Journal of Inorganic Biochemistry - LABEN · Cytotoxicity of Ru(II) piano–stool complexes with chloroquine and chelating ligands againstbreastandlungtumorcells:InteractionswithDNAandBSA

Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

JIB-09769; No of Pages 12

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Cytotoxicity of Ru(II) piano–stool complexes with chloroquine and chelating ligandsagainst breast and lung tumor cells: Interactions with DNA and BSA

Legna Colina-Vegas a,⁎, Wilmer Villarreal a, Maribel Navarro b, Clayton Rodrigues de Oliveira a,Angélica E. Graminha a, Pedro Ivo da S. Maia c, Victor M. Deflon c, Antonio G. Ferreira a,Marcia Regina Cominetti d, Alzir A. Batista a,⁎a Departamento de Química, Universidade Federal de São Carlos— UFSCar, CEP 13565-905 São Carlos, SP, Brazilb Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO, CEP 25250-020 Duque de Caxias, RJ, Brazilc Instituto de Química de São Carlos, Universidade de São Paulo, CEP 13560-970 São Carlos, SP, Brazild Departamento de Gerontologia, Universidade Federal de São Carlos— UFSCar, CEP 13565-905 São Carlos, SP Brazil

⁎ Corresponding authors.E-mail addresses: [email protected] (L. Colina-Ve

(A.A. Batista).

http://dx.doi.org/10.1016/j.jinorgbio.2015.07.0160162-0134/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: L. Colina-Vegas, ebreast and lung tumor cells: Interac..., J. Inor

a b s t r a c t
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Article history:Received 5 April 2015Received in revised form 6 July 2015Accepted 15 July 2015Available online xxxx

Keywords:CytotoxicityChloroquineArene–rutheniumDNABSA

The synthesis and spectroscopic characterization of nine π-arene piano–stool ruthenium (II) complexeswith aromatic dinitrogen chelating ligands or containing chloroquine (CQ), are described in this study:[Ru(η6-C10H14)(phen)Cl]PF6 (1), [Ru(η6-C10H14)(dphphen)Cl]PF6 (2), [Ru(η6-C10H14)(bipy)Cl]PF6 (3), [Ru(η6-C10H14)(dmebipy)Cl]PF6 (4) and [Ru(η6-C10H14)(bdutbipy)Cl]PF6 (5), [Ru(η6-C10H14)(phen)CQ](PF6)2 (6),[Ru(η6-C10H14)(dphphen)CQ](PF6)2 (7), [Ru(η6-C10H14)(bipy)CQ](PF6)2 (8), [Ru(η6-C10H14)(dmebipy)CQ](PF6)2(9): [1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (dphphen), 2,2′-bipyridine (bipy), 5,5′-dimethyl-2,2′-bipyridine (dmebipy), and 4,4′-di-t-butyl-2,2′-bipyridine (dbutbipy)]. The solid state structuresof five ruthenium complexes (1–5) were determined by X-ray crystallography. Electrochemical experimentswere performed by cyclic voltammetry to estimate the redox potential of the RuII/RuIII couple in each case.Their interactions with DNA and BSA, and activity against four cell lines (L929, A549, MDA-MB-231 and MCF-7) were evaluated. Compounds 2, 6 through 9, interact with DNA which was comparable to the one observedfor free chloroquine. The results of fluorescence titration revealed that these complexes strongly quenched theintrinsic fluorescence of BSA following a static quenching procedure. Binding constants (Kb) and the numberof binding sites (n ~ 1)were calculated usingmodified Stern–Volmer equations. The thermodynamic parametersΔG at different temperatures were calculated and subsequently the values of ΔH and ΔS were also calculated,which revealed that hydrophobic and electrostatic interactions play amajor role in the BSA–complex association.TheMTT assay results indicated that complexes 2, 5 and 7 showed cytostatic effects at appreciably lower concen-trations than those needed for cisplatin, chloroquine and doxorubicin.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Chloroquine diphosphate, a derivative of quinine, was discovered in1934 and it has long been commonly used against different human dis-eases [1–3]. Recently several studies have shown that it has extensivebiological effects, such as inhibiting cell growth and/or inducing deathin different kinds of cells [4]. On the other hand, chloroquine complexesof ruthenium [5], platinum [6], palladium [7] and gold [8] have shownantitumor properties against different types of tumor cells.

The development of half-sandwich η6-arene-Ru(II) complexes hasattracted particular attention and have been extensively studied over

gas), [email protected]

t al., Cytotoxicity of Ru(II) piag. Biochem. (2015), http://dx

the past 20 years, as these complexes show applications in differentareas of scientific and industrial interest. More importantly, in somecases, these complexes present higher antitumor activity and lower tox-icity compared to platinum compounds [9], and therefore are potentialchemotherapeutic agents [10]. Several reports on η6-arene Ru(II) com-plexes with the general formula [Ru(arene)(L–L′)(X)]n+ can be foundin the literature, where L–L′ are neutral bidentate ligands of types:N–O [11], N–S [12], P–P [13], or P–N [14]. These classes of compoundshave exhibited activity in vitro and in vivo against A2780 human ovariancancer cells, with promising results [15]. Furthermore, it is well knownthat N∩N-heterocyclic ligands provide good stability for the structure ofRu(II) complexes, due to their π acceptor properties,which allow for theformation of supramolecular structures. They establish slightly direc-tional non-covalent interactions of the π–π type, which are weakerthan hydrogen bonds, but still strong enough to increase the stabilityof the metal complexes, both in solution and in the solid state [16].

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2 L. Colina-Vegas et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

Usingmodels able to reproduce the chemical behavior of metal–bio-molecule interactions is one of the approaches most commonly used inattempting, not only to reproduce the biological activity, but also to ex-plain the interactions between metals and biomolecules. Their interac-tions with blood proteins, DNA (components) or small biomoleculescan have a protective effect and decrease the rate of degradation orside product formation [17]. Albumin, the most abundant protein inthe circulatory system, has been one of the most extensively studiedof all proteins; it serves as a transporter of a range of molecules suchas fatty acids, vitamins and drugs. Binding a drug to albumin results inincreased drug solubility in plasma, decreased toxicity, as well as pro-tection against oxidation [18].

In this paper we report on the syntheses and characterizationof ruthenium arene cationic complexes containing N–N chelate ligands[N–N=1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline(dphphen), 2,2′-bipyridine (bipy), 5,5′-dimethyl-2,2′-bipyridine(dmebipy), 4,4′-di-t-butyl-2,2′-bipyridine (dbutbipy) and CQ(Scheme 1). These species were chosen in order to determine if thereis any influence of the type of N–N ligand on the arene complex thatcould be reflected in its biological properties. Here, we also presentthe interaction study of the synthesized ruthenium complexes withDNA by titration spectroscopy, viscosity measurements and electro-phoreses, their interaction with BSA by fluorescence quenching andtheir cytotoxicity against four cell lines (L929, MCF-7, A549 andMDA-MB-231).

2. Experimental section

All manipulations were carried out under an argon atmosphereusing standard Schlenk techniques. The electronic spectra were ob-tained via scanning on a Hewlett-Packard diode array model 8452Aspectrophotometer. The microanalyses were performed using aFISIONS CHNS, mod. EA 1108 microanalyzer. The IR spectra wererecorded on a FTIR Bomem-Michelson 102 spectrometer in the4000–200 cm−1 region. Conductivity values were obtained using aMeter Lab CDM2300 instrument.

Scheme 1. Structures of piano–stool ruthenium (II) complexes, identi

Please cite this article as: L. Colina-Vegas, et al., Cytotoxicity of Ru(II) piabreast and lung tumor cells: Interac..., J. Inorg. Biochem. (2015), http://dx

2.1. NMR spectroscopy data

All the 1D and 2D NMR experiments (1H, 13C, 1H–1H gCOSY,1H–13C gHSQC, 1H–13C gHMBC, 1H–15N gHMBC) were recorded on a9.4 T Bruker Avance III spectrometer with a 5 mm internal diameterindirect probe with ATMATM (Automatic Tuning Matching), holdingthe temperature stable at 300 K. In general, 20 mg samples of CQ,free ligands and metal–CQ complexes were dissolved in deuterateddimethylsulfoxide (DMSO-d6, Cambridge Isotope Laboratories, Inc.,USA), containing a trace amount of tretramethylsilane (TMS) thatwas used as an internal reference (0 ppm). The 1H and 13C NMR spec-tra were acquired with 8 and 12 K scans; spectral widths (sw) of8012.820 Hz and 24,038.461 Hz; relaxation delays (d1) of 10 s and0.1 s; acquisition times (aq) of 4.1 s and 0.7 s; data points (td) at64 K and 32 K; and 90° and 30° pulse lengths, respectively. The1H–1H gCOSY, 1H–13C gHSQC, 1H–13C gHMBC, and 1H–15N gHMBCspectra were acquired with 8, 40, 72, and 32 scans and spectralwidths of 6127.451 Hz, 6127.451 Hz, 6127.451 Hz, and8012.820 Hz for F2 and 6127.451 Hz, 23,147.572 Hz, 23,148.150 Hz,and 120,481.930 Hz for F1, respectively. The relaxation delay of 1 s,with 256 data points at F1 and 4 K at F2 were the same in all 2DNMR experiments. The chemical shift in nitromethane (380.2 ppm)was used as an internal reference for the 15N dimension in the1H–15N gHMBC experiment.

2.2. Single crystal X-ray structure data analysis

The data collections were performed with Mo-Kα radiation (λ =71.073 pm) on a BRUKER APEX II duo diffractometer. The structureswere solved by direct methods using SHELXS-97 [19] and refined byfull-matrix least-square methods against F2 (SHELXL-97) [20]. All non-hydrogen atoms were refined with anisotropic displacement parame-ters with SHELXL97. The hydrogen atoms' positions were found inFourier maps or calculated in their idealized positions. Crystallographicdata and experimental details of the structural analysis are summarizedin Table 4 and 1S. (See Table 3.)

fied in the scheme for the purpose of NMR spectroscopy studies.



Table 1Electronic spectra data for complexes in dimethylsulfoxide.

Complex 1 2 3 4 5 6 7 8 9

λ(nm)(ε M−1 cm−1)

274(24,000)

276(28,200)

262(18,890)

262(19,490)

262(18,310)

280(24,480)

294(36,200)

296(18,240)

304(18,710)

292(16,200)

294(31,410)

296(21,440)

302(15,820)

294(24,050)

336(16,150)

334(23,070)

318(15,280)

328(25,520)

344(3380)

356(7845)

354(5650)

354(2390)

348(5000)

348(16,870)

348(23,990)

340(8340)

348(16,910)

402(1995)

402(1675)

402(5250)

400(990)

402(2350)

402(1420)

402(4230)

402(2155)

402(1400)

3L. Colina-Vegas et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

2.3. Electrochemical studies

The electrochemical experiments were performed using a BAS-100B/W MF-9063 Bioanalytical Systems Instrument in nitrogen atmo-sphere at room temperature with tetrabutylammoniumperchlorate(TBAP Fluka Purum) as a supporting electrolyte. The electrochemicalcell was equipped with platinum working and auxiliary electrodes andAg/AgCl as the reference electrode in a Luggin capillary probe, amediumin which ferrocene is oxidized at 0.43 V (Fc+/Fc). Voltammogramswere performed at a scan rate of 0.100 V s−1. The half-wave potentialsfor the reversible cyclic voltammetric processes were estimated by theaverage of the anodic and cathodic peak potentials (E1/2 = (Epa +Epc)/2).

2.4. Synthesis of [RuCl(η6-C10H14)(N–N)]PF6, general procedure

The precursors were synthesized according to a previously reportedmethod [21,22]. A solution of [RuCl2(η6-C10H14)]2 (0.200 g, 0.31 mmol)with an excess of the desired N–N ligand (0.75 mmol) in methanol(25 mL) was stirred for 1 h. NH4PF6 (0.30 g; 1.00 mmol) was added tothis solution, also dissolved in methanol (5 mL), and the mixture wasstirred at room temperature for 1 h longer. The orange-yellow solidthat precipitated was filtered off, washed with cold methanol anddiethyl ether, and dried under vacuum.

2.4.1. [RuCl(η6-C10H14)(1,10-phenanthroline)]PF6 (1)Yellow solid, yield 330 mg (83%). Elemental analysis (%) calcd for

C22H22ClF6N2PRu 1/3H2O C 43.90, H 3.80, N 4.65. Found: C 43.97, H3.86, N 4.47. IR (Selected bands, cm−1) ν (C=C) 1519; ν (C=N)1470; ν (Ru–Cl) 297. Molar conductivity in acetone, ΛM = 133.5 ±0.2 Ω−1 cm2 mol−1. 1H NMR δ ppm (m, I): 9.93 (dd, 2H, J = 5.2,1.1 Hz, H15–H20), 8.93 (dd, 2H, J = 8.2, 1.1 Hz, H13–H18), 8.29 (s, 1H,H21–H22), 8.17 (dd, 2H, J = 8.2, 5.2 Hz, H14–H19), 6.34 (d, 2H, J =6.4 Hz, H6–H8), 6.11 (d, 2H, J = 6.4 Hz, H5–H9), 2.61 (hept, 1H, J =6.9 Hz, H1), 2.10 (s, 3H, H10), 0.91 (d, 6H, J = 6.9 Hz, H2–H3).

Table 215NNMR chemical shifts (δ) for chloroquine (CQ), free ligands andmetal–CQcomplexes inDMSO-d6.

15N chemical Shift (ppm)

N-1 N-2 N-3 N-4

CQ 358.5 341.0 335.8 –1 – – – 355.02 – – – 355.33 – – – 355.64 – – – 355.65 – – – 353.16 347.1 (Δδ = 11.4) 343.8 (Δδ = 2.8) 337.0 (Δδ = 1.2) 355.07 347.2 (Δδ = 11.3) 343.8 (Δδ = 2.8) 337.1 (Δδ = 1.3) 355.38 347.0 (Δδ = 11.5) 343.9 (Δδ = 2.9) 337.0 (Δδ = 1.2) 355.69 347.0 (Δδ = 11.5) 343.9 (Δδ = 2.9) 337.2 (Δδ = 1.4) 355.5 (Δδ = 2.4)


2.4.2. [RuCl(η6-C10H14)(4,7-diphenyl-1,10-phenanthroline)]PF6 (2)Orange solid, yield 380 mg (80%). Elemental analysis (%) calcd for

C34H30ClF6N2PRu 1/2H2O C 53.94, H 4.13, N 3.70. Found: C 53.89, H

4.12, N 4.06. IR (Selected bands, cm−1) ν (C = C) 1519; ν (C = N)1475; ν (Ru–Cl) 302. ΛM (acetone) 134.2 ± 0.2 Ω−1 cm2 mol−1. 1HNMR δ ppm (m, I): 10.01 (d, 2H, J = 5.5 Hz, H15–H20), 8.15 (d, 2H,J=5.5Hz, H14–H19), 8.13 (s, 2H, H21–H22), 7.71 (m, 10, H24–H34, phenylgroup), 6.40 (d, 2H, J= 6.4 Hz, H6–H8), 6.19 (d, 2H, J=6.4 Hz, H5–H9),2.73 (hept, 1H, J = 6.5 Hz, H1), 2.20 (s,3H), 1.02 (d, 6H, J = 6.5 Hz,H2–H3).

2.4.3. [RuCl(η6-C10H14)(2,2′-bipyridine)]PF6 (3)Yellow solid, yield 370 mg (97%). Elemental analysis (%) calcd for

C20H22ClF6N2PRu 1/3H2O: C 41.57, H 3.95, N 4.85. Found: C 41.87, H

4.25, N 4.71. IR (Selected bands, cm−1) ν (C=C) 1498; ν (C=N)1469; ν (Ru–Cl) 302. ΛM (acetone) 135.3 ± 3.0 Ω−1 cm2 mol−1. 1HNMR δ ppm (m, I): 9. 53 (d, 2H, J = 7.9 Hz, H15–H20), 8.64 (d, 2H, J =7.9 Hz,H12–H17), 8.29 (dd, 2H, J = 7.9, 7.9 Hz, H13–H18), 7.80 (dd, 2H,J = 7.9, 7.9 Hz,H14–H19), 6.22 (d, 2H, J = 6.3 Hz, H6–H8), 5.98 (d, 2H,J = 6.3 Hz, H5–H9), 2.58 (hept, 1H, J = 6.9 Hz, H1), 2.18 (s, 3H, H10),0.94 (d, 6H, J = 6.9 Hz, H2–H3).

2.4.4. [RuCl(η6-C10H14)(5,5′-dimethyl-2,2′-bipyridine)]PF6 (4)Yellow solid, yield 350 mg (92%). Elemental analysis (%) calcd for

C22H26ClF6PRu: C 44.04, H 4.37, N 4.67. Found: C 44.21, H 4.43, N 4.40.IR (Selected bands, cm−1) ν (C=C) 1506; ν (C=N) 1477; ν (Ru–Cl)295. ΛM (acetone) 123.0 ± 0.6 Ω−1 cm2 mol−1. 1H NMR δ ppm (m, I):9.35 (s, 2H, H15–H20), 8.46 (d, 2H, J = 8.4 Hz, H12–H17), 8.09 (dd, 2H,J = 8.4, 0.8 Hz, H13–H18), 6.25 (d, 2H, J = 6.4 Hz, H6–H8), 5.97 (d, 2H,J = 6.4 Hz, H5–H9), 2.57 (hept, 1H, J = 6.9 Hz, H1), 2.52 (s, 6H,H21–H22), 2.21(s, 3H, H10), 0.91 (d, 6H, J = 6.9 Hz,H2–H3).

2.4.5. [RuCl(η6-C10H14)(4,4′-t-dibutyl-2,2′-bipyridine)]PF6 (5)Orange solid, yield 340 mg (78%). Elemental analysis (%) calcd for

C29H41ClF6N2PRu C 49.82, H 5.91, N 4.01. Found: C 49.93, H 5.54, N4.41. IR (Selected bands, cm−1) ν (C=C) 1548; ν (C=N) 1477; ν(Ru–Cl) 295. ΛM (acetone) 135.3 ± 1.6 Ω−1 cm2 mol−1. 1H NMR δppm (m, I): 9.39 (d, 2H, J = 6.0 Hz, H15–H20), 8.63 (d, 2H, J = 1.7 Hz,H13–H17), 7.71 (dd, 2H, , J = 6.0, 1.8 Hz, H14–H19), 6.16 (d, 2H, J =6.2 Hz, H6–H8), 5.95 (d, 2H, J = 6.2 Hz, H5–H9), 2.60 (hept, 1H, J =6.8 Hz,H1), 2.14 (s, 3H, H10), 1.43 (s, 18H, H22–H28, t-butyl group),0.97 (d, 6H, J = 6.9 Hz, H2–H3).

2.5. Synthesis of [RuCQ(η6-C10H14)(N–N)][PF6]2, general procedure

A solution of the precursor (0.25 mmol) with an excess of NH4PF6(0.50 mmol) in methanol (25 mL) was stirred for 1 h. CQ (0.50mmol), also dissolved in methanol (10 mL), was added to this solution,and the mixture was stirred under reflux for 24 h. The orange-red solu-tion that was obtained was dried under vacuum, dissolved in dichloro-methane, and the precipitate filtered off. The orange-red solution wasdried under vacuum to obtain an orange-red solid, which was washed



Table 3Electrochemical potentials of ruthenium complexes in acetonitrile.

Compound Epa (I) Epc(II) Epa(V) Epa(III) Epc(IV) Ipa/Ipc(III/IV) E1/2(III/IV)

[RuCl2(η6-C10H14)]2 – – 1.447 1.033 0.980 – 1.001 −0.641 −1.084 1.667 1.057 0.991 1.30 1.012 −0.632 −1.042 1.627 1.048 0.948 1.45 0.993 −0.623 −1.089 1.648 1.051 0.971 1.53 1.014 −0.709 −1.185 1.619 1.044 0.935 1.32 0.985 −0.700 −1.189 1.614 1.029 0.948 1.38 0.986 −0.613 −1.051 1.660 1.132 0.984 1.80 1.057 −0.644 −1.055 1.630 1.136 1.010 2.00 1.078 −0.673 −1.088 1.690 1.156 0.997 1.90 1.079 −0.750 −1.125 1.610 1.206 0.967 1.40 1.08


with diethyl ether (3 × 30 mL) and dichloromethane and dried undervacuum.

2.5.1. [RuCQ(η6-C10H14)(1,10-phenanthroline)][PF6]2 (6)Orange solid, yield 180 mg (70%). Elemental analysis (%) calcd for

C40H48ClF12N5P2Ru 11/3CH2Cl2 C 43.60, H 4.49, N 6.15. Found: C 43.94,H 4.80, N 5.92. IR (Selected bands, cm−1) ν (C=C) 1519; ν (C=N)1460. ΛM (acetone) 175.4 ± 1.3 Ω−1 cm2 mol−1. 1H NMR δ ppm (m,I): 9.93 (dd, 2H, J = 5.2, 1.2 Hz, H15–H20), 8.93 (dd, 1H, J = 8.2, 1.2 Hz,H13–H18) 8.87 (d, 1H, J = 8.3, NH–CQ), 8.65 (d, 1H, J = 9.2 Hz,He–CQ), 8.56 (d, 1H, J = 7.2 Hz, Hb–CQ), 8.30 (s, 2H, H21–H22), 8.17(dd, 2H, J = 8.2, 5.2 Hz, H14–H19), 7.93 (d, 1H, J = 2.0 Hz, Hh–CQ),7.82 (dd, 1H, J = 9.2, 2.0 Hz, Hf–CQ), 6.96 (d, 1H, J = 7.2 Hz, Hc–CQ),6.34 (d, 2H, J = 6.4 Hz, H6–H8), 6.11 (d, 2H, J = 6.4 Hz, H5–H9), 4.36(m, 1H, Ha′–CQ), 3.10 (m, 6H, Hd′–e′–CQ), 2.60 (hept, 1H, J = 6.8 Hz,H1), 2.17 (s,3H, H10), 1.60 (m, 4H, Hb′–c′–CQ), 1.32 (d, 3H, J = 6.4 Hz,Ha″–CQ), 1.17 (t, 6H, Hf′–CQ), 0.90 (d, 6H, J = 6.8 Hz, H2–H3).

2.5.2. [RuCQ(η6-C10H14)(4,7-diphenyl-1,10-phenanthroline)][PF6]2 (7)Orange solid, yield 245 mg (83%). Elemental analysis (%) calcd for

C52H56ClF12N5P2Ru 2CH2Cl2 C 48.14, H 4.49, N 5.20. Found: C 48.41, H4.53, N 5.40. IR (Selected bands, cm−1) ν (C=C) 1518; ν (C=N)1459. ΛM (acetone) 180.4 ± 2.2 Ω−1 cm2 mol−1. 1H NMR δ ppm (m,I): 10.01 (d, 2H, J = 5.6 Hz, H15–H20), 8.85 (d, 1H, J = 8.3 Hz, NH–CQ),8.64 (d, 1H, J = 9.0 Hz, He–CQ), 8.57 (d, 1H, J = 6.9 Hz, Hb–CQ), 8.15(d, 2H, J = 5.6 Hz, H14–H19), 8.13 (s, 2H, H21–H22), 7.92 (d, 1H, J =2.0 Hz, Hh–CQ), 7.83 (dd, 1H, J = 9.0, 2.0 Hz, Hf–CQ), 7.70 (m, 10H,H24–H34, phenyl group), 6.97 (d, 1H, J = 6.9 Hz, Hc–CQ), 6.40 (d,

Table 4Selected bond lengths (Å) and angles (°) in [Ru(η6-C10H14)(phen)Cl]∙PF6 (1), [Ru(η6-C10H14)(dpasymmetric unit), [Ru(η6-C10H14)(dmebipy)Cl]∙PF6 (4), and [Ru(η6-C10H14)(dbutbipy)Cl]∙PF6°0

Compound 1 2a

Bond lengthsRu(1)–Cl(1) 2.3992(9) 2.3795(19)Ru(1)–N(1) 2.095(3) 2.085(5)Ru(1)–N(2) 2.096(3) 2.087(5)Ru(1)–C(4) 2.232(4) 2.179(12)Ru(1)–C(5) 2.189(4) 2.182(12)Ru(1)–C(6) 2.176(4) 2.253(10)Ru(1)–C(7) 2.210(4) 2.208(11)Ru(1)–C(8) 2.198(4) 2.153(14)Ru(1)–C(9) 2.178(3) 2.139(14)

Bond anglesN(2)–Ru(1)–N(1) 77.84(11) 77.19(19)N(2)–Ru(1)–Cl(1) 84.41(8) 84.94(14)N(1)–Ru(1)–Cl(1) 84.22(8) 85.97(16)C(4)–Ru(1)–C(5) 36.89(14) 38.4(4)C(4)–Ru(1)–C(7) 80.80(15) 80.6(5)C(4)–Ru(1)–C(8) 67.70(15) 67.6(6)C(4)–Ru(1)–Cl(1) 96.14(10) 158.6(5)C(2)–C(1)–C(3) 110.0(4) 114.8(15)

* Two molecules in the asymmetricunit.a Disordered η6-C10H14 group with higher occupancy factor.


2H, J = 6.4 Hz, H6–H8), 6.20 (d, 2H, J = 6.4 Hz, H5–H9), 4.15 (m, 1H,Ha′-CQ), 3.10 (m, 6H, Hd′–e′–CQ), 2.73 (hept, 1H, J = 6.4 Hz, H1), 2.20(s,3H, H10), 1.70 (m, 4H, Hb′–c′–CQ), 1.32 (d, 3H, J = 6.4 Hz, Ha″–CQ),1.17 (t, 6H, Hf′–CQ), 1.02 (d, 6H, J = 6.4 Hz, H2–H3).

2.5.3. [RuCQ(η6-C10H14)(2,2′-bipyridine)][PF6]2 (8)Orange solid, yield 215 mg (86%). Elemental analysis (%) calcd for

C38H48ClF12N5P2Ru.3CH2Cl2 C 39.20, H 4.33, N 5.58. Found: C 39.39, H4.65, N 5.89. IR (Selected bands, cm−1) ν (C=C) 1553; ν (C=N)1470. ΛM (acetone) 179.1 ± 1.5 Ω−1 cm2 mol−1. 1H NMR δ ppm (m,I): 9.34 (dd, 2H, J = 8.0, 1.0 Hz, H15–H20), 8.30 (m, 4H, He,b–CQ andH12–H17), 8.20 (m, 2H, H13–H18), 7.90 (d, 1H, J = 2.0 Hz, Hh–CQ), 7.72(m, 3H, Hf–CQ and H14–H19), 7.50 (m, 1H, NH–CQ), 6.82 (d, 1H, J =7.2 Hz, Hc–CQ), 5.93 (d, 2H, J = 6.4 Hz, H6–H8), 5.73 (d, 2H, J =6.4 Hz, H5–H9), 4.07 (m, 1H, Ha′–CQ), 3.13 (m, 6H, Hd′–e′–CQ), 2.66 (m,1H, H1), 2.21 (s,3H, H10), 1.80 (m, 4H, Hb′–c′–CQ), 1.40 (d, 3H, J =6.4 Hz, Ha″–CQ), 1.26 (t, 6H, Hf′–CQ), 1.04 (d, 6H, J = 7.2 Hz, H2–H3).

2.5.4. [RuCQ(η6-C10H14)(5,5′-dimethyl-2,2′-bipyridine)][PF6]2 (9)Orange solid, yield 195 mg (76%). Elemental analysis (%) calcd for

C40H52ClF12N5P2Ru.CH2Cl2 C 44.19, H 4.88, N 6.29. Found: C 44.21, H4.84, N 5.90. IR (Selected bands, cm−1) ν (C=C) 1554; ν (C=N)1477. ΛM (acetone) 175.3 ± 0.6 Ω−1 cm2 mol−1. 1H NMR δ ppm (m,I): 9.34 (d, 2H, H15–H20), 8.86 (d, 1H, J = 8.4 Hz, NH–CQ), 8.65 (d, 2H,He,b–CQ and H12–H17), 8.20 (m, 2H, H13–H18), 7.90 (d, 1H, J = 2.0 Hz,Hh–CQ), 7.72 (m, 3H, Hf–CQ and H14–H19), 6.82 (d, 1H, J = 7.2 Hz,Hc–CQ), 5.93 (d, 2H, J= 6.4 Hz, H6–H8), 5.73 (d, 2H, J= 6.4 Hz, H5–H9),4.07 (m, 1H, Ha′–CQ), 3.13 (m, 6H, Hd′–e′–CQ), 2.66 (m, 1H, H1), 2.21

hphen)Cl]∙PF6∙CH3CH2OCH2CH3 (2). [Ru(η6-C10H14)(bipy)Cl]∙PF6 (3, twomolecules in the.5CH2Cl2 (5).

3A/3B* 4 5

2.4004(9)/2.4000(9) 2.3990(9) 2.3955(12)2.087(3)/2.092(3) 2.088(3) 2.073(3)2.091(3)/2.101(3) 2.088(3) 2.076(3)2.233(3)/2.239(3) 2.196(3) 2.199(5)2.207(3)/2.193(3) 2.170(3) 2.147(5)2.195(3)/2.203(3) 2.204(3) 2.192(5)2.209(3)/2.211(3) 2.234(3) 2.225(5)2.163(3)/2.178(3) 2.195(3) 2.201(5)2.202(3)/2.190(3) 2.191(3) 2.169(4)

77.08(11)/76.73(12) 77.17(11) 76.57(12)83.34(8)/84.90(8) 85.41(7) 84.02(10)85.17(8)/85.76(8) 83.81(8) 84.75(10)

37.39(13)/37.57(13) 37.81(13) 37.98(17)80.53(13)/80.57(13) 81.06(12) 80.8(2)67.63(13)/67.94(13) 68.22(12) 67.63(19)90.61(10)/94.77(9) 158.20(9) 170.33(13)111.6(4)/111.4(4) 111.2(3) 110.3(5)



Fig. 1. Synthetic pathway of ruthenium complexes.


(s,3H,H10), 1.80 (m, 4H,Hb′–c′–CQ), 1.40 (d, 3H, J=6.4Hz,Ha″–CQ), 1.26(t, 6H, Hf′–CQ), 1.04 (d, 6H, J = 7.2 Hz, H2–H3).

2.6. DNA interaction studies

All measurements with DNA (calf thymus (ct)) were taken in a Tris–HCl buffer (5 mM Tris–HCl and 50 mMNaCl, pH 7.4). The DNA concen-tration per nucleotidewas determined by absorption spectrophotomet-ric analysis using a molar absorption coefficient of 6.600 mol−1 L cm−1

at 260 nm [23]. The spectroscopic titrations were carried out by addingincreasing amounts of ctDNA to a solution of the complex in a quartz celland recording the UV–vis spectrum after each addition. The bindingaffinities (Kb1 and Kb2) were obtained by using the Scatchard equation,r/Cf = K(n − 1), corresponding to two major binding interactionswith cooperative effects [24], where r is the number of moles of Rucomplex bound to 1 mol of DNA (Cb/CDNA), n is the number of equiv-alent binding sites, and K is the affinity of the complex for those sites.Concentrations of free (Cf) and bound (Cb) complexes were calculatedfrom Cf = C(1 − α) and Cb = C − Cf, respectively, where C is thetotal Ru concentration. The fraction of bound complex (α)was calculatedfrom α=(Af − A)/(Af − Ab), where Af and Ab are the absorbance of thefree and fully bounddrug at the selectedwavelengths, andA is the absor-bance at any given point during the titration. The plot of r/Cf vs. r givesthe binding constant Kb as the slope of each graph.

Fig. 2. 1H–15N HMBC spectra in dmso-d6 for (A) chloroquine, (B) complex 2 and (C) complex 7.4 K (F2) and 256 (F1) data points; 32 scans and the nitromethane signal at 380.2 ppm as an ex


Viscosity measurements were carried out using an Ostwald vis-cometer immersed in awater bathmaintained at 25 °C. The DNA con-centration in buffer Tris–HCl was kept constant in all samples, while thecomplex concentration was increased from 0 to 60 μM. The flow timewas measured at least 5 times with a digital stopwatch and the meanvalue was calculated. Data are presented as (η/η0)1/3 versus the [com-plex]/[DNA] ratio, where η and η0 are the specific viscosities of DNA inthe presence and absence of the complex, respectively. The values of ηand η0 were calculated using the expression (t − tb)/tb, where t is theobserved flow time and tb is the flow time of buffer alone.

Agarose gel electrophoresis studies. 10 μL aliquots of pBR322 plasmidDNA in Tris–HCl buffer were incubated at 37 °C for 20 hwithmolar ratiosof the Ru compounds between 0.5 and 2.0. After incubation, 5 μL of eachsample were separated by electrophoresis in a 1% agarose gel for90 min at 100 V using Tris-borate–EDTA buffer (TBE) and stained withethidium bromide (5 μL ethidium bromide per 50 mL agarose gel mix-ture). Samples of free DNA and DNA+ DMSO were used as controls.TheDNAbandswere visualized by imagingwithUV light transilluminator(ChemiDoc MP, Bio-Rad).

2.7. BSA-binding experiments

Fluorescence spectroscopy is an effective method to explore the in-teractions between small molecules and biomacromolecules. The

Bruker standard pulse sequences; spectral width of 20.02 ppm (F2) and 301.10 ppm (F1);ternal reference.




fluorescence of BSA comes from its tryptophan, tyrosine and phenylala-nine residues, the latter two contributing to its fluorescence to only aminor extent [25]. The protein interaction was examined in 96-wellplates used for fluorescence assays. The concentration of BSA (Sigma Al-drich), prepared by dissolving the protein in Tris–HCl, was determinedby absorption spectrophotometric analysis using a molar absorptioncoefficient of 43.824 mol−1 L cm−1 at 279 nm [18]. The complexeswere dissolved in sterile DMSO. For fluorescence measurements, theBSA concentration in buffer Tris–HCl was kept constant in all samples,while the complex concentration was increased from 100 to 0.78 μM,and quenching of the emission intensity of the BSA's tryptophan residuesat 344 nm (excitation wavelength 295 nm) was monitored at differenttemperatures (295, 305 and 310 K). The experiments were carried outin triplicate and analyzed using the classical Stern–Volmer equation:

F0=F ¼ 1 þ Kqτo Q½ � ¼ 1 þ Ksv Q½ � ð1Þ

where F0 and F are the fluorescence intensities in the absence andpresence of quencher, respectively, [Q] is the quencher concentration,and Ksv Stern–Volmer the quenching constant, which can be written asKq = Ksv/τo, where Kq is the biomolecular quenching rate constant andτo is the average lifetime of the fluorophore in the absence of quencher(6.2 × 10−9 s) [26]. Therefore, Eq. (1) was applied to determine Ksv bylinear regression of a plot of Fo/F vs. [Q].

The binding constant (Kb) and number of complexes bound to BSA(n) were determined by plotting the double log graph of the fluores-cence data using Eq. (2):

log F0−Fð Þ=F½ � ¼ logKb þ n log Q½ � ð2Þ

Fig. 3. Typical cyclic voltammograms with a Pt disc electrode, 100 mV/s and a scan rate of com(c) acetonitrile, (d) solution of 8 representing the electrochemical behavior of 6–9 in acetonitr


The thermodynamic parameters were calculated from the van't Hoffequation:

lnK ¼ −ΔH=RTþ ΔS=R ð3Þ

where K is analogous to the Stern–Volmer quenching constant, Ksv atthe corresponding temperature (the temperatures used were 295, 305and 310 K), and R is the gas constant, from which the ΔH and ΔS ofthe reaction can be determined from the linear relationship betweenln K and the reciprocal absolute temperature. Furthermore, the changein free energy (ΔG) was calculated from the following equation:

ΔG ¼ −RT lnK ¼ ΔH–TΔS ð4Þ

2.8. Partition coefficient (P)

Water–octanol partition coefficientswere determined using the stir-flask method [27]. Each complex was tested in a mixture of equal vol-umes of water and octanol with continuous shaking for 24 h at100 rpm and 37 °C. Then the samples were centrifuged for 5 min at300 rpm and the organic and aqueous phases were separated. The con-centration of drug in each phase was measured spectrophotometricallyin order to determine values of P= [drug](in octanol)/[drug](inwater).The experiments were carried out in triplicate.

2.9. Cell culture, cytotoxicity assays and morphological observations

In vitro cytotoxicity assays on cultured human tumor cell lines stillrepresent the standardmethod for initial screening of antitumor agents.

plex 3 in (a) dichloromethane, (b) dichloromethane with a few drops of acetonitrile andile.



Fig. 4. ORTEP view of complexes 1–5 with the thermal ellipsoids at the 50% probability level. The solvent molecules, PF6 anions and hydrogens have been omitted for clarity.


Thus, as a first step to assess its pharmacological properties, the rutheni-um complexes were assayed against human breast tumor cell linesMDA-MB-231 (ATCC No. HTB-26) and MCF-7 (ATCC No. HTB-22), thehuman lung tumor line A549 (ATCC No. CCL-185), and the normal cellline L929 (ATCC No. CCL-1). The cells were routinely maintained withDulbecco's Modified Eagle's medium (DMEM-for L929 and A549) orRPMI 1640 (for MDA-MB-231 and MCF-7) supplemented with 10%fetal bovine serum (FBS), at 37 °C in a humidified 5% CO2 atm. For thecytotoxicity assay, 1.5 × 104 cells well−1 were seeded in 200 μL of com-plete medium in 96-well plates (Corning Costar). Each complex wasdissolved in sterile DMSO (from 40 to 0.01 mM). One microliter ofeach complex sample was added to 200 μLmedium. Cells were exposedto the complex for a 48 h period. The conversion of MTT to formazan bymetabolically viable cells was monitored by an automated microplatereader at 540 nm. The percent cell viability was calculated by dividingthe average absorbance of cells treatedwith the ruthenium–arene com-plex by that of the control; % cell viability versus drug concentration

Table 5Binding constants for the interactions between the ruthenium complexes and ctDNA.

Compound λmax

(nm)H(%) Red

shift (

Chloroquine 330 18.2 –[Ru(η6-C10H14)(phen)Cl]+ 1 280 1.8 –[Ru(η6-C10H14)(phphen)Cl]+ 2 280 50.1 10[Ru(η6-C10H14)(bipy)Cl]+ 3 315 2.3 –[Ru(η6-C10H14)(mebipy)Cl]+ 4 326 2.7 –[Ru(η6-C10H14)(butbipy)Cl]+ 5 302 4.9 –[Ru(η6-C10H14)(phen)CQ]2+ 6 330 27.4 –[Ru(η6-C10H14)(phphen)CQ]2+ 7 330 19.0 –[Ru(η6-C10H14)(bipy)CQ]2+ 8 330 3.63 –[Ru(η6-C10H14)(mebipy)CQ]2+ 9 326 12.1 –


(logarithmic scale) was plotted to determine the IC50 (drug concentra-tion at which 50% of the cells are viable relative to the control), withits estimated error derived from the average of 3 trials.

For the morphological study MDA-MB-231 breast tumor cells wereseeded at a density of 0.8 × 105 cells/well into 12-well plates. Afterallowing 24 h to adhere, images of cells treated with or without com-plexes 2, 5 and 7 were taken at 0, 2, 4, 6, 24 and 48 h.

3. Results and discussion

3.1. Synthesis and characterization

All complexeswere prepared in good yields, higher than 75%, as yel-low/orange solids, stable in light and in air, soluble dimethylsulfoxide,chloroform and acetonitrile; slightly soluble in water and insoluble indiethyl ether and hexane. Good quality crystals of complexes 1–5were grown by slow diffusion of diethyl ether into dichloromethane

nm)Isosbestic point Kb1

(106 M−1)Kb2

(104 M−1)

350 2.60 ± 0.30 11.00 ± 0.02– 0.06 ± 0.02 0.67 ± 0.03– 0.10 ± 0.01 1.91 ± 0.32– 0.07 ± 0.03 0.84 ± 0.14– 0.08 ± 0.06 1.06 ± 0.25– 1.39 ± 0.15 1.62 ± 0.05350 2.14 ± 0.51 5.75 ± 1.70350 3.32 ± 1.60 7.10 ± 0.55– 1.73 ± 0.33 3.68 ± 0.56350 2.06 ± 0.45 20.20 ± 1.80



Fig. 5. Electrophoresis of plasmid pBR322 DNA incubated in 5 mM Tris/HCl buffer for 18 h at 37 °C treated with 0.5, 1.0 and 2.0 eq. of CQ and complexes 6, 7, 8 and 9. MM: molecularmarkers, lanes 2 and 14: pBR322 DNA; lanes 3 and 15: DNA treated with DMSO.


or acetone solutions. The elemental analyses of all the complexes wereconsistentwith their proposed formulas. The UV–vis absorption spectraof the complexes in dimethylsulfoxide (Fig. 1S and Table 1) were char-acterized by an intense high energy band centered at about 300 nm,which can be assigned to a ligand-localized, intra-ligand π–π* transi-tion. Complexes 1–5 exhibited lower-energy bands in the range of340–400 nm, which can be attributed asmetal to ligand charge transfer(MLCT) transitions from Ru (dπ) to the ligand (π*) [28,29]. Complexes6–9 showed two intense bands between 330 and 340 nm, features ofthe quinolinic chloroquine ring. The IR spectra of the complexesdisplayed characteristic bands in the range of 1506–1540 cm−1,ν(C=C); 1384–1413 cm−1 ν(C=N); 846 and 557 cm−1 ν(PF6−) and295–302 cm−1, ν(Ru–Cl, for complexes 1–5). The molar conductivitiesof complexes 1–5, in acetone, were in the range of 1:1 electrolytes[30] and in the range of 1:2 electrolytes in acetonitrile or dmso,indicating that the complexes can accept one molecule of the solventinto their coordination sphere by substituting the chloride atom [31].Likewise, 6–9 molar conductivities in acetone were in the range of 1:2electrolytes.

All NMR signals were assigned on the basis of 1D (1H and 13C NMR)and 2D experiments such as Correlation Spectroscopy (1H–1H gCOSY),Heteronuclear Single Quantum Coherence (1H–13C gHSQC) andHeteronuclear Multiple Bond Coherence (1H–15 N and 1H–13C gHMBC)for all complexes. The 1H NMR spectra showed characteristic reso-nances of η6-C10H14 and signals for the N∩N ligand in question. Allsignals were consistent with chemical shifts, integrations, signal multi-plicities and coupling constants for these classical ligands; the atom

Fig. 6. (a) Fluorescence quenching spectra of BSAwith different concentrations of complex 7witthe intensity changes upon increasing the concentration of the quencher. (b) Stern–Volmer pl


numbering scheme is based on the X-ray structures and Scheme 1). Inall complexes, after coordinating ruthenium with the N∩N or CQligands, the most remarkable characteristics of the 1H NMR spectra ofthese complexes, compared with the spectra of the free ligands, werethe chemical shifts in the H5, H6, H8 and H9 atoms in the aromatic ringzone. Thus, since the N–N ligands used in this work are relatively goodπ acceptors, they can remove electron density from the metal center,shielding these hydrogen atoms to a lesser extent and causing somechanges in the chemical shifts of these protons [32]. All carbons, includ-ing quaternary, were determined by their correlation with the protonsin the complexes by gHMBC and gHSQC resonance techniques.

The 1H–15N gHMBC experiment (Fig. 2) was performed in order todetermine the mode of bonding of CQ to the metal (for complexes6–9). A characterization of free CQ was performed first, and after thatthe characterization of the metal–CQ complexes. This procedure wasperformed to evaluate the chemical shift variation (Δδ) in each signalof 15N in the free ligand in comparison to the ruthenium–CQ complexes.The values of Δδ (Table 2) suggest that CQ binds to the ruthenium atomthrough the nitrogen of the quinoline ring, as significant changes wereobserved with respect to the free CQ, for N-1 Δδ = ~11.0 ppm, whilefor theN-2 andN-3 atoms theΔδwere ca. 3.0 ppmand 1.5 ppm, respec-tively, for all the ruthenium complexes. (See Fig. 1.)

The complexes were studied by cyclic voltammetry (CV) experi-ments, in dichloromethane or acetonitrile solutions, the complexes3 and 8 are a representation of electrochemical behavior for 1–5 and6–9, respectively. CVs of complex 3 in dichloromethane (Fig. 3a) didnot show any process in the anodic region, but after adding a few

h the excitationwavelength at 280nmat 310 K in a Trizmabuffer, pH7.4. The arrow showsots showing tryptophan quenching in BSA at 310 K.



Table 7In vitro anticancer activity in A549,MDA-MB-231,MCF-7 and L929 cells compared to chlo-roquine, cisplatin and doxorubicin after 48 h.

IC50 (μM)

A549 MDA-MB-231 MCF-7 L929 log P

1 N100 N100 N100 N100 0.402 1.70 ± 0.22 0.40 ± 0.10 1.35 ± 0.22 4.27 ± 0.56 0.503 N100 N100 N100 N100 0.214 N100 N100 N100 N100 0.105 18.00 ± 0.57 6.80 ± 1.46 4.33 ± 0.48 89.50 ± 14.01 1.126 N100 N100 N100 N100 0.457 0.95 ± 0.10 2.30 ± 1.64 0.80 ± 0.10 6.40 ± 1.87 1.208 N100 N100 N100 N100 0.359 N100 N100 N100 N100 0.24Chloroquine 56.53 ± 9.18 N100 82.01 ± 11.62 25.94 ± 4.46 0.60CDDP 17.48 ± 4.46 2.44 ± 0.20 8.91 ± 2.60 16.53 ± 2.38 –Doxorubicin – 3.67 ± 0.15 – –

Table 6Stern–Volmer quenching constant (Ksv, L mol−1), biomolecular quenching rate constant(Kq, L mol−1 s−1), Binding constant (Kb, M−1), the number of binding sites (n), ΔG0

(KJ·mol−1), ΔH0 (KJ·mol−1) and ΔS0 (J·mol−1 K) values for the complex–BSA systemat different temperatures.

Ksva(104) Kq

a(1012) Kba (104) na ΔG0 a ΔH0 ΔS0

1 1.54 ± 0.01 2.50 ± 0.01 1.03 ± 0.01 0.95 −24.75 1.10 84.001.58 ± 0.01 2.54 ± 0.01 1.44 ± 0.01 1.00 −25.201.57 ± 0.01 2.54 ± 0.01 1.50 ± 0.02 1.00 −26.00

2 3.35 ± 0.21 5.40 ± 0.34 2.06 ± 0.01 0.97 −23.15 −2.45 78.353.28 ± 0.20 5.30 ± 0.47 2.47 ± 0.01 0.95 −23.613.20 ± 0.20 5.15 ± 0.34 2.70 ± 0.01 1.00 −24.32

4 0.90 ± 0.03 1.45 ± 0.05 1.31 ± 0.01 1.03 −25.20 2.80 85.300.93 ± 0.02 1.51 ± 0.04 2.00 ± 0.01 1.08 −25.650.95 ± 0.06 1.54 ± 0.01 2.60 ± 0.01 1.10 −26.40

5 1.25 ± 0.18 2.02 ± 0.30 0.34 ± 0.02 0.87 −93.40 −1.00 75.001.25 ± 0.20 2.02 ± 0.32 0.41 ± 0.01 0.90 −95.251.23 ± 0.18 2.00 ± 0.30 1.43 ± 0.01 1.02 −98.10

6 2.75 ± 0.05 4.43 ± 0.08 0.64 ± 0.01 0.84 −26.51 1.63 89.802.80 ± 0.04 4.50 ± 0.03 0.67 ± 0.02 0.84 −26.932.84 ± 0.03 4.57 ± 0.04 0.70 ± 0.01 0.84 −27.82

7 3.80 ± 0.04 6.13 ± 0.06 2.46 ± 0.01 1.28 −27.64 1.75 93.613.86 ± 0.01 6.22 ± 0.02 2.62 ± 0.01 0.95 −28.073.94 ± 0.01 6.35 ± 0.02 2.83 ± 0.01 0.96 −29.01

8 1.86 ± 0.04 3.00 ± 0.06 1.26 ± 0.01 0.95 −26.35 2.22 89.201.90 ± 0.05 3.05 ± 0.08 2.24 ± 0.02 1.02 −26.751.95 ± 0.06 3.13 ± 0.10 2.67 ± 0.01 0.95 −27.64

9 1.58 ± 0.02 2.55 ± 0.02 3.44 ± 0.03 1.10 −24.72 1.00 83.751.58 ± 0.02 2.55 ± 0.02 3.01 ± 0.08 1.15 −25.151.61 ± 0.01 2.60 ± 0.02 4.55 ± 0.04 1.10 −26.00

CQ 1.02 ± 0.01 1.65 ± 0.03 2.00 ± 0.02 1.05 −22.15 −0.52 75.001.02 ± 0.01 1.65 ± 0.02 4.78 ± 0.01 1.15 −22.601.00 ± 0.04 1.64 ± 0.02 5.67 ± 0.02 1.18 −23.30

a The values are in ascending order of temperatures 295, 305 and 310 K.


drops of acetonitrile to the electrochemical cell, the processes (III–IV)were detected (Fig. 3b) suggesting displacement of the Cl− ligand bythe CH3CN molecule. The CVs in acetonitrile (Fig. 3c) show a quasi-reversible process (III/IV) at a potential close to 1.0 V, which correspondto the redox pair Ru(II)/Ru(III), and an irreversible oxidation process(V) at around 1.65 V, studies developed by Bernhard Gollas and co-workers [33,34] indicate that this irreversible process at a potentialaround +1.65 V is due to arene ligand oxidation and it depends onthe substituent groups of this ligand [35]. Complex 3 shows oxidation(I) and reduction (II) processes at negative potentials that also belongto the coordinated η6-C10H14 ligand, as described for similar complexes[36]. Complexes 6–9, containing CQ, revealed cyclic voltammogramssimilar to those represented in Fig. 3d. The CQ exhibited two oxidationprocesses in the anodic region (Fig. 3S), where both processes areirreversible and may be attributed to irreversible oxidation of the N-heterocyclic nitrogen of the aminoquinoline moiety (first peak) andthe nitrogen of the alkylamino side chain group of the chloroquinemol-ecule (second peak) [37]. In complexes 6–9, the first oxidation processwas not observed, confirming its coordination to the Ru metal center.This process was observed only after the first sweep, indicating thatthe CQ was dissociated from the metal center. These data are in accor-dance with reports in the literature for Ru(II) arene complexes withphosphine [38] and pyrazolyl [39].

Crystals of compounds of 1–5 were grown by slowly diffusingdiethyl ether into a concentrated solution of the complexes in CH2Cl2or acetone, yielding yellow or orange crystals, which were investigatedby X-ray crystallography. All complexes adopted the familiar half-sandwich three-legged piano–stool, in which the immediate coordina-tion sphere around the Ru(II) consists of two nitrogen atoms of theN–N heterocyclic chelating ligand, where the chloride ligand is thelegs of the stool, and the six carbon atoms of the η6-C10H14 ligandform the seat. Fig. 4 (a–f) shows the structures of these ruthenium com-plexes. The most important interatomic distances and bond angles arecollected and can be seen in Table 4. Ru(II) to η6-C10H14 distances didnot vary significantly between all the derivatives, ranging from[2.139(14) to 2.153(10) Å]. The Ru–Cl bond lengths in the complexeswere almost the same (~2.400 Å), comparable to the Ru–Cl bond lengthobserved in [RuCl(η6-tetralin)(en)]+ [11], [RuCl(η6-C10H14)(mimid)2]+

and [RuCl(η6-C10H14) (bimid)2]+ [[40] ]. Ru(1)–N(1) and Ru(1)–N(2) bond lengths were ~2.080 Å. The N(1)–Ru(1)–N(2) bond anglesin these complexes did not differ significantly from each other. An inter-esting feature in the crystal structure of these complexes is that,depending on the N–N heterocyclic chelating ligand used, the complexmay adopt different configurations. In the first configuration, adoptedby complex 1 and complex 3, the isopropyl part of the η6-C10H14 ligandis on the same side of the chloride ligand, while in the second configu-ration (adopted by the other complexes) the isopropyl moiety isinverted, occupying the side opposite the chloride ligand. This fact iseasily observed by the C(4)–Ru(1)–Cl(1) angle, which is close to 90 °for 1 and 3, and changes in the range of 158–170 for the other com-plexes. In the crystalline state of 3, the arene adopts two different orien-tations, presenting two different molecules in the asymmetric unit.

In the crystal structure of complex 2, the η6-C10H14 group is disor-dered in two positions (Fig. 4S). In this cation complex, the η6-C10H14

group can adopt two different positions; however, the Ru–C bondlengths are very similar for the two positions.

3.2. Interactions with DNA

The Ru–arene complexes interact with various targets includingDNA and proteins, which is probably the explanation for their antican-cer activity [41]. It is known that free CQ intercalates into DNA, whileits Au–CQ and Ru–CQ complexes bind to DNA through intercalationand electrostatic interactions [6–42]. Experiments of absorption titra-tion, viscosity and electrophoresis in agarose were performed to studythe mode of interactions of the new compounds with DNA. In the


spectroscopic titrations, in the presence of ctDNA, all complexes showeddecreases in absorption (hypochromism-%H) thus determining the con-stant of interaction (Kb2, Kb1) by a nonlinear Scatchard curve (Fig. 14S).The Kb1 and Kb2 (Table 5) are within the accepted range for compoundsconsidered to interactwithDNA [6]. Overall, complexes 1–5present lowhypochromism and also low Kb1 and Kb2 values compared with theiranalogous with CQ, and the overall positive charge could offer an inter-acts electrostatics with the phosphate groups. The constants exhibitedby complexes 6, 7, 8 and 9 were similar to those found for free CQ,and the same order of magnitude for analogous ruthenium–CQ andplatinum–CQ complexes [43].

Viscosity measurements were used successfully to determineintercalation or non-intercalation binding modes of the complexesto DNA [44]. The observed increase in the viscosity of this biomole-cule in the presence of complexes 6 to 9 (Fig. 15S), suggests interca-lation of the complexes with the DNA probably is solely due to the CQmoiety. This behavior is similar to that reported for the complex[Ru(bpy)2(dppz)]2+, which acts like the classical intercalator ethidium



A549

-2 -1 0 1 20

50

100 257

log [ ] µmol.L-1

% C

ell V

iabi

lity

(a)

MCF-7

-1 0 1 20

50

10052

7

log [ ] µmol.L-1

% C

ell V

iabi

lity

(b)

Fig. 7.MTT colorimetric cell viability assay for (a) A549 lung tumor cell line (b) MCF-7 breast tumor cell line, treated with complexes 2, 5 and 7 for 48 h.


bromide [45]. While the viscosity of DNA remains unchanged uponaddition of the ruthenium complexes (1–5), suggesting no interactionbetween these species.

It is well known that plasmid DNA exists in two main forms, asupercoiled form, which migrates faster, and an open circular form,which migrates more slowly. Changes in the mobilities of these bandsare generally interpreted as evidence of DNA binding with another

Fig. 8.Morphological study under an inverted microscope (100×) of MDA-MB-231 control celimages are representative of many pictures taken in n = 3 experiments.


molecule [46]. This prompted a study aiming to evaluate the electro-phoretic mobility of plasmid pBR322 DNA in agarose after incubationof ruthenium complexes for 18 h at 37 °C (compound: DNA ri 0.5 and2.0) compared to free pBR322 and pBR322 treated with CQ (Fig. 5).

The results showed that exposure of pBR322 to increasing complexconcentrations did not induce a noticeable alteration in the tertiarystructure of the plasmid under these conditions, similar to that obtained

ls and cells treated with the IC50 concentrations of complexes 2, 5 and 7. In all panels, the




for CQ and Ru–CQ complexes and commonly associated with thestrength of the DNA interaction. Analyzing all the results of our DNAbinding studies together, we suggest that compounds 6–9 interactwith DNA primarily through intercalation.

3.3. Interactions with BSA

Fluorescence quenching of BSA. Quenching can occur by differentmechanisms, usually classified as either dynamic or static quenching,which can be distinguished by their differing dependence on tempera-ture and viscosity, or preferably by lifetime measurements [47]. Highertemperatures result in faster diffusion and hence a larger degree ofcollisional quenching. Higher temperatures will also result in thedissociation of weakly bound complexes, and hence lead to less staticquenching. As shown in Fig. 6a, BSA showed strong fluorescence emis-sion,while the ruthenium–arene complexes displayed almost no intrin-sic fluorescence under the experimental conditions used for themeasurements. The fluorescence intensity of BSA decreased with in-creasing ruthenium–arene complex concentrations, accompanied by ared shift in the maximum emission wavelengths in the fluorescencespectra. In order to ascertain the fluorescence quenching mechanism,the fluorescence quenching data were measured at different tempera-tures (295, 305 and 310 K). These results show that in all complexesthe KSV values increased when the temperature is increased (see Fig. 6and Table 6), except to 2, 5 and CQ. Moreover, the values of Kq for allcomplexes were in the range of 1.45 × 1012 to 6.35 × 1012 mol·L−1,far higher than 2.0 × 1010 mol·L−1, the maximum possible value fordynamic quenching [48], indicating the existence of static quenchingmechanism.

3.3.1. Determinations of binding constants, numbers of binding sites andthermodynamic parameters

The quenching data for the static quenching process were analyzedaccording to the modified Stern–Volmer equation Eq. (2). The bindingconstants Kb and binding sites, n, were calculated from the slopes ofthe static quenching equation: log(Fo–F)/F vs. log[Q]. The results aresummarized in Table 6. The number of binding sites between BSA andthe Ru/arene complexes is approximately equal to 1. The interactionconstant for all complexes is between 0.34 × 104 and 5.67 M−1, thesame range reported for Ru [49,50], V [51] and Zn [52] complexes,showing hydrophobic interactions with BSA. The interaction forces be-tween drugs and biomolecules may include electrostatic interactions,multiple hydrogen bonds, van der Waals interactions, hydrophobicand steric contacts within the antibody-binding site, etc. [53]. The ther-modynamic parameters, enthalpy change (ΔH), entropy change (ΔS)and free energy change (ΔG), are the main means used to confirm thebinding modes. From the thermodynamic standpoint, ΔH N 0 andΔS N 0 imply a hydrophobic interaction; ΔH b 0 and ΔS b 0 reflectsvan der Waals force or hydrogen bond formation; and ΔH b 0 andΔS N 0 suggests an electrostatic force.

Thehomologous series of ruthenium-containing aromatic dinitrogenchelating ligands utilized in this study provides an opportunity to relatethese substituents to protein association process. To elucidate theprotein association process between these ruthenium–arene complexesand BSA, the thermodynamic parameters (ΔH and ΔS) were calculatedfrom the van't Hoff plots, Eq. (3), ΔG was estimated from Eq. (4) andthe results are presented in Table 6. The negative value of ΔG revealsthat the interaction process is spontaneous. Complexes 1 and 3 and allcomplexes containing chloroquine presents positive ΔH and ΔS valuesindicated hydrophobic association with BSA, while complexes 2 and 5shownegativeΔHandpositiveΔS values indicated an electrostatic asso-ciation, this behavior can be related to the size and electronic density ofthe dinitrogen ligand, where ligands with larger substituents and moreactivators like phenyl and tercbutyl, promotes localization of electronicdensity, which can be related to electrostatic affinity for BSA, in thecase of complex 7 the inclusion of chloroquine in the coordination


sphere favors a delocalization of the electronic density, which is evi-denced by an increase of the oxidation potential, leading to a hydropho-bic association with the BSA. On the other hand, although hydrophobicinteractions may play a key role in their interactions with BSA, electro-static interactions could not be excluded because it was not possible toaccount for the thermodynamic parameters of the Ru/arene–BSA com-pounds on the basis of a single intermolecular force model.

3.4. Partition coefficient (log P)

The partition coefficient between the water or buffer, and n-octanolis the most widely used measure of chemical compound lipophilicity[54] as it is amajor structural factor governing both the pharmacokinet-ics and pharmacodynamics of drugs, because up to a certain limit,compounds with higher lipophilicity have higher permeation across bi-ological membranes (but lower aqueous solubility). In general terms,for an effective anticancer drug to initiate cell death, itmust reach viablecells in a tumor, and be retained at a sufficient concentration on a rele-vant time scale. The partition coefficient (log P-Table 7) values for com-plexes 1–9 are in the range of 0.21 to 1.20, which do not necessarilycorrelate with cytotoxicity, and it can be observed that, in general, thesubstituents in bipy and phen ligands, as well as the coordination ofchloroquine, increase lipophilicity of the ruthenium complexes. Thiscombination of structural effects and enhanced drug lipophilicity resultin increasing accumulation in biological membranes.

3.5. Cytotoxicity assay and morphological observations

Cell respiration as an indicator of cell viability was determined by amitochondrial dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan [34]. The results ob-tained using this assay are listed in Table 7. Only complexes 2, 5 and 7show cytostatic effects at doses appreciably lower in comparison withcisplatin, chloroquine and doxorubicin under the same conditions.Interestingly, complex 7 was the most active, with an IC50 of 0.95, 2.30and 0.80 μM in A549, MDA-MB-231 and MCF-7 cells, respectively,showing that the coordination of CQ enhances the biological activityof the metal complexes, as reported previously [6,8]. Under an invertedmicroscope, cell shape and changes in it can be observed clearly. Asshown in Fig. 8, MDA-MB-231 breast cancer cells appeared phenotypi-cally as spindle shaped in the control group and there were very fewround cells. Cells treatedwith 2, 5 and 7 showed obviousmorphologicalchanges after the first 6 h; cells treated for 48 h showed, in addition tomorphological changes, a loss of adhesion, a spindle shaped form andconfluence, and there was a clear concentration–response tendency,indicating the possibility of apoptosis. (See Fig. 7.)

Complexes 2 and 5 not only showed greater lipophilicity, but alsopresented higher interaction constants with BSA (Ksv), DNA (Kb1), incomparison with those ones obtained for complexes 1, 3 and 4. Thesedesirable biological properties are reflected in the anticancer activityshown by complexes 2 and 5 at lower concentrations. Likewise, theRu–CQ derivatives showed the same tendency 7 N 6 N 9 ≈ 8 for Kb1

(DNA), Ksv (BSA) and log P. In accordance with this trend, complex 7showed the best IC50 values compared to all tumor cell lines tested. Itspotential cytotoxicity can be associated to the combination of its affinityto BSA, an interaction with DNA and lipophilic properties which in-creases cellular uptake. Multi-target properties of complex 7 make thisa promising candidate for the next stages of anticancer evaluation.

4. Conclusions

Piano–stool type complexes of the formulae [RuCl(η6-C10H14)(N–N)]PF6 [N–N=1,10-phenanthroline 4,7-diphenil-1,10-phenanthroline,2,2′-bipyridine, 5,5′-dimethyl-2,2′-bipyridine, 4,4′-di-t-butyl-2,2′-bipyridine and their CQ derivates were synthesized and fully character-ized, both in solution and in the solid state. All complexeswere air stable




and presented distorted octahedral coordination spheres around the ru-thenium center in the solid state, as shown in their X-ray crystal struc-tures. All complexes showed quasi-reversible metal-based RuII/RuIII

electrochemical processes. Complexes 6–9 interact in an intercalativemanner with DNA, to an extent comparable to that of free chloroquine.The experimental results indicated that all ruthenium complexes understudy bind to BSA with moderate affinity and the intrinsic fluorescenceof BSA was quenched through a static quenchingmechanism. The ther-modynamic parameters, negative or positive forΔH, positive forΔS andnegative for ΔG, indicate that the hydrophobic properties of the com-plexes and hydrogen bonding interactions between the complexesand BSA play a major role in the complex/BSA binding process.

Complexes 2, 5 and 7 showed greater lipophilicity and also presentedhigher interaction constants with BSA (Ksv) and DNA (Kb1) in compari-son with the rest of the analogous compounds evaluated in this work.These desirable biological properties are reflected in the growth inhibi-tion on human tumor cell lines at lower concentrations, with complex7 showing the most promising results.

Acknowledgments

The authors gratefully acknowledge the support provided by FAPESP,CNPq, CAPES and CYTED.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2015.07.016.

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Journal of Inorganic Biochemistry - LABEN · Cytotoxicity of Ru(II) piano–stool complexes with chloroquine and chelating ligands againstbreastandlungtumorcells:InteractionswithDNAandBSA