Synthesis , Characterization , Fluorescence , Electrochemical , Antimicrobial and Cytotoxic studies of Cobalt ( II ) , Nickel ( II ) and Copper ( II ) Complexes with N4-Methylphenyl ( 2 , 4-dihydroxyacetophenylideneimine )

N-4-Methylphenyl(2,4-dihydroxyacetophenylideneimine)(NMEPDHAPI) complexes of Co(II), Ni(II) and Cu(II) have been synthesized and characterized. A study on their electrochemical, fluorescence and biological behaviour have also been performed. The complexes have been characterized on account of metal estimation studies, conductance behaviour, magnetic property, IR, UV-Visible, EPR(Liquid nitrogen temperature) spectral studies and thermal analysis. The 1:2 metal : ligand complex formation is authenticated from the analytical data. Based on the various studies, the Co(II) complex has been recommended for octahedral geometry and a square planar geometry has been prompted for Cu(II) and Ni(II) complexes. The electro chemical property of the complexes have been studied by cyclic voltammetry. The Schiff base and its complexes have been scrutinized against bacterial and fungal pathogens. The complexes have been found to be more active than the Schiff base towards most of the pathogens studied. In vitro cytotoxicity of the copper complex of NMEPDHAPI has been screened against breast cancer cell lines (MCF-7). The IC50 value recommend that the copper complex has notable cytotoxicity against breast cancer cell line (MCF-7).


INTROdUCTION
2][3] Biological resemblance of Schiff bases is due to the structural resemblance of peptide bonds in protein. 4Such type of metal complexes exhibit many types of applications like catalysis 5 , pharmaceuticals 6 and molecular based materials 7 .The continuous need for renewed anti-cancer drug has developed chemotherapeutic research depending upon the consume of metallic elements since this must have high potential activity and low side effects. 8A well known anticancer drug is cisplatin.Unfortunatelty cisplatin has number of side effects, which limit the clinical use 9 .Therefore finding a new metal based drug with more activity and less side toxicity is a thurst area of research. 10The present report involves the synthesis of three transition metal complexes based on the Schiff base NMEPDHAPI.All three have been characterized from different spectral studies like IR, UV-Visible and EPR in Liquid nitrogen temperature.The redox behavior from Cyclic voltammetry studies revealed the catalytic application of complexes and fluorescence property indicates the sensor applicability of the complexes.Both the ligand and the complexes have been screened against bacterial and fungal pathogens.Anticancer activity (In vitro cytotoxicity) of the copper complex of NMEPDHAPI has been inquired towards breast cancer cell lines (MCF-7).

Materials and Methods
IR spectra were performed in KBr medium in FT-IR spectrophotometer (model : Shimadzu IR affinity).Measurements were recorded on powder samples at 298K.Thermal analysis was done by using the instrument (Perkin Elmer Diamond TG/DTA) from 40 0 C to 740 0 C at the heating range about 20 0 C/minute.The EPR spectra were recorded using JEOL model JES FA200 ESR spectrometer in liquid nitrogen temperature.The UV-spectra of the Schiff base and its complexes were taken using Perkin Elmer Spectrometer provided with quarts cells in DMSO medium.The electrical conductance of the synthesized complexes were determined in 10 -3 M solution of DMSO using a conductivity bridge of Elico make and a dip type conductivity cell.The percentage of metal was estimated by the standard methods.The magnetic susceptibility were ascertained in a Gouy Balance at room temperature.Electrochemical behaviour were measured at room temperature in an air tight three electrode cell by using glassy carbon electrode as a working electrode, a platinum wire served as the counter electrode and a Ag/AgCl in a saturated KCl solution as reference electrode.The electrochemical reactions were carried out using tetrabutylammonium perchlorate (0.11M) as a supporting electrolyte with a scan rate of 0.2V/s.The fluorescence spectra were analysed by using Jasco Spectro Fluorimeter (Model FP-8200) at room temperature by maintaining the concentration at 10 -4 M in DMSO.

Synthesis of complexes
The Schiff base N-4-methylphenyl-(2,4dihydroxyacetophenylideneimine) (NMEPDHAPI) has been synthesized, characterized through data from spectral studies.Its structure was established by single crystal XRD analysis by the authors in their previous article. 11The ligand NMEPDHAPI (0.2 mmol) and the metal salt (0.1 mmol) (cobalt acetate, Nickel chloride & copper chloride) were dissolved in 100 ml of ethanol and allowed to react for 6 h at 60 o C. The complex formed was filtered, washed with water and dried.The complexes are found to be freely soluble in DMF and DMSO.

Anticancer activities
The human breast adeno carcinoma cell lines (MCF7) was collected from National Centre for Cell Science (NCCS), Pune.The MTT assay method was implemented to evaluate the cytotoxicity. 12,13The % cell inhibition was inclined by applying the formula % Cell Inhibition = 100-Abs (sample)/Abs (control) x100.Nonlinear regression graph was computed between % Cell inhibition and Log concentration.The IC50 was ascertained by employing GraphPad Prism software.

Characterisation of complexes FT-IR spectra
In complexes the band assignable to the azomethine linkage is shifted from 1605 cm -1 in the Schiff base to 1607-1655 cm -1 in their metal complexes.This proves the binding between the metal and azomethine linkage. 14The nitrogen coordination may be further authenticated by the presence of new band in the order of 467-561 in the complexes corresponding to M-N bond which is absent in the ligand. 15,16The another coordination site is phenolic oxygen which is present in the ortho position to the imine group.This is evidenced by the emergence of new band correlating to metaloxygen bond around 600 cm -1 in the complexes. 17,18he C-O stretching vibration of phenolic group is noticed at 1282 cm -1 in the Schiff base NMEPDHAPI.This band is displaced to lower frequency in the spectra of complexes authenticating metal oxygen coordination. 19

Electronic spectra and magnetic susceptibility
The UV spectrum of the Co(II) complex exhibit the transition at 924 nm assignable to 4 T 1g (F) → 4 T 2g , 4 T 1g (F)→ 4 A 2g (F) and 4 T 1g (F)→ 4 A 2g (P) transitions 20 and the magnetic moment of 4.65 BM support the octahedral arrangement around Co(II) ion. 21The band at 527 nm in Ni(II)complex is designated to the transitions from 1 A 1g → 3 A 2g , 1 A 1g → 1 A 2g and 1 A 1g → 1 B 1g respectively 22 correlating to the square planar geometry around Ni(II) and is diamagnetic. 23In the Cu(II) complex the band at 537 nm is ascribed to 2 B 1g → 2 Eg ; 2 B 1g → 2 B 2g and 2 B 1g → 2 A 1g transitions 24 of Cu(II) with square planar geometry.The magnetic moment of1.54 BM substantiate the square planar geometry 25 around Cu(II).The UV spectra of the complexes are given in supplementary data (Figure S1-S3).

EpR spectra
In liquid nitrogen temperature the complex [Cu(NMEPDHAPI) 2 ] exhibits well defined peaks.The ground state of Cu(II) complex can be determined from the g value.In square planar complexes the unpaired electron lies in the d x .As a result the screening effect by the d z 2 electrons is to a greater extent leading to elongation (Jahn teller distortion) finally detachment of two orbitals from the metal ion, resulting in square planor geometry.The epr spectra of the complex is shown in Figure 1.

Thermal Analysis
In ther mo gravimetr ic analysis of [Co(NMEPDHAPI) 2 (H 2 O) 2 ], the first weight loss noticed in the temperature range 101-142 0 C (observed 5.97%, calculated 6.25%) is correlated with the displacement of two water molecules bound Co(II) ion.Another weight loss is in the temperature range of 156-519 0 C ( observed 83.20%, calculated 83.85%) is suitable to the twofold decomposition of binary molecules of ligand NMEPDHAPI.In [Ni(NMEPDHAPI) 2 ] the weight loss ascertained in the temperature range 158-542 0 C (observed 89.67%, calculated 89.45%) is also due to the double step decomposition of two molecules of ligand.The thermogram of [Cu(NMEPDHAPI) 2 ] showed only one weight loss in the temperature range 136-382 0 C ( observed 88.69%, calculated 85.09%) is owed to disintegration of couple of ligand moieties. 29The thermogram of all the complexes finally attains constant mass due to the dissipation of analogous metal oxides.The TGA/DTA curves are given under supplementary data (Figure S4-S6).
From the above analytical data it is may be concluded that the cobalt complex is having octahedral geometry with two coordinated water molecules and nickel and copper complexes are in square planar geometry.The structure assigned for the complexes is depicted in Figures 2 and 3.

Conductance measurements
The molar conductance of the complexes were found to be ranging from 14 ohm -1 cm 2 mol -1 to 22 ohm -1 cm 2 mol -1 .This evidences the non-electrolytic quality of the complexes. 27No change in conductance is observed when determined in different solutions.This further substantiate the neutral quality of all three complexes.

Metal estimation studies
The metal percentage of all the complexes were ascertained through standard methods. 28The amount of cobalt in complex was estimated by pyrolytic method.The quantity of nickel calculated by using titrimetric method.The copper percentage were found by colorimetric method.The values obtained are conforming with the values manipulated for the proposed structure of the complexes.

Study on Redox property
T h e c y c l i c v o l t a m m o g r a m o f [Co(NMEPDHAPI) 2 (H 2 O) 2 ] exhibits two well defined quasi-reversible peaks.First reduction peak found in Ep c = -0.680Vwith an associated oxidation peak placed at Ep a = -0.338Vand second reduction peak appearing in Ep c = 0.132 V is associated with oxidation peak situated at Epa = 0.673 V.The value of ΔE p is 0.342V and 0.541V for first and second redox couples respectively and the ratio between oxidation and reduction peak current indicative of simple quasi-reversible one electron redox processes. 30The voltammogram of [Ni(NMPDHAPI) 2 ] also shows one well denoted redox process.The reduction peak is viewed at EP c = -0.994Vand the oxidation peak is noted at EP a = 0.730V.This couple is quasi-reversible and ratio between oxidation and reduction current suggests the process to be simple one-electron transfer 31 .The voltammogram of [Cu(NMPDHAPI) 2 ] displays one oxidation peak at EP a = -0.715Vand the corresponding reduction peak at EP c = -1.224V.The another oxidation peak found in EP a = 0.603V and the respective reduction peak placed at EP c = 0.120V.The ΔEp values 0.509V and 0.483V shows that the redox processes are quasi-reversible.The correlation of oxidation to reduction peak height suggest that the the process is simple one electron process. 32The cyclic voltammograms are given in Fig 4. The redox property of the complex may find the application in catalytic studies.

Fluorescence property
The fluorescence spectra of all the synthesized compounds are shown in Fig. 5.The emission peaks are noticed in the range of 357-359 nm.Quenching of emission was observed in all the three complexes.This may be due to the predominance of PET process over CHEF effect. 33his quenching may lead to the chemo sensor utility of the Schiff base.

Antimicrobial activities
Antimicrobial activities of NMEPDHAPI and their complexes are given in Table 1.It is ascertained that the complexes are more active than the Schiff base towards almost all pathogens studied, which can be elucidated by Tweedy's chelation theory. 34Lipophilic nature of the metal ion increases by chelation, which could facilitates to cross the lipid layer of the cell membrane.The coordination of metal ion to the Schiff base influence the magnetic property and conductance which may also be a cause for the extensive biological characteristics of the complexes.The azomethine bond also extend contribution for activity of the complexes 35 .The maximum sensitiveness of the complexes is observed with the fungal pathogen candida albicans.The antimicrobial activity images are shown in Figures 6 and 7.

Anticancer activities
The in vitro cytotoxicity of copper complex were examined against breast cancer cell line (MCF-7) by MTT assay.The % of cell inhibition in different concentration shown in the Fig. 7 and Table 2.The IC 50 value of copper complex (27.61µM) compared with cis platin (4.5µM).From the IC 50 value, it may be accomplished that the copper complex possess considerable cytotoxicity for breast cancer cell line (MCF-7).