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Ofloxacin Metal Complexes: Synthesis,Characterization, Analytical Properties, and DNABinding StudiesAysegul Golcua

a Department of Chemistry, Faculty of Science and Letters, University of KahramanmarasSutcu Imam, Campuse of Avsar, Kahramanmaras, TurkeyAccepted author version posted online: 23 Jan 2014.Published online: 02 Jun 2014.

To cite this article: Aysegul Golcu (2014) Ofloxacin Metal Complexes: Synthesis, Characterization, Analytical Properties, andDNA Binding Studies, Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 44:10, 1509-1520, DOI:10.1080/15533174.2013.818020

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Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 44:1509–1520, 2014Copyright C© Taylor & Francis Group, LLCISSN: 1553-3174 print / 1553-3182 onlineDOI: 10.1080/15533174.2013.818020

Ofloxacin Metal Complexes: Synthesis, Characterization,Analytical Properties, and DNA Binding Studies

Aysegul GolcuDepartment of Chemistry, Faculty of Science and Letters, University of Kahramanmaras Sutcu Imam,Campuse of Avsar, Kahramanmaras, Turkey

The present work comprises the synthesis of ofloxacin (OFL)complexes with various transition metals. Two types of complexes[Cu(OFL)Cl2].H2O, [Pt(OFL)CI2].3H2O, [Zn(OFL)2]2CI.4H2O,[Ru(OFL)2ClH2O]2CI.4H2O, and [Fe(OFL)2Cl]2CI.5H2O wereobtained. The complexes were characterized by different physico-chemical, spectroscopic, and elemental analysis. The thermal de-composition behaviors of metal complexes were investigated in ni-trogen atmosphere using TG and DTA techniques. Results suggestthat OFL interacts with the metals as a monoanionic bidentateligand. These complexes were also tested for their antibacterial ac-tivity against nine different microorganisms, and the results werecompared with the parent drug. The electrochemical propertiesof all complexes have been investigated by cyclic voltammetryusing glassy carbon electrode. The oxidation/reduction of metalcomplexes was irreversible/reversible and exhibited diffusion con-trolled process depending on pH. The dependence of intensitiesof currents and potentials on pH, concentration, scan rate, natureof the buffer was investigated. The oxidation/reduction mecha-nism was proposed and discussed. The DNA binding activity ofthe complexes was evaluated by examining their ability to bind tocalf-thymus DNA (CT DNA) with UV spectroscopy. UV studies ofthe interaction of the OFL and complexes with DNA have shownthat these compounds can bind to CT DNA. The binding constantsof the OFL and complexes with CT DNA have also been calculated.

Keywords metal-based drugs, ofloxacin, spectrophotometry, volt-ammetry

INTRODUCTIONSince the discovery of cisplatin and its extensive applica-

tion in chemotherapy, several metal-based drugs have been ex-amined for their potential therapeutic properties. Metal-based

Received 19 April 2013; accepted 11 June 2013.The author thanks M. Cesme (Ph.D.), H. Muslu (Ph.D.), D. Tarinc

(Ph.D.), and M. Vahdettin (M.Sc.) Avcioglu for their great contributionsin experimental studies.

Address correspondence to Aysegul Golcu, Department of Chem-istry, Faculty of Science and Letters, University of KahramanmarasSutcu Imam, Campuse of Avsar 46100, Kahramanmaras, Turkey.E-mail: ag518@ksu.edu.tr

Color versions of one or more of the figures in the article can befound online at www.tandfonline.com/lsrt.

compounds enlarge the possibility of building up moleculesbetter suited for binding to specific biological targets.[1] Thesenew potential metal-based drugs, compared to cisplatin, shouldoffer improved antitumor activity, reduced levels of toxicity, andlimited side effects.[2] For this purpose, the biological signifi-cance of the different metal cations are used for the synthesis ofnew metal-based drugs. Similarly, scarcity of some metal ionscan lead to disease. Well-known examples include perniciousanemia resulting from iron deficiency, growth retardation aris-ing from insufficient dietary zinc, and heart disease in infantsowing to copper deficiency. In addition, copper(II) in biologicalsystems fulfills a range of catalytic functions, most notably inthe antioxidant defense enzyme of copper- and zinc-containingsuperoxide dismutases,[3] in the mitochondrial electron transportenzyme cytochrome c oxidase,[4] and in the copper-containingproteins dopamine β-monooxygenase and peptidylglycineα-hydroxylating monooxygenase, which catalyze the transfor-mation of dopamine to norepinephrine.[5] Less well known thanthe fact that metal ions are required in biology as pharmaceu-ticals. A drug based on metal that has no known natural bi-ological function, Ru(III), is widely used for the treatment ofmice bearing Ehrlich as cites cancer.[6] In addition, compoundsof radioactive metal ions such as 99mTc and complexes of para-magnetic metals such as Gd(III) are now in widespread useas imaging agents for the diagnosis of disease. Many patientsadmitted overnight to a hospital in the U.S. will receive an in-jection of a 99mTc compound for radio diagnostic purposes. Yet,despite the obvious success of metal complexes as diagnosticand chemotherapeutic agents, few pharmaceutical or chemicalcompanies have serious in-house research programs that addressthese important bioinorganic aspects of medicine.[7]

Quinolones, with the term quinolone carboxylic acids or 4-quinolones, are a group of synthetic antibacterial agents contain-ing 4-oxo-1,4-dihydroquinoline skeletons[8] and are extremelyuseful for the treatment of various infections. They have effec-tives against Gram-positive and Gram-negative bacteria throughinhibition of their DNA gyrase.[9]

Ofloxacin (OFL) is a synthetic chemotherapeutic antibi-otic of the fluoroquinolone drug class considered to be asecond-generation fluoroquinolone (Scheme 1).[10, 11] The orig-inal brand, Floxin, was discontinued by the manufacturer in

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N

O

OH

OO

CH3

F

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SCH. 1. Chemical structure of ofloxacin.

the United States on 18 June 2009, though generic equivalentscontinue to be available. It was first patented in 1982 (EuropeanPatent Daiichi) and received approval from the U.S. Food andDrug Administration on December 28, 1990. It is sold undera wide variety of brand names as well as generic drug equiva-lents, for oral and intravenous administration. It is also availablefor topical use, as eye and ear drops (marketed as Ocuflox andFloxin Otic, respectively, in the United States).

Like other quinolones, OFL has been associated with a signif-icant number of serious adverse drug reactions, such as tendondamage (including spontaneous tendon ruptures) and peripheralneuropathy (which may be irreversible); such reactions maymanifest long after therapy is completed, and, in severe cases,may result in lifelong disabilities.[12]

In our previous study, we synthesized and characterized newcomplexes of fluoroquinolone drug pefloxacin, with Cu(II),Zn(II), Pt(II), Ru(III), and Fe(III) by spectroscopic techniquesinvolving UV-vis, IR, 1H-HMR, CHN elemental analysis, elec-trochemical, and thermal behaviors.[13] As you know the em-ployees in this regard, there are many texts in the literature thatbelong to fluoroquinolone drug-metal complexes.[14–18] Espe-cially, around 50 studies that belong to OFL-metal complexesare in the “web of science” data base.[19–30] But, in these studies,the interactions of metal complexes of OFL with DNA have notbeen studied. Whereas, the study of the interactions of transition-metal complexes with DNA has been an active field of research.Interest in this field stems from attempts to gain some insightinto the interaction model between DNA and the complexes,and obtain some information about drug design and tools ofmolecular biology.[31] Transition metal complexes can interactwith DNA through a non-covalent way, such as electrostaticinteraction, groove binding, and intercalation. Binding to DNAthrough an intercalation mode with planar ligands intercalatinginto the adjacent base pairs of DNA correlates to the planarityof the ligand, coordination geometry of the metal ion, and donortype of the ligand.[32] Considerable useful applications of thesecomplexes need to be done to develop the complexes binding toDNA through an intercalation mode with their structures con-taining fully planar intercalating into the adjacent base pairs ofDNA.[27, 28] These applications give valuable information to ex-plore the potential chemotherapeutical agents. Along this line,lots of copper (II) complexes, which possess biologically acces-sible redox potentials and demonstrate high nucleobase affinity,

have been synthesized and their interactions with DNA wereextensively studied.[33, 34]

In continuation of our previous work,[35–54] mononuclearmetal (II/III) complexes of OFL have been synthesized andcharacterized by elemental analyses, Fourier transform infraredspectra (FT-IR), electronic and atomic absorption spectra mo-lar conductance, and magnetic susceptibility data. The thermaldecomposition behavior of the complexes has been investigatedunder nitrogen atmosphere using thermal analysis (TG) and dif-ferential thermal analysis (DTA) techniques. The interactions ofmetal ions with OFL have been studied in an attempt to examinethe mode of binding and possible synergetic effects. The resul-tant mononuclear complexes have been investigated by cyclicvoltammetry (CV). The complex has been tested for its ability tobind to calf-thymus DNA (CT DNA). The binding properties ofthe complexes with CT DNA have been investigated with UVtitration. Also, antibacterial and antifungal properties of OFLand complexes have been studied against both Gram-positiveand Gram-negative bacterias. Minimum concentration of thecompounds required to inhibit the growth of microorganismwas obtained using the double dilution technique.

EXPERIMENTAL

GeneralOFL was kindly provided by Eczacıbası (Istanbul, Turkey).

CT DNA was purchased from Sigma; NaCl, metal salts(CuCl2.2H2O, ZnCl2, FeCl3.6H2O, K2PtCl4, and RuCl3.3H2O),NaCl, 0.2 M phosphate buffer at pH 2.0–12.0, ethidium bro-mide (EB), and Tris-HCl were purchased from Merck. All thechemicals and solvents were reagent grade and were used aspurchased. Elemental analyses (C, H, and N) were performedusing a LECO CHNS 932 elemental analyzer. Infrared spectra ofthe ligands and their metal complexes were obtained using KBrdiscs (4000-400 cm−1) with a Perkin Elmer spectrum 400 FT-IRspectrophotometer. Far spectra of the complexes were recordedusing a Perkin Elmer spectrum 400 FT-IR/FT-FAR instrument.The electronic spectra were obtained in the 200–900 nm rangeby a Perkin Elmer Lambda 45 spectrophotometer. Magneticmeasurements were carried out by the Gouy method usingHg[Co(SCN)4] as a calibrant. 1H-NMR spectra were recordedon a Bruker 400 MHz instrument. Tetramethylsilane (TMS)was used as internal standard, and dimethyl sulfoxide (DMSO)was used as solvent. The metal content of the complex wasdetermined by an Ati Unicam 929 Model AA spectrometer insolutions prepared by decomposing the compounds in aqua re-gia and then subsequently digesting in concentrated HCl. Thethermal analysis studies of the complex were performed on aPerkin Elmer STA 6000 simultaneous Thermal Analyzer undernitrogen atmosphere at a heating rate of 10◦C/min.[38]

Electrochemical MeasurementsAll voltammetric measurements at the glassy carbon

working electrode were performed using a BAS 100W

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(Bioanalytical System, USA) electrochemical analyzer underN2 gas. Glassy carbon working electrode (BAS; �: 3-mm diam-eter), an Ag/AgCl reference electrode (BAS; 3 M KCl) and plat-inum wire counter electrode, and a standard one-compartmentthree-electrode cell of 10-ml capacity were used in all exper-iments. Glassy carbon working electrode was polished man-ually with aqueous slurry of alumina powder (�: 0.01 μm)on a damp smooth polishing cloth (BAS velvet polishingpad), before each measurement. All measurements were real-ized at room temperature. Mettler Toledo MP 220 pH meterswas used for the pH measurements using a combined elec-trode (glass electrode reference electrode) with an accuracy of±0.05 pH.

Synthesis of a Copper(II) and Platinum(II) ComplexOFL (1 mmol, 0.361 g) dissolved in 20-ml EtOH solution.

1 mmol metal salts (CuCl2.2H2O and K2PtCl4) in MeOH (20 ml)were added to solution of OFL. And the mixture was heatedunder reflux for 1 day. At the end of the reaction, determinedby TLC, the precipitate was filtered off, washed with distilledwater, EtOH and dried under vacuum. The proposed formulasof M(II)-OFL complexes is given in Scheme 2.

SCH. 2. Chemical structures of Cu(II) and Pt(II) complexes of OFL (n = 1for Cu(II), and n = 3 for Pt(II)).

Synthesis of a Zinc (II), Ruthenium (III), and Iron(III)Complex

OFL (2 mmol, 0.722 g) dissolved in 20-ml EtOH solution.1 mmol metal salts (ZnCl2, RuCl3.3H2O) in MeOH (20 ml)were added to solution of OFL. And the mixture was heatedunder reflux for 1 day. At the end of the reaction, determinedby TLC, the precipitate was filtered off, washed with distilledwater, EtOH and dried under vacuum. The proposed formulasof complexes are given in Schemes 3 and 4.

Preparation of the Microorganism CulturesThe growth-inhibitory activity of the chemical matter was

tested against nine bacteria (Escherichia coli, Enterobactercloacae, Bacillus megaterium, Bacillus cereus, Pseudomonassp., Brucella melitensis, and Staphylococcus aureus) and fungi

SCH. 3. Chemical structure of Fe(III) and Ru(III) complexes OFL (n = 5 forFe(III), and n = 4 for Ru(III)).

(Candida albicans and Saccharomyces cerevisiae). These mi-croorganisms were provided from Microbiology LaboratoryCulture Collection, Department of Biology, KahramanmarasSutcu Imam University, Kahramanmaras, Turkey. Antimicro-bial activities of the chemical matter were determined using theagar-disc diffusion method as will be described below. The bac-teria were first incubated at 37 ± 0.1◦C for 24 h in nutrient broth(Difco), and the yeasts were incubated in Sabouraud dextrosebroth (Difco) at 25 ± 0.1◦C for 24 h. The cultures of the bacteriaand yeast were injected into the petri dishes (9 cm) in the amountof 0.1 ml. And then, Mueller Hinton agar and Sabouraud dex-trose agar (sterilized in a flOFL and cooled to 45–50◦C) werehomogenously distributed onto the sterilized petri dishes in theamount of 15 ml. Subsequently, the sterilized blank paper discsof 6-mm diameter were saturated with 1200 μg of chemicalmatters per disc. The discs were placed onto the agar plates,which had previously been inoculated with the above organ-isms. In addition, blank paper discs treated with OFL was usedas positive controls. Afterward, the plates combined with thediscs were left at 4◦C for 2 h, the plates injected with yeastwere incubated at 25 ± 0.1◦C for 24 h, and ones injected withbacteria were incubated at 37 ± 0.1◦C for 24 h. After 24 h,inhibition zones appearing around the discs were measured andrecorded in millimeter. The initial number of microorganisms inthe suspension was determined for the total yeasts and bacterialcount during 24 h at 37◦C for bacteria and 48 h at 25◦C foryeasts.[51, 55]

SCH. 4. Chemical structure of Zn(II) complex of OFL.

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TABLE 1Analytical and physical results of ofloxacin and metal complexes

Elemental analysis; calculated (found)Formula weight Yield Melting

Compound (g/mol) Color (%) point (◦C) C H N M

[Cu(OFL)Cl2].H2O 513.83 Green 75 221 42.07 (42.09) 4.323 (3.924) 8.18 (7.863) 12.37 (12.30)[Ru(OFL)2ClH2O]2CI.4H2O 990.21 Black 80 284 42.45 (42.73) 4.89 (4.787) 8.49 (7.836) 10.21 (10.18)[Fe(OFL)2Cl]2CI.5H2O 963.00 Dark Red 70 212 43.65 (43.92) 5.23 (5.095) 8.73 (8.141) 5.80 (5.75)[Pt(OFL)CI2].3H2O 681.40 Brown 65 260 31.73 (31.91) 3.85 (3.013) 6.17 (5.907) 28.63 (28.60)[Zn(OFL)2]2CI.4H2O 901.08 White 78 160 46.65 (46.25) 5.15 (5.029) 9.33 (8.543) 7.26 (7.30)

RESULT AND DISCUSSIONIn this study, metal (II/III) complexes of OFL were pre-

pared and characterized by the analytical and spectroscopicmethods. The isolated complexes are stable in air, insolublein water, and common inorganic solvents, but completely solu-ble in DMSO. The elemental analysis, color, and melting pointtogether for the complexes are given in Table 1 and aggressvery well with molecular formula proposed. The analyticaldata composition of the metal complexes to be [Cu(OFL)Cl2].H2O, [Pt(OFL)CI2].3H2O, [Zn(OFL)2]2CI.4H2O, [Ru(OFL)2

ClH2O]2CI.4H2O, and [Fe(OFL)2Cl]2CI.5H2O. The ratios ofthe metal present in all complexes are determined by atomicabsorption spectroscopy. The complexes were decomposed inHNO3/H2O2 (1/1) and then dissolved in 1.5 N HNO3. Theamounts of metals were determined (Table 1). They supportthe structures given in the Schemes 2–4. Equivalent conduc-tance of [Zn(OFL)2]2CI.4H2O, [Ru(OFL)2ClH2O]2CI.4H2O,and [Fe(OFL)2Cl]2CI.5H2O complexes were carried out in 1× 10−3 M DMSO solutions, and the values were found to bein the 73.7–103.7 �−1cm2mol−1 range, and from these data,I can say that these complexes have the cationic nature andconduct electricity in solution (Table 2). The mononuclear[Cu(OFL)Cl2].H2O and [Pt(OFL)CI2].3H2O complexes werefound to be non-electrolyte in DMSO.[36]

Chloride ions in all complexes were determined by titrationwith AgNO3.

Equivalent conductance of Na[C19H28NO5S2Co]·H2O,[C17H26NO5S2Ni]Cl·H2O, Na[C21H26NO6S2Zn], and Na[C17

H22N3O9S2Cd]·H2O complexes were carried out in 1 × 10−3

M DMSO solutions, and the values were found to be in the7.3–88.3 �−1cm2mol−1 range, and from these data, I can saythat these complexes have the cationic or anionic nature andconduct electricity in solution[35] (see Table 3). The mononu-clear [C34H40N2O4S4Cu]·H2O complex was found to be non-electrolyte in DMSO.[36]

Electronic SpectraElectronic spectra of the metal complexes were investigated

at 1 × 10−3 M in ethanol solution and different pH values(pH 2–12) in phosphate buffer. The spectra are pH dependentbecause of the weak acid character of the metal complexes.They show π→π∗ transitions and n→π∗ transitions between207 and 293 nm and 308 and 342 nm, respectively. All com-plexes except Pt(II) and Zn(II) complexes show d-d∗ transi-tions between 448 and 476 nm. The spectra of the complexesshow bands between 350 and 390 nm assigned to M→L andL→M (L: ligand, M: metal) charge transfer transitions. Cu(II),Pt(II), and Ru(III) complexes absorption maximas were shiftedto longer wavelengths as pH increased, which means that thereis a bathochromic effect for these three complexes. All com-plexes, except Fe(III) and Zn(II), show hyper chromic effect ina 2–12 pH region (Table 2).

IR Absorption StudiesIn order to clarify the mode of bonding and the effect of

the metal ion on the ligand, the FT-IR spectra of the OFL andits metal complexes were compared and assigned on the basisof careful comparison. The FT-IR results of OFL and its metal

TABLE 2UV-vis results of OFL and its metal complexes

Compound �a �- �∗ n- �∗ Charger transfer (M→L or L→M) d-d∗

[Cu(OFL)Cl2].H2O — 225 342 380 —[Ru(OFL)2ClH2O]2CI.4H2O 93.1 293 308, 318 390 452[Fe(OFL)2Cl]2CI.5H2O 103.7 260 314 381 476[Pt(OFL)CI2].3H2O — 244 313 381 424[Zn(OFL)2]2CI.4H2O 73.7 207 308 350 —

a�-1cm2mol−1.

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TABLE 3The intrinsic binding constants (Kb) of complexes with CT

DNA

Compounds Kb

OFL 1.17 × 104

[Cu(OFL)Cl2].H2O 7.12 × 108

[Ru(OFL)2ClH2O]2CI.4H2O 6.90 × 108

[Fe(OFL)2Cl]2CI.5H2O 7.01 × 108

[Pt(OFL)CI2].3H2O 6.85 × 108

[Zn(OFL)2]2CI.4H2O 6.57 × 108

complexes are listed in Table 4. All OFL complexes and itselfshow broad band between 3384 and 3440 cm−1, correspondingto υ(O H) stretching. Between 1614- and 1712-cm−1 frequen-cies, there are characteristic vibrations of υ(C O) group ofOFL and its complexes. However, there are slightly shifting onthese vibrations, they can be explained by the coordination ofthe metal ions to the (C O) group of OFL.[51] In order to inves-tigate the substituent effect upon both δ- and π - contribution tothe total metal-nitrogen, metal-oxygen, and metal-chloride bondstrength, the FT-Far-IR method were used. The infrared spectrain the region 700-30 cm−1 were measured for the complexes,and the maximum absorption frequencies are listed at Table 4.There are new bands between 380 cm−1, and 287–318 cm−1,corresponding to υ(M O), and υ(M Cl), respectively.

The 1H-NMR StudiesThe molecular structures of Pt(II) and Zn(II) complexes were

also examined by proton nuclear magnetic resonance (1H-NMR)spectroscopy. There are slight differences between the spectrumof OFL and its metal complexes. The only clear shift is oncarboxyl proton, which is at 7.5 ppm (Figures 1 and 2).

Thermal AnalysisThermogravimetric analysis is a useful technique for the

determination of the thermal stability and structural elucida-tion of various insoluble and infusible compounds[47]; but todate, only a limited number of reports concerning thermal dataand solution thermochemistry of metal-drug complexes have

FIG. 1. 1H-NMR spectrum of OFL.

appeared in the literature. In this study, the thermal behav-ior of the complexes was characterized using DTA and ther-mogravimetric analysis (TGA)/differential thermogravimetricanalysis (DTG) methods. The DTA/TG measurements of thecomplexes were carried out in the 30–1000◦C range. The com-plexes contain the absorbed and/or hydrated chloride ion andwater molecules. There is one route in removal of the absorbedand/or hydrated water molecules in the 50–250◦C temperaturerange from the complexes. Moreover, the coordinated chlorideion losses from complexes in the 250–500◦C temperature range.At higher temperatures (500–1000◦C), all complexes decom-pose to give the approximate metal oxide. The thermal curves ofthe [Fe(OFL)2Cl]2CI.5H2O and [Pt(OFL)CI2].3H2O are givenin the Figures 3 and 4.

The chelates of OFL with di- and tri-valent metal ions un-dergo thermal decomposition in a series of overlapping re-action with multiple products. The organic part (OFL) to-gether with chloride anion in the moiety of complexes may

TABLE 4FT-IR results of OFL and its metal complexes

Compound ν(OH)H2O ν(OH)COOH (C H)str∗ ν(C O)str ν(M O) ν (M CI)

[Cu(OFL)Cl2].H2O — 3043 2757 1712 — —[Ru(OFL)2ClH2O]2CI.4H2O 3560, 3477 3050 2880 1622 380 287[Fe(OFL)2Cl]2CI.5H2O 3383 2958 2694 1614 380 292[Pt(OFL)CI2].3H2O 3363 2960 2688 1615 380 288[Zn(OFL)2]2CI.4H2O 3419 3043 2850 1705 — 318[Cu(OFL)Cl2].H2O 3383 2844 2694 1614 380 292

∗Stretching.

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FIG. 2. 1H-NMR spectrum of [Zn(OFL)2]2CI.4H2O complex.

decompose in more than two steps with the possibility of theformation of more than one intermediate. These intermediatesmay finally decompose to stable metal oxide. The DTA curvesshow a medium to strong exothermic and endothermic peaks,the position of each peak accompanying the decomposition ofthe anions and OFL molecule different gases like H2O, O2,CO2, etc.[56] From the calculations, it follows that the final de-composition product can be MO/M2O3 (MO is for the Cu(II),

Zn(II), and Pt(II) complexes; M2O3 is for the Ru(III) and Fe(III)complexes).

Antimicrobial StudiesThe susceptibility of certain strains of bacterium toward

OFL and its complexes has been judged by measuring the sizeof inhibition zone diameter. Antibacterial activities of OFLand its complexes have been carried out with three Gram-positive (B. megaterium, B. cereus, S. aureus) and four Gram-negative (E. coli, Enterobacter cloaca, Pseudomonas sp., Bru-cella melitensis) bacterias. And antifungal screenings have beentested against two fungi (C. albicans and Saccharomyces cere-visiae).

The test solutions have been prepared in DMSO. The re-sults of the biological activities are summarized in Figure 5.The synthesized compounds have been found to have re-markable bactericidal and fungicidal properties. Surprisingly,[Ru(OFL)2ClH2O]2CI.4H2O complexes show excellent activ-ity against all type of bacteria and fungi.

Such an increased activity of the metal chelates as comparedto the OFL can be explained on the basis of chelation theory.[57]

Chelation considerably reduces the polarity of the metal ionbecause of the partial sharing of its positive charge with thedonor groups and possible p-electron delocalization over thechelate ring. Such chelation increases the lipophilic character ofthe central metal ion, which subsequently favors the permeationthrough the lipid layer of cell membrane. It is likely that theincreased liposolubility of the ligand up on metal chelation maycontribute to its facile transport into the bacterial cell whichblocks the metal-binding sides in enzyme of microorganism.[58]

FIG. 3. Thermal curves of [Fe(OFL)2Cl]2CI.5H2O complex.

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FIG. 4. Thermal curves of [Fe(OFL)2Cl]2CI.5H2O complex.

Electrochemical StudiesAccording to our research of the literature, the polarographic

and voltammetric behavior of OFL has been studied by variouselectrochemical methods by Zhou and Pan.[59] Their experi-mental results prove that the reduction of OFL is irreversibleand that the peak has adsorption characteristics. In our studies,all complexes were subjected to a cyclic voltammetric study to

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FIG. 5. Biological activity diagram of OFL and its metal complexes.1: C. albicans, 2: Saccharomyces cerevisiae, 3: E. coli, 4: Enterobactercloaca, 5: B. megaterium, 6: B. cereus, 7: Pseudomonas sp., 8: Brucellamelitensis, 9: Staphylococcus aureus. Blue: OFL, red: [Cu(OFL)Cl2].H2O,pale blue: [Pt(OFL)CI2].3H2O, orange: [Zn(OFL)2]2CI.4H2O, green:[Ru(OFL)2ClH2O]2CI.4H2O, and purple: [Fe(OFL)2Cl]2CI.5H2O.

characterize their electrochemical behavior on the glassy carbonelectrode (GCE) compared to metal. Electrochemical behaviorswere studied over a wide pH range (2.0–12.0) with a glassycarbon disc electrode in buffered aqueous media. For example,cyclic voltammetric behavior of Cu(II) complex yielded twowell-defined anodic peaks and one ill-defined cathodic peak al-most at all pH values depending on the pH for GCE (Figure 6).The scanning was started at −1.0 V in the positive directionat pH 2.0 phosphate buffer; anodic oxidation of Cu(II) com-plex did not occur until about −0.29 V for pH 2 (−0.19 Vfor pH 4, −0.13 V for pH 6, −0.11 V for pH 7, −0.07 Vfor pH 9). By reversing at high potentials, oxidation peak cor-responding to the second anodic peaks were observed on theanodic scan for GCE. Typical cyclic voltammograms of 1 ×10−4 M Cu(II) complex at between pH 2 and 9 have been givenin Figure 6a–e. As shown in these voltammograms, the com-plex Cu(II) shows one irreversible and one quasireversible ox-idation peaks on the positive side against Ag+/AgCl, and onequasireversible reduction wave (�E = 183 mV for pH 2, �E= 58 mV for pH 4, �E = 11 mV for pH 6, and �E = 32 mVfor pH 7) on the negative side. The redox process is assigned toCu(II)/Cu(III) and Cu(III)/Cu(IV) oxidations.[60] In the reversescan, Cu(IV)/Cu(III) was observed in the cathode sweep. Onthe other hand, the Cu(II) complex have one irreversible andone quasireversible oxidation and one reduction waves in the1.3–0.07 V range. The irreversibility/quasireversible nature ofthe redox processes in the Cu(II) complex is attributed to thechanges in the coordination geometry or coordination numberupon change of the oxidation state or even to the expulsion ofmetal ions from the coordination sphere.

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FIG. 6. Cyclic voltammograms of 1 × 10−4 M [Cu(OFL)Cl2].H2O at (a) pH 2; (b) pH 4; (c) pH 6; (d) pH 7; (e) pH 9 phosphate buffer. Scan rate 100 mVs−1.

Plots of pH versus Ep and Ip have been investigated using CVtechniques. The pH of the supporting electrolyte has a significanteffect on the electrooxidation/electroreduction of the complexesat the GCE. CV of solid synthesized complexes exhibited oneor two well-defined peaks, and the peaks became sharper inphosphate buffer at pH 2 for Cu(II), Fe(III), or Ru(III) and pH4 for Pt(II) or Zn(II) complexes. The peak potential Ep at the

redox process moved to less positive potential values by raisingthe pH (Figure 7). The plot of the peak potential versus pHshowed one straight line between pH 2.0 and 12.0, which canbe expressed by the following equations in phosphate buffer:

Ep(mV) = 1162.1 − 33.745pH; r :0.9583

for [Cu(OFL)Cl2] H2O

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METAL COMPLEXES OF OFLOXACIN 1517

FIG. 7. Effect of pH on (a) [Pt(OFL)CI2].3H2O, (b) [Cu(OFL)Cl2].H2O, (c) [Fe(OFL)2Cl]2CI.5H2O, and (d) [Ru(OFL)2ClH2O]2CI.4H2O complexes anodicpeak potential; 0.2 M phosphate buffer.

Ep (mV) = 1223.8 − 40.045pH; r :0.9758

for [Pt(OFL)CI2].3H2O

Ep(mV) = 1180.5 − 38.39pH; r :0.9643

for [Zn(OFL)2]2CI.4H2O

Ep (mV) = 1229 − 43.864pH; r :0.9788

for [Ru(OFL)2ClH2O]2CI.4H2O

Ep (mV) = 1205.8 − 36.662 pH; r :0.9829

for [Fe(OFL)2Cl]2CI.5H2O

Scan rate studies were carried out to investigate whether theprocess at the GCE was under diffusion or adsorption control.The effects of the potential scan rate between 5 and 1000 mVs−1

on the peak current and potential of all complexes were eval-uated in pH 2 or 4 phosphate buffer. When the scan rate wasvaried from 5 to 1000 mVs−1 in 1 × 10−5 mol L−1 complexsolutions, a linear dependence of the peak current Ip (μA) uponthe square root of the scan rate v1/2 (mVs−1) was found by GCEdemonstrating diffusional behavior (Figure 8). The equationsare noted below in pH 2.0 or 4.0 phosphate buffer (n = 10 in allstudies).

ip(μA) = 1.4185ν1/2(mVs−1) − 0.919

× (r : 0.9981; n : 10)for [Cu(OFL)Cl2].H2O

ip(μA) = 0.6479v1/2(mVs−1) − 2.0679 (r : 0.9923; n : 10) for

[Pt(OFL)CI2].3H2O

ip(μA) = 0.6270v1/2(mVs−1) − 1.0673 (r : 0.9980; n : 10)

for[Zn(OFL)2]2CI.4H2O

ip(μA) = 1.9016v1/2(mVs−1) − 4.1062 (r : 0.9986; n : 10)

for [Ru(OFL)2ClH2O]2CI.4H2O

ip(μA) = 1.6973v1/2(mVs−1) + 1.5869 (r : 0.9908; n : 10)

for [Fe(OFL)2Cl]2CI.5H2O

The effect of scan rate on peak current was also examined un-der the above conditions with a plot of logarithm of peak currentversus logarithm of scan rate giving a straight line within thesame scan rate range. These linear relationships were obtainedas followed (n = 10 in all studies):

logi(μA) = 0.5471 log v (mVs−1)

+ 0.015 (r : 0.9991; n : 10) for

[Cu(OFL)Cl2].H2O

logi(μA) = 0.7043 log v (mVs−1)

−0.8035 (r : 0.9931; n : 10)

for [Pt(OFL)CI2].3H2O

logi(μA) = 0.6117 log v(mVs−1)

−0.6043 (r : 0.9917; n : 10)

for [Zn(OFL)2]2CI.4H2O

logi(μA) = 0.6872 log v (mVs−1)

−0.233 (r : 0.9943; n : 10)

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FIG. 8. The ip-v1/2 curves for [Ru(OFL)2ClH2O]2CI.4H2O (a) and [Pt(OFL)CI2].3H2O (c); the log ip-log v curves for [Ru(OFL)2ClH2O]2CI.4H2O (b) and[Pt(OFL)CI2].3H2O (d).

for [Ru(OFL)2ClH2O]2CI.4H2O logi(μA)

= 0.4973 log v (mVs−1)

+ 0.2936 (r : 0.9937; n : 10)

for [Fe(OFL)2Cl]2CI.5H2O

The slopes (between 0.49 and 0.70) of the relationship areclose to the theoretically expected (0.5) for an ideal reactionof solution species, so in this case the process had a diffusivecomponent.[13, 50, 51, 54]

DNA Binding StudiesElectronic absorption spectroscopy is an effective method to

examine the binding mode of DNA with metal complexes.[31]

The mutual effect of the complexes with CT DNA has beenstudied with UV spectroscopy in order to investigate the pos-sible binding modes to CT DNA and to calculate the bindingconstants to CT DNA (Kb). In UV titration experiments, thespectra of CT DNA in the presence of each complexes (or OFL)have been recorded for a constant CT DNA concentration indiverse [complex (or OFL)]/[CT DNA] mixing ratios (r). Theintrinsic binding constants, Kb, of the complexes (or OFL) withCT DNA have been determined through the UV spectra of thecomplexes recorded for a constant complex (or OFL) DNAconcentration (1 × 10−5 M) in the absence and presence of CTDNA for diverse r values. The transition metal complexes areknown to bind to DNA via both covalent and/or non-covalentinteractions.[31, 33, 61, 62] In covalent binding, the labile ligand of

the complexes is replaced by a nitrogen base of DNA such asguanine N7. Moreover, the non-covalent DNA interactions in-clude intercalative, electrostatic, and groove (surface) bindingof metal complexes along outside of DNA helix, along majoror minor groove. It has been reported that DNA can providethree distinctive binding sites for all metal complexes; namely,groove binding, electrostatic binding to phosphate group, andintercalation.[63] Figure 9 illustrates that the spectral changes oc-curred in 1 × 10−5 M methanolic solution of [Cu(OFL)Cl2]H2Oupon addition of increasing amounts of CT DNA. Even thoughno appreciable change in the position of the intraligand band ofmetal complexes has been observed by addition of CT DNA, theintensity of the band centered at 258 nm for [Cu(OFL)Cl2]H2Ocomplex has been increased in the presence of DNA up to r = 7,and a blue shift of 6 nm has been observed for higher amountsof DNA.

The hypsochromic effect that has been observed might beascribed to external contact (electrostatic binding)[31, 33] orthat all complexes could uncoil the helix structure of DNAand made more bases embedding in DNA exposed.[62, 63] Theintrinsic binding constant Kb of [Cu(OFL)Cl2].H2O, [Pt(OFL)CI2].3H2O, [Zn(OFL)2]2CI.4H2O, [Ru(OFL)2ClH2O]2CI.4H2O, and [Fe(OFL)2Cl]2CI.5H2O complexes with CTDNA represents the binding constant per DNA base pair, canbe obtained by monitoring the changes in absorbances between264 and 242 nm with increasing concentrations of CT DNAfrom plots [DNA] / εa − εf versus [DNA] and is given by theratio of slope to the y intercept, according to the following

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METAL COMPLEXES OF OFLOXACIN 1519

FIG. 9. UV spectra of [Cu(OFL)Cl2](H2O) in buffer solution in the presenceof CT DNA at increasing amounts. [Cu(OFL)Cl2](H2O) = 1 × 10−5 M. Thearrow shows the intensity changes upon increasing concentration of CT DNA.

equation[63];

[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb(εa − εf ),

where εa = Aobsd/[complex], εa = extinction coefficient for thefree complex, and εb = extinction coefficient for [Cu(OFL)Cl2].H2O, [Pt(OFL)CI2].3H2O, [Zn(OFL)2]2CI.4H2O, [Ru(OFL)2ClH2O]2CI.4H2O, and [Fe(OFL)2Cl]2CI.5H2O in thefully bound form. The high value of Kb obtained for [Cu(OFL)Cl2].H2O, [Fe(OFL)2Cl]2CI.5H2O, [Ru(OFL)2ClH2O]2CI.4H2O, [Pt(OFL)CI2].3H2O, and [Zn(OFL)2]2CI.4H2O, re-spectively, suggest a strong binding of complexes to CTDNA (Table 3). Indeed, it is much higher than Kb calcu-

FIG. 10. UV spectra of (a) 4 ppm EB, (b): a + 15 ppm DNA, (c): b + 2 ppm[Cu(OFL)Cl2](H2O), (d): b + 15 ppm [Cu(OFL)Cl2](H2O).

lated for OFL (=1.17 ± 0.02 × 104 M−1), indicating thatthe coordination of OFL ligand to M(II)/(III) ion enhancesignificantly the ability to bind to CT DNA. This is an im-portant point Kb of [Cu(OFL)Cl2].H2O, [Pt(OFL)CI2].3H2O,[Zn(OFL)2]2CI.4H2O, [Ru(OFL)2ClH2O]2CI.4H2O and[Fe(OFL)2Cl]2CI.5H2O is higher than the EB binding affinityfor DNA (Figure 10) suggesting that electrostatic and intercala-tive interaction may affect EB displacement.[64] Also, the Kb

value of free OFL has been found 1.17 × 104. This value is toosmaller than the complexes Kb values. Thi result clearly showsthe importance of complexation.

CONCLUSIONOFL complexes with Cu(II), Pt(II), Zn(II), Fe(III), and

Ru(III) have been synthesized and characterized by physico-chemical and spectroscopic methods. OFL is bidentate, boundto metal ion carbonyl and carboxylic acid oxygens. In the cyclicvoltammograms of the complexes recorded in acetonitrile/water(1/1, v/v) solution quasireversible waves attributed to redox cou-ples, characteristics for each metal complex, have been recordedat expected potentials. The study of the interactions with CTDNA has been performed with UV spectroscopy, revealing thatthe complexes bind to DNA. [Cu(OFL)Cl2].H2O exhibits muchhigher intrinsic binding constant to CT DNA than the othercomplexes. The results show that changing the metal environ-ment can modulate the binding of the complex with DNA.[65]

DNA adducts of [Pt(OFL)CI2].3H2O could lead to a broaderspectrum of antibacterial activity. Cyclic voltammetric studiesshow that all complexes bind to CT DNA by both intercalationand electrostatic interaction. Antibacterial properties of OFLcomplexes with different metal ions tested for activity againstdiverse microorganisms show antimicrobial activity compara-ble to free fluoroquinolones. In certain examples, the activitieswere increased, for example, norfloxacin complexes with zinc,iron, and silver; the magnesium complex with ciprofloxacinis characterized by a slight decrease in antibacterial activity.Vanadium-ciprofloxacin complex is promising with respect toits insulin-mimetic behavior and its concomitant low toxicity inthe physiological concentration range.[65] According to our bio-logical results, all complexes exhibited higher inhibitory activitythan OFL.

FUNDINGFinancial support for this study was received from the

TUBITAK under the COST action of D39 through project109T020.

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