Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 624–631

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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Synthesis, characterization, electrochemical and biological studieson some metal(II) Schiff base complexes containing quinoxaline moiety

1386-1425/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.saa.2013.09.007

⇑ Corresponding author. Tel.: +91 4651245765.E-mail address: c.justindhanaraj@gmail.com (C. Justin Dhanaraj).

Chellaian Justin Dhanaraj ⇑, Jijo JohnsonDepartment of Chemistry, University College of Engineering Nagercoil, Anna University, Tirunelveli Region, Konam, Nagercoil 629 004, Tamil Nadu, India

h i g h l i g h t s

� Tridentate Schiff base ligand.� Four metal(II) complexes have been

synthesized.� 1H NMR, mass, IR, UV–Vis., ESR, CV,

PXRD, SEM, TG/DTA studies.� Complexes show significant

antimicrobial activity.� Complexes have DNA cleaving

activity.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 May 2013Received in revised form 21 August 2013Accepted 2 September 2013Available online 13 September 2013

Keywords:QuinoxalineSchiff baseComplexesAntibacterial and antifungal activityDNA cleavage

a b s t r a c t

Novel Co(II), Ni(II), Cu(II) and Zn(II) complexes of Schiff base derived from quinoxaline-2,3-(1,4H)-dioneand 4-aminoantipyrine (QDAAP) were synthesized. The ligand and its complexes were characterized byelemental analyses, molar conductance, magnetic susceptibility measurements, FTIR, UV–Vis., mass and1H NMR spectral studies. The X band ESR spectrum of the Cu(II) complex at 300 and 77 K were alsorecorded. Thermal studies of the ligand and its complexes show the presence of coordinated water inthe Ni(II) and Zn(II) complexes. The coordination behavior of QDAAP is also discussed. All the complexesare mono nuclear and tetrahedral geometry was found for Co(II) complex. For the Ni(II) and Zn(II) com-plexes, octahedral geometry was assigned and for the Cu(II) complex, square planar geometry has beensuggested. The grain size of the complexes was estimated using powder XRD. The surface morphology ofthe compounds was studied using SEM analysis. Electrochemical behavior of the synthesized complexesin DMF at room temperature was investigated by cyclic voltammetry. The in vitro biological screening ofQDAAP and its metal complexes were tested against bacterial species Staphylococcus aureus, Escherichiacoli and Pseudomonas aeruginosa. The fungal species include Aspergillus niger, Aspergillus flavus and Can-dida albicans. The DNA cleavage activity of QDAAP and its complexes were also discussed.

� 2013 Elsevier B.V. All rights reserved.

Introduction

Coordination chemistry is the most widely developed field inthe last few decades. Among the complexing ligands Schiff basesare having special interest due to their preparative accessibilityand structural variety. Schiff base are widely designed and

prepared for its high yield and one-step procedure via condensa-tion of amines and aldehydes/ketones [1]. Schiff bases offer oppor-tunities for inducing substrate chirality, tuning the metal centeredelectronic factor, enhancing solubility and either performinghomogeneous or heterogeneous catalyzes and include diversifiedsubjects comprising their various aspects in bio-coordination andbio-inorganic chemistry [2]. Metal complexes of Schiff bases havea wide variety of biological applications such as antitumor [3],

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antimicrobial [4], antioxidant [5,6] and anticancer [7] activities.They are also used as catalysts [8].

Quinoxalines are bicyclic hetero fused systems containing twonitrogen atoms. They are widely distributed in nature. Few Schiffbase metal complexes containing quinoxaline moiety were re-ported [9]. They show biological applications such as antibacterial[10–12], anti diabetic [13] activities. They are also known for theircatalytic activity [14,15]. Schiff bases with an additional donoratom closer to the imino nitrogen form stable chelate with metalions, thus the presence of keto group nearer to the imino nitrogenin 4-aminoantipyrine Schiff bases will lead to the formation of sta-ble metal chelates. In recent years, 4-aminoantipyrine transitionmetal complexes and their derivatives have been extensivelyexamined due to their applications in various fields. A large num-ber of transition metal complexes have been used as cleaving agentfor DNA and also for novel potential DNA targeted antitumor drugs[16]. The new compounds exploit passive and active targetingstrategies to overcome aspects of drug resistance. So, it is essentialto increase the variety of potential metal based drugs [17]. By con-sidering the above facts, in this work we selected quinoxaline-2,3-(1,4H)-dione and 4-aminoantipyrine for ligand synthesis. The aimof the present work is to study the metal(II) complexes of newquinoxaline based Schiff base. The ligand and its complexes havebeen tested for in vitro antimicrobial activity against different bac-teria and different fungi species by minimum inhibitory concentra-tion (MIC). The DNA cleaving properties were also illustrated. Sincethe synthesized complexes have high biological activity they maybe used as therapeutic agents.

Experimental

Materials and reagents

The chemicals used were of AnalaR grade. 4-aminoantipyrinewas purchased from Sigma Aldrich and o-phenylenediamine waspurchased from Loba Chemie India. Oxalic acid and metal(II) ace-tates were obtained from Merck and were used as received withoutany further purification. Solvents were purified and dried beforeuse by standard procedures [18].

Instruments

Elemental analysis of ligand and its metal complexes were car-ried out using Perkin–Elmer elemental analyzer. Molar conduc-tance of the complexes was measured using a coronation digitalconductivity meter. IR spectra were recorded using Jasco FTIR-410 spectrometer in KBr pellets from 400 to 4000 cm�1. 1H NMRspectra were recorded with Brucker 300 MHz spectrometer usingDMSO-d6 solvent for ligand and Zn(II) complex with TMS as inter-nal standard. DART-MS spectrum was recorded on a JEOL-Accu TOFJMS mass spectrometer. Magnetic moments were measured byGuoy method and corrected for diamagnetism of the componentusing Pascal’s constants. Electronic spectra were recorded on Ther-mo Scientific Evolution-200 UV–Vis., spectrophotometer in therange 190–1100 nm. ESR spectrum of the Cu(II) complex wasrecorded at 300 and 77 K in DMSO solution using Varian, USAE-112 ESR spectrometer using tetracyanoethylene (TCNE) asg-marker. Cyclic voltammetry measurements were performed usingelectrochemical analyzer CH instruments electrochemical workstation (Model 650 C) using a glassy carbon working electrode(GCE) with Ag/AgCl reference electrode and platinum counterelectrode. XRD studies were carried out using XPERT-PRO, X-raydiffractometer system. SEM images were recorded in a HitachiSEM analyzer. Thermal analysis were carried out under nitrogen

atmosphere at a heating rate of 10 �C per minute using Perkin El-mer Pyres Diamond TG/DTA analyzer.

Antimicrobial activity

The in vitro antibacterial activity of QDAAP and its complexeswere tested against the bacterial species Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa by agar diffusionmethod [19]. Initially, the stock cultures of bacteria were revivedby inoculating in broth media and grown at 37 �C for 18 h. The agarplates of media (peptone-10 g, NaCl-10 g and yeast extract 5 g,agar 20 g in 1000 ml of distilled water) were prepared and wellswere made in the plate. Each plate was inoculated with 18 h oldcultures (100 ll) and spread evenly on the plate. After 20 min,the wells were filled with compound at 50, 100 and 200 lg/mLconcentrations to determine the minimum inhibitory concentra-tion (MIC) value. The control wells with Gentamycin were also pre-pared. DMSO was used as negative control. All the plates wereincubated at 37 �C for 24 h and the diameter of inhibition zonewere noted.

For antifungal activity, initially the stock cultures were revivedby inoculating in broth media and grown at 27 �C for 48 h. The agarplates of the media (Czapek-Dox agar: composition (g/l) Sucrose-30.0; sodium nitrate-2.0; K2HPO4-1.0, MgSO4.7H2O-0.5; KCl-0.5;FeSO4-0.01; agar-20;) were prepared and wells were made in theplate. Each plate was inoculated with 48 h old cultures (100 ll)and spread evenly on the plate. After 20 min, the wells were filledwith compound at 50, 100 and 200 lg/mL concentrations, to findout the minimum inhibitory concentration (MIC) value. The con-trol wells were filled with Amphoterecin. All the plates were incu-bated at 27 �C for 72 h and the diameter of inhibition zone werenoted.

Cleavage of CT DNA

A solution of CT DNA in the buffer (5 mmol L�1 Tris–HCl/50 mmol L�1 NaCl buffer (pH 7.2)) gave a ratio of UV absorbanceat 260 and 280 nm of about 1.89:1, indicating the CT DNA suffi-ciently free from protein contamination. The CT DNA concentrationwas determined by UV absorption spectroscopy using the molarabsorption coefficient of 6600 M�1 cm�1 at 260 nm. Stock solutionswere kept at 4 �C and used after not more than four days.

The DNA cleavage activity of the complexes was studied usingagarose gel electrophoresis. CT DNA (0.3 lg) dissolved in5 mmol L�1 Tris–HCl/50 mmol L�1 NaCl buffer (pH 7.2), was trea-ted with the complexes. The mixture was incubated at 37 �C for2 h and then mixed with the loading buffer (2 lL) containing 25%bromophenol blue, 0.25% xylene cyanol and 30% glycerol. Eachsample (5 lL) was loaded into 0.8% (w/v) agarose gel. Electropho-resis was undertaken for 2 h at 50 V in Tris–acetate–EDTA (TAE)buffer (pH 8.0). The gel was stained with ethidium bromide for5 min after electrophoresis and then photographed under UV light.To enhance the DNA cleaving activity of the complexes, hydrogenperoxide (100 lmol L�1) was added to each sample.

Synthesis of ligand

The synthesis of quinoxaline-2,3-(1,4H)-dione was doneaccording to the literature [20]. To the hot methanolic solution ofquinoxaline-2,3-(1,4H)-dione (0.005 mol) dry methanolic solutionof 4-aminoantipyrine (0.005 mol) was added drop wise with con-stant stirring at room temperature. The mixture was refluxed for2 h and the solid obtained was filtered, washed with ether anddried in vacou over anhydrous calcium chloride (Scheme 1).

Scheme 1. Schematic representation of preparation of ligand.

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Synthesis of complexes

Methanolic solution of metal(II) acetates (0.002 mol) was addeddrop by drop to a methanolic solution of QDAAP (0.002 mol) andthe mixture was refluxed on a water bath for 3–4 h. The solid com-plex obtained was filtered, washed with hot methanol and dried invacou over anhydrous calcium chloride.

Results and discussion

One of the two dione groups of quinoxaline-2,3-(1,4H)-dionewas used for the condensation of amino group of 4-aminoantipy-rine to synthesize the ligand (QDAAP). The condensation betweenquinoxaline-2,3-(1,4H)-dione and 4-aminoantipyrine lead to theformation of a tridentate ligand. It is stable in air and is partiallysoluble in ethanol, methanol and completely soluble in DMSO,DMF, THF and chloroform.

The metal(II) complexes of QDAAP is non hygroscopic, solublein DMSO, DMF and sparingly soluble in methanol and ethanol.The complexes are stable at room temperature. The elementalanalysis data of QDAAP and its metal complexes along with molarconductance values are given in Table 1.

Molar conductance

The metal(II) complexes of QDAAP (10�3 mol dm�3) were dis-solved in DMF and molar conductivities of the solutions at roomtemperature were measured. The conductance data (Table 1) indi-cate that all the metal complexes are having conductivity values inaccordance with non electrolytes [21,22].

IR spectra

IR spectrum of QDAAP is compared with the IR spectra of corre-sponding metal complexes (Fig. S1(a–e)). The peak observed at1680 cm�1 in quinoxaline-2,3(1,4H)-dione is assigned for (>C@O)group. 4-aminoantipyrine has strong bands at 3432 and3328 cm�1 corresponding to the ANH2 stretching frequency. InQDAAP these peaks are absent, the imine >C@N band is super im-posed with the >C@O group of the pyrazole moiety and appears asa strong band at 1630 cm�1 [23] confirms the formation of QDAAP.The strong band observed at 1630 cm�1 is shifted to lower wavenumber by 15–7 cm�1 in the spectra of metal(II) complexes. Thisindicate the coordination of imino (>C@N) and keto (>C@O) groups

Table 1Elemental analysis and molar conductivity data of QDAAP and its metal complexes.

Compound Yield (%) Color m.p. (�C) Elemental analysis (%) found (

C H N

QDAAP 88 Brown 119–121

65.69(65.59) 4.93(4.88) 20

Co(II) complex 75 Dark brown >300 54.32(54.17) 4.12(4.17) 15Ni(II) complex 80 Pale green >300 50.43(50.18) 4.64(4.37) 14Cu(II) complex 65 Pale blue >300 53.78(53.74) 4.08(4.13) 15Zn(II) complex 73 Brown >300 53.77(53.37) 4.57(4.12) 13

to the metal atom in complexes. QDAAP exist in stable enol formexcept in Cu(II) complex. In Cu(II) complex it exist in keto form evi-denced by the presence of m(C@O) band at 1681 cm�1, assigned forthe >C@O group of quinoxaline moiety, in all other complexes theband due to m(C@O) has not observed due to the coordination ofoxygen through enol mode [13]. The m(OH) band is observed at3422 cm�1 in QDAAP. The absence of this band in Co(II), Ni(II)and Zn(II) complexes indicates the coordination of –OH groupthrough deprotonation [24]. This is further evidenced by the pres-ence of m(CAO) band in these complexes (1286–1279 cm�1),appearing at higher frequencies compared to that of QDAAP(1223 cm�1). In the spectra of QDAAP and its complexes the banddue to ring m(NH) is observed at 3344–3336 cm�1. In Cu(II) com-plex ring m(NH) is observed at 3266 cm�1. In Ni(II) and Zn(II) com-plexes the bands at 3410 and 3409 cm�1 respectively assigned form(OH) of coordinated water. The monodentate coordination of ace-tato group is confirmed by the presence of m(COO�)sym andm(COO�)asym stretching frequencies observed at the region1562–1542 cm�1 and 1323–1318 cm�1 respectively [25]. In the spectraof the complexes, appearance of new bands in the region 501–486 cm�1 and 457–422 cm�1 has been attributed to MAO andMAN bonds respectively [26]. From the IR spectral data it is con-cluded that QDAAP acts as a tridentate ligand in Co(II), Ni(II) andZn(II) complexes, coordinating through phenolic –OH, imino(>C@N) nitrogen atom and through >C@O group of pyrazole moi-ety. In Cu(II) complex it act as a tridentate ligand, coordinatingthrough imino (>C@N) nitrogen atom, through >C@O group of pyr-azole moiety and through one of the ring >NH through deprotona-tion. Important IR spectral bands of QDAAP and its metalcomplexes along with possible assignments are summarized inTable 2.

1H NMR spectra

1H NMR spectra of QDAAP (Fig. 1a) and its Zn(II) complex(Fig. 1b) were analyzed. The singlet observed at 11.92 ppm in thespectrum of QDAAP is assigned to quinoxaline ring ANH proton[27]. In the spectrum of Zn(II) complex this peak remained un-changed. The phenolic AOH proton resonates at 6.61 ppm [28],which is not present in the spectrum of Zn(II) complex indicatingthe participation of phenolic AOH group in chelation throughdeprotonation. The aromatic protons observed in the region7.07–7.48 ppm show small shifts in complex, due to variation inelectron density and steric constraints because of chelation. The

Calculated) Molar conductivity(O�1 cm2 mol�1)

O M

.60(20.67) 9.21(9.36) – –

.08(15.05) 13.78(13.50) 12.69(12.62) 2.5

.00(14.05) 19.19(19.60) 11.74(11.49) 3.9

.93(15.98) 13.65(13.96) 13.55(13.38) 6.5

.82.(13.92) 13.94(13.91) 13.69(13.46) 2.5

Table 2IR spectral data of QDAAP and its complexes in cm�1.

Compound m(NH) m(OH)phenolic m(OH) m(C@N) and m(C@O) m(C@O)Q m(C@N)ring m(MAO) m(MAN)

QDAAP 3336 3422 – 1630 – 1498 – –Co(II) complex 3343 – – 1623 – 1502 488 446Ni(II) complex 3344 – 3410 1621 – 1502 486 433Cu(II) complex 3266 – – 1615 1681 1502 501 422Zn(II) complex 3343 – 3409 1623 – 1504 494 457

Fig. 1a. 1H NMR spectrum of QDAAP.

Fig. 1b. 1H NMR spectrum of Zn complex.

Fig. 2. DART mass spectrum of QDAAP.

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signal for methyl proton attached to pyrazolone ring carbon appearas singlet at 2.10 ppm and the pyrazolone ring nitrogen attachedmethyl protons (>NCH3) appear as a singlet at 3.61 ppm. A newsinglet appeared at 4.37 ppm in the spectrum of Zn(II) complex

indicates the presence of coordinated water molecule in the com-plex [29].

Mass spectra

The mass spectrum of ligand (Fig. 2) and its Co(II), Ni(II), Cu(II)and Zn(II) complexes were recorded and their stoichiometric com-positions were compared. The DART mass spectrum of QDAAP(C19H17N5O2) shows a well defined molecular ion peak atm/z = 347 (Relative Intensity = 5%), which coincides with theformula weight of the Schiff base. In addition, the spectrum ofQDAAP shows a series of peaks at m/z = 331 (100%), 330 (32%),204 (3%), and 99 (4%) corresponding to its various fragments.whereas the molecular ion peaks of Co(II), Ni(II), Cu(II) and Zn(II)complexes were observed at 465 (11%), 500 (6%), 469 (13%) and511 (9%) m/z respectively. Elemental analysis values are inagreement with the values calculated from the molecular formulaeassigned to these complexes which are further supported byDART-mass studies.

Electronic absorption spectra

The electronic spectral data of QDAAP and its complexes alongwith magnetic moment values are tabulated in Table 3.

Electronic spectra of QDAAP and its complexes were recorded inDMF at room temperature in the range 190–1100 nm. QDAAP ex-hibit electronic transitions with strong band at 230–250 nm, as-signed to p–p� transitions, which remain almost unchanged inthe complexes. The transitions at 275–300 and 350 nm are as-signed to n–p� transitions of carbonyl and imine respectively. Incomplexes these bands experiences bathochromic shift due tocoordination of imino nitrogen atom and carbonyl group of 4-ami-noantipyrine moiety. In Co(II), Ni(II) and Cu(II) complexes a shoul-der observed at 400–430 nm is assigned to ligand to metal chargetransfer transitions [30]. The electronic spectrum of Co(II) complexshow absorption band at 730 nm assignable to 4A2 (F) ? 4T1(P),which is characteristic value for the tetrahedral Co(II) complex.The electronic spectrum of the Ni(II) complex show three absorp-tion bands at �1100, 786 and 385 nm assignable to 3A2g

(F) ? 3T2g (F), 3A2g (F) ? 3T1g (F) and 3A2g(F) ? 3T1g (P) transitionsrespectively. This is characteristic of six coordinated octahedralNi(II) complex [31]. The square planar Cu(II) complex display abroad band at 550–700 nm, corresponding to 2B1g ?

2A1g and2B1g ?

2Eg transitions [32]. The Zn(II) complex has d10 electronicconfiguration and exhibit only intra ligand transitions (Fig. S2(a–d)). According to the empirical formula, an octahedral geometryis proposed for this complex.

Magnetic measurements

At room temperature, the magnetic moment values of Co(II)and Ni(II) complexes are 4.62 and 3.23 BM, suggesting four coordi-nate tetrahedral and six coordinate octahedral geometry respec-tively [33]. The leff value for the present Cu(II) complex is 1.93BM, which is characteristic for square planar geometry around

Table 3Electronic spectral data and magnetic moment values of QDAAP and its complexes.

Compound Absorptions(nm)

Transition leff (BM) Geometry

Co(II)complex

730 4A2ðFÞ ! 4T1ðPÞ 4.62 Tetrahedral

Ni(II)complex

�1100 3A2gðFÞ ! 3T2gðFÞ

786 3A2gðFÞ ! 3T1gðPÞ 3.23 Octahedral

385 3A2gðFÞ ! 3T1gðPÞ

Cu(II)complex

550–700 2B1g ! 2A1g 1.93 Square planar

2A1g ! 2Eg

Zn(II)complex

– – Diamagnetic Octahedral

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Cu(II). Spin–orbit coupling followed by lowering of symmetry isresponsible for the increase in observed magnetic moment values.

The spectral and analytical data suggest the following empiricalformula [M(QDAAP)(OAc)] for Co(II) and Cu(II) complexes. ForNi(II) and Zn(II) complexes the empirical formula is[M(QDAAP)(OAc)(H2O)2].

EPR spectroscopy

EPR spectral studies of paramagnetic transition metal(II) com-plexes gives information about the distribution of the unpairedelectrons and hence about the nature of the bonding betweenthe metal ion and its ligands. The X-band EPR spectra of the pres-ent Cu(II) complex was recorded in DMSO at liquid nitrogen tem-perature and at room temperature (Fig. 3). The EPR spectrum ofCu(II) complex at room temperature show one intense absorptionband in the high field and is isotropic due to tumbling motion ofthe molecules. The EPR spectrum of Cu(II) complex at liquid nitro-gen temperature show four well resolved peaks; three peaks withlow intensities in the low field region and one intense peak in thehigh field region. The g values are in the order g|| > g? > 2:0027 cor-responding to d2

x � d2y orbital as ground state. For Cu(II) complex,

g11 is a parameter sensitive enough to indicate covalency. The factthat g|| is less than 2.3 is an indication of significant covalent char-acter to MAL bond [34].

From the spectral data, it is found that, A|| = 157 > A? = 61;g|| = 2.24 > g? = 2.06 > 2 and the EPR parameters of the complexsuggest that the complex have square planar geometry and thesystem is axially symmetric. This is also supported by the fact thatthe unpaired electron lies predominantly in the d2

x � d2y orbital [35].

Fig. 3. EPR spectrum of Cu(II) complex.

Electrochemical behavior

Cyclic voltammetry is the most widely used technique foracquiring qualitative information about electrochemical reactions.It offers a rapid location of redox potentials of the electro activespecies. The cyclic voltammograms of ligand and its metal com-plexes (Fig. S3(a–d)) were recorded at 300 K in DMSO solution inthe potential range �1.0 to1.2 V with scan rate 0.1 V/s.

The ligand shows one irreversible couple with Epc = �0.8 V vsAg/AgCl and the associated anode peak at Epc = 0.15 V. The largeseparation DE = 0.65 V indicates irreversible couple. The Co(II)complex shows two irreversible peak one at Epc = 0.95 V corre-sponding to Co(II)/(I) couple. The other peak at Epc = �0.02 V dur-ing the reverse scan corresponds to Co(II)/(III) couple. The peakto peak separation DE is 0.75 V indicating the process to be irre-versible. Ni(II) complex shows two irreversible peaks one atEpc = 0.8 V and the other at Epc = 0.45 V corresponding to Ni(II)/(I)couple. The Cu(II) complex shows irreversible cathodic peak atEpc = 0.6 V vs Ag/AgCl corresponding to Cu(II)/(III) couple and theassociated anode peak at Epa = �0.58 V corresponding to the for-mation of Cu(II)/I couple. The peak to peak separation DE is1.12 V confirming the process as irreversible. The Zn(II) complexshows quasireversible peak at Epc = 0.35 V vs Ag/AgCl and the asso-ciated anode peak at Epa = 0.55 V. The peak to peak separation va-lue DE = 0.25 V corresponding to quasireversible process [36].

Thermal studies

The thermal stabilities of QDAAP and its metal complexes wereinvestigated using TG and DTA under nitrogen atmosphere with aheating rate of 10 �C per minute from 50 �C to 700 �C. The Schiffbase ligand QDAAP showed single stage decomposition below150 �C, where as the metal complexes showed multi stage decom-position pattern [37]. QDAAP does not show a sharp melting pointbut it undergoes some phase change in the temperature range115–125 �C. DTA showed an endotherm at 120 �C correspondingto this decomposition. The thermograms of Co(II) and Cu(II) com-plexes do not show any weight loss up to 250 �C indicating the ab-sence of water molecules in these complexes. Thermograms ofNi(II) and Zn(II) complexes show weight loss around 140–200 �Cindicates the presence of two coordinated water molecules [25].In DTA, an endothermic peak seen at 150 �C further confirms thisdecomposition. The thermal decomposition of the complexes atthe temperature range 245–400 �C corresponds to the loss of ace-tate groups and the decomposition of quinoxaline moiety ofQDAAP. DTA showed an endotherm at 250 �C corresponding tothe second stage decomposition. An endotherm observed in DTAat 410 �C was due to third stage of decomposition. The representa-tive TG-DTA thermograms are shown in (Fig. S4). Thermal analysisresults are in good agreement with the formula of the complexesarrived from the analytical data.

Based on the above results the proposed structures of the com-plexes are given in Fig. 4.

XRD

The nature of QDAAP and its complexes were also obtained bypowder X-ray diffraction studies. The X-ray diffractions of QDAAPand its complexes are shown in Fig. S5(a–d). From the observeddXRD patterns, the grainsize of the ligand and its complexes werecalculated from Schererr’s formula, dXRD = 0.9k/bCosh, where dXRD

is the particle size, k is the wave length of X-ray radiation, b isthe full-width half maximum and h is the diffraction angle forthe hkl plane. The ligand, Ni(II), Cu(II) and Zn(II) complexes arenanocrystalline with grain sizes 14.1, 4.2, 12.0 and 8.3 nm respec-tively, while the Co(II) complex is amorphous in nature [38].

Fig. 4. Proposed structure of complexes.

Table 4Antibacterial activity of QDAAP and its metal complexes (lg/mL).

Compound S. aureus E. coli P. aeruginosa

QDAAP 20 17 19Co(II) complex 18 13 14Ni(II) complex 08 15 11Cu(II) complex 05 12 04Zn(II) complex 10 12 09Gentamycin 18 13 10

Table 5Antifungal activity of QDAAP and its metal complexes (lg/mL).

Compound C. albicans A. flavus A. niger

QDAAP 13 10 18Co(II) complex 05 11 09Ni(II) complex 07 04 13Cu(II) complex 08 05 12Zn(II) complex 10 09 15Amphoterecin 10 08 12

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SEM

The SEM micrographs of ligand and its complexes are shown in(Fig. S6(a–e)). The SEM micrograph of ligand differs significantlyfrom the complexes. The micrograph of ligand show irregularlyshaped particles. The Co(II) complex shows cauliflower like struc-ture. Agglomerated morphology was seen for the Ni(II) complex.For Cu(II) and Zn(II) complexes faceted microcrystals with layeredmorphology was present.

Antimicrobial analysis

The results of the antibacterial and antifungal activities aresummarized in Tables 4 and 5. The standards used are Gentamycinfor antibacterial and Amphotericin for antifungal activities.

The antibacterial and antifungal activity of QDAAP and its metalcomplexes indicate that the complexes possess higher growth inhi-bition potential compared to those of the ligand. The antimicrobialactivity of the metal complexes increases with increase in concen-tration of the complexes. It is suggested that the complexes havingantibacterial and antifungal activities inhibit multiplication pro-cess of the microbe by blocking their active sites [39]. The mecha-nism of toxic activity of the complexes with the ligands can beascribed to the increase in the lipophilic nature of the complexesarising from chelation. Chelation reduces the polarity of the metalatom mainly because of partial sharing of its positive charge withthe donor groups and possible p electron delocalization within thewhole chelate ring. The chelation also increases the lipophilic nat-ure of the metal atom, which subsequently favors the permeationthrough the lipid layer of cell membrane. The mode of action ofcomplexes involves the formation of hydrogen bonds with the

imino group by the active sites leading to interference with the cellwall synthesis. This hydrogen bond formation damages the cyto-plasmic membrane and the cell permeability may also be alteredleading to cell death [40,41].

In the present work, Cu(II) complex show higher activity in bothantibacterial and antifungal studies. The difference in antimicro-bial activity is due to the nature of metal ions and also the cellmembrane of the microorganisms [42]. The higher bioactivity ofthe complexes compared to that of the ligands is due to the

Fig. 5. Changes in the agarose gel electrophoretic pattern of CT DNA induced byH2O2. (1) DNA alone, (2) DNA + QDAAP + H2O2, (3) DNA + Co(II) complex + H2O2, (4)DNA + Ni(II) complex + H2O2, (5) DNA + Cu(II) complex + H2O2 and (6) DNA + Zn(II)complex + H2O2.

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decrease in the charge localization of the metal ion that leads tomore lipid solubility.

DNA cleavage studies

Transition metals have been seen to inhibit DNA repair en-zymes. When DNA is run on horizontal gel using electrophoresis,the fastest migration will be observed for the open circular form(Form I). If one strand is cleaved a slower-moving linear form(Form II) is observed [43]. Gel electrophoretic experiment usingCT DNA was performed with metal complexes in the presence ofH2O2 as oxidant. From the Fig. 5, it is clear that the complexescleave DNA more effectively in the presence of H2O2. This maybe due to the formation of hydroxyl free radicals. These hydroxylradicals participate in the oxidation of the deoxyribose moiety, fol-lowed by hydrolytic cleavage of the sugar-phosphate backbone.

The DNA cleavage efficiency of the complex was due to the dif-ferent binding affinity of the complex to DNA. Control experimentusing DNA alone does not show any significant cleavage even onlonger exposure time. In the present study, Ni(II) complex com-pletely cleaved the DNA. Co(II) and Cu(II) complexes exhibited en-hanced DNA cleaving activity than QDAAP and Zn(II) complex.Probably this is due to the formation of redox couple of the metalions and its behaviour. Co(II) complex reacts with H2O2 to formOH�, OH� and Co(III) [44]. In the case of Cu(II) complex, the redoxcycle between Cu(II) and Cu(I) can catalyze the production ofhighly reactive hydroxyl radicals [45]. It is observed that mostcleavage cases are caused by copper ions reacting with H2O2 toproduce the diffusible hydroxyl radical or molecular oxygen, whichmay damage DNA through Fenton type chemistry. In the case ofgenomic DNA (CT DNA) circular form is converted into linear form.The DNA cleavage efficiency was calculated by determining theability of the complex to convert open circular form (Form I) to lin-ear form (Form II).

Conclusion

Co(II), Ni(II), Cu(II) and Zn(II) complexes of QDAAP have beensynthesized and characterized on the basis of various spectro-ana-lytical data. QDAAP act as tridentate ligand with ONO donor inCo(II), Ni(II) and Zn(II) complexes. The coordination sites are phe-nolic -OH, imino nitrogen and ketonic oxygen groups. It acts as tri-dentate ONN donor with imino nitrogen, oxygen atom in ketogroup of pyrazole moiety and one of the ring nitrogen throughdeprotonation in Cu(II) complex. Co(II) complex shows tetrahedralgeometry and Cu(II) complex shows square planar geometry. Thepresent Ni(II) and Zn(II) complexes possesses octahedral geometry.The powder XRD study reveals that the Co(II) complex is amor-

phous where QDAAP, Ni(II), Cu(II) and Zn(II) complexes are nano-crystalline. SEM measurements indicated cauliflower likemorphology and agglomerated morphology for the Co(II) and Ni(II)complexes respectively. For Cu(II) and Zn(II) complexes facetedmicrocrystals with layered morphology was present. The in vitroantibacterial and antifungal studies of of QDAAP and its metalcomplexes have also been evaluated. The thermal studies indicatethat the metal complexes are thermally more stable compared toQDAAP. Among the metal complexes, Cu(II) complex exhibitedhigher antimicrobial activity. CT DNA cleavage activity of QDAAPand its metal complexes was performed by agarose-gel electropho-retic assay and the complexes exhibited effective DNA cleavage viahydrolytic mechanistic path way.

Acknowledgements

We are thankful to Council of Scientific and Industrial Research(CSIR), New Delhi, India for the financial support in the form of aresearch project (Scheme No: 01(2453)/11/EMR-II).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.saa.2013.09.007.

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