Synthesis , Characterization , Biological Activity and DNA Binding Studies of Metal Complexes with 4-Aminoantipyrine Schiff Base Ligand

A new series of transition metal complexes of Cu(II),Ni(II),Co(II), Zn(II) and VO(IV) have been synthesized from the Schiff base ligand (L) derived from 4-amino antipyrine and 5bromo salicylaldehyde. The structural features of Schiff base and metal complexes were determined from their elemental analyses, thermogravimetric studies, magneticsusceptibility, molar conductivity, ESI-Mass, IR, UV-VIS, H NMR and ESR spectral studies. The data show that the complexes have composition of ML2 type. The UV-VIS, magnetic susceptibility and ESR spectral data suggest an octahedral geometry around the central metal ion. Biological screening of the complexes reveals that the Schiff base transition metal complexes show significant activity against microorganisms. Binding of Co(II) complex with calf thymus DNA (CT DNA) was studied by spectral methods.


Introduction
Antipyrine derivatives are reported to exhibit analgesic and anti -infammatory effects [1][2][3][4] , antiviral 5 , antibacterial 6 activities and have also been used as hair colour additives 7 and to potentiate the local anesthetic effect of lidocaine 8 .These compounds have been used in spectrophotometric determination of metal ions.Many of these reagents give intense colours with transition metal ions, providing sensitive probes 9 and some of them can also coordinate to rare earth ions to form metal complexes with interesting structures 10 .Antipyrine Schiff base derivatives can serve as antiparasitic agents and their complexes with platinum(II) and cobalt(II) ions have been shown to act as antitumour substances 11 A search through the literature reveals that no work has been done on the transition metal complexes of Schiff base formed by the condensation of 4-aminoantipyrine with 5-bromo salicylaldehyde.In the present context, we synthesized tridentate ligand system from 4-aminoantipyrine and 5-Br salicylaldehyde.The synthesized compound and its ligation behaviour with metal ions was studied.

Experimental
All the chemicals used were of analar grade.4-Aminoantipyrine, metal salts and 5-bromo salicylaldehyde were obtained from Sigma Aldrich.Solvents such as ethanol, methanol, petroleum ether were purified by standard procedures.

Instruments
Elemental analysis of the ligands and their metal complexes was carried out using a Perkin-Elmer 240C (USA) elemental analyser.Molarconductances of the metal complexes were measured in a DMSO solution using digisun electronic conductivity meter.Magnetic susceptibilities of the complexes were determined on Guoy balance, model 7550 using Hg [Co(NCS) 4 ] as standard.The diamagnetic corrections of the complexes were done using Pascal's constants.TGA studies were carried on Shimadzu DTG -60 H system in the temperature range of 0-1000 0 C. IR spectra were recorded in KBr discs on Brucker FTIR spectrophotometer from 400 to 4000 cm -1 .Electronic spectra were recorded with an Elico SL 159 UV visible spectrometer from 200 -1100 nm. 1 H NMR & 13 C NMR spectra were recorded using CDCl 3 , at Brucker 400MHZ spectrometer.The mass spectra were recorded by ESI technique on LCQ ion trap (thermo Finningan Sanjose CA (USA) mass spectrometer instrument.The ESR spectra of the Cu(II) complexes were recorded on Jeol, JES -A 200 ESR spectrometer at room temperature.

Synthesis of Schiff base(5-Br SALAAP) ligand
The 5-bromo salicylidene-4-aminoantipyrine(5-Br SALAAP) was prepared by using the reported method 12 .4-Aminoantipyrine (2.03 g 0.01 mmol) and 5-bromo salicylaldehyde (2.01 g 0.01 mmol) were dissolved in a methanol solution.The mixture was stirred for 30 min at room temperature to give a yellow precipitate.The mixture was filtered and the solid obtained was recrystallised from methanol solution.

Synthesis of metal complexes
In the preparation of the metal complexes, the metal and the ligands were mixed in 1:2 molar ratio using required quantities of ethanol.Hot ethanolic solution of ligand (0.01 mol) and hot ethanolic solution of corresponding metal salts (0.01/0.005 mol) (MX 2 where M=Cu(II),Ni(II),Co(II),Zn(II) and VO(IV) X=Cl; for VO(IV);X=SO 4 ) were mixed together, refluxed for 2-3 h and left for evaporation at room temperature for 3 days, coloured solid metal complexes were obtained.The products were filtered, washed with cold ethanol and dried under vacuum over calcium chloride.

DNA binding activity
Absorption spectra were reorded on a UV-Visible spectrophotometer using 1 cm quartz micro cuvettes.Absorption titrations were performed by keeping the concentration of the complex constant (100 µM) and by varying the concentration of CT -DNA from 0-40 µM.The binding constant (K b ) for the binding of the complex with DNA, has been determined from the spectroscopic titration data using the following equation.
[DNA]/(ɛ a -ɛ Where [DNA] is the concentration per nucleotide,the apparent extinction coefficient ɛ a , was obtained by calculating A obs / [Co(II)], ɛ f extinction coefficient of the complex in presence of DNA and ɛ b the extinction coefficients for the fully Co(II) complex in the fully bound form.K b (binding constant) the ratio of the slope to the intercept ,was obtained from a plot of [DNA] /(ɛ a -ɛ f ) versus [DNA], a slope 1/(ɛ b -ɛ f ) and an intercept 1/K b (ɛ b -ɛ f ).

Infrared spectra
The infrared spectra of the ligand exhibited a broad meduim intensity band around 3434 cm -1 is due to intramolecular hydrogen bonded OH group.A very strong band occurring at 1637 cm -1 is attributed to υ C=O of pyrazolone ring.A band at 1595 cm -1 in the ligand spectrum is possibly due to the vibrational stretching frequency of azomethine υ C=N group.

Mass spectra
In the ESI-mass spectrum of ligand (Figure 3), the molecular ion peak was observed at m/z 386 which is in good agreement with the molecular weight of the proposed structure.

Characterization of metal complexes
We have prepared and characterized the complexes of Cu(II),Ni(II),Co(II) and Zn(II) and VO(IV) with 5-Br SALAAP ligand.All the complexes are stable at room temperature and non hygroscopic, insoluble in water, but soluble in DMSO and Ethanol.
Based on the data of elemental analysis & ESI mass, the composition assigned to the complexes, are formulated and presented in Table 1.The conductance values for all complexes were measured using DMSO solvent, the obtained low values (6-11 Ohm -1 cm 2 mol -1 ) suggest the presence of a non-electrolytic nature of complexes in the solvent and that no anions are present outside the co-ordination sphere.

Infrared spectra
The IR spectra of the complexes are compared with that of the free ligand to determine the changes that might have taken place during the complexation, all data are listed in Table 2.The band assigned to the azomethine group in the free Schiff base ligand was observed at υ C=N 1595 cm -1 and shifted to lower or higher wave numbers ranging from 1562-1617 cm -1 in the spectra of all complexes.This indicates the participation of the nitrogen atom of the azomethine group in coordination [13][14][15][16] .The band at υ C=O = 1637 cm -1 is characteristic of the carbonyl group present in the free Schiff base ligand.This group was shifted to lower or higher wave numbers ranging from 1591-1641 cm -1 in all complexes, which indicates the involvement of the carbonyl oxygen in coordination [13][14][15]17 . Thepresence of IR broad bands of metal complexes are in the range of 2931 -3419 cm -1 indicate the presence of coordinated/lattice water molecules supported from thermal analysis 13,18 .The υ C-O (phenolic) stretching frequency of ligand is seen at 1342 cm -1 , which gets shifted to higher (1355-1384 cm -1 ) region in the complexes indicating coordination of phenolic oxygen 19 .The IR spectra of complexes show new bands at 552-573 cm -1 and 429 -473 cm -1 assigned to υ (M-O) and υ (M-N) respectively 20 .The peak observed at 987 cm -1 complexes of VO are due to υ (V=O) .

Electronic spectra and magnetic susceptibility
The electronic spectral data and magnetic moments (µ eff B.M) of the complexes are presented in Table 3.The electronic absorption spectra of ligand and metal complexes were recorded in DMSO, in the range of 200-1100 nm.The electronic spectrum of free Schiff base showed three bands at 297 nm, 347 nm and 407 nm are due to π→π * and n-π * transitions 13 .Cu(II) complex displayed four bands at 302 nm, 357 nm, 427 nm and 580 nm.The first two peaks are most probably due to ligand →metal charge -transfer transitions.The last two peaks are due to d -d transitions, which is assigned 2 E g → 2 T 2g suggesting tetragonal geometry (distorted octahedral geometry) 21 .The magnetic moment of Cu(II) was seen at 1.72 B.M. On the basis of electronic spectra and magnetic susceptibility measurements, a distorted octahedral geometry around Cu(II) is suggested.
The electronic spectra of Ni(II) complex displayed four bands at 302 nm, 347 nm, 457 nm and 705 nm.The first two peaks are most probably due to ligand→metal chargetransfer transitions.The last two peaks are due to d-d transitions and are assigned as 3 A 2g (F) → 3 T 1g (P) and 3 A 2g → 3 T 1g (F) respectively.This suggests an octahedral geometry [21][22][23][24] around Ni(II) ion.
The magnetic moment of Ni(II) complex was found to be 2.87 B.M which are in the normal range observed for octahedral Ni(II) complexes.The electronic spectrum of the Co(II) complex exhibited four bands at 297 nm, 347 nm, 437 nm and 634 nm.The first two intense maxima are due to intra-ligand transitions of the organic moiety.
The other two transitions may be assigned to 4 T 1g → 4 T 1g (P) and 4 T 1g (F)→ 4 T 2g (F) respectively, suggesting an octahedral geometry around Co(II) ion 25 .The magnetic moment of Co(II) complex which was found to be 5.14 B.M. is also an indicative of octahedral geometry 26 .
The Zn(II) complex has been found to be diamagnetic in nature.On the basis of analytical, conductance and spectral data, the Zn complex, assigned to be an octahedral geometry 27 .The observed magnetic moment value for VO(IV) complex is 1.91 B.M. which suggests octahedral geometry for this complex 28 .In the present studies, VO(IV) complex shows four bands at 307 nm, 347 nm, 452 nm and 527 nm.The first two intense maxima are due to intra-ligand transitions of the organic moiety.The other two transitions may be due to 2 B 2 → 2 B 1 and 2 B 2 → 2 A 1. The low frequency band corresponding to 2 B 2 → 2 E is not clearly resolved in the spectrum.Based on the two bands observed, octahedral geometry is proposed for the above complex 29 .

ESR spectra
The ESR spectra of the Cu(II) complex (Figure 4) was recorded on Jeol, JES -FA 200 ESR spectrometer at R.T.The observed data for complex showed that g Π =2.07-2.543and g ┴ = 2.22.This complex showed g Π > 2.3 which is characteristic of anion environment.The trend g Π > g ┴ > 2.0023 observed for the complexes indicates that unpaired electron is localised in d x 2 -y 2 orbital of the Cu(II) ion.Thus a tetragonal geometry is proposed for the complex.G =(g Π -2) /(g ┴ -2), which measure the exchange interaction between the metal centres in a polycrystalline solid has been calculated.Acc to Hathaway 30 if G>4 the exchange interaction is negligible if G< 4 indicates considerable exchange interaction in the solid complexes.The above reported complex showed G values < 4 indicating the exchange interaction in complex.Furthr more, Masa cesi et al 31 reported that g Π is 2.4 for copperoxygen bonds and 2.3 for copper -nitrogen bonds.for the complex reported here, g Π values between 2.3 -2.4 which further confirms ,the presence mixed copper-nitrogen and copperoxygen bonds in these chelate complex.

Thermal analysis
In the present investigation heating rates were suitably controlled at 10 0 C min -1 under nitrogen atmosphere and the weight loss was measured from the ambient temperature up to 1000 0 C. Water molecules in complexes are generally of two types lattice water and coordinated water 32

Mass spectra
Mass spectra of complexes provides a vital clue for elucidating the structure of compounds.The ESI mass of the ligands and its metal complexes recorded at room temperature were used to compare their stoichiometry.
The molecular ion peaks of Cu(II),Ni(II) and Co(II), (Figure 7) Zn(II) and VO(IV) complexes were observed respectively at 834, 828, 830,835 and 838 and which were in good agreement with the molecular weight of the proposed structures and few un identified ions were also observed.

Antimicrobial activity
In the present investigation, biological activity of the ligand and their complexes with Cu(II),Ni(II),Co(II),Zn(II) and VO(IV) have been screened for antimicrobial activity against bacteria (E.coli and S.aureus) by paper disc method.The concentration for these samples maintained as 200 µg/mL, 100 µg/mL and 50 µg/mL in DMSO.In the present study the zones of inhibition of antibacterial activity have been presented in Table 4.The results indicate that copper and cobalt complexes show activity against E.coli and S. aureus at 200 µg/mL (Figure 8).

DNA binding activity
In general, complexes with aromatic moieties which bind to DNA through intercalation usually results in hypochromism and bathochromism, due to the stacking interaction between aromatic chromophore of the complexes and the base pairs of DNA.The absorption spectra of the complex, Co(II)-5-Br SALAAP in the absence and presence of calf thymus DNA are illustrated in Figure 9.In the presence of DNA, decrease of peak intensities were observed in the absorption spectra of complexes.Hypochromism was suggested to be due to the interaction between the electronic state of the intercalating chromophore and that of the DNA bases [33][34][35][36][37] .

Molecular modelling studies
The possible geometries of metal complexes were evaluated (Figure 11 & 12) using the molecular calculation with arguslab 38 software.Initially the atomic charges of each complex were calculated by a semiempirical method.The structures using the standard force field included in the package, further refinement was done for the complexes.The refined structures were screened on the basis of the lowest strain energy.The energies of the resulting systems were calculated using UFF [38][39][40][41][42][43][44] .

Conclusion
The metal chelates of ligand have been structurally characterized.The complexes of the ligand are non electrolytes in DMSO.This ligand act as tridentate, coordinating through nitrogen of azomethine, phenolic oxygen and carbonyl of antipyrine ring respectively.Geometries of the metal complexes are assigned,based on analytical, conductance, magnetic and electronic spectral data.Biological studies of these complexes reveal that they show better activity when compared to that of the ligand.

The 1 H
NMR spectrum of the Ligand (Figure1) displays the following signals: C 6 H 5 as a multiplet at 6-8 ppm.=C -CH 3 at 2.4 ppm, -N-CH 3 at 3.2 ppm and azomethine proton at-CH=N-at 9.8 ppm, intramolecular hydrogen bonded OH group at 13.4 ppm.

Figure 4 .
Figure 4. ESR spectra of Cu(II) complex . The lattice water will be lost at low temperature (60-120 0 C) where as the loss of coordinated water molecule is observed at high temperatures (150-200 0 C).In the thermograms of DTA and TGA of complexes [Co(II)(5-BrSALAAP) 2 ]H 2 O, [VO(IV) (5-Br SALAAP) 2 ] H 2 O, (Figure 5 & 6) a small weight loss in the range of 60-120 0 C is assigned to loss of lattice water, maximum and gradual weight loss in the range of 300-1000 0 C is and attributable to decomposition of ligand moiety.The residue at 1000 0 C indicates the non volatile metal component present in the complex.

Figure 9 .Figure 10 .
Figure 9. Absorption spectra of [Co(II) (5-Br SALAAP) 2 ] in the absence and presence of increasing amounts of DNA, [Co] =100 µM, with increasing DNA concentration from 0-40 µM.(Arow (↓) shows the absorbance changes upon increasing DNA concentration.In side: Linear plot for the calculation of the intrinsic DNA binding constant K b )

Table 1 .
Analytical data of complexes

Table 2 .
IR spectral data of ligand and its complexes in cm-1

Table 3 .
Electronic spectra and magnetic susceptibility data for ligand and complexes

Table 4 .
Antimicrobial acivity of complexes (inhibition of growth)