effect of ligand substitution in pyrazolone based binary and ternary cu(ii) complexes on dna...

Polyhedron xxx (2014) xxx–xxx

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Polyhedron

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Effect of ligand substitution in pyrazolone based binary and ternaryCu(II) complexes on DNA binding, protein binding and anti-canceractivity on A549 lung carcinoma cell lines

0277-5387/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.poly.2013.12.037

⇑ Corresponding author. Tel.: +91 265 2795552x30; fax: +91 27 82567760.E-mail address: [email protected] (R.N. Jadeja).

Please cite this article in press as: K.M. Vyas et al., Polyhedron (2014), http://dx.doi.org/10.1016/j.poly.2013.12.037

Komal M. Vyas a,d, R.N. Jadeja a,⇑, Dipak Patel b, R.V. Devkar b, Vivek K. Gupta c

a Department of Chemistry, Faculty of Science, The M.S. University of Baroda, Vadodara 390 002, Indiab Division of Phytotherapeutics and Metabolic Endocrinology, Faculty of Science, The M.S. University of Baroda, Vadodara 390 002, Gujarat, Indiac Post-Graduate Department of Physics & Electronics, University of Jammu, Jammu Tawi 180 006, Indiad Institute of Infrastructure, Technology, Research And Management, Ahmedabad 380 026, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 September 2013Accepted 31 December 2013Available online xxxx

Keywords:Acyl pyrazoloneSchiff baseCopper complexesDNA and protein bindingAnti-cancer activity

A new series of 4-acyl pyrazolone based ternary Cu(II) complexes [Cu(TPMP)(Phen)NCS] (1), [Cu(TPMP)(-Bipy)NCS] (2) and binary Cu(II) complex [Cu(TPMP-BA)2] (3) has been synthesized and characterized bystructural, analytical and spectral methods, in order to investigate the influence of ligand substitution onstructure and pharmacological properties. In all of the complexes, the pyrazolone based ligand is coordi-nated to the Cu(II) ion in a neutral fashion as bidentate ligand. The single-crystal X-ray study of binarycomplex (3) exhibits a square planar structure, while ternary complexes (1) and (2) revealed slightly dis-torted square-pyramidal structures. The interaction of the compounds with calf thymus DNA (CT-DNA)has been explored by absorption and emission titration methods, which revealed that compounds 1–3could interact with CT-DNA through intercalation. The interaction of the compounds with bovine serumalbumin (BSA) was also investigated using fluorescence spectroscopic method. The results indicated thatall of the compounds could quench the intrinsic fluorescence of BSA in a static quenching process. Fur-ther, the cytotoxic effect of the compounds 1–3 examined on human lung cancerous cell line (A549)and noncancerous rat cardiomyoblasts (H9C2) cell lines showed that all three complexes exhibited sub-stantial cytotoxic activity. All the pharmacological investigations support the fact that there exists astrong influence of ligand substitution on pharmacological activities.

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1. Introduction

Cancer is one of the main health concerns confronting humanityand one of the primary targets in therapeutic chemistry. So, themost rapidly developing area of pharmaceutical research is the dis-covery of drugs for cancer therapy. In this regard, cisplatin (cis-diamminedichloroplatinum(II)) is a promising and well-knownmetal-based drug for cancer therapy, but it has its own limitationsbecause of the development of resistance in tumour cells and seri-ous side effects such as nausea, kidney and liver failure typical ofheavy metal toxicity [1–4]. So, more-efficient, less toxic and tar-get-specific noncovalent DNA binding anticancer drugs are to bedeveloped. In general, anticancer agents that are approved for clin-ical use are molecules which damage DNA, block DNA synthesisindirectly through inhibition of nucleic acid precursor biosynthesisor disrupt hormonal stimulation of cell growth [5]. Therefore,considerable attempts are being made to replace this drug with

suitable alternatives and hence numerous transition metalcomplexes have been synthesized and tested for their anticanceractivities.

Cu(II) complexes are regarded as the most promising alterna-tives to cisplatin as anticancer drugs; an idea supported by a con-siderable number of research articles describing the synthesis,DNA binding and cytotoxic activities of numerous Cu(II) complexes[6–9]. Also, synthetic Cu(II) complexes have been reported to act aspharmacological agents and as potential anticancer and cancer-inhibiting agents [10–15]. Very recently, certain mixed-ligandCu(II) complexes, which strongly bind and cleave DNA, exhibitprominent anticancer activities and regulate apoptosis are re-ported [7,16–19]. Therefore, designing suitable Cu(II) complexesfor DNA binding and anti-cancer activities, depending upon therecognition of elements in the ligand, is of remarkable importancein considering the advantages of processes that produce fragmentssimilar to those formed by restriction enzymes [20,21].

On the other hand, drug interactions at the protein binding levelsignificantly affect the apparent distribution volume and theirelimination rate. Therefore, the interactions of metal complexes

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2 K.M. Vyas et al. / Polyhedron xxx (2014) xxx–xxx

with serum albumins have received much attention in the scien-tific community by studying antitumoral, metallopharmaceutical,pharmacokinetics and structure–activity relationships [22]. Bovineserum albumin (BSA) is the most extensively studied serum albu-min, due to its structural homology with human serum albumin(HSA). This fact further supports the value of studying the interac-tion behaviour of the metal complexes with bovine serum albumin(BSA) protein, while evaluating their anticancer properties.

On moving into the ligand point of view, it can be said that thepyrazolones as well as their derivatives are a class of ligands pre-senting a wide range of biological applications. Specifically, 4-acylpyrazolones and their derivatives have been screened for theiranti-inflammatory, anti-bacterial, anti-microbial, anti-cancer, anti-oxidant, anti-tumour and analgesic activities [23–27]. Interest-ingly, the activity of these ligands might be enthused bycoordination to some metal ions. Particularly, Cu(II) complexescontaining pyrazolone ligands have displayed a wide spectrum ofbiological properties [28–30].

Though there have been a large number of metal complexes re-ported along with their pharmacological properties, any attemptmade to explain the factors that are responsible or affect theiractivity is relatively less existent. Also there are very few reportson the anti-cancer activity of pyrazolone based metal complexes.In this regard, we have found and reported that the C(4)–pyrazo-lone substitution not only altered the mode of coordination ofsubstituted pyrazolones but also enhanced the activities such asDNA binding protein binding and anti-cancer activity of its Cu(II)complex with [31]. Moreover, only a little attention has been paidto the coordination chemistry of ternary Cu(II) complexes with 4-acyl pyrazolone based ligands. These Cu(II) complexes have beenshown to have interesting pharmacological properties [30]. Andalso, no attempts were made to explore the biological properties,mainly anti-cancer properties of pyrazolone based ligands andtheir ternary metal complexes with polypyridyl ligands. All of theabove facts have stimulated our interest in the present work onsynthesizing new binary and ternary Cu(II) complexes of 4-acylpyrazolone derivatives to better understand the effect of ligandsubstitution on DNA binding, Protein binding and Anti-canceractivities on A549 human lung cancer cell lines of the resultedCu(II) complexes.

2. Experimental

2.1. Materials

The compound 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one(PMP) was obtained from Nutan Dye Chem., Sachin, Surat, India.Dioxane was obtained from E. Merck (India) Ltd. Calcium hydrox-ide, Cu(OAc)2�H2O, Cu(NO3)2�3H2O, ammonium thiocyanate, 1,10-phenanthroline and 2,20-bipyridyl were obtained from LOBA Chem.Pvt. Ltd., Mumbai and used as supplied. p-toluoyl chloride was ob-tained from Shiva Pharmachem. Ltd., Baroda as free gift samples. p-Bromo aniline was obtained from Sisco Research Lab. Pvt. Ltd.,Mumbai, India. Absolute alcohol was obtained from Baroda Chem.Industry Ltd. and was used after distillation. Methanol was ob-tained from Spectrochem., Mumbai, India and was used after distil-lation. CT-DNA (Calf Thymus DNA) and BSA (Bovine SerumAlbumin) were purchased from Sigma Aldrich. All the chemicalsused were of AR grade. Ethidium bromide (EB), Dulbeco’s ModifiedEagle Medium (DMEM), Trypsin Phosphate Versene Glucose(TPVG) solution Trypsin and methylthiazolyldiphenyl-tetrazoliumbromide (MTT) were purchased from HiMedia Laboratories Pvt.Ltd. (Bombay, India). Fetal bovine serum (FBS) was purchased fromBiosera (Ringmer, East Sussex UK) and dimethyl sulfoxide (DMSO)was purchased from the Sisco Research Laboratories Pvt. Ltd.

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(Mumbai, India). Rhodamine 123, 40,6-diamidino-2-phenylindole(DAPI) and 20,70-dichlorofluoresceindiacetate (DCFDA) were pur-chased from Sigma (Delhi, India). Dulbeco’s Modified Eagle Med-ium (DMEM), Trypsin Phosphate Versene Glucose (TPVG)solution Trypsin and methylthiazolyldiphenyl-tetrazolium bro-mide (MTT) were purchased from HiMedia Laboratories Pvt. Ltd.(Bombay, India). Fetal bovine serum (FBS) was purchased from Bio-sera (Ringmer, East Sussex UK) and dimethyl sulfoxide (DMSO)was purchased from the Sisco Research Laboratories Pvt. Ltd.(Mumbai, India). Rhodamine 123, 40,6-diamidino-2-phenylindole(DAPI) and 20,70-dichlorofluoresceindiacetate (DCFDA) were pur-chased from Sigma (Delhi, India).

2.2. Characterization techniques

The synthesized compounds were characterized using differenttechniques. Elemental analyses (C, H, N) of the synthesized com-pounds were performed on a model 2400 Perkin-Elmer elementalanalyzer. Infrared spectra (4000–400 cm�1, KBr discs) of the sam-ples were recorded on a model RX1 FTIR Perkin-Elmer spectropho-tometer. 1H NMR spectra of the ligands were recorded with BrukerAV 400 MHz using CDCl3 as a solvent and TMS as an internal refer-ence. Mass spectra of the ligands were recorded on Trace GC ultraDSQ II. The electronic spectra (in DMF at room temperature) in therange of 400–800 nm were recorded on a model Perkin ElmerLambda 35 UV–Vis spectrophotometer. Fluorescence spectra wererecorded on a model JASCO, FP-6300 fluorescence spectrophotom-eter. Mass spectra (EI)/(FAB) of the complexes were recorded atSAIF, CDRI, Lucknow. Molar conductivity of 10�3 M solution ofthe complexes in DMF was measured at room temperature witha model Elico CM180 digital direct reading deluxe digital conduc-tivity meter. Copper content was determined by EDTA afterdecomposing the complexes with HNO3.

2.3. Preparation of ligands

2.3.1. 5-Methyl-4-(4-methyl-benzoyl)-2-phenyl-2,4-dihydro-pyrazol-3-one [TPMP]

Toluoylation reaction of pyrazolone, PMP (3-methyl-1-phenyl-1H-pyrazol-5(4H)-one) was carried out by following the standardmethod reported in our previous articles [31].

TPMP is cream crystalline solid. Yield 80.85% m.p. 120 �C Anal.Calc. for C18H16N2O2 M.W.: 292.33, C, 73.95; H, 5.52; N, 9.52.Found: C, 73.68; H, 5.21; N, 9.51%. 1H NMR (CDCl3): d ppm 2.16(s, 3H, PZ C–CH3), 2.47 (s, 3H, N-TL C–CH3), 7.28–7.46 (m, 5H,Ph), 7.49–7.59 (m, 4H, TL), 7.88–7.90 (s, 2H, PZ ring). IR (KBr,cm�1): 1598(s) (C@N, cyclic), 1641(m) (C@O, pyrazolone ring);13C NMR (CDCl3) d ppm: 16.04 [C–CH3, PZ], 21.70 [C–CH3, TL],103.56 [C@O], 120.72–142.73 [substituted benzene rings]; MS:m/z = 292.07 [C18H16N2O]+, 214.05 [C12H11N2O2]+, 200.03[C11H9N2O2]+, 119.04 [C8H7O]+, 91.06 [C7H7]+.

2.3.2. 4-[(4-Bromo-phenylimino)-p-tolyl-methyl]-5-methyl-2-phenyl-2,4-dihydro-pyrazol-3-one [TPMP-BA]

Equimolar (10 mmol) ethanolic solution (50 mL) of 5-methyl-4-(4-methyl-benzoyl)-2-phenyl-2,4-dihydro-pyrazol-3-one (TPMP,0.292 g, 1 mmol) and p-bromo aniline (0.172 g, 1 mmol) was re-fluxed for 6 h in round bottom flask. During the reflux a microcrys-talline yellow compound [TPMP-BA] was separated, which wasisolated by filtration and dried in air and finally crystallized inappropriate solvent.

TPMP-BA is yellow crystals. Yield 71.59% m.p. 194 �C Anal. Calc.for C24H20BrN3O M.W.: 446.35, C, 64.57; H, 4.48; N, 10.76. Found:C, 64.50; H, 4.42; N, 10.71%. 1H NMR (CDCl3) d ppm: 1.61 (s, 3H,PZC–CH3), 2.44 (s, 3H, N–TLC–CH3), 12.89 (s, 1H, –NH), 6.66–8.04(m, 13H, Ph); IR (KBr) m(cm�1): 3125(b) (–NH/–OH), 1570 (m)

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K.M. Vyas et al. / Polyhedron xxx (2014) xxx–xxx 3

(C@N, cyclic), 1618 (C@N, azomethane), 1270 (m) (PZ–C–CH3),1229 (C@O, pyrazolone ring); 13C NMR (CDCl3-d6) dppm: 16.19,21.58 [methyl groups], 119.34–132.07 [substituted benzene ring];MS: m/z = 446 [C24H20N3OBr, MIP]+, 366 [C24H20N3O]+, 340 [C16H10-

N3OBr]+, 275 [C17H12N3O]+, 260 [C16H10N3O]+, 197 [C11H7N3O]+, 91[C7H7]+, 80 [C3N2O]+.

2.4. Synthesis of Cu(II) complexes

2.4.1. [Cu(TPMP)(Phen)NCS] (1)To the solution of Cu(NO3)2�3H2O (0.241 g, 1 mmol) in methanol

(5 mL), a solution of TPMP (0.292 g, 1 mmol) in methanol (10 mL)was added while stirring. To this, a solution of 1,10 phenanthroline(1 mmol, 0.198 g) in methanol (5 mL) was added, followed by asolution of NH4NCS (2 mmol, 0.152 g) in warm methanol (5 mL).The pH of the reaction mixture was maintained around 7.5 by add-ing a 10% methanolic solution of ammonia. The resultant mixturewas refluxed for 3 h. The solid green crystalline product obtainedwas filtered off, washed with methanol and dried. The solid prod-uct was dissolved in hot DMSO and was allowed to crystallize atRT. Green crystals of single crystal X-ray quality was obtained in10–15 days.

Synthesis of complex 1 is summarized in Scheme 1.[Cu(TPMP)(Phen)NCS]: Dark green crystals; yield

89.99%,m.p > 250 �C. Anal. Calc. for C31H23CuN5O2S: M.W.: 593.16,C, 62.77; H, 3.91; N, 11.81; Cu, 10.27. Found: C, 62.66; H, 3.86; N,11.73; Cu, 10.71%. IR (KBr, cm�1): 1581(s) (C@N, cyclic), 1469(m)(C–O), 1157(m) (N–N), 846(s) (NC@S), 2083 (N@CS), 476(s) (Cu–N), 430(s) (Cu–O), 420(s) (Cu–S); kmax/nm 713 (dxz,dyz?dx2�y2 ,Square pyramidal; Molar conductance (DMF, X�1 cm2 mol�1):12.00; 534.2 [C30H23CuN4O2, MIP, Base peak]+, 535.3 [C30H23CuN4-

O2, M+1]+, 537.3 [C30H23CuN4O2, M+3]+, 303.1 [C11H7CuN3O2S]+,263.1 [C11H9CuN2O2]+, 245.1 [C6H5CuN3O2S]+, 243.1 [C12H8CuN2]+,123 [C6H6N2O]+.

NN

O

H3C CO

H3C

Cu(NO3)2·3H

Constant stirring

NN

H3C

O

H3C

Scheme 1. Synthesi


2.4.2. [Cu(TPMP)(Bipy)NCS] (2)To the solution of Cu(NO3)2�3H2O (0.241 g, 1 mmol)in methanol

(5 mL), a solution of TPMP (0.292 g, 1 mmol) in methanol (10 mL)was added while stirring. To this, a solution of 2,20 bipyridyl(1 mmol, 0.156 g) in methanol (5 mL) was added, followed by asolution of NH4NCS (2 mmol, 0.152 g) in warm methanol (5 mL).The pH of the reaction mixture was maintained around 7.5 by add-ing a 10% methanolic solution of ammonia. The resultant mixturewas refluxed for 3 h. The solid green crystalline product obtainedwas filtered off, washed with methanol and dried. The solid prod-uct was dissolved in hot DMSO and was allowed to crystallize atRT. Green crystals of single crystal X-ray quality was obtained in10–15 days.

Synthesis of complex 2 is summarized in Scheme 2.[Cu(TPMP)(Bipy)NCS]: Dark green crystals; yield 86.78%,

m.p > 250 �C. Anal. Calc. for C29H23CuN5O2S: M.W.: 569.14, C,61.20; H, 4.07; N, 12.31; Cu, 11.17. Found: C, 61.09; H, 4.16; N,12.23; Cu, 11.43%. IR (KBr, cm�1): 1580(s) (C@N, cyclic), 1469(m)(C–O), 1157(m) (N–N), 843(s) (NC@S), 2088 (N@CS), 415(s) (Cu–N), 479(s) (Cu–O), 410(s) (Cu–S); kmax/nm 673 (dxz,dyz?dx2�y2 ,Square pyramidal; Molar conductance (DMF, X�1 cm2 mol�1): 9.00.

2.4.3. [Cu(TPMP-BA)2] (3)Known amount of TPMP-BA (0.446 g, 1 mmol) was dissolved in

hot dry ethanol. Equimolar amount of Cu(OAc)2�H2O (0.199 g,1 mmol) was dissolved in dry ethanol and then added to the dis-solved ligand. After the complete addition, the reaction mixturewas refluxed for 4 h. A dark greenish microcrystalline solid wasseparated, which was isolated by filtration, washed with hot waterand finally with ethanol and dried under a vacuum. The solid prod-uct was dissolved in hot MeCN and allowed to crystallize at roomtemperature. Green crystals of single-crystal X-ray quality wereobtained in 2–4 days.

Synthesis of complex 3 is summarized in Scheme 3.

2O

Constant stirring

1,10 Phenanthroline+NH4NCS

O N

N

Cu

N

C

S

PH=7.5

Reflux, 3 hr

s of complex 1.

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Scheme 2. Synthesis of complex 2.

Scheme 3. Synthesis of complex 3.


[Cu(TPMP-BA)2]: Green crystals; yield 61.86%, m.p > 250 �C.Anal. Calc. for C48H38Br2CuN6O2: M.W.: 954.21, C, 60.35; H, 4.07;N, 8.31; Cu, 6.17. Found: C, 60.42; H, 4.01; N, 8.81; Cu, 6.66%. IR(KBr, cm�1): 1563(s) (C@N, cyclic), 1476(m) (C–O), 1214 (C@N,azomethane), 534(s) (Cu–N), 483(s) (Cu–O); kmax/nm 714(2B1g?

2A1g, square planar; Molar conductance (DMF, X�1 cm2

mol�1): 4.00; Mass: 954 [C48H38Br2CuN6O2, MIP]+, 951 [C48H38Br2

CuN6O2, M�3]+, 922 [C46H32Br2CuN6O2]+, 862 [C47H37BrCuN6O2]+,831 [C45H31BrCuN6O2]+, 800 [C36H28Br2CuN6O]+, 669 [C32H23

BrCuN6O2]+, 612 [C34H28CuN6O2]+, 582 [C32H18CuN6O2]+, 770 [C34

H23Br2CuN6O2]+, 538 [C28H22CuN6O2]+, 509 [C20H11BrCuN6O2]+,445 [C24H19BrN3O]+, 391 [C20H13BrN3O]+, 306 [C19H17N3O]+, 154[C7H11N3O, basepeak]+.

2.5. Single crystal X-ray studies

Crystals having good morphology were chosen for three-dimen-sional intensity data collection. X-ray intensity data of the com-pounds was collected at room temperature on Bruker CCD area-detector diffractometer equipped with graphite monochromatedMoKa radiation (a = 0.71073 Å). The crystals used for data collec-tion were of suitable dimensions 0.30 � 0.20 � 0.10 mm. The unitcell parameters were determined by least-squares refinement of16728 reflections. Data were corrected for Lorentz, polarizationand multi-scan absorption correction [32]. The structure wassolved by direct methods using SHELXS97 [33]. All non-hydrogenatoms of the molecule were located in the best E-map. Full-matrixleast-squares refinement was carried out using SHELXL97 [33].Hydrogen atoms were placed at geometrically fixed positions andallowed to ride on the corresponding non-H atoms with C–H = 0.93–0.96 Å, and Uiso = 1.5 Ueq of the attached C atom formethyl H atoms and 1.2 Ueq for other H atoms. Atomic scatteringfactors were taken from International Tables for X-ray Crystallog-


raphy (1992, Vol. C, Tables 4.2.6.8 and 6.1.1.4). An ORTEP [34] viewof the complexes 1–3 with atomic labelling are shown in Fig. 1.The packing diagrams of the complexes 1–3 are shown in Fig. S1(Supporting information). The geometry of the molecules has beencalculated using the software PLATON [35] and PARST [36]. The crystal-lographic data for the complexes are summarized in Table 1. Theimportant bond lengths and bond angles of the complexes arelisted in Table S1.

2.6. DNA binding

All of the experiments involving the binding of complex withCT-DNA were carried out in double distilled water with trisodiumcitrate (Tris, 15 mM) and sodium chloride (150 mM) and adjustedto pH 7.05 with hydrochloric acid. The DMF solution of thecomplex was used throughout the study. The concentration ofCT-DNA per nucleotide was estimated from its known extinctioncoefficient at 260 nm (6600 M�1 cm�1) [37]. Solutions of CT-DNAin Tris buffer gave a ratio of UV absorbance at 260 and 280 nm(A260/A280) 1.8–1.9 indicating that the DNA was sufficiently freeof protein. Absorption titration experiments were performed bymaintaining constant metal complexes concentration (20 lM),while gradually increasing the concentration of DNA (5–100 lM).While measuring the absorption spectra, an equal amount ofDNA was added to both the test solution and the reference solutionto eliminate the absorbance of DNA itself.

The data were then fit to Eq. (1) [38] to obtain intrinsic bindingconstant Kb.

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

where [DNA] is the concentration of DNA in base pairs, ea is theextinction coefficient observed for the MLCT absorption band atthe given DNA concentration, ef is the extinction coefficient of the

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Fig. 1. ORTEP Views of the (A) complex 1, (B) complex 2 and (C) complex 3 with displacement ellipsoids drawn at 50%. Solvent molecule has been removed for clarity.

Table 1Summary of crystallographic data of the complexes.

Complex 1 2 3

Chemical formula C31H23CuN5OS,C2H3N

C29H23CuN5O2S,0.5CH3OH

C48H38Br2CuN6O2

Formula weight 634.20 585.15 954.20a (Å) 11.0580(5) 24.894(2) 14.2362(10)b (Å) 12.2614(5) 8.8679(6) 22.5728(14)c (Å) 12.9478(5) 25.108(2) 14.3589(12)a (�) 113.881(4) 90.00 90.00b (�) 109.248(4) 101.798(9) 108.347(8)c (�) 90.425(3) 90.00 90.00Z 2 8 4V (Å3) 1495.20(11) 5425.7(8) 4379.7(5)Reflections

collected21841 11018 16510

Individualreflections

5244 5319 7690

Rint 0.0357 0.0343 0.0619No. of parameters 391 359 542Crystal system triclinic monoclinic monoclinicSpace group P�1 C2/c P21/cDcalcd (g cm�3) 1.409 1.433 1.447Absorption

coefficient, l(cm�1)

0.841 0.921 2.369

F(000) 654 2416 1932T (�C) 23 23 23Goodness-of-fit

(GOF) on F21.008 1.027 1.030

R1/wR2 ([I > 2r(I)] 0.0366/0.0894 0.0522/0.1274 0.0636/0.0997R1/wR2 (all data) 0.0465/0.0946 0.0886/0.1502 0.1592/0.1373CCDC 842200 865376 868303


complex free in solution and eb is the extinction coefficient ofthe complex when fully bound to DNA. A plot of [DNA]/[ea � ef]versus [DNA] gave a slope 1/[ea � ef] and Y intercept equal to(1/Kb)[eb � ef], respectively. The intrinsic binding constant Kb isthe ratio of the slope to the intercept [6].

Competitive studies of compounds with ethidium bromide (EB)have been investigated with fluorescence spectroscopy in order toexamine whether the compounds can displace EB from its DNA�EBcomplex. The DNA�EB complex was prepared by adding 3.3 lM EBand 4.2 lM CT-DNA in buffer (150 mM NaCl and 15 mM trisodiumcitrate at pH 7.05). The intercalating effect of the compound withthe DNA�EB complex was studied by adding a certain amount ofa solution of the compound step by step (0–30 lM) into the solu-tion of the DNA�EB complex. The influence of the addition of each


compound to the DNA�EB complex solution has been obtained byrecording the variation of fluorescence emission spectra. The emis-sion spectra were monitored by keeping the excitation of the testcompound at 546 nm and the emission was monitored in the rangeof 550–750 nm. The emission was observed at 610 nm.

Commonly, fluorescence quenching can be described by the fol-lowing Stern–Volmer equation (Eq. (2)) [6].

F0=F ¼ 1þ Ksv½Q � ¼ 1þ kqs0½Q � ð2Þ

where F0 and F are the steady-state fluorescence intensities in theabsence and presence of quencher, respectively, Ksv is the Stern–Volmer quenching constant, obtained from the slope of the plotF0/F versus [complex], [Q] is the total concentration of quencher,kq is the bimolecular quenching constant, and s0 is the average life-time of protein in the absence of quencher, and its value is 10�8 s.

The apparent DNA binding constant (Kapp) values of the com-plexes were obtained from this fluorescence spectral measure-ment. The Kapp values were obtained from the equation:

Kapp � ½complex�50 ¼ KEB � ½EB� ð3Þ

where Kapp is the apparent binding constant of the complex studied,[complex]50 is the concentration of the complex at 50% quenchingof DNA-bound ethidium bromide emission intensity, KEB is thebinding constant of ethidium bromide (KEB = 1.0 � 107 M�1) and[EB] is the concentration of ethidium bromide (3.3 lM) [38].

2.7. Protein binding studies

Similar experimental procedure was followed for tryptophanquenching study as DNA binding studies. Quenching of the trypto-phan residues of BSA was performed using complex as quencher.To solutions of BSA in buffer, increments of the quencher wereadded and the emission signals at 343 nm (excitation wavelengthat 296 nm) were recorded after each addition of the quencher [6].

The data were fitted in the Stern–Volmer eq 2. The Stern–Vol-mer constant was obtained from the slope of the plot F0/F versus[compound].

2.8. Anticancer activity

2.8.1. Cell cultureAll the reagents used herein were filtered through 0.22 lm filter

(Laxbro Bio-medical Aids Pvt. Ltd) prior to their use for the exper-iment. Human lung carcinoma (A549) cells were obtained from

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National Centre for Cell Science, Pune, India. These cells were thenseeded (1 � 105 cells/T25 flask) and cultured in DMEM containing10% FBS and 1% antibiotic–antimycotic solution at 37 �C with 5%CO2 in forma II water Jacketed CO2 incubator (Thermo scientific,Germany). Cells were sub-cultured every third day by trypsiniza-tion with TPVG solution. A549 cells were maintained for a periodof 24 h in absence of presence of metal complexes at a cell densityof 5.0 � 103 cells/well in 96 well plate for MTT and LDH assay and1 � 105 cells/well in 6 well plate for LDH release assay, mitochon-drial membrane potential assay, DCFDA and AO/EB staining, cellcycle analysis and Annexin V-PI staining assays.

2.8.2. Cell viability (MTT) assayA549 and H9C2 cells (7 � 103 cells/well) were maintain in 96-

well culture plates as mentioned above for 24 h in absence andpresence of metal complexes and later, 10 ll of 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 5 mg/ml) wasadded to the wells. Plates were incubated again at 37 �C for 4 h,culture media was discarded and wells were washed with Phos-phate Buffer Saline (Hi-media, India, Pvt. Ltd.). To each well,150 ll of DMSO was added and incubated for 30 min. Colour inten-sity was measured at 540 nm in ELX800 Universal MicroplateReader [39].

2.8.3. Cellular integrity (LDH release) assayA549 cells were maintained in 96 well plates for 24 has men-

tioned above. Later supernatant from each well was collectedand activity levels of LDH were assayed with commercially avail-able kit (Reckon diagnostics Ltd., Mumbai, India) usingMerckmicrolab L 300 semi-auto-analyzer and% cytotoxicity was calcu-lated [40].

2.8.4. Mitochondrial membrane potential (DWmt)The changes in mitochondrial membrane potential were mea-

sured using the fluorescent cationic dye Rhodamine 123 (RHO123). Cells were treated with the metal complexes for 24 h and la-ter, cells were incubated with 1 lM RHO 123 for 10 min at 37 �C.The fluorescence was measured (excitation-485 and emission-530 nm) using spectroflurometer (Jasco FP-6300, Japan) and ex-pressed as fluorescence intensity units (FIU) [41].

2.8.5. Intracellular ROS generation (DCFDA staining)A549 cells were cultured as mentioned above and after 18 h of

treatment with metal complexes, cells were incubated with 7.5 lM2,7-dichlorodihydrofluoroscein diacetate (CM–H2–DCFDA) at 37 �Cfor 30 min. Cells were observed in a Leica DMRB fluorescent micro-scope [42].

2.8.6. Nuclear morphology assay (DAPI staining)Cells (5 � 104 cells/well) were plated into 6-well plate and al-

lowed to achieve 80% confluence. Later, cells were treated withor without different concentrations of the metal complexes at37 �C for 24 h. Single-cell suspensions of treated cells were washedwith PBS and fixed with 70% ethanol, re-washed with PBS andincubated with DAPI (0.6 lg/mL in PBS) for 5 min. Treated cellswere examined to assess changes in nuclear morphology (con-densed/fragmented nuclei) and photographed under Leica DMRBfluorescence microscope.

2.8.7. Assessment of apoptosis2.8.7.1. AO/EB staining. A549 cells (1 � 105 cells/well) were plated 6well plates for 24 h and later on treated as mentioned earlier. Atthe end of experiment period, cells were collected using TPVG solu-tion. Later, 9 ll of cell suspension (0.5 � 106 cells/ml) was taken ona clean microscope slide and stained with dye mixture (1 mg/ml


AO and 1 mg/ml EB in PBS) and photographed under Leica DMRBfluorescence microscope.

2.8.7.2. FITC Annexin-V/PI Staining. Annexin- V FITC/Propidium io-dide double staining assay was used to quantify apoptosis, accord-ing to the manufacturer’s protocol (Invitrogen, UK). Afterincubation, cells were harvested using TPVG solution and washedwith ice-cold PBS and suspended in 100 ll of 1� binding buffer(10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). To thismixture, 5 ll of annexin V-FITC conjugate and 1 ll of propidium io-dide solution were added to each cell suspension and incubated for15 min at room temperature in the dark. Later, samples were ana-lyzed on flow cytometer (BD FACS Aria III, USA) using Flow Jo (Ore-gon, USA). Double staining of cells with FITC Annexin-V and PIenables the discrimination of live cells (FITC � PI�), early apoptotic(FITC + PI�), late apoptotic (FITC + PI+) or necrotic cells (FITC � PI+).

2.8.8. Cell cycle analysisCells (1 � 106 cells/well) were cultured and treated as men-

tioned earlier for 24 h. Later, cells were washed once in ice-coldPBS and subjected to cell cycle analysis [43]. Briefly, 1 � 105 cellswere fixed in 4.5 ml of 70% (v/v) cold ethanol for 30 min, centri-fuged at 400g for 5 min. Supernatant was removed and cells werewashed with 5 ml of PBS. Cells were then re-suspended in 0.5 ml ofPBS and 0.5 ml of DNA extraction buffer (Mix 192 ml of 0.2 M Na2-

HPO4 with 8 ml of 0.1% Triton X-100 v/v) was added. The pH wasadjusted to 7.8. Cells were incubated at room temperature for5 min and then centrifuged at 400 g for 5 min. Supernatant wasdiscarded and cells were re-suspended in 1 ml of DNA stainingsolution (200 mg of PI in 10 ml of PBS + 2 mg of DNase free RNase).Cells are then incubated for at least 30 min at room temperature inthe dark and the cell cycle distribution was then analyzed on a flowcytometer (BD FACS Aria III, USA) using FlowJo (Oregon, USA).

2.8.9. Statistical analysisData was analyzed for statistical significance using one way

analysis of variance (ANOVA) followed by Bonferroni’s multiplecomparison test and results were ex-pressed as mean ± SEM usingGraph Pad Prism version 3.0 for Windows, Graph Pad Software, SanDiego, California, USA.

3. Results and discussion

3.1. Synthesis and characterization of ligands

On the basis of the relate Ref. [31], 4-toluoyl pyrazolone wassynthesized in a facile large-scale from commercially available 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (PMP) and p-toluoyl chlo-ride in the presence of Ca(OH)2 with the conventional C4-acylationunder acidic conditions afforded 5-methyl-4-(4-methyl-benzoyl)-2-phenyl-2,4-dihydro-pyrazol-3-one (TPMP) in reasonably goodyield. Further, Schiff base ligand (TPMP-BA) was also preparedaccording to established synthetic procedure by the condensationof TPMP with p-bromo aniline in an ethanolic medium [31(c)].Both the ligands were structurally characterized by elementalanalyses, IR, 1H NMR, 13C NMR and EI-Mass, which were in goodagreement with the proposed structures. The Schiff base ligandTPMP-BA was characterized by single-crystal X-ray study [31(c)].The different spectra of the ligands are presented in Figs. S2–S6.

3.2. Synthesis and characterization of Cu(II) complexes

The present work stems from our interest in designing new bin-ary and ternary Cu(II) complexes. On the basis of the results of ourprevious work, here we have synthesized two new ternary and one

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binary Cu(II) complexes. The ternary complexes 1 and 2 were syn-thesized according to the new synthetic procedure. Reaction ofTPMP and 1,10-phenanthroline (for complex 2)/2,20-bipyridyl (forcomplex 3) with Cu(NO3)2�3H2O in presence of NH4NCS in metha-nolic medium at pH � 7.5 gave neutral ternary Cu(II) complexeswith high yield. Further, the binary complex was prepared by themodified method where, the reaction was carried out betweenTPMP-BA and Cu(OAc)2�H2O in a dry ethanolic medium to getgreenish microcrystalline solid complex 3.

The complexes 1–3 are air-stable, nonhygroscopic, green crys-talline solids, soluble in common organic solvents like methanol,ethanol, dichloromethane, chloroform, dimethyl formamide,dimethylsulfoxide and insoluble in diethyl ether, petroleum etherand hexane. Characterization of the complexes has been achievedby satisfactory elemental analyses, metal estimation, molar con-ductance and spectral analyses [IR, ESI-Mass, UV–Vis]. Further,structures of all the three complexes have been authenticated byX-ray single crystal analyses. FT-IR spectra exhibited characteristicbands assignable to m(C@N, cyclic) along with bands due to othermoieties and suggested coordination of pyrazolone based ligandswith respective metal centre. A new band that appears at around1470 cm�1, is assigned to the v(C–O) in the IR spectrum of all thecomplexes, which supports the observation of its enolization ofC@O of pyrazolone ring during coordination. These overall datasuggest that the azomethane-N and enol-O groups are involvedin coordination with the Cu(II) ion during complexation. In thelow frequency region, spectrum of the complex exhibits new bandswhich are not present in the spectrum of the ligand. These bandsare recognized to v(Cu–N) and v(Cu–O). Bands at ca. 1518, 1425and 721 cm�1 in the ternary complexes 2 and 3, assigned tov(C@N), v(C@C) and out-of-plane –CH stretching vibrations of 1,10-phenanthroline and 2,20-bipyridyl, respectively confirm thepresence of phen/bipy ligand in the coordination sphere of thecomplex. Formation of metal complex was also confirmed by ESI-Mass spectrometry. The mass spectra of complex 1 showed peakswith m/z 534.2 [MIP, Basepeak]+, 535.3 [M+1]+ and 537.3 [M+3]+-

indicating that one thiocyanate ligand was easily disassociated(Fig. S7). The mass spectrum of complex 3 showed peaks at m/z954 [MIP]+, 951 [M�3]+, 154 [Basepeak]+ which were attributedto [C48H38Br2CuN6O2]+, [C48H38Br2CuN6O2]+ and [C7H11N3O]+,respectively (Fig. S8). In addition to these peaks, other peaks wereattributed to the further fragmentation of the complexes. All thecomplexes exhibited a band in the Visible region (400–800 nm)attributed to the d–d transitions.

3.3. Crystal structure description of complexes 1–3

3.3.1. Ternary complexes 1 and 2The molecular structure of complexes 1 and 2 together with the

atom-numbering scheme is illustrated in Fig. 1A and B,respectively.

To obtain the quantitative degree of distortion of the copperpolyhedron the ratio between the two basal angles, defined ass = [(h � u)/60], that represents the trigonal distortion from squarepyramidal geometry [44] was used. For an ideal SP, s is 0 while foran ideal TBP s is 1. As these complexes are pentacoordinated, s val-ues have been calculated for all the complexes. Therefore we canfind out the degree of distortion of the polyhedron from SP to TBP.

The structure of the complex 1 consists of two discrete mono-meric Cu(II) species in the unit cell of the triclinic crystal systembelonging to the space group P1 with the metal in a (4 + 1)square-pyramidal CuN3O2 coordination geometry and two latticeacetonitrile molecules. The TPMP and phen ligands are coordinatedin a bidentate fashion to the metal ion Cu1 through oxygen (O1 andO2 of pyrazolone) and nitrogen (N1 and N2 of phen) atoms formingthe equatorial plane. The NCS- molecule is coordinated to Cu1 via


nitrogen atom (Cu1� � �N5 = 2.139(2) Å) at the axial position, gener-ating neutral distorted square pyramidal complex in the samecrystal lattice. Unlike in crystals of the complex, there is no H-bondnetwork, as there are no solvent water molecules or anions in thelattice. The bond angles and bond lengths are in good agreementwith the earlier reports for square pyramidal Cu(II) complexes[45,46].

The structure of the complex 2 also consists of two discretemonomeric Cu(II) species in the unit cell of the monoclinic crystalsystem belonging to the space group C2/c with the metal in a(4 + 1) square-pyramidal CuN3O2 coordination geometry. TheTPMP and bipy ligands are coordinated in a bidentate fashion tothe metal ion Cu1 through oxygen (O1 and O2 of pyrazolone)and nitrogen (N21 and N32 of bipy) atoms forming the equatorialplane. The NCS-molecule is coordinated to Cu1 via nitrogen atom(Cu1� � �N3 = 2.442(4) Å) at the axial position, generating neutralsquare pyramidal complex in the same crystal lattice. The bond an-gles and bond lengths are in good agreement with the earlier re-ports for square pyramidal Cu(II) complexes [45,46].

For complex 1 (h = 162.13 and u = 160.79) and complex 2(h = 168.88 and u = 167.04) the relevant angles yield s values of0.022 and 0.018, respectively which are almost near to 0 and itindicates a geometry close to SP. The coordination spheres are ofthe type Cu1–N1–N2–N5–O2–O1 (complex 1) and Cu1–N21–N32–N3–O2–O1 (complex 2), exhibiting geometry near to squarepyramidal (SP).

3.3.2. Binary complex 3ORTEP diagram of complex 3 with the atom numbering scheme

is shown in Fig. 1(C). In the complex 3, the four-coordinated copperatom is arranged in a distorted square-planar geometry where thetwo ligands acting as monoanionicbidentate N,O-chelators lie inthe trans-conformation to create two stable delocalized six-mem-bered chelate rings (Cu–O–C–C–C–N), with O–Cu–O and N–Cu–Nangles 147.74(16) and 148.28(19) Å, respectively [47].

X-ray data indicates that the oxygen atom of both pyrazolonerings is oriented toward the metal centre (Cu). However, the inter-atomic distances C@O1� � �Cu and C@O2� � �Cu of 1.907(4) and1.917(4) Å, respectively [48] are little large to be considered a coor-dination bond (Fig. 1C). Furthermore, the tetrahedral geometry ishighly distorted, as evidenced by the 41 Å dihedral angle betweenthe planes formed by the Cu(1)–O(1)–N(1) and Cu(1)–O(2)–N(4)planes, being more precisely an intermediate structure between atetrahedral and a square planar arrangements.

3.4. DNA binding studies

3.4.1. Absorption titrationElectronic absorption spectroscopy is one of the most widely

used techniques to follow the interaction of metal complexes withDNA. The binding of metal complexes with DNA usually takes placethrough both covalent and/or noncovalent interactions [49,50]. Incovalent interactions the labile group of complexes is replaced bya nitrogen donor atom from the nucleotide, while noncovalentinteractions occur via intercalative, electrostatic, and groove bind-ing. It has been observed that strong stacking interaction betweenthe complexes and DNA base pairs/contraction of DNA helix/con-formational changes lead to hypochromism. On the other handhyperchromism in absorption bands indicate minor groove bind-ing, unwinding of DNA double helix, simultaneous exposure ofthe DNA bases and damage to the DNA double helix [49,51]. There-fore, absorption titration studies have been made to investigate thebinding mode, intrinsic equilibrium binding constant (Kb) andbinding site size (s) for the complexes with CT-DNA [49,52].

The absorption spectra of 1–3 (20 lM) in the presence of differ-ent concentrations of CT-DNA (5–100 lM) are shown in Fig. 2 and

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Table 2DNA and protein binding parameters.

DNA binding parameters (M�1)

Compound Kb Ksv kq Kapp

TPMP – 0.9 � 103 1.0 � 1011 –TPMP-BA – 1.0 � 103 1.0 � 1011 –Phen – 0.8 � 103 1.0 � 1011 –Bipy – 0.7 � 103 1.0 � 1011 –Complex 1 2.1 � 10�4 6.0 � 103 6.0 � 1011 3.80 � 1011

Complex 2 1.8 � 10�4 5.8 � 103 5.8 � 1011 2.20 � 1011

Complex 3 3.2 � 10�4 8.0 � 103 8.0 � 1011 4.95 � 1011

Protein binding parameters

Ksv M�1 Kbin M�1 n

TPMP 0.83 � 105 0.85 � 106 0.698TPMP-BA 0.55 � 105 0.92 � 106 0.741Phen 0.37 � 105 0.91 � 106 0.562Bipy 0.45 � 105 0.84 � 106 0.423Complex 1 0.21 � 106 5.35 � 106 0.993Complex 2 0.19 � 106 4.16 � 106 0.842Complex 3 0.23 � 106 8.81 � 106 1.042


the resulting data summarized in Table 2. In the UV spectra of thecomplexes, the intense absorption band observed is attributed tothe intraligand transition of the characteristic groups of the coordi-nated ligand. Any interaction between complex and DNA couldperturb the intraligand-centered spectral transitions as observedin the UV spectra of a 20 lM solution of the complexes upon addi-tion of DNA at different concentrations. In the UV region, complexexhibitedintense absorption bands around 270 nm, which was as-signed to p?p⁄ transition of aromatic chromophore. With increas-ing concentration CT-DNA (5–100 lM), the absorption bands ofcomplex are affected, exhibiting hyperchromism in p?p⁄ transi-tion of complex. A strong hyperchromic effect in p?p⁄ transitionwas observed for complexes, suggesting that these complexes pos-sess higher propensity for DNA binding.

Overall results suggested that although 1–3 interact consider-ably with CT DNA, the extent of interaction is different for 1 and2 (may be weaker) relative to 3. Intrinsic equilibrium binding con-stants (Kb) have also been derived (2.1 � 10�4, 1.8 � 10�4 and3.2 � 10�4 for complexes 1–3, respectively) and these are consis-tent with other reports [50]. High binding constant (Kb) for 3 indi-cated its greater affinity for DNA that may be attributed to sterichindrance imposed by coordinated ligands. Lower values also indi-cated partial intercalative binding and/or surface binding of thecomplexes to DNA. The intrinsic binding constants suggested thatbinding affinity of complexes lies in the order of 3 > 1 > 2.

3.4.2. Competitive binding with Ethidium Bromide [EB]Steady-state competitive binding experiments using ligands

and their complexes as quenchers were undertaken to get finalproof for the binding of the compounds to DNA via intercalation.Ethidium bromide (EB) is a planar cationic dye which is widelyused as a sensitive fluorescence probe for native DNA. EB emits in-tense fluorescent light in the presence of DNA due to its strongintercalation between the adjacent DNA base pairs. The displace-ment technique is based on the decrease of fluorescence resultingfrom the displacement of EB from a DNA sequence by a quencher,and the quenching is due to the reduction of the number of bindingsites on the DNA that are available to the EB. The fluorescencequenching spectra of DNA-bound EB by ligands and their com-plexes shown in Fig. 3 illustrate that, as the concentration of thecompounds increases, the emission band at 610 nm exhibited hyp-ochromism of the initial fluorescence intensity for the ligands andtheir complexes. The observed decrease in the fluorescence inten-sity clearly indicates that the EB molecules are displaced from theirDNA binding sites and are replaced by the compounds under inves-tigation [6(b),53]. Quenching data were analyzed according to theStern–Volmer equation.

Fig. 2. Changes in the electronic absorption spectra of complexes (20 lM) with increachanges on addition of the CT-DNA.


The Ksv value is obtained as a slope from the plot of F0/F versus[Q]. The quenching plots illustrate that the quenching of EB boundto CT-DNA by free ligands and the Cu(II) complexes are in goodagreement with the linear Stern–Volmer equation. In the Stern–Volmer plots of F0/F versus [Q] (Fig. 4), the Ksv values for TPMP,TPMP-BA, Bipy, Phen, 1–3 were found to be 0.9 � 103 M�1,1.0 � 103 M�1, 0.7 � 103 M�1, 0.8 � 103 M�1, 6.0 � 103 M�1,5.8 � 103 M�1 and 8.0 � 103 M�1, respectively. Further, the appar-ent binding constant (Kapp) values obtained for the compoundsusing the equation (where the compound concentration has thevalue at a 50% reduction of the fluorescence intensity of EB,KEB = 1.0 � 107 M�1 and [EB] = 3.3 lM) were found to be3.80 � 1011 M�1, 2.20 � 1011 M�1 and 4.95 � 1011 M�1 for 1–3,respectively. These data suggested that the interaction of the Cu(II)complexes with CT-DNA is stronger than that of the free ligands,which is consistent with the above absorption and emission spec-tral observations. Since these changes indicate only one kind ofquenching process, it may be concluded that all of the compoundsbind to CT-DNA via the same mode. Furthermore, such quenchingconstants and binding constants of the ligands and Cu(II) com-plexes suggest that the interaction of all of the compounds withDNA should be intercalation [54–56].

On the basis of all of the spectroscopic studies, we concludedthat the free ligands and Cu(II) complexes can bind to CT-DNA inan intercalative mode and that Cu(II) complexes bind to CT-DNAmore strongly than the free ligands. Among the two ligands, the

sing concentrations (5–100 lM) of CT-DNA (Tris, pH 7.04). The arrow shows the

x.doi.org/10.1016/j.poly.2013.12.037


Fig. 3. Emission spectra (kem = 610 nm) of DNA–EB complex in buffer solution in the absence and presence of the increasing amount of ligands and their complexes The arrowshows the changes of intensity upon the increasing amount of compound.


one which has relatively more planar area and aromatic conjuga-tion due to the presence of a phenyl rings in the C(4) position ofTPMP-BA showed greater binding affinity to DNA over the other li-gand, TPMP. The same phenomenon has also been used to obtainthe reason for the higher binding activity of complex 3 over 1and 2, because the two ligands are coordinated as a monoanionic-bidentate to Cu(II) in complex 3. Hence, more hydrophobicity ofcomplex 3 is likely to be the reason for the observed highest affin-ity of 3 with DNA over 1 and 2.

Fig. 4. Plot of F0/F vs. concentration for complexes and ligands in buffer solution on in(kem = 610 nm).


3.5. Protein binding studies

Qualitative analysis of the binding of chemical compounds toBSA is usually detected by inspecting the fluorescence spectra.Generally, the fluorescence of BSA is caused by two intrinsic char-acteristics of the protein, namely tryptophan and tyrosine. Changesin the emission spectra of tryptophan are common in response toprotein conformational transitions, subunit associations, substratebinding or denaturation. Therefore, the intrinsic fluorescence of

creasing amount of DNA (150 mM NaCl and 15 mM trisodium citrate at pH 7.04)

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Fig. 5. Changes in the fluorescence spectra of BSA in buffer solution on increasing amount of complexes and ligands (150 mM NaCl and 15 mM trisodium citrate at pH 7.04).


BSA can provide considerable information on their structure anddynamics and is often utilized in the study of protein folding andassociation reactions[6(b)].The interaction of BSA with our ligandsand complexes (1–3) was studied by fluorescence measurement atroom temperature. A solution of BSA (5 lM) was titrated with var-ious concentrations of the compound (0–20 lM). Fluorescencespectra were recorded in the range of 300–500 nm upon excitationat 296 nm. The effects of the compound on the fluorescence emis-sion spectrum of BSA are shown in Fig. 5. The addition of the abovecompounds to the solution of BSA resulted in a significant decreaseof the fluorescence intensity of BSA at 343 nm, up to 75% from theinitial fluorescence intensity of BSA. This result suggested a defi-

Fig. 6. Plot of F0/F vs. concentration of BSA in buffer solution on increasing amount o


nite interaction of all of the compounds with the BSA protein[56,57].

Quenching can occur by different mechanisms, which are usu-ally classified as dynamic quenching and static quenching; dy-namic quenching refers to a process in which the fluorophoreand the quencher come into contact during the transient existenceof the excited state. Static quenching refers to fluoro-phore�quencher complex formation in the ground state.

To study the quenching process further, fluorescence quenchingdata were analyzed with the Stern–Volmer equation and Scatchardequation. The quenching constant (Ksv) can be calculated using theplot of F0/F versus [Q] (Fig. 6). If it is assumed that the binding of

f complexes and ligands (150 mM NaCl and 15 mM trisodium citrate at pH 7.04).

x.doi.org/10.1016/j.poly.2013.12.037


LDH release

0 0.079 0.1570

25

50

75

100123

ns ns ns

******

**

***

***

***

Concentration (µM)

Enzy

me

activ

ity

Fig. 8. Effect of Complexes 1–3exposed to A549 cells on LDH release. Results areexpressed as mean ± SEM for n = 3 (replicates). Where, ns p > 0.05, ⁄p < 0.05,⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to 0 lM [complex].

Mitochondrial membranepotential

0 0.079 0.1570

100

200

300

400

500123

ns ns ns

******

***

******

***

Concentration (µM)

Fluo

resc

ent i

nten

sity

Fig. 9. Effect of complexes exposed to A549 cells on mitochondrial membranepotential. Results are expressed as mean ± SEM for n = 3 (replicates). Where, nsp > 0.05, ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to 0 lM complexes.


compounds with BSA occurs at equilibrium, the equilibrium bind-ing constant can be analyzed according to the Scatchard equation:

log½ðI0 � IÞ=I� ¼ log Kbin þ n log½Q �

where Kbin is the binding constant of the compound with DNA and nis the number of binding sites. From the plot of log(I0 � I)/I versuslog[Q] (Fig. 6), the number of binding sites (n) and the binding con-stant (Kbin) have been obtained. The calculated Ksv, Kbin, and n val-ues are given in Table 2. The calculated value of n is around 1 forall of the complexes, indicating the existence of just a single bindingsite in BSA for all of the complexes. The values of Ksv and Kbin for allof the compounds suggested that the complexes interact with BSAmore strongly than the ligands. Among the three Cu(II) complexes,the binary complex 3 has better interaction with BSA than the othertwo ternary complexes (1 and 2). This can explained by the fact thatthe hydrophobicity of 3 having phenyl substitution in its terminalnitrogen is greater than that of 1 and 2; among the Cu(II) com-plexes, the complex of Schiff base pyrazolone (3) have more hydro-phobicity over that of complexes 1 and 2.

3.6. Evaluation of in vitro cytotoxicity of the synthesized metalcomplexes

Cytotoxic activity of the synthesized metal complexes was as-sessed against A549 lung carcinoma cells and H9C2 cardiomyo-blasts cells (Fig. 7). Results indicated that complexes 1–3 showeddose dependent cytotoxicity against A549 cells with complex 3accounting for maximum mortality at 0.157 lM. Complexes 1and 2 also recorded <50% mortality at the same dose. These resultsindicated that complex 3 was a potent cytotoxic agent againstA549 lung cancer cells. These results were well corroborated withthe LDH (Lactate Dehydrogenase) assay performed with A549 cellsthat indicated maximum release of LDH enzyme following com-plex 3 treatment at the highest dose. Release of LDH is a well-known indicator of cell damage and it was observed in our studythat all three test complexes 1–3 could induce significantly ele-vated LDH release (Fig. 8).

Previous studies have shown that pyrazolone based ligands ac-count for cytotoxic properties [58]. In the present study, chelationof Cu(II) by pyrazolone based ligands can be the possible causativeagent for the observed cytotoxicity. Same set of experiments withthe synthesized metal complexes 1–3 was also performed usingH9C2 rat cardiomyoblasts. These results showed a maximum of5% cytotoxicity at 0.157 lM. A comparison of these results con-firms that the synthesized metal complexes are very specific forcancer cells but are nontoxic to the normal somatic cells as ob-served herein. The major challenge of synthesizing new chemo-therapeutic agents is to achieve selective cytotoxicity towardscancer cells with minimal damage to the normal cells. Results

0.0100 0.0520 0.1040 0.15700

25

50

75

100123

MTT assay A549 cells

***

******

***

***

******

***

*ns ns ns

Concentration (µM)

% M

orta

lity

Fig. 7. Effect of Complexes 1–3 exposed to A549 and H9C2 cells on cell


obtained in the present study established the selectivity of synthe-sized metal complexes against cancer cells thus, making them suit-able for a detailed study to assess their potential as anti-canceragents. The overall cytotoxic behavior of the complexes is verysimilar to that of the DNA binding activity by them discussed ear-lier, indicating better activity for 3.

3.6.1. Apoptosis detection by mitochondrial membrane potentialanalysis

Apoptosis (programme cell death) is associated with normalgrowth and development and its rate varies in response to variousexternal and internal physiological stimuli in a controlled manner[11]. Apoptosis differs from necrosis, which is accidental andevidenced by lysis of cell. Hence, ability of a drug or the synthesized

MTT assay H9C2 cells

0.0100 0.0520 0.1040 0.15700

5

10

15

20123

ns ns nsns

nsnsns

nsns

ns ns ns

Concentration (µM)

% m

orta

lity

viability. Results are expressed as mean ± SEM for n = 3 (replicates).

x.doi.org/10.1016/j.poly.2013.12.037


Fig. 10. Florescence photomicrographs of A549 cells exposed to complexes 1–3. DCFDA stained cells with more fluorescent intensity indicates higher oxidative stress.

Fig. 11. Florescence photomicrographs of assessment of nuclear morphology using DAPI staining of A549 cells exposed to complexes 1–3.

Fig. 13. Annexin V-Alexafluro 488/PI staining of A549 cells exposed to complex 3.


metal complexes to induce apoptosis is prerequisite for establish-ing its efficacy as anti-tumor or anti-cancer agent [59]. In the pres-ent study, alteration in mitochondrial membrane potential wasassessed by Rhodamine 123 stain. Mitochondria, a key organellethat controls energy metabolism of a cell undergoes loss of itsmembrane integrity resulting in translocation of cytochrome C intocytoplasm. Such a scenario leads to a decrement in its membranepotential that is assessed in the form of reduced fluorescenceintensity on a fluorimeter. All the synthesized metal complexes1–3 showed significant decrement in Mitochondrial Membrane Po-tential at 0.157 lM (Fig. 9). Other reports have shown that cyto-chrome C binds APAF1 protein to form an apoptosome complexthat activates Caspase protein cascade and apoptosis [11]. Obser-vations recorded in our study clearly indicate induction of apopto-sis induced cell death.

3.6.2. Fluorescence staining methods to assess cell deathTo gain a deeper insight in the mechanism of cell death, the

control and treated cancer cells were stained with a variety of fluo-rescent stains to establish the possible underlying mechanism.Generation of intracellular ROS resulting due to the synthesizedmetal complexes treatment was studied using a fluorescent probeDCFH-DA. Results showed that all three metal complexes showedstrong fluorescence intensity of DCF indicating at the synthesized

Fig. 12. Florescence photomicrographs (AO/EtBr Stained cells) of A549 cells exposed toorange and red cells indicates loss of viability. (Color online.)


metal complexes induced heightened intracellular oxidative stress(Fig. 10). The control and treated cells were then stained with DAPI,a nucleus specific dye that provides visual evidences of possibleblebbing/fragmentation/distortion of nuclei. In the present study,the synthesized metal complexes were instrumental in inducingclear distortion of nuclear characteristics compared to the controlcells (Fig. 11). Control and treated cells were also stained with Acri-dine orange/Ethidium bromide (AO/EB) to observe alterations incytological and nuclear morphologies. Viable nuclei appear uni-form green fluorescence with highly organized nuclear characteris-tics. However, the late apoptotic cells show orange to redfluorescence in their nuclei with a condensed chromatin [60]. Inthe present study, the treated cells showed prominent apoptoticnuclei as compared to the control cells (Fig. 12).

complexes 1–3. Green fluorescence in cells indicates viability whereas, yellowish

x.doi.org/10.1016/j.poly.2013.12.037


Fig. 14. Cell cycle analysis of A549 cellsexposed to complexes 1–3.


Results obtained in the staining techniques clearly indicatedthat complex 3 was the most potent in inducing apoptosis as com-pared to 1 and 2. Hence, complex 3 was chosen for Annexin V-Alexafluoro 488/PI staining to differentiate the early, late apoptoticand necrotic cells. Results revealed that complex 3 was instrumen-tal in triggering apoptosis as evidenced by large population of lateapoptotic (green) cells (Fig. 13).

3.6.3. Flow cytometric analysisBased upon encouraging results obtained in fluorescent staining

of cells, it was thought pertinent to assess the cell cycle of A549cancer cells (Fig. 14). Results revealed that the control cells showedhealthy population (93.8%) of cells in G0/G1 phase indicating attheir viability and rate of multiplication. However, complexes 1and 2 showed significantly less (22.2% and 24.6%, respectively)cells in G0/G1 phase at 0.157 lM dose whereas, a larger populationof cells (77.1% and74.5%, respectively) were found to be in sub G0/G1 phase indicating programmed cell death. Complex 3 recordedmaximum (98.3%) cells in sub G0/G1 phase that further impliesto its ability to induce apoptosis in cancer cells. A superior cyto-toxic property of complex 3 may be attributed to its extended pla-nar structure induced by the p–p⁄ conjugation resulting from thechelation of Cu(II) with the pyrazolone ligand.

4. Conclusion

The pyrazolone based ligands and their binary as well as ternarycomplexes (with phen and bipy) have been prepared and wellcharacterized by analytical, spectral and structural analyses. Thecrystal structure of all the complexes show the ligands are coordi-nated to Cu(II) in a bidentate manner. The DNA binding propertiesof the free ligands and three Cu(II) complexes were investigated byabsorption and fluorescence measurements. The results supportedthe fact that the compounds bind to CT-DNA via intercalation. Thebinding constants showed that the DNA binding affinity increased


in the order 2 < 1 < 3. The protein binding properties of the com-pounds examined by the fluorescence spectra suggested that thebinding affinity increases in the Schiff base moiety in Cu(II) com-plexes, which could be explained on the basis of the interactionsof the compounds with BSA. These results also show the order ofbinding is 2 < 1 < 3. The synthesized metal complexes exhibitanti-cancer activities towards human lung cancer cells (A549 cells)as compared to the normal cardiomyoblasts cells (H9C2 cells).Anti-cancer property of complexes 1–3 was established througha series of fluorescent staining and cell cycle analysis protocols. Itcan be concluded that the synthesized metal complexes 1–3 couldalter the Mitochondrial Membrane Potential causing mitochondrialdysfunction. Also, these complexes could elevate intracellular oxi-dative stress, alter nuclear morphology and induce apoptosis. Con-clusive evidence on apoptosis were established through cell cycleanalysis that confirmed maximum percentage of cells in sub G0/G1 phase. This study also revealed that complex 3 was most potentas an anti-cancer agent as compared to the complexes 1 and 2.These results are encouraging and warrant a detailed in vivo studyto establish anti-cancer property of these complexes.

Acknowledgments

Financial assistance received from the University Grants Com-mission in terms of major research project to RNJ, New Delhi isgratefully acknowledged. We would like to thank Head, Depart-ment of Chemistry and Head, Department of Zoology for providingnecessary facilities required to carry out this work.

Appendix A. Supplementary data

CCDC 842200, 865376 and 868303 contain the supplementarycrystallographic data for complexes 1–3, respectively. These datacan be obtained free of charge via http://www.ccdc.cam.ac.uk/con-ts/retrieving.html, or from the Cambridge Crystallographic Data

x.doi.org/10.1016/j.poly.2013.12.037

http://www.ccdc.cam.ac.uk/conts/retrieving.html

http://www.ccdc.cam.ac.uk/conts/retrieving.html



Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223-336-033; or e-mail: [email protected]. Supplementary dataassociated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.poly.2013.12.037.

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