ic c ant bide e cy E , Trivand en’s Un g r a p h i c a l a b s t r a c t Accepted 15 August 2013 Antibacterial activity ral bidentate, coor- ar conductance val- above observations I), Cu(II) and Zn(II); complex were sub- rophoresis m lexes were been tested gram negative and gram positive bacteria. � 2013 Elsevier B.V. All rights re Introduction Schiff bases form a significant class of compounds in medicinal and pharmacological chemistry due to their varied biological appli- cations such as antibacterial [1–6], antifungal [3–6] and antitumor [7,8] agents. Generation of different heterocyclic derivatives of ⇑ Corresponding author. Tel.: +91 471 2418782, mobile: +91 9447696794. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 154–161 Contents lists availab Spectrochimica Acta P Biomolecular S E-mail address:
[email protected] (K. Mohanan). Available online 31 August 2013 Keywords: Schiff base EPR spectrum XRD DNA cleavage Superoxide dismutase mass and EPR spectral studies. The spectral data revealed that the ligand acts as neut dinating to the metal ion through the carbonyl oxygen and azomethine nitrogen. Mol ues adequately support the electrolytic nature of the complexes. On the basis of the the complexes have been formulated as [M(ISAP)2]X2, where M = Mn(II), Co(II), Ni(I X = Cl, OAc; ISAP = 2-[N-indole-2-one]aminopyrimidine. The ligand and copper(II) jected to X-ray diffraction studies. The DNA cleavage study was monitored by gel elect The superoxide dismutase (SOD) mimetic activities of the ligand and the metal comp using NBT assay. The in vitro antibacterial activity of the synthesized compounds has 1386-1425/$ - see front matter � 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.08.075 ethod. checked against served. a r t i c l e i n f o Article history: Received 26 May 2013 Received in revised form 10 August 2013 a b s t r a c t Complexes of manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) with a Schiff base, formed by the condensation of isatin with 2-aminopyrimidine have been synthesised and characterised through ele- mental analysis, molar conductance measurements, magnetic susceptibility, IR, UV–Vis, 1HNMR, FAB h i g h l i g h t s � A novel potentially bidentate heterocyclic Schiff base has been synthesised. � The Schiff base has been used as a promising chelating agent for some 3D metal ions. � XRD patterns revealed the crystalline nature of the ligand and copper(II) complex. � DNA cleavage and SOD activities of the metal complexes have been studied. � Antibacterial activities of the ligand and metal complexes have also been examined. Synthesis, spectroscop dismutase activity and complexes of a novel and 2-aminopyrimidin L.P. Nitha a, R. Aswathy a, Nie aDepartment of Chemistry, University of Kerala bDepartment of Chemistry, Mother Teresa Wom haracterisation, DNA cleavage, superoxidase ibacterial properties of some transition metal ntate Schiff base derived from isatin lsa Mathews a, B. Sindhu kumari b, K. Mohanan a,⇑ rum, 695581 Kerala, India iversity, Kodaikanal, 624101 Tamilnadu, India journal homepage: www.elsevier .com/locate /saa le at ScienceDirect art A: Molecular and pectroscopy transition metal complexes with DNA has been examined mainly because of the fact that many compounds expose their antitumor activity through binding to DNA and can cause DNA damage of The plasmid DNA (pUC19) cleavage activity of the ligand, 2-[N- A quantity (1.47 g) of isatin was dissolved in ethanol (40 mL) lar a cancer cells by blocking the cell division of cancer cells and result- ing in cell death [15]. The superoxide anion radical is a highly reac- tive toxic species in many biological systems. This reactive superoxide is controlled by one of the enzymes Cu, Zn-Superoxide dismutase. The use of SOD enzyme is limited because of its insta- bility and low membrane permeability, resulting from its high molecular weight. Many low molecular weight complexes of cop- per and other metals have also been reported to exhibit SOD mi- metic activities [16]. Experimental Materials and methods All the chemicals used were of Analytical Grade. Commercial solvents were distilled and used for synthesis. For physico-chemi- cal measurements, the solvents were purified by standard meth- ods. Melting points reported were determined by open capillary method and were uncorrected. Carbon, hydrogen and nitrogen analyses were performed using Elementar Systeme Vario EL III CHN analyzer. The electronic spectra of the complexes were re- corded on a Hitachi 320 UV–Vis spectrophotometer. Infrared spec- tral studies were carried out using KBr discs on a Shimadzu FT-IR 8000 spectrophotometer. Proton NMR spectra of the ligand and zinc(II) complex were recorded on a JEOL GSX 400 MHz FT-NMR spectrometer employing TMS as internal reference and DMSO-d6 as solvent. Far IR spectra were recorded on a polytec FIR 30 Fourier spectrometer using CsI discs. X-ray diffraction studies were per- formed using a Siemens D 5005 model X-ray Spectrometer. Molar conductance measurements were conducted using 10�3 M solu- tions of the complexes in DMSO at room temperature using a Sys- tronic model 304 digital conductivity meter. Magnetic susceptibility values of the complexes were measured at room temperature with a Magway MSBMk1 magnetic susceptibility bal- Schiff bases has created a boom in the field of medicinal chemistry. Isatin is an important heterocyclic compound, which is biologically active and finds significant application in medicinal chemistry [9]. Isatin is a resourceful endogenous heterocyclic molecule identified in human being and rat tissues. In recent years, isatin and its deriv- atives have acquired conspicuous significance due to their wide spectrum biological activities [10]. Therefore the research interest on isatin has expended widely. Similarly aminopyrimidines are molecules of increasing interest, because several therapeutically important compounds contain the pyrimidine ring system [11,12]. This structural motif is a constituent of several nucleo- bases (cytosine, uracil and thymine) and is present in large number of products [13] such as vitamin B1 [14]. The complexing ability of 2-aminopyrimidine derivatives with transition metal ions is of great interest. Transition metal complexes containing pyrimidine ligand are commonly found in biological media and play important roles in catalysis of drug interaction with biomolecules. In view of the biological significance of the parent compounds, a Schiff base ligand was prepared by the condensation of isatin and 2-aminopy- rimidine, and its metal complexes have been synthesised and char- acterised on the basis of various physicochemical and spectroscopic methods. In this communication, we also report the DNA cleavage, SOD mimic and antibacterial activities of the synthesised ligand and its metal complexes. The interaction of L.P. Nitha et al. / Spectrochimica Acta Part A: Molecu ance. FAB mass spectrum of the ligand and copper(II) complex were recorded on a JEOL SX-102 FAB mass spectrometer. The EPR spectrum of the copper(II) complex was recorded in the solid state and was added slowly to 2-aminopyrimidine (0.95 g) dissolved in hot ethanol (10 mL). The resulting mixture was refluxed for 3 h In vitro antibacterial activity The ligand and the metal complexes were screened for their antibacterial activities against Escherichia coli, Staphylococcus aur- eus, Klebsiella pneumoniae, Salmonella typhi and Pseudomonas aeru- ginosa by the minimum inhibitory concentration (MIC) method. MIC is the lowest concentration of solution to inhibit the growth of a test organism and the minimum inhibitory concentrations (MIC) were detected by the serial dilution method [20]. The lowest concentration of compound, which inhibits the growth of bacteria after 24 h incubation at 37 �C was taken as the MIC. The stock solu- tion was prepared by dissolving the compound in DMSO and the solution was diluted to different concentration in the same solvent in order to find the MIC values. Synthesis of 2-[N-indole-2-one]aminopyrimidine Determination of superoxide dismutase activity In order to evaluate the SOD activity of complexes towards superoxide in aqueous buffer, NBT assay was used. The assay is based on competition for the superoxide reaction between NBT and the complex with SOD activity. In vitro SOD activity was mea- sured using alkaline DMSO as a source of superoxide radical (O2��) and nitroblueterazolium (NBT) as O2�� scavenger [18,19]. Sample to be assayed was added to a solution containing 0.2 M potassium phosphate (2.1 mL) buffer (pH 8.6) and 56 lM NBT (1 mL). The tubes were kept in ice for 15 min and then alkaline DMSO (1.5 mL) solution was added while stirring. The SOD activity is in- versely related to the amount of formazan obtained by the reduc- tion of NBT and the absorbance was then monitored at 540 nm. The activity is expressed as IC50 which is the concentration (lM) of the complex or the enzyme required to dismutase 50% of the evolved superoxide radical anion (O2��). indole-2-one]aminopyrimidine and its manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II) complexes in the presence of H2O2 as an oxidant was monitored by agarose gel electrophoresis method. The reaction mixture was prepared as follows: Hydrogen peroxide (2 ll of 5 mM) was added in the mixture of 0.5 ll of super coiled plasmid DNA (0.25 lg/ll) and 5 ll of 50 lM of each sample dissolved in CHCl3 followed by dilution with 50 mM tris-HCl buffer (pH = 7.2) to a total volume of 20 ll. All the reaction mixtures were incubated at 35 �C for 1.5 h before the electrophoresis experiment. The samples were then electrophoresed at 80 V for 2 h on 1.25% agarose gel using tris-aceticacid-EDTA buffer (pH = 8.1). After elec- trophoresis, the gel was stained using 1 lg/cm3 ethidium bromide (EB). The cleavages were visualized by viewing the gel under UV light and photographed [17]. and also in DMSO at liquid nitrogen temperature using a Varian E- 112 EPR spectrometer employing TCNE as reference material. Gel electrophoresis nd Biomolecular Spectroscopy 118 (2014) 154–161 155 on a water-bath and it was concentrated to about one-third of its initial volume and allowed to cool. The orange crystals formed were filtered, washed, dried and recrystallized from ethanol. Synthesis of the metal complexes A series of divalent transition metal complexes of the azome- thine derivatives were synthesized according to the following gen- eral procedure. An ethanolic solution (15 mL) of the metal salt (0.005 mol) was added gradually in small amounts to a hot ethanolic solution of 2- [N-indole-2-one]aminopyrimidine (0.01 mol). The pH of the solu- tion was maintained between 6.5 and 7.0 by adding 1:1 alcoholic ammonia solution. The reaction mixture was refluxed for about 3–4 h and then cooled to room temperature. The metal complex separated out was filtered off, washed successively with ethanol, ether and finally dried in vacuum over P4O10. Infrared spectrum IR spectrum of the free ligand exhibited a medium intensity band at 3215 cm�1 which can be assigned to the t(NAH) stretching vibrations of the indole ring system [25]. In addition to this, two strong bands observed at 1730 cm�1 and 1619 cm�1, assignable to t(C@O) and t(C@N) vibrations of isatin moiety and azomethine group respectively [26]. The strong band at 1577 cm�1 could be very safely assigned to t(C@N) of pyrimidine ring system [27]. Apart from all the above bands, vibrations characteristic of the pyrimidine ring have been observed in the range, 1200– 1462 cm�1 and 635–860 cm�1 [28]. 1H NMR and FAB mass spectra The 1H NMR spectrum of the ligand recorded in DMSO-d6 exhibited a singlet signal at 9.76 ppm due to NH proton of indole l da 156 L.P. Nitha et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 154–161 Table 1 Analytical data and other details of the metal complexes. Complex Colour Yield (%) Analytica M ISAP Orange 82 – [Mn(ISAP)2](OAc)2 Brown 60 10.74 (10.91) [Co(ISAP)2]Cl2 Brown 62 11.20 (11.62) [Ni(ISAP)2]Cl2 Reddish brown 64 11.74 (11.57) [Cu(ISAP)2]Cl2 Pale green 72 12.64 (12.41) [Zn(ISAP)2]Cl2 Red 68 12.23 Result and discussion Formation of metal complexes can be represented by the fol- lowing general equation: MX2 þ 2 ISAP ! ½MðISAPÞ2�X2 M ¼ MnðIIÞ;CoðIIÞ;CuðIIÞ;NiðIIÞ or ZnðIIÞ; X ¼ Cl;OAc All the complexes are analytically pure, stable at room temperature and possess good keeping qualities. They are non hygroscopic sol- ids. The ligand is soluble in common organic solvents, but the com- plexes are found to be soluble in DMSO and DMF. Formulation of these complexes has been done on the basis of elemental analysis, molar conductance and magnetic susceptibility measurements. Mo- lar conductance values of the complexes in DMSO (10�3) solution adequately confirm the electrolytic nature of the complexes [21]. All the metal complexes exhibit 1:2 metal to ligand stoichiometry (Table 1). Structure of the ligand Analytical data indicated that the condensation of isatin and 2- aminopyrimidine occurred in 1:1 ratio to form 2-[N-indole-2- one]aminopyrimidine. The purity of the ligand was checked by CHN analysis and TLC (silica gel). The structure of the ligand was confirmed by elemental analysis, IR, UV–Vis, 1H NMR and FABmass spectral data. UV–Visible spectrum The ultraviolet spectrum of the ligand recorded in ethanol shows the characteristic n? p� transition of the C@N chromo- phore at 327 nm [22]. Besides, the band at 360 nm corresponds to the electronic transitions of the pyrimidine moiety [23,24]. (12.73) a Calculated values are given in brackets. ring [29]. The multiplets observed in the region 6.62–8.32 ppm were ascribable to the aromatic protons of the indole and pyrimi- dine rings [30,31]. The FAB mass spectrum of the ligand (Fig. 1) showed a well de- fined molecular ion peak at m/z = 224.36. This is consistent with the proposed molecular formula of the ligand. Thus, the IR, 1H NMR and FAB mass spectra support an azomethine structural form for the ligand (Fig. 2). Structure of the metal complexes The elemental analyses are consistent with 1:2 metal-ligand stoichiometry for all the complexes. The UV spectral bands charac- teristic of the ligand was only marginally red shifted in the spectra of the metal complexes, indicating that the same structural form of the ligand persists in the metal complexes also. Infrared spectra Comparison of the diagnostic bands in the infrared spectra of the ligand and its metal complexes adequately support the mode of coordination of the ligand with the metal ions. Tentative assign- ments of these bands are given in Table 2. The band due to indolic NH is almost remain unaffected in the spectra of the metal com- plexes confirming that the nitrogen atom of the isatin moiety is not coordinated to the metal ion [32]. The t(C@O) of isatin has been shifted to lower frequency by about 40 cm�1 indicating the involvement of this group in coordination [33]. Coordination through the azomethine nitrogen atom is confirmed by the nega- tive shift of t(C@N) stretching frequency to the extent of 20– 25 cm�1 in the complexes [34]. The band due to t(C@N) of the pyrimidine ring remains unchanged in the spectra of the metal complexes indicating the non-involvement of the ring nitrogen atoms in bond formation [35]. Moreover the vibrational character- taa (%) Molar conductance (X�1 cm2 mol�1) in DMSO C H N 64.42 3.40 24.68 – (64.28) (3.60) (24.99) 57.96 3.44 22.94 93.6 (57.26) (3.20) (22.26) 56.24 3.20 22.48 94.2 (56.81) (3.18) (22.09) 56.66 3.38 22.58 95.4 (56.84) (3.18) (22.10) 56.88 3.32 21.66 96.6 (56.30) (3.15) (21.89) 56.70 3.22 21.90 90.4 (56.10) (3.14) (21.81) ANH proton has not registered any appreciable change compared to that of the free ligand. All the aromatic protons resonate nearly at the same region experiencing a slight down field shift (0.10– 0.20 ppm) compared to that of the free ligand. The FAB mass spectrum of the copper(II) complex showed the molecular ion peak at m/z = 512.15, which suggest the monomeric L.P. Nitha et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 154–161 157 istics of the pyrimidine ring remained almost unaltered in the spectra of the complexes confirming the non-participation of the heterocyclic ring nitrogen during chelation [36]. Far IR spectra Conclusive evidence for the coordination of the ligand with the metal ions is provided by the appearance of broad and medium in- tense non-ligand bands in the region 530–520 cm�1 and 460– 450 cm�1 which can be assigned to t(MAO) and t(MAN) vibra- tions respectively [37]. 1H NMR spectra and FAB mass spectra In conformity with the UV and IR spectral data, the proton NMR spectrum of the zinc(II) complex suggests that the signal due to Fig. 1. FAB mass spectrum of the ligand. Fig. 2. Structure of the ligand. Table 2 Infrared and far infrared spectral data of the ligand and its metal complexes (KBr) (cm�1) ISAP [Mn(ISAP)2](OAc)2 [Co(ISAP)2]Cl2 [Ni(ISAP)2]Cl2 3215 3216 3214 3218 1730 1692 1694 1691 1619 1596 1598 1594 1577 1579 1576 1578 – 520 526 523 – 452 453 458 nature of the complex and confirms the proposed formula. Thus, the IR, 1H NMR and FAB mass spectral data are in good agreement with the neutral bidentate bonding mode of the ligand to the metal ions through the azomethine nitrogen atom and the carbonyl oxygen on the isatin moiety. Electronic spectra and magnetic moment values Visible spectral data along with magnetic susceptibility mea- surements gave adequate support in establishing the geometry of the metal complexes. These data along with the tentative assign- ments are presented in Table 3. The intense charge transfer band due to the ligand totally masks the weak forbidden dAd bands of the manganese(II) complex. The magnetic moment value of the complex is found to be 5.92 BM indicating a high-spin complex with five unpaired electrons. These observations support the assumption of a tetrahedral geometry for the manganese(II) com- plex [38]. The absorption band at 16,800 cm�1 in the cobalt(II) complex corresponding to 4A2g? 4T1g transition, which is charac- teristic of a tetrahedral geometry around the cobalt(II) ion. The magnetic moment value of the cobalt(II) complex (4.38 BM) gives added support to this observation [39]. Nickel(II) complex is dia- magnetic and it exhibits two absorption bands at 13,500 cm�1 and 18,830 cm�1 assignable to 1A1g? 1A2g and 1A1g? 1B1g transi- tions respectively. These values are consistent with a square-pla- nar geometry [40]. The electronic spectrum of the copper(II) complex exhibits a broad band centered at 13,800 cm�1 corre- sponding to 2B1g? 2A1g transition and its magnetic moment value of 1.83 BM suggest a distorted square-planar geometry around the metal ion [41]. Zinc(II) complex is diamagnetic and a tetrahedral geometry is most preferable for four coordinated zinc(II) complex [42]. EPR spectrum The X-band EPR spectrum of the [Cu(ISAP)2]Cl2 complex (Fig. 3) was recorded in solid state at room temperature and in DMSO at 77 K using TCNE free radical as the ‘g’ marker. The spectrum ob- tained for the complex at a microwave frequency of 9.425 GHz with a field strength of 3200 G is characteristic of copper(II) ion in axial ligand field symmetry, i.e., gx = gy– gz. The analysis of the spectrum gives gk = 2.1133, g\ = 2.0341. The trend gk > g\ > ge ob- served for the complex indicates the tetragonal elongation along z axis and the presence of the unpaired electron in the dx2�y2 orbi- tal. It has been reported that gk values are sensitive to the covalent nature of the metal-ligand bond; the values above 2.3 show ionic character and lower values reveal covalent character [43]. From these values, it is evident that metal-ligand bonds have consider- able covalent character [44]. From the values of the g factors, gav estimated is 2.0605. The four peaks in the spectrum are evidently due to the cou- pling of the electron spin of the 63Cu nucleus (I = 3/2). The peaks . [Cu(ISAP)2]Cl2 [Zn(ISAP)2]Cl2 Tentative assignments 3215 3217 t(NAH) of indole ring 1690 1693 t(C@O) of indole ring 1593 1597 t(C@N) 1577 1578 t(C@N) of pyrimidine ring 528 525 t(MAO) 456 460 t(MAN) Table 3 Electronic spectral data and magnetic moment values of the metal complexes. Complex Absorption band (cm�1) [Mn(ISAP)2](OAc)2 – [Co(ISAP)2]Cl2 16,800 [Ni(ISAP)2]Cl2 13,500 18,830 [Cu(ISAP)2]Cl2 13,800 D: diamagnetic. 158 L.P. Nitha et al. / Spectrochimica Acta Part A: Molecular a are broad and have the appearance of ill-resolved triplets. The breadth and triplet appearance can be attributed to hyperfine split- ting by the nitrogen atom (I = 1) of the ligand. The triplet appear- ance is adduced as an evidence for nitrogen coordination. Based on this observation, a distorted square planar geometry is pro- posed for the complex. The EPR study of the copper(II) complex has provided supportive evidence to the conclusion obtained on the basis of electronic spectrum and magnetic moment value. The main features of all spectral data of the metal complexes lead us to suggest the most probable structure as shown in Fig. 4. X-ray diffraction study The ligand and its copper(II) complex has been subjected to X- ray diffraction study. The diffractogram of the ligand (Fig. 5) has re- corded 15 reflections of 2h ranging from 14.7485� to 49.1931�. The maximum recorded is at 17.6442, which corresponds to a d-spac- ing of 5.0225 Å. The observed sin2 h value and 2h values obtained have been compared with the calculated values. The indexing of the X-ray diffraction powder photograph for the ligand indicates that it has an orthorhombic crystal lattice [45]. The unit cell dimensions have been calculated to be: a = 9.4780 Å, b = 7.1065 Å, c = 4.2910 Å and unit cell volume = 289.0220 Å3. The diffractogram of copper(II) complex showed 14 reflections be- Fig. 3. EPR spectrum of [Cu(ISAP)2]Cl2. tween 2h ranging from 12.6319 to 54.0527 with maxima peak at 2h = 12.6319 which corresponds to interplanar distance, d = 7.0663 Å. The sin2 h values have been compared with calculated values, these values shows good agreement between the calculated and observed values. The complex was successfully indexed to an orthorhombic system with lattice constants, a = 12.7103 Å, b = 6.8059 Å, c = 4.4382 Å and unit cell volume, V = 383.9226 Å3. cause of its labile coordination sphere and their ability to interca- late between the bases of DNA. One possible explanation for different reactivity of the complexes is the redox property of the central metal ions where zinc is redox-inactive but others are re- Fig. 4. Structure of the 1: 2 metal complexes. dox-active [47]. Hence zinc(II) complex does not shows any cleav- age activity. However, the structure of the complexes and binding affinity towards DNA alone do not explain the DNA cleavage effi- From the XRD pattern of the copper(II) complex (Fig. 6), it is clear that the crystallinity of the ligand was retained even after on com- plexation with the metal ion. DNA cleavage activity The ability of the ligand and its metal complexes to effect oxida- tive cleavage in the presence of H2O2 has been investigated and the result obtained is shown in Fig. 7 (Lanes 1–7). The cleavage effi- ciency of the complexes compared to the control is due to their efficient DNA binding ability. In the control experiment using DNA alone (Lane 1), no significant cleavage of DNA was observed on long exposure time. It is evident from the figure that ligand (Lane 2) and zinc(II) complex (Lane 7) fail to show any apparent cleavage of pUC 19 DNA. All the complexes except zinc(II) complex, showed enhanced activity than the ligand in presence of H2O2. The increased activity may be due to the increased production of hy- droxyl radicals and is produced by reacting the H2O2 with metal ions. This OH. attack at the C-3 position of sugar moiety and finally cleave the DNA. The selected metal complexes are able to convert super coiled DNA into open circular form. The general oxidative mechanisms account for DNA cleavage by hydroxyl radicals via abstraction of a hydrogen atom from sugar units and the release of specific resi- dues arising from transformed sugars depending on the position from which the hydrogen atom is removed. Major factors that af- fect the extent of DNA cleavage by the metal complexes include the concentration of the complex, nature of the metal ion and addi- tion of hydrogen peroxide as exogenous reagents [46]. Cobalt(II), manganese(II) and nickel(II) complexes (Lanes 3, 4 and 6 respectively) show less cleavage efficiency as compared to the copper(II) complex. The cleavage efficiency of copper(II) is be- Tentative assignments Magnetic moment (BM) – 5.92 4A2g? 4T1g 4.38 1A1g? 1A2g D 1A1g? 1B1g 2B1g? 2A1g 1.83 nd Biomolecular Spectroscopy 118 (2014) 154–161 ciency order of the different metals. The complex which has feeble hydrogen abstraction shows less cleavage activity. Basically a mol- ecule may bind to DNA via non covalent interactions by intercala- tive binding, groove binding or external electrostatic binding [48]. Among these interactions intercalation and groove binding are the most important DNA binding modes as they invariably lead to cel- lular degradation. To know the involvement of hydroxyl radical in oxidative DNA cleavage, the experiment is also carried out by adding radical scav- enger (DMSO) to complex-DNA mixture. Upon the addition of DMSO to complex inhibition of cleavage was observed, suggestive the cleavage is oxidative and hydroxyl radical is generated from the complexes in the presence of H2O2, mediates the cleavage reac- tion [49]. lar and Biomolecular Spectroscopy 118 (2014) 154–161 159 sit y 14000 15000 16000 17000 18000 19000 20000 21000 22000 23000 24000 25000 L.P. Nitha et al. / Spectrochimica Acta Part A: Molecu Superoxidasedismutase (SOD) activity The in vitro SOD activity is measured using alkaline DMSO as a source of superoxide radical (O2��). The ligand and corresponding metal complexes have been tested for SOD activity and the results were given in Table 4. The results have been compared with the native Cu, Zn–SOD (0.04 lM). The results strongly suggest that the copper(II) complex is of highly active for the SOD activity. The zinc(II) and cobalt(II) complexes also exhibit excellent activity than the free ligand. While the ligand fails to show SOD mimetic activity. The manganese(II) and nickel(II) complexes shows less 2-Theta 3 10 20 30 40 50 6 In te n 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 Fig. 5. XRD pattern 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 15000 2-Thet 3 10 20 30 40 50 In te ns ity Fig. 6. XRD pattern o Lane Fig. 7. Gel electrophoresis diagram showing the DNA cleavage. Lane 1: control DNA. Lane 2: DNA + Ligand + H2O2. Lane 3: DNA + CoL2 + H2O2. Lane 4: DNA + MnL2 + H2O2. Lane 5: DNA + CuL2 + H2O2. Lane 6: DNA + NiL2 + H2O2. Lane 7: DNA + ZnL2 + H2O2. - Scale 0 70 80 90 100 110 120 of the ligand. activity. In addition, the ligand stabilize the copper(I) complex formed during superoxide dismutation reaction which further re- acts with superoxide ion to give hydrogen peroxide. Due to the sig- nificant SOD-mimic activity of the tested complexes, we expect the redox mechanism of superoxide dismutation, similar to the Cu, Zn– SOD. The dismutation of superoxide anion by both the native en- zyme SOD or the copper complex is involving redox cycling of cop- per(II) ion as following [50]: CuðIIÞ þ O2�� ! CuðIÞ þ O2 CuðIÞ þ O2�� þ 2H2 ! CuðIIÞ þH2O2 a - Scale 60 70 80 90 100 110 120 f [Cu(ISAP)2]Cl2. Table 4 Superoxide dismutase activity of ligand and complexes. Compound IC50 (lM)a ISAP 0.62 [Mn(ISAP)2](OAc)2 0.58 [Co(ISAP)2]Cl2 0.26 [Ni(ISAP)2]Cl2 0.54 [Cu(ISAP)2]Cl2 0.14 [Zn(ISAP)2]Cl2 0.22 Native Cu,Zn–SOD 0.04 a IC50 was defined as 50% inhibition concentration of NBT reduction. lar a Hydrogen peroxide formed by this reaction is destroyed in vivo by the enzyme catalase. The difference in activity of the synthesised complexes may be attributed to the coordination environment and the redox properties. Generally the redox properties of the complex depend on several factors such as the chelate ring size, ax- ial ligation, degree and distribution of unsaturation and substitution pattern in the chelate ring [51]. The relatively high SOD activity of the complexes under investigation may be explained in terms of limited steric hindrance to the approach of superoxide anion to coordination site. Several complexes containing transition metals [52–54] are known to give good SOD activity, although their struc- tures are totally unrelated with native enzyme [54]. These results render the complexes particularly attractive as catalytic antioxi- dants for pharmacological purposes. In vitro antibacterial activity The ligand and the metal complexes were screened against the bacterial species such as E. coli, S. aureus, K. pneumoniae, S. typhi and P. aeruginosa in order to compare their antibacterial activity and their results were presented in Table 5. The results infer that the tested compounds show remarkable biological activities against different type of bacteria (E. coli, S. aureus, K. pneumoniae, S. typhi, and P. aeruginosa). All the compounds were found to have maximum inhibitory activity against S. aureus and weakly active against E. coli. A comparative study of the ligand and their com- plexes (MIC values) indicates that the metal complexes exhibit higher antibacterial activity than the free ligand. Thus the en- hanced antibacterial activities of the metal complexes are due to the change in the structure and reactivity of the ligand on coordi- nation [55]. Such increased activity of the complexes can be ex- plained with respect to Overtone’s concept [56] and Tweedy’s chelation theory [57]. According to Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only the lipid soluble materials whose liposolubility is an important fac- tor, which controls the antibacterial activity [58]. On chelation, the polarity of the metal ion is reduced to a great extent due to the overlap of the ligand orbital and partial sharing of the positive Table 5 Minimum inhibitory concentration of ISAP and its metal complexes. Compound Minimum inhibition concentration (lg/ml) E. coli K. pneumoniae ISAP 60 50 [Mn(ISAP)2](OAc)2 34 20 [Co(ISAP)2]Cl2 38 24 [Ni(ISAP)2]Cl2 36 20 [Cu(ISAP)2]Cl2 30 16 [Zn(ISAP)2]Cl2 32 18 Ampicillin 14 08 160 L.P. Nitha et al. / Spectrochimica Acta Part A: Molecu charge of the metal ion with donor groups. Further, it increases the delocalization of p-electrons over the whole chelate ring and enhances the lipophilicity of the complexes. This may lead to the breakdown of the permeability barrier of the cell resulting in inter- ference with normal cell processes and blocking of the metal bind- ing sites in the enzymes of microorganisms. These complexes also disturb the respiration process of the cell and thus block the syn- thesis of proteins, which restricts further growth of the organism. Furthermore, the mode of action of the compounds may involve formation of a hydrogen bond through the azomethine group with the active centers of cell constituents, resulting in interference with the normal cell process. The variations in the activity of different complexes against dif- ferent organisms depend either on the impermeability of the cells of the microbes or difference in ribosomes of the microbial cells. The low activity of some complexes can be attributed to low lipid solubility. As a result of this, the metal ion cannot reach the desir- able site of action of the cell wall to interfere with the normal cell activity. Although chelation dominantly affects the biological behaviour of the compounds, other important factors such as nature of the metal ion, nature of the ligand, coordinating sites, geometry of the complex, concentration, hydrophilicity, lipophilicity and pres- ence of co-ligands have considerable influence on the antibacterial activity [59]. 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Nitha et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 118 (2014) 154–161 161 Synthesis, spectroscopic characterisation, DNA cleavage, superoxidase dismutase activity and antibacterial properties of some transition metal complexes of a novel bidentate Schiff base derived from isati Introduction Experimental Materials and methods Gel electrophoresis Determination of superoxide dismutase activity In vitro antibacterial activity Synthesis of 2-[N-indole-2-one]aminopyrimidine Synthesis of the metal complexes Result and discussion Structure of the ligand UV–Visible spectrum Infrared spectrum 1H NMR and FAB mass spectra Structure of the metal complexes Infrared spectra Far IR spectra 1H NMR spectra and FAB mass spectra Electronic spectra and magnetic moment values EPR spectrum X-ray diffraction study DNA cleavage activity Superoxidasedismutase (SOD) activity In vitro antibacterial activity Acknowledgements References