Synthesis , Spectral and Biological Studies of Ni ( II ) , Pd ( II ) , and Pt ( IV ) Complexes with New Heterocyclic ligand Derived from Azo-Schiff Bases Dye

New heterocyclic azo-schiff base dye (L2) have been synthesized from a reaction of 4(dimethylamino)benzaldehyde with (E)-5-((1H-benzo[d]imidazol-2-yl) diazenyl) -2amine-4,6-dimethylpyridin (L1) . A new class of Ni(II), Pd(II), and Pt(IV) complexes of azoschiff base of the type [Ni(L2)2Cl2], [Pd(L2) Cl2] , and [Pt(L2)2Cl2]Cl2 where L2=(E)-5((E)-(1H-benzo[d]imidazol-2-yl)diazenyl)-N-(4-(dimethylamino)benzylidene) -2amine4,6-dimethylpyridin. Both ligand and its metal complexes were characterized by the spectroscopic and analytical methods such as mass spectral, 1HNMR, FTIR, UV-Visb.,Xray, TGA and SEM, elemental analysis, molar conductance measurements and magnetic susceptibility., spectral studies suggest the mole ratio [M:L] was [1:2] for Ni(II) and Pt(IV) metal ions, but [1:1] for Pd(II) metal ion. The structures of theses complexes were elucidated on the basis of different techniques suggest the structures of the prepared metal complexes octahedral geometry for the Ni(II) and Pt(IV) complexes and square planar geometry for the Pd(II) complex. The antibacterial activity of the ligand and prepared metal complexes was also studied against gram positive Staphylococcus aureus and gram negative bacteria Escherichia coli, the antifungal activity of the azo-schiff base and metal complexes against the fungi Alternaria. It is found that some of the complexes are quite effective against fungi Alternaria. In this study the cytotoxicity of Pd(II)-complex on human (PC3) cancer and normal cells were studied using MTT assay. Pd(II)-complex showed selective cytotoxicity against cancer cell line with IC50 = 450 μg/ml, while it was very safe on normal cells line with IC50 = 14554885 μg /ml, respectively for human cells .The results indicate undoubtedly the possibility of using them as antitumor drugs in the field of medicine and Pharmacy against prostate cancer.


INTRODUCTION
Azo-schiff dyes derivatives are important class of organic complexing compounds.This compound is more activity and less toxic antimicrobial agents result to the increase in resistance to the available antimicrobial drugs [1].Azoschiff base compounds are famous for exhibit the wide range of applications in antimicrobial, pharmaceutical, industrial and analytical reagents uses [2,3].These compounds are also a kind of multipurpose ligands in coordination chemistry [4][5][6].Also transition metal complexes of azo schiff bases are found to be useful as anticancer agents, antibacterial [7][8][9][10], as excellent homogeneous and heterogeneous-phase catalysts [11], and as chemical sensors [12].Cancer, known as the malignant tumor or malignant neoplasm, is the kind of diseases characterized by out-of-control cell growth [13].Around a world, big number of people are diagnosed with cancer each year, and most of a patients die from it [14].Several strategies are being developed to fight against cancer [15][16][17].Cis-Pt(II) complex is the first number of platinum-containing anti-cancer drug [18].Despite greatly effective in treating the Al-Adilee & Dakheel / Synthesis, Spectral and Biological Studies of Ni(II), Pd(II), and Pt(IV) Complexes … 2 / 17 various types of cancers, cis-Pt(II) complex has the number of side-effects such as nephrotoxicity, neurotoxicity and ototoxicity etc [19,20].Side-effect problems of cis-Pt(II) have stimulated chemists to develop alternative anticancer drugs based on different metals.Several studies indicated that copper complexes had showed some promising results [21].In this study we report the synthesis, spectra characterization of azo -schiff base ligand and its metal complexes with Ni(II), Pd(II), and Pt(IV) ions derived from (E)-5-((E)-(1H-benzo[d]imidazol-2-yl)diazenyl)-N-(4-(dimethylamino)benzylidene) -2-amine -4,6-dimethylpyridin (L2).The antibacterial activity of the prepared complexes was also studied against gram positive Staphylococcus aureus and gram negative bacteria Escherichia coli and antifungal activity of the azo-schiff base and metal complexes against the fungi Alternaria.Also in this study the cytotoxicity of Pd(II)-complex on human (PC3) cancer and normal cells were studied using MTT assay.

Materials and Measurements
All chemicals and solvents used in the present work were high purity provided from multiple companies such as Fluk, BDH, Aldrich and Sigma and used without further purification.Doubly distilled water was used in all experiments.Elemental analysis of azo-schiff base dyes.Ligand and its metal complexes were carried out by mean of micro analytical unit of EA 300 C.H.N Element analyzer.Mass spectra were obtained using a shimadzu Agilent Technologies 5973C at 70 e and MSD energy using a direct insertion probe (Acq method 10 W energy) at temperature 90-110 °C. 1 H-NMR spectra were recorded on a model Bruker 500 MHZ spectro photometer using DMSO-d6 as a solvent and TMS as an internal standard.Electronic spectra were measured in absolute ethanol as a solvent (10 -3 M) in the range (200-1100) nm by using a UV-Visb.T80-PG spectrophotometer.Infrared spectra were recorded in KBr medium as dics using a shimadzu 8400S FT-IR spectrophotometer in wave number at range (4000-400) cm -1 .X-ray diffraction were measured using Bestec Germany Aluminum anode model X pertpro, wavelength of X-ray beam(Cu kα) 1.54 angstrom, Anod material=Cu, the Voltage = 40KV and current = 30mA.The magnetic susceptibility for the prepared metal complexes were measured at room temperature using faraday method.For this purpose, Burker Magnet (B.M) had been employed and the diamagnetic correction were made by pascals constants.TGA, DSC and DTG analysis were measured with England PL-TG using Rheometric scientific TGA-1000.SEM images of ligand and Pd (II)-complex, were taken using micrograph kyky 3200.Electric molar conductivity measurements were carried out at room temperature in DMF (10 -3 M) solution by using conductivity bridge model 31A.The melting points of ligand (L2) and its complexes were measured by using electro thermal melting point 9300.The pH solutions were measured on a PW 9421 pH meter (±0.001).The chloride ion content in Pt(IV)-complex was determined by more.

Synthesis of Azo-Schiff Base Ligand (L2)
Azo-schiff base ligand (L2) was synthesized following the analogous procedure Al-Adilee etal [22,23] with some modification in a two-step process as described below.In the first step, 2-Amino benzimidazole (0.133 gm, 1mmoc) was dissolved in mixture of 3 ml concentrated hydrochloric acid and 30 mL distilled water with continuously (Scheme 1) cooing and stirring until reach the temperature (0-5) °C and diazotized below 5 °C with solution of sodium nitrite NaNO2 (0.75 gm,1 mmol, dissolved in 20 ml distilled water) was added in drops.The diazonium chloride compound was then coupled with 2-amine-4,6-dimethylpyridin in alkaline media below 5 °C.The pH value during the coupling was maintained between 6-7, coupling to the 2-amine-4,6-dimethylpyridin occurred in basic media at the para-position to the amine group.(E)-5-((1H-benzo[d]imidazol-2-yl) diazenyl) -2-amine -4,6dimethylpyridin (L1) in the second step, the ligand (L2) was synthesized according to the known condensation method.About (0.266 g, 1 mmol) of (L1) in absolute ethanol (20 ml) was added drop-wise to hot absolute ethanol (50 ml) solution of 4-(dimethylamino)benzaldehyde (0.149 g, 1 mmol) with few drops of glacial acetic acid as a catalyst (Scheme 1).The mixture was refluxed and stirred continuously for 3 h at 80 °C.After cooling, the solid product was filtered off, and washed several times with cold distilled water then dried in vacuum for several hours and recrystallized twice from hot ethanol to afford red crystals coloured product and stored in a desiccator over anhydrous calcium chloride.Yield: 76%; m.p. 152 °C The purity was confirmed by the elemental analysis and thin layer chromatography TLC techniques.The structure of azo schiff base ligand (L2) was clucidated by 'H-NMR, mass spectrum, IR and UV-visb.spectra.The structural of the ligand (L2) as shown in Scheme 1.

Synthesis of Metal Complexes
The Ni(II)-complex was prepared by dissolving (0.397 gm , 1 mmol) from azo-schiff base ligand (L2) in hot ethanol than added in drops with stirring continuously to (1 mmol), 1:2 [M;L], for Ni(II) chloride salt dissolved in 20 ml hot buffer solution (ammonium acetate) at PH=7.0, the resulting solutions were refluxed for 1 hour and then the volume of the solution was reduced to one-half by evaporation.The obtained solution was left for 1 hour in the freezer; compounds were filtered off, washed with absolute ethanol and recrystallized in ethanol [24][25][26].
The Pd(II) and Pt(IV) -complexes were prepared by dissolving (0.397 gm , 1 mmol) from azo-schiff base ligand (L2) in hot methanol than added 20 ml methanolic solution of Pd(II) and Pt(IV) chloride salt (Pd(II) chloride salt 1 mmol, but Pt(IV) chloride salt 0.5 mmol) and refluxed for 1 hour.The precipitate was filtered off, washed with distilled water and washed with 5 ml methanol to remove any traces of unreacted materials and dried in air [27][28][29].The % yield, m.p, pH, color, molecular formula, M.wt and element analysis data (C.H.N) of ligand and its metal complexes are collected in Table 1.

Characterization of Azo Schiff Base Ligand (L2) and its Metal Complexes
The metal complexes derived from azo -schiff base ligand (L2) are stable toward air and insoluble in water and some common organic solvents but soluble in ethanol, methanol, dimethylformamide, dimethylsulfoxide and acetone.The physical characteristics and microanalytical data of the ligand (L2) and their metal complexes are given in experimental section.The structures of the obtained ligand and their respective metal complexes were clucidated

H-NMR Spectra
The 1 H-NMR spectral results, obtained for Azo-schiff base ligands (L2) and Pd(II) complex at ambient temperature in DMSO-d6, with TMS as an internal reference is assigned as follows: the characteristic singlet at = 8.668 ppm is due to the azomethine proton (-CH=N), and multiplets around  = 6.246-7.767ppm are assigned to aromatic protons and three signal at  =2.745,3.156,3.703ppm is ascribed to methyl group.A singlet at =5.492ppm is attributed to the (NH) group in imidazole ring of the Azo-schiff base.A signal at  =2.450 ppm assigned to solvent proton [30][31][32].
The 1 H-NMR spectrum of pd(II)-complex shows change in a signal at  = 6.857 ppm (imidazole proton) due to the presence of NH group in the spectrum of the pd(II)-complex is shifted down field compared to the free ligand, suggesting deshielding of the amine group due to the coordination with metal ion singlet at  =9.267 ppm is due to the azomethine proton (-CH=N), and multiplets around  = 6.557-9.003ppm are assigned to aromatic protons and three signal at  = 3.123,3.452and 3.961 ppm is ascribed to methyl group.A signal at  =2.509 ppm assigned to solvent proton [33,34].

Mass Spectra
The recorded mass spectrum of the ligand (L2) (Scheme 2, Figure 3) and the molecular ion peaks have been used to confirm the proposed formula.The mass spectrum of ligand (L2) showed several peaks attributed to the molecular ions at (m/z + ) 397.48 , 353.41 , 222.10 , 207.10 , 117.2 and 76.05 were due to various fragments ions respectively.This data is in good agreement with the corresponding molecular formulae [35].
The molecular ion peaks of Pd(II) -complex shows different m/z + values with different intensities.The mass spectrum contain molecular ion peaks at (m/z + ) 574.80, 528.5, 494.5 459,106.42,121.17,91.9 and 51.0 were due to and [C4H4] respectively .This data is in good agreement with the corresponding molecular formulae [36].

Infrared Spectra of Azo-schiff Base Ligand and their Metal Complexes
The IR spectrum of the ligand (L2) is compared with the metal complexes in order to determine the coordination sites that may be involved in chelation.There are some guide bands, in the spectrum of the free ligand (L2), which are of great help for achieving this goal.The position or the intensities of these bands are expected to be changed upon complex formation.The characteristic bands of the free ligand (L2) and its metal complexes are discussed here.The IR spectrum of the ligand (L2) exhibited a broad strong the band at 3442 cm -1 , which assigned to υ(N-H) stretching vibration of benzimidazol moiety, bands at 1635 cm -1 and 1558 cm -1 due to υ(C=N) in the new azo-schiff  Eurasian J Anal Chem 7 / 17 base ligand and υ(C=N) of imidazole molecule vibrations, respectively.The appearance of a medium band in the IR spectrum of the ligand (L2) at 1411 cm -1 , is ascribed to the stretching vibration of azo group (-N=N-).In comparison with the spectra of the metal complexes exhibited increase shift of about 15-25 cm -1 in the band of -(C=N) indicating the participation of azomethine nitrogen atom in coordination with the metal ions [34][35][36].The most change in the schiff base spectral features when coordinated to metal ion is the change in shape, intensity and shifted frequencies of υ (N=N) stretching, indicates that azo group of the ligand (L2) in coordinate to the metal ion through nitrogen atom, forming metal-nitrogen bond.The stretching vibration of the -(N=C) group of azo-schiff base ligand was not shifted more in the spectrum of metal complexes, thereby, indicating the non-participation of in complex formation.Furthermore, the appearance of new absorption bands at (513-523) cm -1 in the spectra of the metal complexes, indicated the formation M-N bonds, respectively [37].The infrared spectra of new azo-schiff base ligand (L2) and its metal complexes are shown in Figure 5.

Magnetic Susceptibility and Electronic Spectra Measurements
The UV-Visib.spectra were recorded in absolute ethanol solution (10 -3 M) and absorption bands given in Table 3 and Figure 6.The spectra of metal complexes are dominated by intense intra-ligand charge transfer bands.The spectrum of ligand (L2) shows an intense absorption band at 417 nm (2390) cm -1 assigned to n→π* transition of azo and azomethine groups [38].The geometries are supported by their electronic spectra.

Thermal Analysis
The thermal degradation of the azo-schiff base ligand and its metal complexes were investigated by thermogravimetric (TG) analysis, differential thermogravimetric (DTG) and differential scanning calorimetry (DSC) in the temperature range (22-705) ˚C.A representative TG, DTG, DSC diagram is given in Figure 7.The thermal stability data are listed in Table 4.The data from the thermogravimetric analysis clearly indicated that the decomposition of the complexes proceeds in two, three or four steps.Water molecules were lost in first step between 50 -200 °C and metal oxides were formed above 600 °C for metal complexes.The decomposition was complete at ≥600 °C for all metal complexes.
From the TG curve of the Ni(II)-complex, it can be seen that decomposition starts at 90 ˚C and shows almost a continuous weight loss in the temperature range of 90-635 ˚C.Based on the percentage of mass losses and the DTG curve, four-step decomposition is proposed for this complex, which is similar to the decomposition pattern reported previously in the literature [43].According to literature the azo bonds in the azo schiff base-metal complexes breakdown when the temperature is higher than 250 ˚C resulting in the endothermic peaks [44].The first   The Pd(II)-complex is thermally decomposed in four successive decomposition steps within the temperature range (22.5-705.0)˚C.The first decomposition step of estimated mass loss 13 % within the temperature range (210-318)˚C may be attributed to the loss of the 2Cl atoms (calcd.12.19%).DTG peaks corresponding to this stage are observed at 230 ˚C.Simultaneously, the recorded DSC curve reveals a broad endothermic peak at 223˚C.The second step at the temperature range (318-400)˚C with the mass loss 34% corresponds to the loss of the C7H6N2 benzimidazol molecule (calcd.32.19%).This decomposition process shows a broad peak at 346 and 341 ˚C in the DTG and DSC curve respectively.The third step found within the temperature range 400-580˚C with an estimated mass loss 79% which may be attributed to the loss of residual ligand molecule and PdO as a final product.(calcd.75.98%).The broad DTG peak corresponding to this step is observed at 531˚C.The recorded DSC curve shows peak at 529 ˚C.
The Pt(VI)-complex is thermally decomposed in four decomposition steps.The first decomposition step of estimated mass loss 7% within the temperature range (100-150) ˚C with an estimated mass loss of the Cl molecule outer-sphere coordination (calcd.6.27%).The DTG sharp peak corresponding to this stage is observed at 131˚C.The recorded DSC curve peak at 139 ˚C.The second step occurs within the temperature range (150-300)˚C with a DTG peak at 251 ˚C, may be attributed to the loss 14% (calcd.12.54%), which may be due to loss of the 2Cl molecule inner-sphere coordination, The complex also shows third step displays a mass loss of 33% (calcd.33.75%), within the temperature range 300-455˚C with a DTG peak at 382˚C, which may be attributed to the loss of the 2(C8H11N ) N,N-dimethylaniline molecule.The recorded DSC curve reveals an endothermic peak at 380˚C.The final decomposition step in the temperature range (455-611) ˚C exhibits a loss in mass of 80% (calcd.81.19%), which may be attributed to the complete decomposition of a part of ligand molecule.This step shows obvious peaks in the DTG and DSC curves 531 ˚C and 529 ˚C respectively.
According to these results and discussed the data through different techniques suggest the structural formula of prepared metal complexes in this work may be proposed in Figures 8 and 9.  6.

SEM Studies
Scanning electron microscopy (SEM) of the azo-schiff base ligand (L2) and Pd(II)-complex studies the surface morphology and shape of the particles and aggregation, in addition to the distribution of these particles.The SEM   11 and 12. SEM image shows the ligand (L2) have form of peripheral spherical shape with average size 91.22 nm with a ratio of less than aggregation.The SEM image of Pd(II)-complex appeared in the form of heterogeneous the surface with average particle size 86.64 nm.According to the images of SEM different characteristic shapes of Pd(II)-complex was identified and these SEM images were different from that of azo-schiff base ligand (L2).We can be seen that Pd(II)-complex gave a aggregation less than azo-schiff base ligand.

Antibacterial Activity
In this study we investigated antibacterial activity of the azo-schiff base and its series of metal complexes Ni(II),Pd(II) and Pt(IV) were screened for antibacterial activity against microorganisms representing Gram-positive bacteria [Staphylococcus aureus ] and Gram -negative bacteria [Escherichia coli] ,by agar diffusion method.DMSO is  Eurasian J Anal Chem 13 / 17 used as solvent.All the compounds were inoculated using a loop onto plates containing Nutrient Agar (NA) media and incubated at 32 o C for 24 hours.To carry out agar diffusion assay the bacterial suspensions were prepared in sterile distilled water [47][48][49].The ligand and its metal complexes not have activity against both bacterial.The results of the antimicrobial activity of the ligand and its metal complexes against all tested bacterial are shown in Figure 13 and Table 6.

Antifungal
The antifungal activity of ligand and its metal complexes have been studied by agar diffusion method against the fungi Alternaria.Flucanozole is used as standard antibiotic and DMSO is used as solvent [50][51][52].The antifungal activity of the azo-schiff base and metal complexes against the fungi Alternaria are shown in Table 6 and Figure 13.

Cell Viability and Cytotoxicity Assay
In this analytical method that was primarily developed by Mosmsnn [53][54][55], the density of cells can be determined based on cell color in small volumes after converting spheroids to single cells, two different densities of single cells including 8000 and 10,000 cells in different plates were selected for MTT assay.After a period of 24 hrs.dissolved MTT in PBS with final density of 0.5 mg/ml was added and after incubation for 4 hrs.MTT solution was extracted and DMSO was then added and was shaken for 20 min.Finally, absorption of the samples was read by regulating 570 nm filter as the main wavelength and also 630 nm filter as the referenced wavelength.The absorption rate of the wells without cells was diminished from absorption of the wells with cells to obtain pure cellular absorption.According to the following equation, there is a direct association between pure absorption and rate of viable cells: (Mean absorption of sample / mean absorption of referenced)×100=percentage of viable cells.The in vitro cytotoxicity of the Pd(II)-complex, on human cell lines PC3 was used to determine the antiproliferative effect of synthesized compound.The selectivity index (SI), which indicates the cytotoxic selectivity of the compound against cancer cells and its safety towards the normal cells, was determined from the ratio of the *IC50 values were calculated for each cell line Table 7 and Figures 14 and 15, Pd(II)-complex showed selective cytotoxicity against cancer cell line with IC50 = 425 μg/ml, while it was very safe on normal cells line with IC50 = 14554885 μM/ml, respectively for human cells.

Treating Tumors or Tumor Metastases
Killing cancer cells because of DNA binding cannot account for a selective mechanism of cell recognition and of discriminating toxicity.Provided that tumor cells differ from their healthy counterparts for the activated genes rather than for DNA (which is almost identical in terms of nucleotide content, e.g. the number of guanines and their sequence, in any cell of the body including the tumor mass), the question remains open why, besides the high sensitivity of testicular tumors that can be cured, other tumors towards which platinum drugs are applied in a number of combination chemotherapeutic regimens show [56].The mechanism of tumor cell killing pladinum drugs have been illustrated in Figure 16.,but for metal ion Pd(II) was found to be [1:1] and, thus, azo-schiff base ligand appears bidentate coordinated with the metals complexes.Crystal structures of the azo-schiff base ligand and its metal complexes were successfully determined by single-crystal X-ray diffraction.In the biological and antifungal activity studies, some synthesized compounds exhibit antimicrobial properties.The cytotoxicity of Pd(II)-complex on human (PC3) cancer and normal cells were studied using MTT assay.Pd(II)-complex showed higher selective cytotoxicity against cancer cell line, while it was very safe on normal cells line.The results indicate undoubtedly the possibility of using them as prostate cancer drugs in the field of medicine.

Figure 10 .
Figure 10.X-ray diffraction patterns for ligand (L2) and its metal complexes

Figure 16 .
Figure 16.Schematic representation of the mechanism of tumour cell killing by pladinum drugs.The pladinum ion enters tumour cells, reaches their nucleus and binds to two adjacent guanines (making bridges on the same strand or between both strands).If repair mechanisms have no success on the adduct Pd-DNA then cell death occurs

Table 1 .
Analytical and physical data of ligand (L2) and its metal complexes

Table 4 .
Thermal analyses data for (TG,DTG,DSC) of ligand and metal complex

Table 5 .
Inter planar distances , 2θ value, FWHM and Crystallite Size of each peak, relative intensity for ligand and complexes

Table 6 .
Antibacterial activity of ligand (L2) and its complexes

Table 7 .
IC50 (μg/ml) values and percentage (%) of the synthesized compound and standard drugs