Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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

New water-soluble copper (II) complexes including4,7-dimethyl-1,10-phenanthroline and L-tyrosine: Synthesis,characterization, DNA interactions and cytotoxicities q

http://dx.doi.org/10.1016/j.saa.2014.09.0931386-1425/� 2014 Elsevier B.V. All rights reserved.

q This study is a part of a MSc thesis of the first author accepted on 16.01.2013 bythe Graduate School of Natural and Applied Sciences of Uludag University.⇑ Corresponding author. Tel.: +90 224 2941729; fax: +90 224 2941898.

E-mail address: rahmiye@uludag.edu.tr (R. Aydın).

Duygu _Inci a, Rahmiye Aydın a,⇑, Dilek Yılmaz b, Hasene Mutlu Gençkal a, Özgür Vatan b, Nilüfer Çinkılıç b,Yunus Zorlu c

a Department of Chemistry, Faculty of Arts and Sciences, Uludag University, 16059 Bursa, Turkeyb Department of Biology, Faculty of Arts and Sciences, Uludag University, 16059 Bursa, Turkeyc Department of Chemistry, Faculty of Science, Gebze Institute of Technology, 41400 Kocaeli, Turkey

h i g h l i g h t s

�Water soluble new copper(II)complexes and the diquarternary saltof dmphen have been synthesizedand characterized.� Single crystals X-ray study confirms

the structure of the copper(II)complexes.� DNA binding studies reveal that these

compounds bind to CT-DNA viaintercalation.� The copper(II) complexes efficiently

cleavage the super-coiled pBR322plasmid DNA.� XTT assay of the copper(II) complexes

show prominent cytotoxic activityagainst the selected human tumorcell lines.

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

Binary and ternary Cu(II) complexes (1–2) containing 4,7-dimethyl-1,10-phenanthroline (dmphen) andL-tyrosine (tyr) and the diquarternary salt of dmphen (dq-dmphen) have been synthesized and character-ized by elemental analysis, 1H NMR, 13C NMR and IR spectroscopy, thermal analysis and single crystalX-ray diffraction techniques. They have been tested for their in vitro DNA binding activities by the spec-troscopic methods. Binding studies of CT DNA with these complexes and dq-dmphen show 49–62% ofhypochromicity and a minor red-shift (�2 nm) in the charge transfer band. The apparent binding constant(logKapp) and Stern–Volmer quenching constant (logKsv) values of these complexes and dq-dmphen wereobtained from fluorescence measurements. The results showed that the magnitudes of the calculatedlogKapp and logKsv values of all of the compounds were in the order complex 1 > complex 2 > dq-dmphen.The DNA denaturation experiment shows a minor shift in the melting temperature (Tm). The cleavage activ-ity of super-coiled pBR322 plasmid DNA under aerobic condition reveals moderate activity with complex 1and complex 2 while dq-dmphen shows no activity. Complexes 1 and 2 exhibit high cytotoxicity with lowIC50 values against selected human tumor cell lines (Caco-2, A549 and MCF-7) and healthy cells (BEAS-2B).Also, the dq-dmphen and Cu2+ solutions exhibit much less activity against these cancer cells than doescomplexes 1 and 2 and cisplatin.

762 D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770

a r t i c l e i n f o

Article history:Received 28 May 2014Received in revised form 18 July 2014Accepted 22 September 2014Available online 30 September 2014

Keywords:Copper(II)4,7-Dimethyl-1,10-phenanthrolineL-tyrosineSingle crystal X-ray diffractionDNA binding and cleavageCytotoxicity

a b s t r a c t

Two new water-soluble copper(II) complexes, [Cu(dmphen)2(NO3)]NO3 (1), [Cu(dmphen)(tyr)(H2O)]NO3�H2O (2) and the diquarternary salt of dmphen (dmphen = 4,7-dimethyl-1,10-phenanthroline andtyr = L-tyrosine), have been synthesized and characterized by elemental analysis, 1H NMR, 13C NMRand IR spectroscopy, thermal analysis and single crystal X-ray diffraction techniques. The CT-DNA bind-ing properties of these compounds have been investigated by absorption, emission spectroscopy andthermal denaturation measurements. The supercoiled pBR322 plasmid DNA cleavage activity of thesecompounds has been explored by agarose gel electrophoresis. The cytotoxicity of these compoundsagainst MCF-7, Caco-2, A549 cancer cells and BEAS-2B healthy cells was also studied by the XTT method.Complexes 1 and 2 exhibit significant cytotoxicity, with lower IC50 values than those of cisplatin.

� 2014 Elsevier B.V. All rights reserved.

N N

CH3H3CCH2 CH C

NH2

O

OHHO

4,7-dimethyl-1,10-phenanthroline

(dmphen)

L-tyrosine

(tyr)

CH3H3C

+ N N

+

dq-dmphen

Fig. 1. Structures of the ligands used in this study and the diquarternary salt ofdmphen (dq-dmphen).

Introduction

DNA plays an important role in the life process because it con-tains all genetic information required for cellular function [1]. Thebinding of metal complexes with DNA base pairs has been a majorfocus in the study of bioinorganic chemistry [2]. These metalcomplexes are known to bind to DNA via both covalent and non-covalent interactions. In covalent binding, the labile ligand of thecomplexes is replaced by a nitrogenous base of DNA such as guan-ine N7. However, non-covalent DNA interactions include intercala-tive, electrostatic and groove (surface) binding of cationic metalcomplexes along the outside of the DNA helix, along the major orminor groove [3–5].

Copper(II) is known as to have a significant role in biologicalsystems and pharmacological agents. Because of its biologicalrelevance, a large number of copper(II) complexes have been syn-thesized and explored for their biological activities [6–8]. Amongthese copper complexes, attention has been mainly focused onthe copper(II) complexes of 1,10-phenanthroline (phen) due totheir high nucleolytic efficiency, anti-tumor, anticandidal, andantimicrobial activities [9–11]. Many metal complexes have beensynthesized using various modified phen ligands with the purposeof enhancing their interaction with DNA [12,13], and their DNAcleavage activity has also been investigated [14–19]. However,most studies have focused primarily on metal complexes contain-ing fully planar ligands, while metal complexes containing substi-tuted ligands such as methyl groups have rarely been reported[20]. In fact, some of these complexes also exhibit very interestingproperties upon binding to DNA [21–23]. A variation in the natureand position of the substituents at the binding site of the ligand cancreate some interesting differences in the space configuration andelectron density distribution of the metal complexes, resulting indifferences in spectral profiles, DNA binding properties, enantiose-lectivities, and even DNA cleavage activities [15]. Studies of suchdifferences can be very useful to thoroughly understand the bind-ing mechanism of metal complexes to DNA [20].

Amino acids have been widely used in the production of agro-chemicals, racemic drugs, fragrances and pharmaceuticals [24].Furthermore, cancer chemotherapy has also involved transitionmetal complexes with amino acids as subsidiary ligands [25].L-tyrosine (Fig. 1) has an important role in the synthesis of neuro-transmitters, dopamine and noradrenaline, as well as the thyroxinthyroid hormone [26]. L-tyrosine forms coordination compoundswith metal ions using carboxylate, amino and/or hydroxyl groups.However, the most popular coordination mode is a chelating oneinvolving ACOO� and ANH2 groups [27]. Copper complexes con-taining amino acids have been studied as models for the behaviorof copper enzymes [28], and some copper complexes with aminoacid ligands have been reported to exhibit potent antitumor andartificial nuclease activities [29–31].

Although interaction of copper complexes of some 1,10-phenanthroline derivatives as primary ligands and some amino acidsas a secondary ligands with DNA has been studied [12–20], inter-action of copper complexes of 4,7-dimethyl-1,10-phenanthrolineand L-tyrosine with DNA has not been shown yet. We previouslyinvestigated the stability constants of copper(II) complexes withsubstituted 1,10-phenanthroline groups and L-amino acids inaqueous solution [32]. Therefore, in this study, we synthesizedand characterized two water-soluble Cu(II) complexes that includedmphen (Fig. 1) and tyr, [Cu(dmphen)2(NO3)]NO3 (1) and [Cu(dmphen)(tyr)(H2O)]NO3�H2O (2), and the diquarternary salt ofdmphen (dq-dmphen). The CT-DNA binding properties of thesecompounds were investigated by UV–vis, fluorescence and thermaldenaturation experiments. Their cleavage behavior toward pBR322plasmid DNA and the cytotoxicities of these compounds againstMCF-7 (breast adenocarcinoma), Caco-2 (colon adenocarcinoma),A549 (lung adenocarcinoma) and BEAS-2B (bronchial epithelium)cell lines were obtained.

Experimental

Materials

All reagents were obtained from commercial sources and usedas received. Copper(II) nitrate trihydrate and 4,7-dimethyl-1,10-phenanthroline were purchased from Merck and Alfa-Aesar,respectively. L-tyrosine, methanol, KOH, NaCl, tris-(hydroxy-methyl)aminomethane-HCl, CT-DNA, agarose (molecular biologygrade), ethidium bromide, bromophenol blue, xylene cyanol,

D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770 763

RPMI-1640, fetal calf serum, PBS and penicillin–streptomycin wereprovided from Sigma–Aldrich. Tris Borate-EDTA buffer solution(TBE, 10X) were purchased from Sigma. All solutions were pre-pared with analytical grade water (R = 18 MX) using grade Aglassware.

XTT cell proliferation assays were obtained from BiologicalIndustries, Israel. H2O2 was used as a positive control at a singleconcentration of 100 lM. In cytotoxicity studies, sterile distilledwater was used as a solvent control at a maximum concentrationof 0.5% (v/v). Cisplatin was purchased from Santa Cruz Biotechnol-ogy, Santa Cruz, CA, USA. The pBR322 DNA was obtained fromBiolabs, UK.

Instrumentation

Elemental analysis. C, H, and N elemental analyses were carriedout at the Technical and Scientific Research Council of Turkey,TUBITAK Bursa Test, and Analysis Laboratory.FTIR spectroscopy. Infrared spectra were recorded in the4000–400 cm�1 region with a Thermo-Nicolet 6700 Fourier-Transform Infrared Spectrometer by using KBr pellets.NMR. The 1H NMR and 13C NMR solution spectra were recordedat 25 �C on a Varian Mercury Plus 400 MHz spectrometer, utiliz-ing deuterated dimethylsulfoxide (DMSO-d6) as a solvent.Thermal analysis. Thermal analysis curves (TGA and DTA) wereobtained using a Seiko Exstar 6200 thermal analyzer in dry airat a heating rate of 10 �C min�1 from 25 to 1000 �C using plati-num crucibles.UV�vis spectroscopy. Spectrophotometric studies were per-formed using a GBC Cintra 303 UV–Visible spectrophotometerconnected with a Peltier thermocell.Fluorescence spectroscopy. Emission intensity measurementsused a Jasco FP-750 spectrofluorometer.Gel electrophoresis. DNA bands were visualized under UV light,and photographs were taken.Cytotoxicity. Absorbance measurements used an ELx800 Micro-plate Reader (Bio-Tek, Winooski, VT, USA).

Synthesis of dq-dmphen

4,7-Dimethyl-1,10-phenanthroline (0.104 g, 0.5 mmol) and1,2-dibromoethane (2.5 mL) were gently refluxed for 5 h. Aftercooling, the precipitate was collected by filtration and washed withcold EtOH and Et2O. The solid that remained was dissolved inwater (50 mL), and filtered to remove any remaining solid. A 3:1molar equivalent of ammonium hexafluorophosphate was addedto the aqueous filtrate, and the precipitate was collected, washedwith cold EtOH and finally dried in air [33]. Yield: 0.156 g, 59%(grey solid). Anal. calcd. for C16H16N2P2F12 (526.24 gmol–1) (%): C,36.52; H, 3.06; N, 5.32. Found: C, 37.73; H, 2.86; N, 5.33. FT-IR(KBr pellet, m cm�1): 3124–2848 (CAHaromatic and CAHaliphatic);835 (PF6

�). 1H NMR (DMSO-d6, d ppm J Hz): 9.58 (d, J = 6.0, Haromatic,2H); 8.81 (s, Haromatic, 2H); 8.54 (dd, J = 6.0; 0.8, Haromatic, 2H); 5.45(s, ACH2A, 4H); 3.18(s, ACH3, 6H). 13C NMR (DMSO-d6, d ppm):159.74 (AC@NA); 148.38, 130.35, 129.10, 127.79, 126.32(Caromatic); 52.21 (ACH2A); 20.64 (ACH3).

Synthesis of complex 1

A 0.24 g (1.0 mmol) quantity of copper(II) nitrate trihydrate wastreated with 2.0 mmol of dmphen in methanol (60 ml) understirring conditions for 0.5 h at 25 �C. The product was isolated,washed with ethanol and set aside for crystallization by slow evap-oration at room temperature. Green crystals were crystallized fromthe solution after a week. The yield was 85%. Anal. calcd. forC28H24CuN6O6 (604.07 gmol–1) (%): C, 55.67; H, 4.00; N, 13.91.

Found: C, 56.58; H, 4.08; N, 13.91%. IR (KBr phase): 3427br,3088br, 1581 m, 1384 m (NO3

�), 851 m, 724 m, 539w cm�1 (br,broad; w, weak; m, medium, s, strong).

Synthesis of complex 2

Complex was prepared by a general synthetic method in whichan aqueous solution of Cu(NO3)2�3H2O (1.0 mmol) was initiallyreacted with an aqueous solution of L-tyrosine (1.0 mmol) treatedwith NaOH (1.0 mmol), followed by slow addition of a methanolsolution of the heterocyclic base (1.0 mmol dmphen). The reactionmixture was stirred at 60 �C for 2 h and filtered. The resulting solu-tion was filtered and set aside for crystallization by slow evapora-tion at room temperature. The blue crystalline solid separated outwas collected by suction filtration, washed with small amounts ofcold methanol, and dried in vacuum. Slow evaporation of thesolvent gave a crystalline product in 81% yield. Anal. calcd. forC23H26CuN4O8 (550.02 gmol�1) (%): C, 49.73; H, 4.29; N, 10.18.Found: C, 50.22; H, 4.03; N, 10.19%. IR (KBr phase): 3480br,3220br, 1608s, 1456 m, 1381 m (NO3

�), 856 m, 724 m, 535w,420w cm�1 (br, broad; w, weak; m, medium, s, strong).

X-ray data collection and structure refinement

Single crystal data for complexes 1 and 2 were collected at120(2) K on a Bruker APEX II QUAZAR three-circle diffractometerwith monochromatized Mo Ka radiation (k = 0.71073 Å) using a uand x technique. Indexing was performed using APEX2 [34]. Dataintegration and reduction were accomplished with SAINT [35]. Anabsorption correction was performed by a multi-scan methodimplemented in SADABS [36]. The structures were solved usingthe direct-methods procedure in SHELXS-97 [37] and then refinedby full-matrix least-squares refinements on F2 using the SHELXL-97[37]. All non-hydrogen atoms were refined anisotropically usingall reflections with I > 2r(I). In complexes 1 and 2, aromatic and ali-phatic C-bound H atoms were positioned geometrically and refinedusing a riding mode. In complex 2, the water and aromatic hydroxylH atom coordinates were located in a difference Fourier map; theOAH distances were restrained to be 0.84 Å from O atom usingDFIX, and their positions were constrained to refine on their parentO atoms with Uiso(H) = 1.5Ueq(O). N-bound H atoms were locatedfrom the difference Fourier map and restrained to be 0.89 Å fromthe N atom using DFIX, and their positions were constrained torefine on their parent N atoms with Uiso(H) = 1.2Ueq(O). C16A–C23A and C16B–C23B atoms of the tyrosine moiety in complex 2are disordered over two sites with occupancies of 0.50:0.50. Forcomplex 2, a check using PLATON-ADSYM on the P1 refinementresulted in an achiral space group P � 1. Although a chiral aminoacid (L-tyrosine) has been used as a starting material, complex 2has undergone racemization under reaction conditions. It shouldbe noted that similar racemization phenomena have been reportedpreviously [38,39]. Crystallographic data and refinement details ofthe data collection for complexes 1 and 2 are given in Table 1.The selected bond lengths and bond angles for complexes 1 and 2are given in Table 2. The final geometrical calculations and molec-ular drawings were carried out with the PLATON [40] and MER-CURY programs [41].

DNA binding experiments

Absorption spectra measurementsThe absorption spectral titration experiments were performed

in Tris–HCl buffer (5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2)by maintaining a constant Cu(II) complex concentration of 25 lMthroughout the experiment. The nucleotide (CT-DNA) concentra-tion was varied between 0 and 200 lM, keeping the total volume

Table 1Crystal data and structure refinement for complexes 1 and 2.

1 2

CCDC 914900 914899Empirical formula C28H24CuN6O6 C23H26CuN4O8

Formula weight (g mol�1) 604.07 550.02Temperature (K) 120(2) 120(2)Wavelength (Å) 0.71073 0.71073Crystal system Triclinic TriclinicSpace group P – 1 P � 1a (Å) 7.9667(2) 7.0084(2)b (Å) 10.8253(3) 10.8038(3)c (Å) 16.2247(4) 15.7613(4)a (�) 71.190(2) 103.3510(10)b (�) 86.528(2) 96.8410(10)c (�) 74.385(2) 92.4440(10)Crystal size (mm) 0.04 � 0.09 � 0.20 0.03 � 0.05 � 0.12V (Å3) 1275.11(6) 1149.84(5)Z 2 2qcalcd (mg.m�3) 1.573 1.589l (mm�1) 0.914 1.009F (000) 622 570h range for data collection (�) 1.33–26.73 1.34–27.50h/k/l �10/10, �13/13,

�20/20�9/9, �14/14, �20/20

Reflections collected 20924 39914Independent reflections 5412 5264Absorption correction Multi-scan Multi-scanTmin and Tmax 0.84 and 0.97 0.89 and 0.97Data/restraints/parameters 5412/0/374 5264/19/421Goodness-of-fit on F 2 (S) 1.022 1.302Final R indices [I > 2r(I)]a R1 = 0.0489,

wR2 = 0.1224R1 = 0.0520,wR2 = 0.1247

R indices (all data) R1 = 0.0676,wR2 = 0.1339

R1 = 0.0577,wR2 = 0.1267

Largest diff. peak and hole(e �3)

1.224 and �0.333 0.493 and �0.511

a R1 = R||Fo| � |Fc||/R|Fo|; wR2 = {R[w(Fo2 � Fc

2)2]/R[w(Fo2)2]}1/2.

764 D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770

of the reaction mixture constant (4.0 mL). After each addition ofCT-DNA to the Cu(II) complex, the resulting solution was allowedto equilibrate at 25 �C for 2 min before recording absorption pat-terns. Binding constants (Kb) were calculated from the spectro-scopic titration data using the following equation [42]:

½DNA�=ðea � efÞ ¼ ½DNA�=ðeb � ef Þ þ 1=Kbðeb � efÞ ð1Þ

where [DNA] is the concentration of DNA in terms of base-pairs, ea

is the apparent extinction coefficient obtained by calculatingAobs/[complex], ef corresponds to the extinction coefficient of thecomplex in its free form and eb refers to the extinction coefficientof the complex in its bound form. Each set of data, when fitted tothe above equation, yielded a straight line with a slope of 1/(eb � ef)and a y-intercept of 1/Kb(eb � ef). Finally, Kb was determined fromthe ratio of the slope to intercept.

Fluorescence spectra measurementsRelative binding of the complexes to CT-DNA was studied

by the fluorescence spectral method using ethidium bromide(EB)-bound CT-DNA solution in Tris–HCl buffer (5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2). For fluorescence experiments,DNA was pretreated with EB. Both the copper(II) complexes anddq-dmphen were then added to this mixture, and their effect onthe emission intensity at approximately 595–605 nm was mea-sured. The fluorescence intensity was measured after each additionuntil a 50% reduction in intensity had occurred. The apparent bind-ing constant (Kapp) [43] was calculated from the followingequation:

KEB � ½EB� ¼ Kapp � ½complex� ð2Þ

where [complex] is the concentration of the ternary complex at 50%reduction of fluorescence intensity (KEB = 1.0 � 107 M�1,

[EB] = 1.3 lM) [44]. The quenching plots followed the Stern–Volmerrelationship of the form [45].

Io=I ¼ 1þ Ksv � ½complex� ð3Þwhere Io and I are the fluorescence intensities of the excitedCT-DNA-EB in the absence and presence of the complexes, respec-tively. Ksv is the Stern–Volmer quenching constant, and [complex]is the concentration of the quencher.

Thermal denaturationDNA thermal denaturation experiments were carried out in

Tris–HCl buffer solution (pH 7.2) using CT-DNA (120 lM) by vary-ing the temperature from 20 to 100 �C, both in the absence andpresence of the complexes and dq-dmphen (30 lM). These studieswere performed using a GBC Cintra 303 UV–visible spectropho-tometer connected with a Peltier thermocell.

DNA cleavage experiments

The efficiency of DNA cleavage was measured by determiningthe ability of the complexes to form open circular (OC) and nickedcircular (NC) DNA from its super-coiled (SC) form using agarose gelelectrophoresis. In reactions using super-coiled pBR322 plasmidDNA (300 ng), Tris–HCl buffer (50 mM) was diluted with 50 mMNaCl (pH 7.2). pBR322 plasmid DNA was treated with differentdose-complexes – (40, 80, 160, 320, 640, 1280, 2560, 5120,10240 lM) – and diluted with the Tris–HCl buffer. Samples wereincubated for 3 h at 37 �C. After incubation, gel-loading bufferwas added, and electrophoresis was performed at 40 V for 1 h indiluted Tris-Boric Acid-EDTA (diluted TBE, 1X)) buffer using 1% aga-rose gel containing 1.0 lg/mL ethidium bromide. The gel was visu-alized by photographing the fluorescence of intercalated ethidiumbromide under a UV illuminator. The cleavage efficiency wasmeasured by determining the ability of the complex to convertsuper-coiled (SC) DNA to its nicked circular (NC) and linear (LC)forms.

Cytotoxicity assays (XTT)

The cytotoxicities of the two copper(II) complexes, dq-dmphenand cisplatin were evaluated against 3 cancer cell lines, includingA549, MCF-7, Caco-2 and one healthy cell line BEAS-2B (kindlyprovided by Feray KOCKAR), by using the XTT assay. Cells werecultured in RPMI-1640 medium supplemented with 15% fetal calfserum (FCS), penicillin (100 IU/mL) and streptomycin (100 lg/mL), 10 mM L-glutamine, 10 mM non-essential amino acids andsodium pyruvate. Cells were maintained at 37 �C in a humidifiedatmosphere containing 5% CO2. Cells were grown in 75 cm2 flasksand sub-cultured once a week.

Growing cells were harvested and counted; A549 and BEAS-2Bcells were seeded at 5 � 103 cells/well, and Caco-2 and MCF-7 cellswere seeded at 8 � 103 cells/well, all in sterile 96-well flat-bot-tomed plates in triplicate and incubated overnight at 37 �C in ahumidified atmosphere containing 5% CO2. The plates includedblanks that contained complete medium without cells. The copper(II) complexes were dissolved in sterile distilled water. Cells werethen treated with various concentrations of copper (II) complexesfor 24 h. Under the same experimental conditions, cisplatin was alsoevaluated as a reference metallodrug. Cytotoxicity was measuredusing an XTT Cell Viability Kit. XTT, a tetrazolium salt, is reducedby metabolic active cells to orange formazan dye. After treatmentfor 24 h, wells were washed with PBS, and the medium wasreplaced with fresh medium (100 lL). The activated XTT solution(50 lL) was added to each well according to the manufacturer’sinstructions (Biological Industries). The plate was incubated foran additional 3 h in the CO2 incubator at 37 �C. Absorbance wasread using a Microplate Reader at a wavelength of 450 nm, and a

D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770 765

cell proliferation curve was drawn. After blank subtraction, thepercent growth inhibition of cells was calculated as follows:

ð1� Aexp group=AcontrolÞ � 100 ð4Þ

Table 2Selected bond lengths (Å) and bond angles (�) for complexes 1 and 2.

Complex 1

Bond lengths (Å)Cu1AN1 2.037(2) Cu1AN4 2.129(2)Cu1AN2 1.987(2) Cu1AO1 2.678(2)Cu1AN3 1.998(3) Cu1AO2 2.084(2)

Bond angles (�)N2ACu1AN3 178.42(10) N2ACu1AN1 81.65(10)N3ACu1AN1 98.17(10) N2ACu1AO2 90.36(9)N3ACu1AO2 90.68(9) N1ACu1AO2 144.52(9)N2ACu1AN4 98.41(10) N3ACu1AN4 80.29(10)N1ACu1AN4 118.36(10) O2ACu1AN4 96.95(9)N5AO2ACu1 108.05(17) C1AN1ACu1 130.7(2)O1ACu1AO2 52.43(8) O1ACu1AN3 88.03(9)O1ACu1AN(1) 93.42(9) O1A Cu1AN4 147.26(8)O1ACu1AN2 93.54(9) C12AN1ACu1 112.1(2)C11AN2ACu1 113.9(2) C10AN2ACu1 127.7(2)C15AN3ACu1 126.9(2) C26AN3ACu1 115.1(2)C24AN4ACu1 131.1(2) C25AN4ACu1 111.23(19)

Complex 2

Bond lengths (Å)Cu1AO1 2.252(3) Cu1AN1 2.001(3)Cu1AO2 1.940(3) Cu1AN2 2.010(3)Cu1AN3 1.992(3)

Bond angles (�)O2ACu1AN3 84.95(12) O2ACu1AN1 93.25(12)N3ACu1AN1 176.61(14) O2ACu1AN2 161.56(12)N3ACu1AN2 99.02(13) N1ACu1AN2 81.81(12)O2ACu1AO1 102.64(11) N3ACu1AO1 92.45(12)N1ACu1AO1 90.75(11) N2ACu1AO1 95.21(11)C15AO2ACu1 114.6(2) C1AN1ACu1 128.5(3)C12AN1ACu1 113.2(2) C10AN2AC11 117.3(3)C10AN2ACu1 129.9(3) C11AN2ACu1 112.8(2)C16AAN3ACu1 110.2(5) C16BAN3ACu1 102.8(5)

Results and discussion

Structures of complexes 1 and 2

The molecular structure of complex 1 is shown in Fig. 2.Complex 1 crystallizes in the P� 1 centrosymmetric space group. Theasymmetric unit consists of one Cu(II) atom, two 4,7-dimethyl-1,10-phenanthroline (dmphen) ligands, and one coordinated –and – one uncoordinated NO�3 anion. In complex 1, the Cu(II) atomin [Cu(dmphen)2(NO3)]+ is basically five-coordinated with fourN-donor atoms of two chelating dmphen ligands and one O-atomof the coordinated counter nitrate ion, but a second oxygen atom(O1) from the nitrate anion is at a distance of 2.678(2) Å. The dihe-dral angle between two phenanthroline ligands is 61.02(6)�. Thestructure of complex 1 can be best described as the intermediatebetween a distorted trigonal bipyramidal (the value of the tau-descriptor (s5) parameter introduced by Addison [46] is 1.00) anda cis-distorted octahedron, giving (4 + 1 + 1⁄)-type coordination.It should be noted that the geometry of CuN4O2 in complex 1 is clo-sely comparable to those of previously reported Cu(II) complexes[47,48]. In the CuN4O2 coordination sphere, CuAN bond lengthsrange from 1.987(2) to 2.129(2) Å. The CuAO2 bond length of2.084(2) Å is in the normal range. The NACuAN bond angles rangefrom 80.29(10)� to 178.42(10)�.

The molecular structure of complex 2 is shown in Fig. 3. Theasymmetric unit for complex 2 consists of one Cu(II) atom, one4,7-dimethyl-1,10-phenanthroline ligand (dmphen), one tyrosine(tyr), one uncoordinated nitrate anion, one coordinated and onelattice water molecule. The central Cu(II) atom in complex 2 hasa distorted five-coordinate square-pyramidal geometry, in whichthe value of the tau-descriptor (s5) parameter introduced by Addi-son is 0.25 [46]. Equatorial bond lengths [Cu1AN1 = 2.001(3) Å,Cu1AN2 = 2.010(3) Å, Cu1AN3 = 1.992(3) Å, Cu1AO2 = 1.940(3)Å] and weakly bound axial bond lengths [Cu1AO1 = 2.252(3)]agree well with those reported for corresponding square-pyrami-dal Cu(II) complexes [49–52]. As shown in Fig. 3, complex 2 exhib-its no intramolecular aromatic ring-stacking interactions betweenthe side-chain aromatic ring of tyrosine and the 4,7-dimethyl-1,10-phenanthroline ligand. The aromatic ring position of tyrosineis approximately perpendicular to the coordination plane formedby CuN3O atoms with an angle of 64.61�.

Structure of dq-dmphen

The diquarternary salt of dmphen was isolated using a methodadapted from the synthesis by Phillips et al. [33] and characterizedby 1H NMR, 13C NMR, IR and elemental analysis. The IR spectrumdisplays a broad band in the range 3124–2848 cm�1 which can beassigned to the stretching vibration of CAHaromatic and CAHaliphatic.The very strong broad peak in the �835 cm�1 region suggests dis-crete hexafluorophosphate anions. The 1H NMR and 13C NMR spec-tra of dq-dmphen were measured at room temperature in DMSO-d6

solution. The 1H NMR spectrum of the diquarternary salt of dmphencan be easily compared to those of free ligand, dmphen, in which theprotons on the phenanthroline ring undergo deshielding of about1 ppm due to decreasing electron density of this ring system uponquaternization. In 1H NMR spectrum, the additional singlet formethylene protons at 5.45 ppm prove that quaternization on thenitrogen atoms took place. In the 13C NMR spectrum of dq-dmphen,the AC@NA (phenanthroline ring) of the dmphen ligand occurs atapproximately 159.74 ppm, whereas the chemical shifts for the

phenyl carbons are observed in the range from 126.32 to148.38 ppm. We are expected it to be 16 H in the 1H NMR spectrum,but there are only 8H and also we are expected it to be 16 C in the 13CNMR spectrum, but there are only 8C. These data indicate that thestructure of dq-dmphen is symmetrical. Formula of dq-dmphen isC16H16N2P2F12. The elemental analyses further confirm the struc-tures. The dq-dmphen is soluble in water, besides it is stable inthe solid and solution phases.

Spectral and thermal properties of complexes 1 and 2

Two medium to weak peaks appear in the region of �3300 cm�1

for the N–H stretching of the aminoacidato groups. For complex 2,containing H2O molecules, an OAH stretching peak was observedin the 3500 cm�1 region. These peaks are usually broad and weak,suggesting water of crystallization in complex 2. The strong peaksappearing in the �1600 cm�1 region were assigned to m(COO) butwere interfered by m(C@N) and d(NH) peaks. Tyr is coordinated tothe metal ion as a N,O-bidentate ligand, which is in agreementwith the result obtained by single crystal X-ray diffraction for com-plex 2. The IR spectra of complexes 1 and 2 displayed peaks at 851and 724 cm�1 for complex 1 and 856 and 724 cm�1 for complex 2.These peaks were assigned as coordinated mphen skeletal vibrations.The very strong broad peaks in the �1380 cm�1 region suggest dis-crete nitrate anions, in agreement with the X-ray structure of com-plexes 1 and 2.

The electronic absorption spectrum of the complexes intris–HCl buffer solution presents important absorption bands;those, at 270–280 nm can be attributed to the p–p⁄ transitions ofphen, dmphen and tyr ligands, and the broad weak absorptionband at 609–678 nm is due to the d-d transition of the centralcopper(II) ion.

Fig. 2. Molecular structure of complex 1 with displacement ellipsoids drawn at the 50% probability level. H-atoms are shown as small spheres of arbitrary radii.

Fig. 3. Molecular structure of complex 2, showing only the major orientation of the disordered part. One uncoordinated water molecule was omitted for clarity Displacementellipsoids are drawn at the 50% probability level. H-atoms are shown as small spheres of arbitrary radii.

766 D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770

Thermal decomposition behaviors of complexes 1 and 2 wereinvestigated by thermogravimetric analysis (TG) and differentialthermal analysis (DTA) in air in the temperature range25–1000 �C. The TG curve clearly shows that complex 1 is anhy-drous and begins to decompose at approximately 224 �C. The massloss of 89.3% in the temperature range 224–433 �C corresponds tothe mass loss of both dmphen and nitrate (calcd. 86.4%). Three

exothermic DTA peaks at 269, 357 and 379 �C are associated withthe decomposition of both dmphen and nitrate. The final residuesof complex can be attributed to a mixture of CuO and Cu (obsd.10.8/calcd. 13.2%).

The thermal decomposition of complex 2 has a dehydrationstep that occurs between 62 and 115 �C and corresponds to the lossof all water molecules (obsd. 6.8%/calcd. 6.6%); second and third

Wavelength, nm200 250 300 350 400

A

0,0

0,2

0,4

0,6

0,8

1,0

Complex 1

Fig. 4. UV–Vis absorption spectra of complex 1 upon addition of CT-DNA. [Complex1] = 25 lM, [DNA] = 0–200 lM.

[DNA], 105

[DN

A] /

( ε a

- εf )

, 109

0

2

4

6

8

10

12dq-dmphenComplex 1Complex 2

5 1510 20

Fig. 5. A plot of [DNA]/(ea � ef) vs [DNA] for the titration of DNA to dq-dmphen andcomplexes 1 and 2.

D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770 767

weight losses occur between 217 and 480 �C that are attributed tothe release of one nitrate anion, one dmphen ligand and one tyrligand (obsd. 81.2%/calcd. 77.2%). The final residues can be attrib-uted to a mixture of CuO and Cu (obsd. 16.3%/calcd. 14.5%). TheDTA curves show one endothermic event at 107 �C due to dehydra-tion. Four exothermic DTA peaks at 256, 311, 366 and 442 �C forcomplex 2 are associated with the decomposition of dmphen, tyrand nitrate ligands.

4. DNA binding studies

Absorption spectroscopyThe interaction of dq-dmphen and complexes 1 and 2 with

CT-DNA has been studied with UV spectroscopy to investigatethe possible binding modes of CT-DNA and to calculate the bindingconstants (Kb) to CT-DNA.

The electronic absorption spectra of complex 1 in the absenceand presence of CT-DNA appear in Fig. 4. There exists a band at267 nm for complex 1. With increasing concentrations ofCT-DNA, the absorption bands of complex 1 exhibited significanthypochromism of 61.6%. In addition, the absorption bands of com-plex 2 and dq-dmphen exhibited hypochromism of 52.1% and49.1%, respectively (Table 3).

Complexes 1 and 2 show minor bathochromic shift of the spec-tral band of �2 nm, and dq-dmphen is accompanied by a small redshift of less than 1 nm. From the absorption data, a plot was drawnof [DNA]/(ea � ef) values vs DNA concentrations for the titration ofDNA to dq-dmphen and complexes 1 and 2 using Eq. (1), and Kb

values were calculated (Fig. 5). The logarithms of Kb values (logKb)for dq-dmphen and complexes 1 and 2 were 5.15, 5.25 and 5.19,respectively. These outcomes are listed in Table 3.

Previous reports have suggested that a compound binding toDNA through intercalation usually results in hypochromism andbathochromism due to an intercalation mode involving strong

Table 3Absorption spectral properties of dq-dmphen and complexes 1 and 2 bound to CT-DNA.

Compounds kmax (nm) Red shift Change in absor

dq-dmphen 281 1 HypcComplex 1 267 2 HypcComplex 2 273 2 Hypc

Hypc: hypochromism.

p–p stacking interactions between the aromatic chromophoreand DNA base pairs [53,54]. It seems to be generally accepted thatthe extent of hypochromism in the UV–vis band is consistent withthe strength of intercalative interaction [53,54]. From our absorp-tion spectroscopy results, we can suggest that dq-dmphen andcomplexes 1 and 2 can interact with CT-DNA through this interca-lation mode.

Fluorescence spectroscopyFluorescence quenching measurements have been confirmed to

be effective for monitoring the binding nature of metal complexesto DNA and comparing binding abilities of these complexes. EB isweakly fluorescent in aqueous solution, but its emission intensityin the presence of DNA is greatly enhanced because of its strongintercalation between adjacent DNA base pairs [55–57]. Competi-tive DNA binding of dq-dmphen and complexes 1 and 2 wasexpected to displace the bound EB. As a consequence, the emissionintensity of EB is likely to decrease. The fluorescent emission titra-tion of dq-dmphen and complexes 1 and 2 are illustrated inFig. 6a–c. The apparent binding constant [43] (Kapp) is calculatedusing Eq. (2) and is the concentration of the compound in mediumat 50% reduction of the fluorescence intensity of EB. The logKapp

values were calculated as 3.43, 5.53 and 4.20 for dq-dmphen andcomplexes 1 and 2, respectively. With the addition of the com-pound to DNA, there was a noticeable decrease in the emissionintensity due to the replacement of EB by complexes. From theexperimental data, a plot of emission intensity (I/Io) vs. compoundconcentration was prepared (Fig. 6a–c). These behaviors can beanalyzed with Eq. (3) and logKsv values were calculated to be4.42, 6.74 and 5.04 for dq-dmphen and complexes 1 and 2,respectively [45]. In addition, the Stern–Volmer quenching con-stant (Ksv) and Kapp are illustrated in Table 4. The quenching ofthe fluorescence of the compounds clearly indicated that the bind-ing of DNA to these molecules changed the environment of EB. A

bance Hypc (%) Kb (M�1) logKb

49.1 1.42 � 105 ± 0.04 5.15 ± 0.0161.6 1.77 � 105 ± 0.02 5.25 ± 0.0152.1 1.56 � 105 ± 0.03 5.19 ± 0.02

Wavelength, nm

Fluo

resc

ence

Inte

nsity

0

50

100

150

200

EB-DNA

[Complex] x 104 M

I o/I

0,00,51,01,52,02,53,0(a)

Wavelength, nm

Fluo

resc

ence

Inte

nsity

0

50

100

150

200

EB-DNA

[Complex] x 106 M

I o/I

0,00,51,01,52,02,53,0(b)

Wavelength, nm

Fluo

resc

ence

Inte

nsity

0

50

100

150

200

EB-DNA

[Complex] x 105 M

I o/I

0,0

0,5

1,0

1,5

2,0

2,5(c)

540 560 580 600 620 640 660 680 700

0 1 2 3 4 5

540 560 580 600 620 640 660 680 700

0 1 2 3 4 5

540 560 580 600 620 640 660 680 700

0 2 4 6 8 10 12

Fig. 6. Effect of addition of the compounds on the emission intensity of CT-DNAbound ethidium bromide at different concentrations in a Tris–HCl buffer (pH 7.2);(a) dq-dmphen, (b) complex 1 and (c) complex 2. Insets: Stern–Volmer plot of thefluorescence data.

Table 4Binding parameters for the effect of dq-dmphen and complexes 1 and 2 on thefluorescence of EB in the presence of CT-DNA.

Compounds Kapp logKapp Ksv logKsv

dq-dmphen 3.18 � 103 ± 0.04 3.43 ± 0.09 2.60 � 104 ± 0.01 4.42 ± 0.01Complex 1 3.43 � 105 ± 0.03 5.53 ± 0.01 5.47 � 106 ± 0.01 6.74 ± 0.01Complex 2 1.77 � 104 ± 0.07 4.20 ± 0.02 1.08 � 105 ± 0.01 5.04 ± 0.01

Table 5Tm and DTm values for dq-dmphen and complexes 1 and 2.

Compounds Tm (�C) DTmc (�C)

dq-dmphen 70.4 0.8Complex 1 75.0 5.4Complex 2 71.0 1.4EB 81.8 12.2

c Change in the CT- DNA melting temperature.

768 D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770

remarkable reduction in emission intensity was observed as thecompounds was added to the EB-DNA system, indicating that someEB molecules were replaced by the compounds and released intosolution from EB-DNA adduct, characteristic for the intercalativebinding of the compounds to DNA. The fluorescence quenching

results are in good agreement with the UV spectroscopic resultsfor complexes 1 and 2.

Thermal denaturationThe thermal behavior of DNA in the presence of metal com-

plexes can give insight into their conformational changes whentemperature is raised and information about the interactionstrength of the complexes with DNA. Intercalation of natural orsynthesized organics and metallointercalators generally results ina considerable increase in melting temperature (Tm) [58]. Classicalintercalators such as EB are known to show significantly high DTm

values (11.2 �C) [59]. In this experimental medium, EB displaysremarkable DTm values of 12.2 �C (Table 5). The DNA denaturationexperiment shows a minor shift in the melting temperature (Tm),yielding a DTm value of 0.8–5.4 �C on addition of the compoundsto CT-DNA (Fig. 7).

DNA cleavage studies

The DNA cleavage property of the complex against super-coiledpBR322 plasmid DNA was demonstrated through agarose gel elec-trophoresis. Cleavage was confirmed by comparing the bandingpatterns of untreated and treated (with compounds) plasmidDNA. The influence of the complexes on the tertiary structure ofDNA was determined by their ability to modify the electrophoreticmobility of the super-coiled form (Form I, SC) to the nicked circular(Form II, NC) or linear open circular forms (Form III, LC). Complexes1 and 2 were found to promote the cleavage of pBR322 plasmidDNA from supercoiled Form (I) to nicked Form (II). The gel electro-phoretic separation, showing the cleavage of plasmid pBR322 DNAinduced by the compounds under identical conditions, is shown inFigs. 8 and 9. Under comparable experimental conditions, the com-pounds exhibited different cleaving efficiency for the plasmid DNA.

With increased compound concentration, the amount of Form I(supercoil form) of pBR322 DNA diminished gradually, whereas theamount of Form II (nicked form) increased significantly. Thesecompounds can cleave plasmid DNA in a dose-dependent manner.When the concentration reached 1280 lM, complex 1 promoted acomplete conversion from Form I to Form II (Fig. 8). At 10240 lM,complex 2 shows complete conversion of Form I to Form II of DNA.Control experiments with plasmid DNA (Line 1) show no apparentconversion of Form I to Form II (Fig. 9).

In summary, complex 1 had a greater DNA cleavage effect thatwas shown at lower doses than complex 2. DNA cleavage activityoutcomes demonstrate that these compounds efficiently cleavesuper-coiled pBR322 plasmid DNA.

Temperature, °C

A260

1,2

1,4

1,6

1,8 CT-DNA

Temperature, °C

A260

1,2

1,4

1,6

1,8

2,0

2,2

2,4 EB + CT-DNA

Temperature, °C

20 40 60 80 100

20 40 60 80 100

20 40 60 80 100

A260

1,2

1,4

1,6

1,8

2,0

2,2 dq-dmphen + CT-DNAComplex 1 + CT-DNAComplex 2 + CT-DNA

Fig. 7. DNA denaturation temperature curves of CT-DNA (120 lM) in the absenceand presence of dq-dmphen, complexes 1, 2 and EB (30 lM) in Tris–HCl buffersolution (pH 7.2).

Fig. 8. Agarose gel electrophoresis diagrams. Cleavage of pBR322 plasmid DNAinduced by complex 1. Lane 1: Control, Lanes 2–10: DNA-complex 1, complex 1: 40,80, 160, 320, 640, 1280, 2560, 5120 and 10240 lM, respectively.

D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770 769

Cytotoxicity

It is commonly believed that many anticancer agents act bybinding to DNA. From experiments of electronic absorption bind-ing, competitive binding with EB, thermal denaturation and cleav-age activity, we know that the newly synthesized compounds canreact with DNA.

To determine whether the newly synthesized compounds haveanticancer activity, we performed cytotoxicity in vitro tests againstselected human tumor cell lines (Caco-2, A549 and MCF-7) andhealthy cells (BEAS-2B). In parallel, the influence of a widely usedanticancer drug, cisplatin, has also been assayed. The IC50 valuesare listed in Table 6.

As shown in Table 6, the IC50 values for complex 1 ranged from0.70 to 2.10 lM, those for complex 2 ranged from 2.21 to 3.77 lM,those for dq-dmphen ranged from 550 to 598 lM, and those forCu2+(aq) ranged from 111 to 1085 lM, indicating that all com-pounds exhibited antitumor activity against all cell lines in differ-ent degrees.

From Table 6, complexes 1 and 2 were found to be cytotoxic toselected human tumor cell lines. These complexes exhibited highercytotoxic effects on these cancer cells with lower IC50 values,

Fig. 9. Agarose gel electrophoresis diagrams. Cleavage of pBR322 plasmid DNAinduced by complex 2. Lane 1: Control, Lanes 2–10: DNA-complex 2, complex 2: 40,80, 160, 320, 640, 1280, 2560, 5120 and 10240 lM, respectively.

Table 6Cytotoxic effects of metal solution, dq-dmphen, complexes 1 and 2 and cisplatinagainst selected human tumor cells and healthy cells.

Compounds IC50 (lM)

MCF-7 Caco-2 A549 BEAS-2B

Cu2+ (aq) 521 ± 13 111 ± 30 875 ± 60 1085 ± 45dq-dmphen >570 598 ± 17 550 ± 16 598 ± 21Complex 1 0.95 ± 0.11 0.70 ± 0.02 0.76 ± 0.03 2.10 ± 0.06Complex 2 2.21 ± 0.46 2.78 ± 0.81 3.05 ± 0.10 3.77 ± 0.21Cisplatin 31.1 ± 3.2 22.7 ± 2.3 22.2 ± 0.4 18.9 ± 0.9

IC50

( μμM

)

0

5

10

15

20

25

30 Complex 1Complex 2cisplatin

MCF7 CACO2 A549 BEAS-2B

Fig. 10. IC50 values of complexes 1 and 2 and cisplatin against different humantumor cells.

770 D. _Inci et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 136 (2015) 761–770

indicating their efficiency in killing cancer cells even at low con-centrations compared with cisplatin (Fig. 10). Furthermore, thesensitivity ranking of the tumor cell lines for complex 1 wasCaco-2 > A549 > MCF-7, and the ranking for complex 2 wasMCF-7 > Caco-2 > A549. The dq-dmphen and Cu2+ solutions exhibitmuch less activity against these cancer cells than cisplatin does.

Obviously, complex 1 is more active toward cell lines than iscomplex 2. We suggest that the cytotoxicity profile has changeddue to the difference between the coordinated modes of complexes1 and 2.

Conclusion

To summarize, the diquarternary salt of dmphen and complexes1 and 2 have been synthesized and characterized. Crystalstructures of complexes 1 and 2 were determined by single-crystalX-ray diffraction. The DNA binding properties of these compoundshave been investigated by UV–vis spectroscopy, fluorescence spec-troscopy and thermal denaturation studies. The results obtainedindicate that these compounds bind to DNA via an intercalationbinding mode. The studies also confirm that the binding abilityof the compounds is an important precondition for efficient DNAcleavage activity. The cleavage capability of pBR322 DNA by thesecompounds indicates that these compounds exhibit efficient DNAcleavage. The cytotoxicity of these compounds against differentcancer cell lines (A549, Caco-2 and MCF-7) and a healthy cell line(BEAS-2B) was investigated. Complexes 1 and 2 exhibit high cyto-toxicity with low IC50 values (<4 lM) and show selective cytotox-icity. Complex 1 exhibits higher cytotoxicity than does cisplatinagainst the Caco-2 cell line; complex 2 shows higher cytotoxicitythan does cisplatin against the MCF-7 cell line. The significant cyto-toxicity of complexes 1 and 2 may be a result of their effectiveinteractions with DNA.

Supplementary materials

Additional crystallographic data have been deposited at theCambridge Crystallographic Data Centre, CCDC reference numbersare 914900 for complex 1 and 914899 for complex 2. The data canbe obtained available free of charge from www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic DataCentre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223336033; email: deposit@ccdc.cam.ac.uk.

Acknowledgement

We thank the Research Fund of Uludag University for the finan-cial support given to the research project (Project Numbers: UAP(F)-2011/71).

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