Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

Spectral characterization, electrochemical and anticancer studieson some metal(II) complexes containing tridentate quinoxalineSchiff base

http://dx.doi.org/10.1016/j.saa.2014.02.0751386-1425/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +91 4651245765.E-mail address: c.justindhanaraj@gmail.com (J.D. Chellaian).

Justin Dhanaraj Chellaian ⇑, Jijo JohnsonDepartment of Chemistry, University College of Engineering Nagercoil, Anna University, Tirunelveli Region, Konam, Nagercoil 629004, Tamil Nadu, India

h i g h l i g h t s

� Tridentate ONO donor quinoxalineSchiff base ligand.� Complexes have antimicrobial

activity.� Complexes show DNA binding and

DNA cleaving abilities.� The metal complexes possesses

anticancer activity.

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

NHN

ON

NO

O NNH

ON

N

O

O

M

(Where M = Co(II), Ni(II), Cu(II) and Zn(II))

a r t i c l e i n f o

Article history:Received 3 January 2014Received in revised form 6 February 2014Accepted 13 February 2014Available online 26 February 2014

Keywords:Schiff baseONO donorMetal complexesAntimicrobialDNAMTT assay

a b s t r a c t

Co(II), Ni(II), Cu(II) and Zn(II) complexes of a tridentate ONO donor Schiff base ligand derived from 3-(2-aminoethylamino)quinoxalin-2(1H)-one were synthesized. The ligand and its metal complexes werecharacterized using elemental analysis, molar conductance, IR, 1H NMR, mass, magnetic susceptibility,electronic spectra and ESR spectral studies. Electrochemical behavior of the synthesized compoundswas studied using cyclic voltammetry. The grain size of the synthesized compounds was determinedby powder XRD. The Schiff base and its complexes have been screened for their antimicrobial activitiesagainst the bacterial species E. coli, K. pneumoniae, P. aeruginosa and S. aureus; fungal species include,A. niger, and C. albicans by disc diffusion method. The results show that the complexes have higher activ-ity than the free ligand. The interaction of the complexes with calf thymus DNA (CT DNA) has been inves-tigated by electronic absorption method. Furthermore, the DNA cleavage activity of the complexes wasstudied using agarose gel electrophoresis. In vitro anticancer studies of the ligand and its complexes usingMTT assay was also done.

� 2014 Elsevier B.V. All rights reserved.

Introduction preparative accessibility, diversity and structural variability [1].

Metal chelate Schiff base complexes have continued to play therole of one of the most important stereochemical models in maingroup and transition metal coordination chemistry due to their

Metal based and metal binding agents are used for design of inor-ganic drugs. The use and significance of inorganic compounds hadbeen important to the medical field [2]. Schiff base compounds arewell known to exhibit a wide range of applications in pharmaceu-tical, antimicrobial, anticarcinogenic reagents, industrial and ana-lytical fields [3]. Many transition metal complexes have beenutilized as probes of DNA structure, as agents for mediation of

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404 397

strand scission of duplex DNA and as chemotherapeutic agents [4].Coordination of organic compounds with metal ions causes drasticchange in the biological properties of the ligand and the metal ionmoieties. Nitrogen containing heterocyclic molecules constitutesthe largest portion of chemical entities, which are part of manynatural products, fine chemicals and biologically active pharma-ceuticals vital for enhancing the quality of life [5]. Transition metalcomplexes with their varied coordination environments and tun-able redox and spectral properties are able to interact with duplexDNA in various ways [6]. One of the potential approaches in anti-cancer chemistry is focused on the design of new metal com-pounds with different substituents and labile sites which mayincrease their cytotoxicity [7]. Studies on synthesis of quinoxa-line-based Schiff base complexes have considerable importancebecause of their interesting chemical and biological properties.Quinoxaline derivatives are widely distributed in nature and havea variety of biological applications. Quinoxalines are also impor-tant in material science, as magnetic materials, organic light emit-ting diodes and non-linear materials [8].

The present study describes the synthesis of Schiff base derivedfrom 3-(2-aminoethylamino)quinoxalin-2(1H)-one and o-vanillin(1). Subsequently, the complexes of Co(II), Ni(II), Cu(II) and Zn(II)ions with 1 were synthesized and characterized.

Experimental

Materials

The chemicals used were of AnalaR or synthesis grade, o-vanil-lin was obtained from Spectrochem PVT. LTD., Mumbai, India ando-phenylenediamine was purchased from Loba Chemie India andwas purified by recrystallization. Oxalic acid, ethylenediamineand metal(II) chlorides were obtained from Merck and were usedas received without any further purification. Solvents were purifiedand dried before use by standard procedures [9].

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 usingCDCl3 solvent for ligand and its Zn(II) complex with TMS as inter-nal standard. DART-MS spectrum was recorded by 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–Visible 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 tetracyano etthylene (TCNE) asg-marker. Cyclic voltammetry measurements were performedusing electrochemical analyzer CH instruments electrochemicalwork station (Model 650 C) using a glassy carbon workingelectrode (GCE) with Ag/AgCl reference electrode and platinumcounter electrode. Powder XRD studies were carried out usingXPERT-PRO, X-ray diffractometer system.

Biological studies

In vitro antimicrobial activity

In vitro antibacterial and antifungal activities of the ligand andits complexes were tested against the bacterial species E. coli, K.

pneumoniae, P. aeruginosa and S. aureus; also the fungal species,A. niger, and C. albicans by disc diffusion method [10]. Chloram-phenicol was used as standard antibacterial agent whereas Nysta-tin was used the standard antifungal agent. The test organismswere grown on nutrient agar (Muller Hinton agar for bacteriaand antimytotic agar for fungi) medium in petri plates. The com-pounds were prepared in DMF and soaked in filter paper disc of5 mm diameter and 1 mm thickness. The discs were placed onthe previously seeded plates and incubated at 37 �C and the diam-eter of inhibition zone around each disc was measured after 24 hfor bacterial and 72 h for fungal species.

DNA binding experiment

Electronic absorption titrations were performed in Tris–HCl/NaCl buffer (5 mmol L�1 Tris–HCl/50 mmol L�1 NaCl buffer pH7.2) using DMF (10%) solution of metal complexes at room temper-ature. The concentration of CT-DNA was determined from theabsorption intensity at 260 nm with e value of 6600 (mol L�1)�1 -cm�1. Absorption titration experiments were made using differentconcentrations of CT-DNA, keeping the complex concentrationconstant. Correction was made for absorbance of the CT-DNA itself.Metal-DNA solutions were allowed to incubate for 5 min before theabsorption spectra were recorded. For metal(II) complexes,the intrinsic binding constant (Kb) was determined by monitoringthe changes of absorption in the MLCT band with increasingconcentration of DNA using the following equation [11,12].

½DNA�=ðea � efÞ ¼ ½DNA�=ðeb � efÞ þ 1=Kbðeb � efÞ

where [DNA] is the concentration of DNA in base pairs. Theapparent absorption coefficients ea, ef and eb correspond to Aobsd/[complex], the extinction coefficient of the complex when fullybound to equilibrium binding constant in (mol L�1)�1 respectively.Each sample solution was scanned from 200 to 500 nm.

Cleavage of pUC18 DNA

The extent of cleavage of super coiled pUC18 DNA to its nickedcircular form was studied using agarose gel electrophoresis. pUC18 DNA (0.3 lg) dissolved in 5 mmol L�1 Tris–HCl/50 mmol L�1

NaCl buffer (pH 7.2), was treated with the complexes. The mixturewas incubated at 37 �C for 2 h and then mixed with the loadingbuffer containing 25% bromophenol blue, 0.25% xylene cyanoland 30% glycerol. Each sample (10�3 M, 0.5 lL) was loaded into1% (w/v) agarose gel. Electrophoresis was undertaken for 2 h at50 V in Tris–acetate–EDTA (TAE) buffer (pH 8.0). The gel wasstained with ethidium bromide for 5 min after electrophoresisand then photographed under a UV transilluminator. To enhancethe DNA cleaving activity of the complexes, hydrogen peroxide(100 lmol L�1) was added to each sample. The DNA cleavage effi-ciency of the complexes was measured by determining the abilityof the complexes to convert the super coiled DNA to nicked circularform and linear form.

In vitro anticancer study

The human cervical cancer cell line (HeLa) was obtained fromNational Centre for Cell Science (NCCS), Pune and grown in EaglesMinimum Essential Medium (EMEM) containing 10% fetal bovineserum (FBS). All cells were maintained at 37 �C, 5% CO2, 95% airand 100% relative humidity. The monolayer cells were detachedwith trypsin-ethylenediamine tetraacetic acid (EDTA) to makesingle cell suspensions and viable cells were counted using ahemocytometer and diluted with a medium containing 5% FBS togive final density of 1 � 105 cells/mL. 100 lL per well of cell sus-pension were seeded into 96-well plates at plating density of

398 J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404

10,000 cells per well and incubated at 37 �C, 5% CO2, 95% air and100% relative humidity. After 24 h the cells were treated with serialconcentrations of the test samples. They were initially dissolved inneat dimethylsulfoxide (DMSO) and diluted to twice the desiredfinal maximum test concentration with serum free medium. Addi-tional four, 2-fold serial dilutions were made to provide a total offive sample concentrations. Aliquots of 100 lL of these differentsample dilutions were added to the appropriate wells alreadycontaining 100 lL of medium, gave the required final sample con-centrations. Following drug addition, the plates were incubated foran additional 48 h at 37 �C, 5% CO2, 95% air and 100% relativehumidity. The medium without samples were served as controland triplicate was maintained for all concentrations.

3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT)assay

3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide(MTT) is a yellow water soluble tetrazolium salt. A mitochondrialenzyme in living cells, succinate-dehydrogenase, cleaves the tetra-zolium ring, converting the MTT to an insoluble purple formazan.Therefore, the amount of formazan produced is directly propor-tional to the number of viable cells. After 48 h of incubation,15 ll of MTT (5 mg/ml) in phosphate buffered saline (PBS) wasadded to each well and incubated at 37 �C for 4 h. The mediumwith MTT was then flicked off and the formazan crystals formedwere dissolved in 100 lL of DMSO and absorbance at 570 nmwas measured using micro-plate reader. The % cell inhibition wasdetermined using the following formula [13,14].

% Cell inhibition ¼ 100� Abs ðsampleÞ=Abs ðcontrolÞ� 100: ð1Þ

Nonlinear regression graph was plotted between % cell inhibi-tion and log concentration and IC50 was determined using GraphPad Prism software.

Synthesis of ligand

3-(2-Aminoethylamino)quinoxalin-2(1H)-one was prepared bythe condensation of quinoxaline-2,3(1,4H)-dione and ethylenedia-mine. To the hot aqueous solution of 3-(2-aminoethylamino)qui-noxalin-2(1H)-one (2 mmol), methanolic solution of o-vanillin(2 mmol) was added drop by drop. Immediately after the additionof o-vanillin an orange red solid (1) was formed. The reaction mix-ture was heated with stirring at 50–60 �C for 2 h. The solid sepa-rated out was cooled to room temperature, filtered and washedwith petroleum ether. Then the compound was recrystallised frommethanol and dried in vacuum over anhydrous calcium chloride(Scheme 1).C18H18N4O3: Orange red solid; Yield (%): 87; Anal. Calcd(%): C (63.89), H (5.36), N (16.56), O (14.19); Found (%): C (63.68), H(5.43), N (16.60), O (14.19); IR ˆ(cm�1): 2847 (free NH), 3415(o-vanillin AOH), 3041 (quinoxaline AOH), 1614 (HC@NA), 1247(CAO); 1H NMR (CDCl3) d (ppm): 13.19 (S, 1H, o-vanillin AOH),13.59 (S, 1H, quinoxaline AOH) 9.14 (S, 1H, ACH@N), 3.91 (S, 1H,

NH

NHN

ONH2

+O

HO

Oo-vanillin3-(2-aminoethylamino)quinoxalin-2(1H)-one

Scheme 1. Synthe

AOACH3), 3.95 (S, 1H, free ANH), 6.75–8.63 (m, Ar–H); DART-MS(m/z): 339.

Synthesis of metal complexes

Methanolic solution of metal(II) chlorides (2 mmol) was addedin drops to a methanolic solution of the ligand (1) (4 mmol) 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 invacuum over anhydrous calcium chloride.

Co(II) complex (C36H32CoN8O6 (2)): Brownish pink solid; Yield(%): 62; Anal. Calcd (%): C (59.10), H (4.41), N (15.32), O (13.12),Co (8.06); Found (%): C (59.30), H (4.84), N (15.40), O (13.18), Co(8.32); IR ˆ(cm�1): 2945 (ring NH), 1605 (HC@NA), 1239 (CAO),485 (MAN), 609 (MAO); DART-MS (m/z): 732; Molar conductance(O�1 cm2 mol�1): 18.3.

Ni(II) complex (C36H32N8NiO6 (3)): Pale green solid; Yield (%): 73;Anal. Calcd. (%): C (59.12), H (4.41), N (15.32), O (13.13), Ni (8.02);Found (%): C (59.34), H (4.84), N (15.04), O (13.28), Ni (8.17); IRˆ(cm�1): 2947 (ring NH), 1608 (HC@NA), 1234 (CAO), 492(MAN), 628 (MAO); DART-MS (m/z): 733; Molar conductance(O�1 cm2 mol�1): 14.9.

Cu(II) complex (C36H32N8CuO6 (4)): Dark green solid; Yield (%):67; Anal. Calcd. (%): C (58.73), H (4.38), N (15.22), O (13.04), Cu(8.63); Found (%): C (58.79), H (4.25), N (15.35), O (13.43), Cu(8.79); IR ˆ(cm�1): 2940 (ring NH), 1603 (HC@NA), 1236 (CAO),490 (MAN), 581 (MAO); DART-MS (m/z): 737; Molar conductance(O�1 cm2 mol�1): 29.2.

Zn(II) complex (C36H32N8O6Zn (5)): Yellow solid; Yield: 59%, Anal.Calcd. (%): C (58.58), H (4.37), N (15.18), O (13.01), Zn (8.86); Found(%): C (58.79), H (4.47), N (15.32), O (13.23), Zn (8.73); IR ˆ(cm�1)2934 (ring NH), 1605 (HC@NA), 1231 (CAO), 496 (MAN), 590(MAO), 1H NMR (CDCl3) d (ppm): 9.25 (S, 1H, ACH@N), 3.91 (S,1H, AOACH3), 11.12 (S, 1H, ring ANH), 6.38–7.83 (m, Ar–H),DART-MS (m/z): 739. Molar conductance (O�1 cm2 mol�1): 30.1.

Results and discussion

The ligand 1 was prepared by the condensation of 3-(2-amino-ethylamino)quinoxalin-2(1H)-one with o-vanillin (Scheme 1). Thesolid complexes obtained by the reaction between 1 and metal(II)chlorides were filtered, washed with hot methanol and dried invacou over anhydrous calcium chloride.

The metal(II) complexes of 1 are non-hygroscopic, soluble inDMSO, DMF and sparingly soluble in chloroform. The complexesare stable at room temperature. The elemental analysis data ofthe ligand 1 and its metal complexes (2–5) indicate that the metalto ligand ratio is 1:2.

Molar conductance

The metal(II) complexes of ligand 1 (10�3 mol dm�3) were dis-solved in DMF and molar conductance of the solutions at roomtemperature was measured. The molar conductance data indicate

N

N

OH

HN

N

HO

O(1)

tic route of 1.

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404 399

that, all the metal complexes are having conductance values inaccordance with non-electrolytes [15,16].

IR spectra

The IR spectrum of the ligand 1 exhibited characteristic azome-thine band ˆ(AHC@NA) at 1614 cm�1. The strong band observed at1614 cm�1 is shifted to lower wave number by 11–9 cm�1 in thespectra of metal(II) complexes. The lowering of wave numbermay be attributed to the decrease in electron density on the nitro-gen atom of the azomethine group [17]. This indicates the coordi-nation of azomethine (AHC@NA) group to the metal ion incomplexes. The presence of bands observed at 3415 and3041 cm�1 in 1 is attributed to ˆ(OH) stretching frequencies ofo-vanillin and quinoxaline moiety respectively. The absenceof these bands in the complexes 2–5 indicates the coordinationof AOH group through deprotonation [18]. This is furtherevidenced by the ˆ(CAO) band in these complexes (1236–1231 cm�1), appearing at lower frequencies compared to that ofthe ligand 1 (1247 cm�1) [19]. In the spectra of complexes the banddue to ring ˆ(NH) was observed at 2947–2934 cm�1 region. Thefree ˆ(NH) stretching frequency was observed in ligand 1 at2847 cm�1. This band is absent in the spectra of complexes dueto the conversion of free ANHA group into AN@C< group and ring>C@NA group into ANHAC@N in complexes. In the spectra of thecomplexes, appearance of new bands in the region 496–485 cm�1

and 628–581 cm�1 has been attributed to MAN and MAO bondsrespectively [20,21]. From the IR spectral data it is clear that the li-gand 1 acts as a tridentate ligand, coordinated with metal ion viatwo phenolic AOH groups through deprotonation and through azo-methine (AHC@NA) nitrogen atom.

1H NMR spectra

1H NMR spectra of 1 (Fig. 1a) and its Zn(II) complex 5 (Fig. 1b)were recorded in CDCl3. The signal for azomethine proton(ACH@NA) in the ligand 1 appears as a singlet at 9.14 ppm [22].In the spectrum of the complex 5, this signal shows small shift,indicates the coordination of azomethine nitrogen atom. The sin-glet peak observed at 13.19 and 13.59 ppm in the spectrum of 1is assigned to AOH protons of o-vanillin and quinoxaline moiety[23] respectively. In the spectrum of the complex 5 these peaksdisappeared, indicating the participation of phenolic AOH groupsin chelation through deprotonation. The aromatic proton signalsobserved in the region 6.75–8.63 ppm show small shifts in

Fig. 1a. 1H NMR s

complex, due to variation in electron density due to chelation.Singlets appeared at 3.95 and 3.91 ppm in the spectrum of ligandare attributed to the free >NH and AOACH3 group of o-vanillinmoiety respectively. The singlet peak due to free ANH proton isabsent in the spectrum of 5 and new singlet peak appeared at11.12 ppm. This confirms the conversion of free ANHA group intoAN@C group and ring >C@NA group into ANHAC@N in complexes.

Mass spectra

Mass spectra of l and its complexes gave vital clues for elucidat-ing the structure and to determine the stoichiometry of the com-pounds. The DART mass spectrum of 1 (C18H18N4O3) shows awell defined molecular ion peak at m/z = 339, which coincides withthe formula weight of the Schiff base. The molecular ion peaks ofcomplexes 2, 3, 4 and 5 were observed at 732, 733, 737 and739 m/z respectively. Along with the molecular ion peaks, spectrashow some other peaks which are attributed to molecular cationsof various fragments. Elemental analysis and mass spectral studiesconfirm the metal to ligand ratio as 1:2.

Magnetic susceptibility and electronic spectra

The UV–Vis. spectra of 1–5 (10�3 mol L�1) were recorded inDMF in the wavelength range 190–1100 nm. The magnetic suscep-tibility and UV–Vis. spectral data are presented in Table 1. Ligand 1exhibits electronic transitions with strong bands at 250–270 nm,assigned to p–p* transitions, which remain almost unchanged inthe complexes. The transition at 306 nm is assigned to n–p* tran-sition of azomethine group. Due to the coordination of azomethinenitrogen this band shows shift in the electronic spectra of all com-plexes. The complexes 2, 3 and 4 show a shoulder like absorptionpeak at 330–400 nm region, which is assigned to the charge-trans-fer transition from the filled pp orbital of the phenolic oxygenatoms to vacant d orbital of the metal [24]. The complex 2 exhib-ited an absorption band at 485 nm, for d–d transition correspond-ing to 4T1g(F) ? 4T1g(P) transition, suggesting an octahedralgeometry. It has a magnetic moment of 5.18 BM indicating a highspin octahedral structure for the complex. Complex 3 exhibits a leff

value of 3.21 BM. Presence of spin allowed electronic transition at510 nm corresponding to 3A2g(F) ? 3T1g (F) transition suggests anoctahedral geometry for the complex. Complex 4 exhibits a mag-netic moment of 1.89 BM corresponding to one unpaired electron.The band observed at 464 nm is assigned to 2B1g ?

2E1g transition,indicating the complex to have distorted octahedral geometry

pectrum of 1.

Fig. 1b. 1H NMR spectrum of 5.

Table 1Electronic spectral data and magnetic moment values of 2–5.

Complex kmax (nm) Band assignments Magnetic moment (BM) Geometry

2 485 4T1g (F) ? 4T1g (P) 5.18 Octahedral338 INCT

3 510 3A2g (F) ? 3T1g (F) 3.21 Octahedral390 INCT

4 464 2B1g ?2E1g 1.89 Distorted octahedral

385 INCT

5 239 INCT Diamagnetic Octahedral299 INCT

NHN

ON

N

O

O NNH

ON

N

O

O

M

(Where M = Co(II), Ni(II), Cu(II) and Zn(II))

Fig. 2. Proposed structure of metal complexes.

400 J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404

[25,26]. The complex 5 is diamagnetic and two intra ligand bandswere observed in the electronic spectrum at 239 and 299 nm. Asper the empirical formula, an octahedral geometry is proposedfor this complex.

ESR spectra

The X-band ESR spectra of the present Cu(II) complex (4) wasrecorded in DMSO at liquid nitrogen temperature (77 K) and atroom temperature (300 K). The X-band ESR spectrum of the com-plex 4 at room temperature exhibits a single isotropic broad signalin the high field. The ESR spectrum of the complex 4 at 77 K showsa well resolved hyperfine splitting and exhibits two different g val-ues indicating magnetic anisotropy in the complex. The groundstate of the complex 4 is derived from the g-tensor values. In thepresent Cu(II) complex the g-tensor values are g|| (2.18) > g\(2.05) > 2.0027, suggests that this complex has a distorted octahe-dral geometry with axial symmetry and the unpaired electron liesin the d2

x � y2 orbital [27]. The small g|| value can be attributed tothe large covalent interaction in MAL bond. The g values are re-lated to the exchange interaction coupling constant (G) by theexpression, G = (g|| � 2)/(g\ � 2). If G > 4.0 suggests that the localtetragonal axes are only slightly misaligned and the exchangeinteractions between Cu(II) centers in the solid state are negligible.The absence of a half field signal in the spectrum at 1600 G due toDms = ±2 transitions, ruled out any Cu–Cu interaction [28].

Based on the above results the proposed structure of the com-plexes is given in Fig. 2.

Electrochemical studies

The cyclic voltammograms of 1 and its metal complexes (2–5)were recorded at 300 K in DMSO solution. The cyclic voltammogram

of 1 is recorded in the potential range �0.8 to 0.6 V, for 2–5 it isrecorded in the potential range �1.0 to 1.0 V with scan rate0.005 V/s.

Fig. 4. Powder XRD pattern of (a) 2 and (b) 3.

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404 401

The ligand shows one irreversible peak at Epc = 0.35 V vs Ag/AgCl. The complex 2 exhibits two irreversible peaks one atEpc = 0.6 V corresponding to Co(II)/(I) couple. The correspondingoxidation peak at Epa = �0.7 V during the reverse scan correspondsto Co(II)/(III) couple. The peak to peak separation DE is 1.3 V indi-cating that the process is irreversible. The complex 3 shows oneirreversible peak at Epc = 0.6 V. The complex 4 (Fig. 3) exhibitedtwo redox processes; each reduction is associated with a singleelectron transfer at room temperature [29]. Among the two redoxprocesses, one is irreversible with reduction peak at Epc = 0.55 Vand the corresponding oxidation peak at Epa = �0.55 V. The peakto peak separation DE is 1.1 V. The second process is quasi revers-ible, the peaks observed at Epc = �0.19 V and Epa = �0.79 with peakseparation value DE = 0.6 V. The complex 5 shows irreversible peakat Epc = 0.65 V vs Ag/AgCl and the associated anode peak at Epa =�0.70 V. The peak to peak separation value DE = 1.35 V corre-sponding to irreversible process.

Powder XRD

The powder XRD patterns of the compounds 1–5 were recordedover the 2h = 0–80 Å range and are given in Fig. 4. From the ob-served data, all the complexes except 2 show sharp peaks indicat-ing their crystalline nature. The complex 2 is amorphous. Theaverage crystallite sizes of the complexes dXRD were calculatedusing Scherrer’s formula [30]. The ligand 1 and the complexes 3,4 and 5 have an average grain size of 21, 14, 08 and 16 nm, respec-tively, suggesting that the ligand and complexes are in a nanocrys-talline phase.

Biological studies

In vitro antimicrobial studies

The in vitro antimicrobial activity of the ligand and its com-plexes are given in Table 2. The standard error for the experimentis ±0.001 mm and the experiment is repeated three times undersimilar conditions. DMF is used as negative control and Chloram-phenicol is used as positive standard for antibacterial and Nystatinfor antifungal activities. The variation in the effectiveness of thedifferent compounds against different organisms depends on theirimpermeability of the microbial cells or on the difference in theribosome of the microbial cells [31]. The metal(II) complexesexhibited higher activity. The increase in the antimicrobial activityof the metal chelates is due to the effect of metal ion on normal cell

Fig. 3. Cyclic voltammogram of 4.

process. Such increase activity of the metal chelates can beexplained on the basis of Overtone’s concept [32] and Chelationtheory [33].

In the present study, the complex 4 exhibited higher antibacte-rial and antifungal activities. Though the Schiff base ligand and itsmetal(II) complexes possess activity, it could not reach theeffectiveness of the standard drugs. The variation in the effective-ness of the different compounds against different organismsdepends either on the impermeability of the cells of the microbesor differences in ribosomes of microbial cells [34,35].

DNA binding studies

The interaction of the metal(II) complexes (2–5) with DNA wasinvestigated by electronic absorption titration to evaluate theirbinding affinities. Electronic absorption spectroscopy was an effec-tive method to examine the binding mode of DNA with metalcomplexes. Intercalative mode of binding usually results in hypo-chromism and red shift because of the strong stacking interactionbetween an aromatic chromophore and the base pairs of DNA. Theextent of the spectral change is related to the strength of binding[36]. The electronic absorption titration of the complex 3 is shownin Fig. 5. The absorption band of the complex 2 at 396.8 nm exhib-ited hypochromism of 4.1% and bathochromism of 4 nm. Theabsorption band of 3 at 257.6 nm exhibited hypochromism of18.9% and bathochromism of 4.8 nm. The absorption band of 4 at389.6 nm exhibited hypochromism of 10.9% and bathochromismof 2.4 nm. The complex 5 at 329.6 nm exhibited hypochromismof 3.2% and bathochromism of 2.5 nm. These results suggest anassociation of the complexes (2–5) with DNA and interacting withDNA through intercalation. The intrinsic binding constant (Kb) val-ues were also calculated (2.5 � 105, 3.2 � 105, 3.5 � 105 and2.6 � 105 M�1 for the complexes 2, 3, 4 and 5 respectively).

DNA cleavage studies

The DNA cleavage activity of the compounds (1–5) was studiedin the presence of H2O2 as an oxidizing agent using pUC18 DNA byagarose gel electrophoresis. The cleavage efficiency was noted by

Table 2Antimicrobial activity of 1–5.

Compound Inhibition zone (mm)

Bacterial species Fungal species

K. pneumoniae E. coli P. aeruginosa S. aureus C. albicans A. niger

1 6 6 6 8 6 62 10 8 6 6 13 63 6 12 6 6 6 84 12 6 11 10 10 145 8 6 6 6 10 6Chloramphenicola 24 22 23 24 – –Nystatina – – – – 23 22

a Standard.

Fig. 5. Electronic absorption spectrum of (a) complex 3 in the absence of CT DNAand (b) in the presence of increasing amount of CT DNA.

Fig. 6. Changes in the agarose gel electrophoretic pattern of pUC18 DNA induced byH2O2. (1) Marker DNA; (2) Control DNA; (3) pUC18 DNA + H2O2 + 1; (4) pUC18DNA + H2O2 + 2; (5) pUC18 DNA + H2O2 + 3; (6) pUC18 DNA + H2O2 + 4; (7) pUC18DNA + H2O2 + 5.

Table 3Anticancer activity of 1–5.

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measuring the ability of the complex to convert the super coiledDNA into nicked open circular form or linear form. When circularplasmid DNA is subjected to electrophoresis, the fastest migrationwill be observed for the supercoiled form (Form I). If one strand iscleaved, the supercoils will relax to produce a slower-moving opencircular form (Form II). If both strands are cleaved, a linear form(Form III) will be generated that migrates in between [37].

Control experiments with H2O2 as an oxidizing agent did notshow any cleavage of the supercoiled DNA under similar experi-mental conditions. It is evident from Fig. 6, there is a considerableincrease in the intensity of bands for open circular form (Form II) inthe case of Cu(II) complex (4) (lane 6). This suggests that the com-plex 4 has nicking activity [38]. There is no appreciable level of in-crease in the intensity of bands of open circular form for the DNAsamples treated with the ligand 1 (lane 3), complexes 3 (lane 5)and 5 (lane 7). Shearing of pUC18 DNA is evident in lane 4, wherethe plasmid DNA was treated with the Co(II) complex (2). Oxida-tive cleavage was observed in the presence of H2O2. The complexescleaved DNA more efficiently in the presence of an oxidant, due tothe formation of hydroxyl free radicals.

Compound IC50 (lM�1)

1 69.512 66.833 33.234 35.615 17.67

In vitro anticancer studies

Anticancer activity of newly synthesized ligand (1) as well ascorresponding metal complexes (2–5) was investigated on human

cervical carcinoma (HeLa) cells by MTT assay and the results areexpressed in terms of IC50 values. The MTT cell proliferation assayhas been widely accepted as a reliable way to measure the cellproliferation rate [39]. The compounds were applied in the concen-tration range 0.1–100 lM. The data obtained by the MTT assayshow that all complexes have very good inhibitory effect on thegrowth of human cervical carcinoma (HeLa) cells. Table 3 illus-trates the IC50 values for the compounds tested. The IC50 valuesof the investigated compounds ranges from 17.67 to 69.59 lM.The complex 5 exhibited higher activity with IC50 value 17.67 lM.The complexes 3 and 4 were also exhibited good anticancer activitywith IC50 values 33.23 and 35.61 lM respectively. The complex 2gave IC50 value at 66.83 lM. The ligand has lowest growth inhibi-tory activity against HeLa cells (69.51 lM). From the results (Fig. 7)

Fig. 7. Cytotoxic profiles of (a) 1, (b) 2, (c) 3, (d) 4 and (e) 5 towards human cervical cancer cell line (HeLa).

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404 403

it is clear that the metal chelates have higher anticancer activitythan their corresponding ligand.

Conclusion

Co(II), Ni(II), Cu(II) and Zn(II) complexes with ligand 1 havebeen synthesized and characterized based on elemental analysis,molar conductance, magnetic moment and spectral data. The Schiffbases acts as tridentate ONO donor ligand. The complexes are non-electrolytes. All complexes have octahedral geometry except Cu(II)complex (4), which has distorted octahedral geometry. The metalcomplexes show redox behavior. All compounds except 2 werefound to be nanocrystalline. In vitro antimicrobial study indicatesthat metal chelates have higher activity than ligand. DNA bindingstudies revealed that all the complexes of 1 exhibits stronger bind-ing affinity to DNA through intercalation mode. DNA cleavageactivity of the compounds (1–5) was performed by agarose gelelectrophoretic assay. The complex 4 exhibited effective DNAcleavage. The complexes also showed oxidative hydroxyl radicalcleavage in the presence of H2O2 as an oxidant. The synthesized

metal chelates (2–5) have enhanced anticancer activity than theligand.

Acknowledgement

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).

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