Synthesis, Structural Characterization, and Biological Activity Studies of Ni(II) and Zn(II) Complexes

Ni(II) and Zn(II) complexes were synthesized from tridentate 3-formyl chromone Schiff bases such as 3-((2-hydroxyphenylimino)methyl)-4H-chromen-4-one (HL1), 2-((4-oxo-4H-chromen-3-yl)methylneamino)benzoic acid (HL2), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL3), and 3-((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL4). All the complexes were characterized in the light of elemental analysis, molar conductance, FTIR, UV-VIS, magnetic, thermal, powder XRD, and SEM studies. The conductance and spectroscopic data suggested that, the ligands act as neutral and monobasic tridentate ligands and form octahedral complexes with general formula [M(L1–4)2]·nH2O (M = Ni(II) and Zn(II)). Metal complexes exhibited pronounced activity against tested bacteria and fungi strains compared to the ligands. In addition metal complexes displayed good antioxidant and moderate nematicidal activities. The cytotoxicity of ligands and their metal complexes have been evaluated by MTT assay. The DNA cleavage activity of the metal complexes was performed using agarose gel electrophoresis in the presence and absence of oxidant H2O2. All metal complexes showed significant nuclease activity in the presence of H2O2.


Introduction
Metal complexes of O, S, and N containing Schiff bases have been the subject of current and growing interest because it has wide range of pharmacological activities [1]. In particular Schiff bases with 2-amino thiophenol, 2-amino phenol, 2amino benzoic acid, and 2-amino 3-hydroxy pyridine exhibit various biological activities such as antimicrobial activity, protein tyrosine phosphatases inhibition, and nuclease activity [2,3]. Several aromatic amine Schiff bases have been investigated but few works deal with chromone skeleton derivatives. Chromones are a group of naturally occurring compounds that are ubiquitous in nature especially in plants. Molecules containing the chromone skeleton have extensive biological applications including antimycobacterial, antifungal, anticancer, antioxidant, antihypertensive, and antiinflammatory applications and tyrosinase and protein kinase C inhibitors [4][5][6][7][8][9][10][11][12][13].
DNA plays an important role in the life process since it contains all the genetic information for the cellular function. However, DNA is the primary intracellular target of anticancer drugs, damaged under various conditions such as interactions with some small molecules, which cause DNA damage in cancer cells blocking the division of cancer cells and resulting in cell death. Metal complexes such as cobalt, nickel, copper, and zinc with Schiff base ligands have shown excellent binding and cleavage activities [14][15][16]. There is substantial literature supporting the DNA binding studies of chromone Schiff bases and their metal complexes [17][18][19].
Compounds showing the properties of effective binding as well as cleaving double stranded DNA under physiological conditions are of great importance since these could be used as diagnostic agents in medicinal and genomic research.
Fluorescent transition metal centres are particularly attractive moieties because they often possess distinctive electrochemical or photophysical properties, thus enhancing the functionality of the binding agent [20].
In previous papers we presented synthesis, detailed characterization, and biological activity studies of 3-formyl chromone Schiff bases such as 3-((2-hydroxy phenylimino)methyl)-4H-chromen-4-one (HL 1 ), 2-((4-oxo-4H-chromen-3-yl)methylneamino)benzoic acid (HL 2 ), 3-((3-hydroxypy- standards [27], was introduced onto the surface of sterile nutrient agar plate and evenly spread by using a sterile glass spreader. Sterile antibiotic discs (6 mm in diameter, prepared using Whatmann number 1 paper) were placed over the nutrient agar medium. Each disc was spread by 100 g of the compounds (initially dissolved in DMSO). The plates were incubated with bacterial cultures for 24 h at 37 ∘ C and fungal cultures at 25 ∘ C for 48 h. The activity of the compounds was determined by measuring the diameter of inhibition zone in "millimetres" and compared with standard antibiotics. DMSO (which has no activity) and standard antibiotics were used as negative and positive controls for antimicrobial activity studies. The activity results are calculated as a mean of triplicates. Minimum inhibitory concentrations (MIC) of the complexes which showed antimicrobial activity were determined using the literature method [28]. All the compounds that were diluted within the range of 100-10 g/mL were mixed in nutrient broth and 0.1 mL of active inoculums was added to each tube. The tubes were incubated aerobically at 37 ∘ C for bacteria and 25 ∘ C for fungi up to 24 h. The lowest concentration of the compound that completely inhibited bacterial growth (no turbidity) in comparison to control was regarded as MIC.

Nematicidal
Activity. Root knot nematode, Meloidogyne incognita, is major plant parasitic nematodes affecting quantity and quality of the crop production in many annual and perennial crops. Meloidogyne nematode can develop galls and lesions in the roots, thereby causing stunted growth of the plants. Some of the chemicals can be used to control nematodes [29].
Nematicidal activity of the complexes was carried out on Meloidogyne incognita. Fresh egg masses of Meloidogyne incognita are collected from stock culture maintained on tomato (Lycopersicon esculentum) root tissues and kept in water for egg hatching. The eggs suspensions were poured on a cotton wool filter paper and incubated at 30 ∘ C to obtain freshly hatched juveniles (J2). Juveniles collected within 48 h were used for screening nematicidal activity of the compounds.
The compounds were initially dissolved in dimethyl sulfoxide (DMSO) and then in distilled water to make dilutions of 250, 150, and 50 g/mL. Experiments were performed under laboratory conditions at 30 ∘ C. About 100 freshly hatched second stage juveniles were suspended in 5 mL of each diluted compound and incubated. Distilled water with nematode larvae was taken as control. The dead nematodes were observed under an inverted binocular microscope. After an incubation of 24 and 48 h, percentage of mortality was calculated. Nematodes were considered dead if they did not move when probed with a fine needle [30].
2.6. DPPH Radical Scavenging Activity. The free radical scavenging activities of the metal complexes were determined by using DPPH free radical scavenging method according to the literature [31]. DPPH is a stable free radical containing an odd electron in its structure and usually utilized for detection of the radical scavenging activity in chemical analysis. In the spectrophotometric assay the ability to scavenge the stable free radical DPPH is measured by decrease in the absorbance at 517 nm. Each compound was dissolved in methanol (10 mg/10 mL) and it was used as stock solution. From the stock solutions, 1 mL of each compound solution with different concentrations (0.25 g-1.00 g) was added to the 3 mL of methanolic DPPH (0.004%) solution. After 30 min, the absorbance of the test compounds was taken at 517 nm using UV-VIS spectrophotometer. BHT was used as standard, DPPH solution was used as control without the test compounds, and methanol was used as blank. The percentage of scavenging activity of DPPH free radical was measured by using the following formula: where is the absorbance of the control and is the absorbance of the sample. were obtained from the National Centre for Cell Science (NCCS), Pune, and grown in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS), amphotericin (3 g/mL), gentamycin (400 g/mL), streptomycin (250 g/mL), and penicillin (250 units/mL) in a carbon dioxide incubator at 5% CO 2 . About 700 cells/well were seeded in 96-well plate using culture medium; the viability was tested using trypan blue dye with help of haemocytometer and 95% of viability was confirmed. After 24 h, the new medium with compounds in the concentration of 100, 10, and 1 g/mL were added at respective wells and kept in incubation for 48 h. After incubation MTT assay was performed.

MTT Assay.
After 48 h of the drug treatment the medium was changed again for all groups and 10 L of MTT (5 mg/mL stock solution) was added and the plates were incubated for an additional 4 h. The medium was discarded and the formazan blue, which was formed in the cells, was dissolved with 50 L of DMSO. The optical density was measured on microplate spectrophotometer at a wavelength of 570 nm. The percentage of cell inhibition was calculated by using the following formula [32]: where is the absorbance of the sample and is the absorbance of the control. IC 50 values were determined using Graph Pad Prism software.
2.8. DNA Cleavage Activity. The DNA cleavage activity of metal complexes was monitored by agarose gel electrophoresis. pUC19 plasmid was cultured, isolated, and used as DNA for the experiment. Test samples (1 mg/mL) were prepared in DMF. 25 g of the test samples was added to the isolated plasmid and incubated for 2 h at 37 ∘ C. After incubation, 30 L of plasmid DNA sample mixed with bromophenol blue dye (1 : 1) was loaded into the electrophoresis chamber wells along with the control DNA, 5 M FeSO 4 (treated with DNA), and standard DNA marker containing TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L). Finally, it was loaded on to an agarose gel and electrophoresed at 50 V constant voltage up to 30 min. After the run, gel was removed and stained with 10.01 g/mL ethidium bromide and image was taken in Versadoc (Biorad) imaging system. The results were compared with standard DNA marker. The same procedure was followed in the presence of H 2 O 2 also.

Results and Discussion
All the Ni(II) complexes were colored, stable, and nonhygroscopic in nature. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The elemental analysis showed that the complexes have 1 : 2 stoichiometry of the type [M(L 1-4 ) 2 ]⋅ H 2 O, where L stands for singly deprotonated ligands. Molar conductance of the complexes was measured in DMF. The conductance values, which are presented in the Table 1, indicate the nonelectrolytic nature of the complexes [33].

Determination of the Metal Content of the Complexes.
Known amount (0.150 g) of complexes was decomposed with concentrated nitric acid. This process was repeated till the organic part of the complexes got completely lost. The excess nitric acid was expelled by evaporation with concentrated sulphuric acid. The Ni(II) and Zn(II) contents of the complexes were determined as per the procedure available in the literature [34].

FTIR Spectra.
The FTIR spectra of the complexes are compared with those of the ligands in order to determine the coordination sites that may involve in chelation. The position and/or the intensities of bands are expected to be changed while the coordination. The most important IR bands of the metal complexes with probable assignments are given in Table 2. The Schiff base ligands showing a band around 1650-1620 cm −1 is assigned to the (C=O) group of the chromone system. Upon complexation the ](C=O) group is shifted to 20-35 cm −1 lower wavenumber region [35]. The ligands show the most characteristic ](C=N) bands in the region 1605-1563 cm −1 . In the spectra of their corresponding metal complexes, this band appears at 25-45 cm −1 to lower wavenumber region indicating the coordination of the azomethine nitrogen atom to the metal ion [36]. A broad band appeared in HL 1 and HL 3 at 3241 and 3246 cm −1 , respectively, and is attributed to the ](O-H) group. The absence of the band in the spectra of their corresponding metal complexes is due to the involvement of oxygen atom of (OH) group coordination to the metal ion. In HL 2 ligand, a strong band appeared at 1365 cm −1 and is due to the ](C-O) of carboxylic group. In its metal complexes it is shifted to 18-47 cm −1 lower wavenumber region [37]. Since SH stretching frequency band is very weak, the peak corresponding to SH is not clearly observed in the case of HL 4 ligand [38]. Presence of band around 3400 cm −1 in all the complexes is the indication of water molecules present in the complexes. The presence of oxygen and nitrogen in the coordination sphere is further confirmed by the presence of ](M-N) and ](M-O) bands at 400-600 cm −1 region. The FTIR results show that all Schiff base ligands act as tridentate chelating ligands.

Electronic Spectra and Magnetic
Moments. The electronic absorption spectra of the Schiff base metal complexes in solid state were recorded at room temperature and the band positions of the absorption maxima, band assignments, ligand field parameters, and magnetic moment values are listed in Table 3. The electronic spectra of [Ni(L 4 ) 2 ]⋅H 2 O and [Zn(L 4 ) 2 ]⋅H 2 O complexes are depicted in Figure 2. The electronic spectra of Ni(II) complexes displayed three absorption bands in the range 8000-9000, 14000-16000, and 20000-24000 cm −1 . Thus, these bands may be assigned to the three spin allowed transitions 3 respectively, characteristic of octahedral geometry. The values of transition ratio ] 2 /] 1 and lie in the range of 1.70-1.80 and 0.89-0.95, respectively, providing further evidence for octahedral geometry of Ni(II) complexes [39]. The B values for the complexes are lower than the free ion value, thereby indicating orbital overlap and delocalisation of d-orbitals. The -values obtained are less than unity suggesting the covalent character of the metal-ligand bonds. All Ni(II) complexes are paramagnetic and the magnetic movement values at room temperature are in the range of 2.91-3.25 B.M which is well agreed with the reported octahedral Ni(II) complexes [40]. All Zn(II) complexes showed two bands around 25000 and 30000 cm −1 and are attributed to the → * and → * transitions, respectively. Zn(II) complexes that are in d 10 configuration are diamagnetic and do not show any d-d transitions.
3.4. Thermal Analysis. The thermogravimetric analysis (TGA) of the metal complexes was carried out within the temperature range from room temperature to 1000 ∘ C. The TG-DTA graphs of [Ni(L 4 ) 2 ] and [Zn(L 4 ) 2 ]⋅H 2 O complexes are given in Figure 3. The TGA data and their assignments of all the metal complexes are listed in Table 4. Metal complexes decompose gradually with formation of respective metal oxides. The TG graphs of [Ni( in two to three steps. The first step corresponds to the loss of lattice water molecule in the temperature range between 30 and 140 ∘ C with a weight loss of 2-3%. In the second and third steps, the total loss of ligand molecules was observed in the temperature range between 160 and 850 ∘ C leaving behind metal oxide as residue. The thermal decomposition of remaining metal complexes ([Ni( and [Zn(L 3 ) 2 ]) occurs in two to three steps. Upon starting heating, these metal complexes losses of the organic moieties of Schiff base ligands were observed in two to three successive steps within the temperature range of 150-850 ∘ C. In all the complexes the final mass loss is due to the formation of metal oxides as residue.
On the basis of all spectral data (elemental analysis, FTIR, electronic spectra, and thermal analysis), the suggested structures of the complexes are shown in Figure 4.
where is the X-ray wavelength, is the full width at half maximum of prominent intensity peak, and is the diffraction angle.        3.6. Fluorescence Spectra. The fluorescence characteristics of metal complexes were studied at room temperature in solid state. In metal complexes metal to ligand coordination may lead to significant changes of the fluorescence properties of the ligand, including increase or decrease of the intensity, emission wavelength shift, quenching of the fluorescence, or appearance of new emissions [42]. The fluorescence spectra of HL 1 ligand and its metal (Ni(II) and Zn(II)) complexes were depicted in Figure 7.   -----HL 2  80  80  80  80  80  HL 3  -----HL 4 -  Increase in the activity of the complexes compared to that of ligands can be explained on the basis of Overtone's concept and Tweedy's chelation theory. The theory states that the polarity of the metal ion is reduced on complexation due to the partial sharing of its positive charge with donor groups. Consequently, the positive charge is delocalized over the whole ring, which causes the improved lipophilicity of the compound through cell membrane of the pathogen [43]. The negative results can be attributed either to the inability of the complexes to diffuse into the bacteria cell membrane and hence they become unable to interfere with its biological activity or they can diffuse and become inactivated by unknown cellular mechanism, that is, bacterial enzymes [44]. complexes exhibited moderate activity indicating 47% and 51% mortality, respectively, after 48 h exposure in 250 g/mL concentration. However, the activity of the metal complexes depends on concentration and time, that is, activity was higher at high concentrations and increased with time.
3.9. DPPH Radical Scavenging Activity. The antioxidant activity of the complexes was tested by measuring their ability to donate an electron to the free radical compound     mechanisms of the DNA cleavage studies were reported by several research groups [46][47][48]. Many literature reports infer that the compound was to cleave the DNA; it can be concluded that the compound inhibits the growth of the pathogenic organism by cleaving the genome [49].

Conclusions
Ni(II) and Zn(II) complexes have been synthesized using 3formyl chromone Schiff bases and characterized by various analytical and spectral data. Based on the electronic spectra, magnetic moment, and elemental analysis data, octahedral geometry was proposed for Ni(II) and Zn(II) complexes. The well-defined crystalline and homogeneous nature of the metal complexes is observed from powder XRD and SEM analyses.