and dipped with the help of these different solution. The susceptibility of the certain strains of bacterial towards the nickel (II) was determined by measuring the size of inhibition diameter. The growth inhibitory effects were observed against all the bacterial/fungal strains. Both bacteria are pathogens for humans, which cause dysentery and food poisoning, respectively. Complexes 1 and 2 were tested for their antibacterial and antifungal activity. The antibacterial activity of [Ni(L 1 ) 2 ](ClO 4 ) 2 1 is graphically presented in Figure 2. Results of these antimicrobial and antifungal assessments of complexes are presented in Tables 3 and 4. The area of zone of inhibition is less in the concentration of 5 mM in both microorganisms and more in 15 mM concentration. This kind of observation is suggestive of that these complexes are effective against both pathogens. In case of complex 1 diameter of inhibition zone (20 nm) is highest for E.coli. It was noted that complex 1 was more effective against E. coli than Streptococcus aureus. Similar observations were found for complex 2. Among fungal species, two isolates were taken into consideration and were Aspergillus and Penicillium sp.; similar trends were observed as in the case of bacteria. It was noted that Penicillium sp. was highly susceptible against complex 1. Another fungi Aspergillus sp. showed least effectiveness against complex 2 but comparatively more  susceptible towards complex 1. Similar antimicrobial results were reported by Tarafder et al. [33] and also by Patel School [34] on simple nickel (II) binary and ternary complexes. It is observed from these test that metal chelates have a higher activity than the free ligands such that increased activity of the metal chelated can be explained on the basis of Tweedy's chelation theory [35] . These complexes also disturb the respiratory processes of the cell and thus block the synthesis of protein, which restricts further growth of the organisms. The magnetic and spectroscopic data of complexes 1-5 indicate square planar geometry. The SOD activity of reported complexes in the increasing order 2 < 4 < 5 < 3 < 1. Complexes 1 and 2 have shown excellent antibacterial activity against E. coli comparable to that of Streptococcus aureus.