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

Article abstract of DOI:10.1016/j.jinorgbio.2015.07.016

The synthesis and spectroscopic characterization of nine π-arene piano-stool ruthenium (II) complexes with 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 structures of five ruthenium complexes (1-5) were determined by X-ray crystallography. Electrochemical experiments were performed by cyclic voltammetry to estimate the redox potential of the Ru^II/Ru^III 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 observed for free chloroquine. The results of fluorescence titration revealed that these complexes strongly quenched the intrinsic fluorescence of BSA following a static quenching procedure. Binding constants (K_b) and the number of binding sites (n ~ 1) were calculated using modified Stern-Volmer equations. The thermodynamic parameters ΔG at different temperatures were calculated and subsequently the values of ΔH and ΔS were also calculated, which revealed that hydrophobic and electrostatic interactions play a major role in the BSA-complex association. The MTT assay results indicated that complexes 2, 5 and 7 showed cytostatic effects at appreciably lower concentrations than those needed for cisplatin, chloroquine and doxorubicin.

Full text of DOI:10.1016/j.jinorgbio.2015.07.016

Cytotoxicity of Ru(II) piano-stool complexes with chloroquine and chelating
ligands against breast and lung tumor cells: Interactions with DNA and BSA
Legna Colina-Vegas, Wilmer Villarreal, Maribel Navarro, Clayton Ro-
drigues de Oliveira, Ang e´ lica E. Graminha, Pedro Ivo da S. Maia, Victor M.
Deflon, Antonio G. Ferreira, Marcia Regina Cominetti, Alzir A. Batista
PII:
DOI:
Reference:
S0162-0134(15)30042-8
doi: 10.1016/j.jinorgbio.2015.07.016
JIB 9769
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
5 April 2015
6 July 2015
15 July 2015
Please cite this article as: Legna Colina-Vegas, Wilmer Villarreal, Maribel Navarro,
Clayton Rodrigues de Oliveira, Ang ´e lica E. Graminha, Pedro Ivo da S. Maia, Victor M.
Deflon, Antonio G. Ferreira, Marcia Regina Cominetti, Alzir A. Batista, Cytotoxicity
of Ru(II) piano-stool complexes with chloroquine and chelating ligands against breast
and lung tumor cells: Interactions with DNA and BSA, Journal of Inorganic Biochemistry
(2015), doi: 10.1016/j.jinorgbio.2015.07.016
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ACCEPTED MANUSCRIPT
Cytotoxicity of Ru(II) piano-stool complexes with chloroquine and chelating ligands against breast
and lung tumor cells: interactions with DNA and BSA
†
†
&
†
Legna Colina-Vegas *, Wilmer Villarreal , Maribel Navarro , Clayton Rodrigues de Oliveira ,
†
‡
‡
†
Angélica E. Graminha , Pedro Ivo da S. Maia , Victor M. Deflon , Antonio G. Ferreira , Marcia
§
†
Regina Cominetti , Alzir A. Batista *
†
Departamento de Química, Universidade Federal de São Carlos – UFSCar, CEP 13565-905; São
&
Carlos – SP, Brazil. Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO, CEP
‡
2
5250-020; Duque de Caxias - RJ - RJ, Brazil. Instituto de Química de São Carlos, Universidade de
§
São Paulo, CEP 13560-970, São Carlos - SP, Brazil. Departamento de Gerontologia, Universidade
Federal de São Carlos – UFSCar, CEP 13565-905; São Carlos – SP, Brazil.
*
Corresponding authors: Alzir A. Batista. daab@ufscar.br; Legna Colina-Vegas
esleg_24@hotmail.com
Fax: +55 (16) 3351-8350
Phone: +55 (16) 3351-8285
1

ACCEPTED MANUSCRIPT
ABSTRACT
The synthesis and spectroscopic characterization of nine π-arene piano-stool ruthenium (II)
complexes with aromatic dinitrogen chelating ligands or containing chloroquine (CQ), are described
6
6
6
in this study: [Ru(η -C H )(phen)Cl]PF (1), [Ru(η -C H )(dphphen)Cl]PF (2), [Ru(η -
10
14
6
10 14
6
6
6
C H )(bipy)Cl]PF (3), [Ru(η -C H )(dmebipy)Cl]PF (4) and [Ru(η -C H )(bdutbipy)Cl]PF
6
10
14
6
10 14
6
10 14
6
6
6
(
5), [Ru(η -C H )(phen)CQ](PF )
(6), [Ru(η -C H )(dphphen)CQ](PF )
(7), [Ru(η -
10
14
6 2
10 14
6 2
6
C H )(bipy)CQ](PF )
(8), [Ru(η -C H )(dmebipy)CQ](PF ) (9): [1,10-phenanthroline
10
14
6 2
10 14
6 2
(phen), 4,7-diphenyl-1,10-phenanthroline (dphphen), 2,2’-bipyridine (bipy), 5,5’-dimethyl-2,2’-
bipyridine (dmebipy), 4,4’-di-t-buthyl-2,2’-bipyridine (dbutbipy)] The solid state structures of five
ruthenium complexes (1-5) were determined by X-ray crystallography. Electrochemical experiments
II
III
were performed by cyclic voltammetry to estimate the redox potential of the Ru /Ru 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 was
comparable to the one observed for free chloroquine. The results of fluorescence titration revealed
that these complexes strongly quenched the intrinsic fluorescence of BSA following a static
quenching procedure. Binding constants (K ) and the number of binding sites (n~1) were calculated
b
using modified Stern–Volmer equations. The thermodynamic parameters ΔG at different
temperatures were calculated and subsequently the values of ΔH and ΔS were also calculated, which
revealed that hydrophobic and electrostatic interactions play a major role in the BSA–complex
association. The MTT assays results indicated that complexes 2, 5 and 7 showed cytostatic effects at
appreciably lower concentrations than those needed for cisplatin, chloroquine and doxorubicin.
Keywords: Cytotoxicity, Chloroquine, Arene-Ruthenium, DNA, BSA.
2

ACCEPTED MANUSCRIPT
1
. INTRODUCTION
Chloroquine diphosphate, a derivative of quinine, was discovered in 1934 and it has long
been commonly used against different human diseases [1,2,3]. Recently several studies have shown
that it has extensive biological effects, such as inhibiting cell growth and/or inducing death in
different kinds of cells [4]. On the other hand, chloroquine complexes of ruthenium [5], platinum [6],
palladium [7] and gold [8] have shown antitumor properties against different types of tumor cells.
6
The development of half-sandwich η -arene-Ru(II) complexes has attracted particular
attention and have been extensively studied over the past 20 years, as these complexes show
applications in different areas of scientific and industrial interest. More importantly, in some cases,
these complexes present higher antitumor activity and lower toxicity compared to platinum
6
compounds [9], and therefore are potential chemotherapeutic agents [10]. Several reports on η -arene
n+
Ru(II) complexes with the general formula [Ru(arene)(L-L')(X)] can be found in 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 compounds have exhibited activity in vitro and in vivo against A2780 human ovarian
cancer cells, with promising results [15]. Furthermore, it is well known that N∩N-heterocyclic
ligands provide good stability for the structure of Ru(II) complexes, due to their π acceptor
properties, which allows for the formation of supramolecular structures. They establish slightly
directional non-covalent interactions of the π-π type, which are weaker than hydrogen bonds, but still
strong enough to increase the stability of the metal complexes, both in solution and in the solid state
[16].
Using models able to reproduce the chemical behavior of metal-biomolecule interactions is
one of the approaches most commonly used in attempting, not only to reproduce the biological
activity, but also to explain the interactions between metals and biomolecules. Their interactions with
blood proteins, DNA (components) or small biomolecules can have a protective effect and decrease
the rate of degradation or side product formation [17]. Albumin, the most abundant protein in the
3

ACCEPTED MANUSCRIPT
circulatory system, has been one of the most extensively studied of all proteins; it serves as a
transporter of a range of molecules such as fatty acids, vitamins and drugs. Binding a drug to
albumin results in increased drug solubility in plasma, decreased toxicity, as well as protection
against oxidation [18].
In this paper we report on the syntheses and characterization of 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-
buthyl-2,2’-bipyridine (dbutbipy) and CQ (Scheme 1). These species were chosen in order to
determine if there is any influence of the type of N-N ligand on the arene complex that could be
reflected in its biological properties. Here, we also present the interaction study of the synthesized
ruthenium complexes with DNA by titration spectroscopy, viscosity measurements and
electrophoreses, their interaction with BSA by fluorescence quenching and their cytotoxicity against
four cell lines (L929, MCF-7, A549 and MDA-MB-231).
Scheme 1. Structures of piano-stool ruthenium (II) complexes, identified in the scheme for the
purpose of NMR spectroscopy studies.
4

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2
. EXPERIMENTAL SECTION
All manipulations were carried out under an argon atmosphere using standard Schlenk
techniques. The electronic spectra were obtained via scanning on a Hewlett–Packard diode array
model 8452A spectrophotometer. The microanalyses were performed using a FISIONS CHNS, mod.
EA 1108 microanalyzer. The IR spectra were recorded on a FTIR Bomem-Michelson 102
−
1
spectrometer in the 4000–200 cm region. Conductivity values were obtained using a Meter Lab
CDM2300 instrument.
1
13
1
1
1
13
2
.1 NMR spectroscopy data. All the 1D and 2D NMR experiments ( H, C, H- H gCOSY, H- C
1
13
1
15
gHSQC, H- C gHMBC, H- N gHMBC) were recorded on a 9.4 T Bruker Avance III
TM
spectrometer with a 5 mm internal diameter indirect probe with ATMA
(Automatic Tuning
Matching), holding the temperature stable at 300 K. In general, 20 mg samples of CQ, free ligands
and metal-CQ complexes were dissolved in deuterated dimethylsulfoxide (DMSO-d , Cambridge
6
Isotope Laboratories, Inc., USA), containing a trace amount of tretramethylsilane (TMS) that was
1
13
used as an internal reference (0 ppm). The H and C NMR spectra were acquired with 8 and 12 K
scans; spectral widths (sw) of 8012.820 Hz and 24038.461 Hz; relaxation delays (d1) of 10 s and 0.1
s; acquisition times (aq) of 4.1 s and 0.7 s; data points (td) at 64 K and 32 K; and 90º and 30º pulse
1
1
1
13
1
13
1
15
lengths, respectively. The H- H gCOSY, H- C gHSQC, H- C gHMBC, and H- N gHMBC
spectra were acquired with 8, 40, 72, and 32 scans and spectral widths of 6127.451 Hz, 6127.451 Hz,
6
1
127.451 Hz, and 8012.820 Hz for F2 and 6127.451 Hz, 23147.572 Hz, 23148.150 Hz, and
20481.930 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 2D NMR experiments. The chemical shift in nitromethane (380.2 ppm)
1
5
1
15
was used as an internal reference for the N dimension in the H- N gHMBC experiment.
5

ACCEPTED MANUSCRIPT
2
.2 Single crystal X-ray structure data analysis. The data collections were performed with Mo-K
radiation ( = 71.073 pm) on a BRUKER APEX II duo diffractometer. The structures were solved by
2
direct methods using SHELXS-97 [19] and refined by full-matrix least-square methods against F
(SHELXL-97) [20]. All non-hydrogen atoms were refined with anisotropic displacement parameters
with SHELXL97. The hydrogen atoms’ positions were found in Fourier maps or calculated in their
idealized positions. Crystallographic data and experimental details of the structural analysis are
summarized in Table 4 and 1S.
2
.3 Electrochemical studies. The electrochemical experiments were performed using a BAS-
1
00B/W MF-9063 Bioanalytical Systems Instrument in nitrogen atmosphere at room temperature
with tetrabutylammoniumperchlorate (TBAP Fluka Purum) as a supporting electrolyte. The
electrochemical cell was equipped with platinum working and auxiliary electrodes and Ag/AgCl as
the reference electrode in a Luggin capillary probe, a medium in which ferrocene is oxidized at 0.43
−
1
V (Fc+/Fc). Voltammograms were performed at a scan rate of 0.100 V s . The half-wave potentials
for the reversible cyclic voltammetric processes were estimated by the average of the anodic and
cathodic peak potentials (E1/2 = (Epa + Epc)/2).
6
2
.4 Synthesis of [RuCl(η -C H )(N-N)]PF , general procedure. The precursors were synthesized
10
14
6
6
according to a previously reported method [21,22]. A solution of [RuCl (η -C H )] (0.200 g, 0.31
2
10 14 2
mmol) with an excess of the desired N-N ligand (0.75 mmol) in methanol (25 mL) was stirred for 1
h. NH PF (0.30 g; 1.00 mmol) was added to this solution, also dissolved in methanol (5 mL), and
4
6
the mixture was stirred at room temperature for 1 h longer. The orange-yellow solid that precipitated
was filtered off, washed with cold methanol and diethyl ether, and dried under vacuum.
6
[
RuCl(η -C H )(1,10-phenanthroline)]PF (1). Yellow solid, yield 330 mg (83%). Elemental
10
14
6
.
1
analysis (%) calcd for C H ClF N PRu / H O C 43.90, H 3.80, N 4.65. Found: C 43.97, H 3.86, N
2
2
22
6
2
3
2
-1
4
.47. IR (Selected bands, cm ) ν (C=C) 1519; ν (C=N) 1470; ν (Ru-Cl) 297. Molar conductivity in
-1
2
-1 1
acetone, Λ = 133.5 ± 0.2 ohm cm mol . H NMR δ ppm (m, I): 9.93 (dd, 2H, J = 5.2, 1.1 Hz,
M
6

ACCEPTED MANUSCRIPT
H -H ), 8.93 (dd, 2H, J = 8.2, 1.1 Hz, H -H ), 8.29 (s, 1H, H -H ), 8.17 (dd, 2H, J = 8.2, 5.2 Hz,
1
5
20
13
18
21
22
H -H ), 6.34 (d, 2H, J = 6.4 Hz, H -H ), 6.11 (d, 2H, J = 6.4 Hz, H -H ), 2.61 (hept, 1H, J = 6.9
1
4
19
6
8
5
9
Hz, H ), 2.10 (s, 3H, H ), 0.91 (d, 6H, J = 6.9 Hz, H -H ).
1
10
2
3
6
[
RuCl(η -C H )(4,7-diphenyl-1,10-phenanthroline)]PF (2). Orange solid, yield 380 mg (80%).
10 14 6
.
1/
2
Elemental analysis (%) calcd for C H ClF N PRu H O C 53.94, H 4.13, N 3.70. Found: C 53.89,
3
4
30
6
2
2
-1
H 4.12, N 4.06. IR (Selected bands, cm ) ν (C=C) 1519; ν (C=N) 1475; ν (Ru-Cl) 302. Λ (acetone)
M
-1
2
-1 1
1
34.2 ± 0.2 ohm cm mol . H NMR δ ppm (m, I): 10.01 (d, 2H, J = 5.5 Hz, H -H ), 8.15 (d, 2H,
15 20
J = 5.5 Hz, H -H ), 8.13 (s, 2H, H -H ), 7.71 (m, 10, H -H , phenyl group), 6.40 (d, 2H, J = 6.4
1
4
19
21
22
24
34
Hz, H -H ), 6.19 (d, 2H, J = 6.4 Hz, H -H ), 2.73 (hept, 1H, J = 6.5 Hz, H ), 2.20 (s,3H), 1.02 (d,
6
8
5
9
1
6
H, J = 6.5 Hz, H -H ).
2 3
6
[
(
(
RuCl(η -C H )(2,2’-bipyridine)]PF (3). Yellow solid, yield 370 mg (97%). Elemental analysis
10 14 6
.1/
%) calcd for C H ClF N PRu H O: C 41.57, H 3.95, N 4.85. Found: C 41.87, H 4.25, N 4.71. IR
2
0
22
6
2
3
2
-1
-1
Selected bands, cm ) ν (C=C) 1498; ν (C=N) 1469; ν (Ru-Cl) 302. Λ (acetone) 135.3 ± 3.0 ohm
M
2
-1 1
cm mol . H NMR δ ppm (m, I): 9. 53 (d, 2H, J = 7.9 Hz, H -H ), 8.64 (d, 2H, J = 7.9 Hz,H -
1
5
20
12
H ), 8.29 (dd, 2H, J = 7.9, 7.9 Hz, H -H ), 7.80 (dd, 2H, J = 7.9, 7.9 Hz,H -H ), 6.22 (d, 2H, J =
1
7
13
18
14
19
6
0
.3 Hz, H -H ), 5.98 (d, 2H, J = 6.3 Hz, H -H ), 2.58 (hept, 1H, J = 6.9 Hz, H ), 2.18 (s, 3H, H ),
6 8 5 9 1 10
.94 (d, 6H, J = 6.9 Hz, H -H ).
2
3
6
[
RuCl(η -C H )(5,5’-dimethyl-2,2’-bipyridine)]PF (4). Yellow solid, yield 350 mg (92%).
10 14 6
Elemental analysis (%) calcd for C H ClF PRu: C 44.04, H 4.37, N 4.67. Found: C 44.21, H 4.43,
2
2
26
6
-1
N 4.40. IR (Selected bands, cm ) ν (C=C) 1506; ν (C=N) 1477; ν (Ru-Cl) 295. Λ (acetone) 123.0 ±
M
-
1
2
-1 1
0
8
.6 ohm cm mol . H NMR δ ppm (m, I): 9.35 (s, 2H, H -H ), 8.46 (d, 2H, J = 8.4 Hz, H -H ),
15 20 12 17
.09 (dd, 2H, J = 8.4, 0.8 Hz, H -H ), 6.25 (d, 2H, J = 6.4 Hz, H -H ), 5.97 (d, 2H, J = 6.4 Hz, H -
1
3
18
6
8
5
H ), 2.57 (hept, 1H, J = 6.9 Hz, H ), 2.52 (s, 6H, H -H ), 2.21(s, 3H, H ), 0.91 (d, 6H, J = 6.9
9
1
21
22
10
Hz,H -H ).
2
3
7

ACCEPTED MANUSCRIPT
6
[
RuCl(η -C H )(4,4’-t-dibuthyl-2,2’-bipyridine)]PF (5). Orange solid, yield 340 mg (78%).
10 14 6
Elemental analysis (%) calcd for C H ClF N PRu C 49.82, H 5.91, N 4.01. Found: C 49.93, H
2
9
41
6
2
-
1
5
1
=
.54, N 4.41. IR (Selected bands, cm ) ν (C=C) 1548; ν (C=N) 1477; ν (Ru-Cl) 295. Λ (acetone)
M
-1
2
-1 1
35.3 ± 1.6 ohm cm mol . H NMR δ ppm (m, I): 9.39 (d, 2H, J = 6.0 Hz, H -H ), 8.63 (d, 2H, J
15
20
1.7 Hz, H -H ), 7.71 (dd, 2H, , J = 6.0, 1.8 Hz, H -H ), 6.16 (d, 2H, J = 6.2 Hz, H -H ), 5.95 (d,
13
17
14
19
6
8
2
H, J = 6.2 Hz, H -H ) 2.60 (hept, 1H, J = 6.8 Hz,H ), 2.14 (s, 3H, H ), 1.43 (s, 18H, H -H , t-
5 9 , 1 10 22 28
buthyl group), 0.97 (d, 6H, J = 6.9 Hz, H -H ).
2
3
6
2
.5 Synthesis of [RuCQ(η -C H )(N-N)][PF ] , general procedure. A solution of the precursor
10 14 6 2
(0.25 mmol) with an excess of NH PF (0.50 mmol) in methanol (25 mL) was stirred for 1 h. CQ
4 6
(0.50 mmol), also dissolved in methanol (10 mL), was added to this solution, and the mixture was
stirred under reflux for 24h. The orange-red solution that was obtained was dried under vacuum,
dissolved in dichloromethane, and the precipitate filtered off. The orange-red solution was dried
under vacuum to obtain an orange-red solid, which was washed with diethyl ether (3x 30 ml) and
dichloromethane and dried under vacuum.
6
[
RuCQ(η -C H )(1,10-phenanthroline)][PF ] (6). Orange solid, yield 180 mg (70%). Elemental
1
0
14
6 2
.
1/
3
analysis (%) calcd for C H ClF N P Ru 1 CH Cl C 43.60, H 4.49, N 6.15. Found: C 43.94, H
4
0
48
12
5
2
2
2
-1
-
4
.80, N 5.92. IR (Selected bands, cm ) ν (C=C) 1519; ν (C=N) 1460. Λ (acetone) 175.4 ± 1.3 ohm
M
1
2
-1 1
cm mol . H NMR δ ppm (m, I): 9.93 (dd, 2H, J = 5.2, 1.2 Hz, H -H ), 8.93 (dd, 1H, J = 8.2, 1.2
15
20
Hz, H -H ) 8.87 (d, 1H, J = 8.3, NH-CQ), 8.65 (d, 1H, J = 9.2 Hz, H -CQ), 8.56 (d, 1H, J = 7.2 Hz,
13
18
e
H -CQ), 8.30 (s, 2H, H -H ), 8.17 (dd, 2H, J = 8.2, 5.2 Hz, H -H ), 7.93 (d, 1H, J = 2.0 Hz, H -
b
21
22
14
19
h
CQ), 7.82 (dd, 1H, J = 9.2, 2.0 Hz, H -CQ), 6.96 (d, 1H, J = 7.2 Hz, H -CQ), 6.34 (d, 2H, J = 6.4 Hz,
f
c
H -H ), 6.11 (d, 2H, J = 6.4 Hz, H -H ), 4.36 (m, 1H, H -CQ), 3.10 (m, 6H, Hd’-e’-CQ), 2.60 (hept,
6
8
5
9
a’
1
H, J = 6.8 Hz, H ), 2.17 (s,3H, H ), 1.60 (m, 4H, H -CQ), 1.32 (d, 3H, J = 6.4 Hz, H -CQ), 1.17
1 10 b’-c’ a’’
(
t, 6H, H -CQ), 0.90 (d, 6H, J = 6.8 Hz, H -H ).
f’ 2 3
8

ACCEPTED MANUSCRIPT
6
[
RuCQ(η -C H )(4,7-diphenyl-1,10-phenanthroline)][PF ] (7). Orange solid, yield 245 mg
10 14 6 2
.
(
83%). Elemental analysis (%) calcd for C H ClF N P Ru 2CH Cl C 48.14, H 4.49, N 5.20.
52 56 12 5 2 2 2
-1
Found: C 48.41, H 4.53, N 5.40. IR (Selected bands, cm ) ν (C=C) 1518; ν (C=N) 1459. Λ
M
-1
2
-1 1
(
acetone) 180.4 ± 2.2 ohm cm mol . H NMR δ ppm (m, I): 10.01 (d, 2H, J = 5.6 Hz, H -H ),
15 20
8
8
1
6
.85 (d, 1H, J = 8.3 Hz, NH-CQ), 8.64 (d, 1H, J = 9.0 Hz, H -CQ), 8.57 (d, 1H, J = 6.9 Hz, H -CQ),
e b
.15 (d, 2H, J = 5.6 Hz, H -H ), 8.13 (s, 2H, H -H ), 7.92 (d, 1H, J = 2.0 Hz, H -CQ), 7.83 (dd,
1
4
19
21
22
h
H, J = 9.0, 2.0 Hz, H -CQ), 7.70 (m, 10H, H -H , phenyl group), 6.97 (d, 1H, J = 6.9 Hz, H -CQ),
f
24
34
c
.40 (d, 2H, J = 6.4 Hz, H -H ), 6.20 (d, 2H, J = 6.4 Hz, H -H ), 4.15 (m, 1H, H -CQ), 3.10 (m, 6H,
6
8
5
9
a’
Hd’-e’-CQ), 2.73 (hept, 1H, J = 6.4 Hz, H ), 2.20 (s,3H, H ), 1.70 (m, 4H, Hb’-c’-CQ), 1.32 (d, 3H, J =
1
10
6
.4 Hz, H -CQ), 1.17 (t, 6H, H -CQ), 1.02 (d, 6H, J = 6.4 Hz, H -H ).
a’’ f’ 2 3
6
[
RuCQ(η -C H )(2,2’-bipyridine)][PF ] (8). Orange solid, yield 215 mg (86%). Elemental
1
0
14
6 2
.
analysis (%) calcd for C H ClF N P Ru 3CH Cl C 39.20, H 4.33, N 5.58. Found: C 39.39, H
38
48
12
5
2
2
2
-1
-
4
.65, N 5.89. IR (Selected bands, cm ) ν (C=C) 1553; ν (C=N) 1470. Λ (acetone) 179.1 ± 1.5 ohm
M
1
2
-1 1
cm mol . H NMR δ ppm (m, I): 9.34 (dd, 2H, J = 8.0, 1.0 Hz, H -H ), 8.30 (m, 4H, H -CQ
1
5
20
e,b
and H -H ), 8.20 (m, 2H, H -H ), 7.90 (d, 1H, J = 2.0 Hz, H -CQ), 7.72 (m, 3H, H -CQ and H -
1
2
17
13
18
h
f
14
H ), 7.50 (m, 1H, NH-CQ), 6.82 (d, 1H, J = 7.2 Hz, H -CQ), 5.93 (d, 2H, J = 6.4 Hz, H -H ), 5.73
1
9
c
6
8
(
d, 2H, J = 6.4 Hz, H -H ), 4.07 (m, 1H, H -CQ), 3.13 (m, 6H, H -CQ), 2.66 (m, 1H, H ), 2.21
5 9 a’ d’-e’ 1
(
s,3H, H ), 1.80 (m, 4H, H -CQ), 1.40 (d, 3H, J = 6.4 Hz, H -CQ), 1.26 (t, 6H, H -CQ), 1.04 (d,
10 b’-c’ a’’ f’
6
H, J = 7.2 Hz, H -H ).
2 3
6
[
RuCQ(η -C H )(5,5’-dimethyl-2,2’-bipyridine)][PF ] (9). Orange solid, yield 195 mg (76%).
10 14 6 2
.
Elemental analysis (%) calcd for C H ClF N P Ru CH Cl C 44.19, H 4.88, N 6.29. Found: C
4
0
52
12
5
2
2
2
-
1
4
0
8
3
4.21, H 4.84, N 5.90. IR (Selected bands, cm ) ν (C=C) 1554; ν (C=N) 1477. Λ (acetone) 175.3 ±
M
-1
2
-1 1
.6 ohm cm mol . H NMR δ ppm (m, I): 9.34 (d, 2H, H -H ), 8.86 (d, 1H, J = 8.4 Hz, NH-CQ),
15
20
.65 (d, 2H, H -CQ and H -H ), 8.20 (m, 2H, H -H ), 7.90 (d, 1H, J = 2.0 Hz, H -CQ), 7.72 (m,
e,b
12
17
13
18
h
H, H -CQ and H -H ), 6.82 (d, 1H, J = 7.2 Hz, H -CQ), 5.93 (d, 2H, J = 6.4 Hz, H -H ), 5.73 (d,
f
14
19
c
6
8
9

ACCEPTED MANUSCRIPT
2
H, J = 6.4 Hz, H -H ), 4.07 (m, 1H, H -CQ), 3.13 (m, 6H, H -CQ), 2.66 (m, 1H, H ), 2.21 (s,3H,
5
9
a’
d’-e’
1
H ), 1.80 (m, 4H, H -CQ), 1.40 (d, 3H, J = 6.4 Hz, H -CQ), 1.26 (t, 6H, H -CQ), 1.04 (d, 6H, J
1
0
b’-c’
a’’
f’
=
7.2 Hz, H -H ).
2 3
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 mM NaCl, pH 7.4). The DNA concentration per nucleotide was
determined by absorption spectrophotometric analysis using a molar absorption coefficient of 6.600
-1.
.
-1
mol Lcm at 260 nm [23]. The spectroscopic titrations were carried out by adding increasing
amounts of ctDNA to a solution of the complex in a quartz cell and recording the UV–Vis spectrum
after each addition. The binding affinities (Kb and Kb ) were obtained by using the Scatchard
1
2
equation, r/C =K(n−1), corresponding to two major binding interactions with cooperative effects
f
[
24], where r is the number of moles of Ru complex bound to 1 mol of DNA (C / CDNA), n is the
b
number of equivalent binding sites, and K is the affinity of the complex for those sites.
Concentrations of free (C ) and bound (C ) complexes were calculated from C =C(1–α) and
f
b
f
C =C−C , respectively, where C is the total Ru concentration. The fraction of bound complex (α) was
b
f
calculated from α=(A −A)/(A −A ), where A and A are the absorbance of the free and fully bound
f
f
b
f
b
drug at the selected wavelengths, and A is the absorbance at any given point during the titration. The
plot of r/C vs. r gives the binding constant Kb as the slope of each graph.
f
Viscosity measurements were carried out using an Ostwald viscometer immersed in a water bath
maintained at 25 °C. The DNA concentration in buffer Tris–HCl was kept constant in all samples,
while the complex concentration was increased from 0 to 60 μM. The flow time was measured at
least 5 times with a digital stopwatch and the mean value was calculated. Data are presented as
1
/3
(
η/η0) versus the [complex]/[DNA] ratio, where η and η are the specific viscosities of DNA in the
0
presence and absence of the complex, respectively. The values of η and η were calculated using the
0
expression (t - t )/t , where t is the observed flow time and t is the flow time of buffer alone.
b
b
b
1
0

ACCEPTED MANUSCRIPT
Agarose gel electrophoresis studies. 10 μL aliquots of pBR322 plasmid DNA in Tris-HCl buffer
were incubated at 37 °C for 20 h with molar ratios of the Ru compounds between 0.5 and 2.0. After
incubation, 5 µL of each sample were separated by electrophoresis in a 1% agarose gel for 90 min at
1
00 V using Tris-borate–EDTA buffer (TBE) and stained with ethidium bromide (5 μL ethidium
bromide per 50 mL agarose gel mixture). Samples of free DNA and DNA+ DMSO were used as
controls. The DNA bands were visualised by imaging with UV light transilluminator (ChemiDoc
MP, Bio-Rad).
2
.7 BSA-binding experiments. Fluorescence spectroscopy is an effective method to explore the
interactions between small molecules and biomacromolecules. The fluorescence of BSA comes from
its tryptophan, tyrosine and phenylalanine residues, the latter two contributing to its fluorescence to
only a minor extent [25]. The protein interaction was examined in 96-well plates used for
fluorescence assays. The concentration of BSA (Sigma Aldrich), prepared by dissolving the protein
in Tris–HCl, was determined by absorption spectrophotometric analysis using a molar absorption
-1.
.
-1
coefficient of 43.824 mol Lcm at 279 nm [18]. The complexes were dissolved in sterile DMSO.
For fluorescence measurements, the BSA 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 residues at 344 nm (excitation wavelength 295 nm) was
monitored at different temperatures (295, 305 and 310 K). The experiments were carried out in
triplicate and analyzed using the classical Stern–Volmer equation:
F /F = 1 + K τ [Q] = 1 + K [Q]
(1)
0
q o
sv
where F and F are the fluorescence intensities in the absence and presence of quencher, respectively,
0
[
Q] is the quencher concentration, and Ksv Stern–Volmer the quenching constant, which can be
written as K = K /τ , where K is the biomolecular quenching rate constant and τ is the average
q
sv
o
q
o
-9
lifetime of the fluorophore in the absence of quencher (6.2x10 s) [26]. Therefore, Eq. (1) was
applied to determine K by linear regression of a plot of F /F vs. [Q].
sv
o
1
1

ACCEPTED MANUSCRIPT
The binding constant (K ) and number of complexes bound to BSA (n) were determined by plotting
b
the double log graph of the fluorescence data using Eq. (2):
log [(F -F)/F] = log K + nlog[Q]
(2)
The thermodynamic parameters were calculated from the van’t Hoff equation:
ln K = -ΔH/RT + ΔS/R (3)
where K is analogous to the Stern-Volmer quenching constant, K at the corresponding temperature
0
b
sv
(the temperatures used were 295, 305 and 310 K), and R is the gas constant, from which the ΔH and
ΔS of the reaction can be determined from the linear relationship between ln K and the reciprocal
absolute temperature. Furthermore, the change in free energy (ΔG) was calculated from the
following equation:
ΔG = -RT lnK = ΔH – TΔS
(4)
2
.8 Partition coefficient (P)
.
Water–octanol partition coefficients were determined using the stir-
flask method [27]. Each complex was tested in a mixture of equal volumes of water and octanol with
continuous shaking for 24 h at 100 rpm and 37 °C. Then the samples were centrifuged for 5 min at
3
00 rpm and the organic and aqueous phases were separated. The concentration of drug in each
phase was measured spectrophotometrically in order to determine values of P= [drug](in
octanol)/[drug](in water). 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 still represent the standard method for initial screening of antitumor
agents. Thus, as a first step to assess its pharmacological properties, the ruthenium complexes were
assayed against human breast tumor cell lines MDA-MB-231 (ATCC No. HTB-26) and MCF-7
(ATCC No. HTB-22), the human lung tumor line A549 (ATCC No. CCL-185), and the normal cell
line L929 (ATCC No. CCL-1). The cells were routinely maintained with Dulbecco’s Modified
Eagle’s medium (DMEM-for L929 and A549) or RPMI 1640 (for MDA-MB-231 and MCF-7)
supplemented with 10% fetal bovine serum (FBS), at 37 °C in a humidified 5% CO atmosphere. For
2
1
2

ACCEPTED MANUSCRIPT
4
-1
the cytotoxicity assay, 1.5x10 cells well were seeded in 200 µL of complete medium in 96-well
plates (Corning Costar). Each complex was dissolved in sterile DMSO (from 40 to 0.01 mM). One
microliter of each complex sample was added to 200 µL medium. Cells were exposed to the complex
for a 48 h period. The conversion of MTT to formazan by metabolically viable cells was monitored
by an automated microplate reader at 540 nm. The percent cell viability was calculated by dividing
the average absorbance of cells treated with the ruthenium-arene complex by that of the control; %
cell viability versus drug concentration (logarithmic scale) was plotted to determine the IC (drug
5
0
concentration at which 50% of the cells are viable relative to the control), with its estimated error
derived from the average of 3 trials.
For the morphological study MDA-MB-231 breast tumor cells were seeded at a density of
5
0
.8×10 cells/well into 12-well plates. After allowing 24 h to adhere, images of cells treated with or
without complexes 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 complexes were prepared in good yields, higher than 75%, as yellow/orange solids, stable in light
and in air, soluble dimethylsulfoxide, chloroform and acetonitrile; slightly soluble in water and
insoluble in diethyl ether and hexane. Good quality crystals of complexes 1-5 were grown by slow
diffusion of diethyl ether into dichloromethane or acetone solutions. The elemental analyses of all
the complexes were consistent with their proposed formulas. The UV-Vis absorption spectra of the
complexes in dimethylsulfoxide (Figure 1S and Table 1) were characterized by an intense high
energy band centered at about 300 nm, which can be assigned to a ligand-localized, intra-ligand π-π*
transition. Complexes 1–5 exhibited lower-energy bands in the range of 340–400 nm, which can be
attributed as metal to ligand charge transfer (MLCT) transitions from Ru (dπ) to the ligand (π*) [28-
2
9]. Complexes 6–9 showed two intense bands between 330 and 340 nm, features of the quinolinic
1
3

ACCEPTED MANUSCRIPT
chloroquine ring. The IR spectra of the complexes displayed characteristic bands in the range of
-1
-1
-1
-
-1
1
506–1540 cm , ν(C=C); 1384–1413 cm ν(C=N); 846 and 557 cm ν(PF ) and 295-302 cm ,
6
ν(Ru-Cl, for complexes 1–5). The molar conductivities of 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 solvent into their coordination sphere by
substituting the chloride atom [31]. Likewise, 6–9 molar conductivities in acetone were in the range
of 1:2 electrolytes.
1
13
All NMR signals were assigned on the basis of 1D ( H and C NMR) and 2D experiments
1
1
1
such as Correlation Spectroscopy ( H- H gCOSY), Heteronuclear Single Quantum Coherence ( H-
1
3
1
15
1
13
C gHSQC) and Heteronuclear Multiple Bond Coherence ( H- N and H- C gHMBC) for all
1
6
complexes. The H-NMR spectra showed characteristic resonances of η -C H and signals for the
10
14
N∩N ligand in question. All signals were consistent with chemical shifts, integrations, signal
multiplicities and coupling constants for these classical ligands; the atom numbering scheme is based
on the X-ray structures and Scheme 1). In all complexes, after coordinating ruthenium with the N∩N
1
or CQ ligands, the most remarkable characteristics of the H NMR spectra of these complexes,
compared with the spectra of the free ligands, were the chemical shifts in the H , H , H and H atoms
5
6
8
9
in the aromatic ring zone. Thus, since the N-N ligands used in this work are relatively good π
acceptors, they can remove electron density from the metal center, shielding these hydrogen atoms to
a lesser extent and causing some changes in the chemical shifts of these protons [32]. All carbons,
including quaternary, were determined by their correlation with the protons in the complexes by
gHMBC and gHSQC resonance techniques.
1
15
The H- N gHMBC experiment (Figure 2) was performed in order to determine the mode of
bonding of CQ to the metal (for complexes 6-9). A characterization of free CQ was performed first,
and after that the characterization of the metal-CQ complexes. This procedure was performed to
1
5
evaluate the chemical shift variation (Δδ) in each signal of N in the free ligand in comparison to the
1
4

ACCEPTED MANUSCRIPT
ruthenium-CQ complexes. The values of Δδ (Table 2) suggest that CQ binds to the ruthenium atom
through the nitrogen of the quinoline ring, as significant changes were observed with respect to the
free CQ, for N-1 Δδ = ~ 11.0 ppm, while for the N-2 and N-3 atoms the Δδ were ca. 3.0 ppm and 1.5
ppm, respectively, for all the ruthenium complexes.
The complexes were studied by cyclic voltammetry (CV) experiments, in dichloromethane or
acetonitrile solutions, the complexes 3 and 8 are a representation of electrochemical behavior for 1-5
and 6-9, respectively. CVs of complex 3 in dichloromethane (Figure 3a) did not show any process in
the anodic region, but after adding a few drops of acetonitrile to the electrochemical cell, the
-
processes (III-IV) were detected (Figure 3b) suggesting displacement of the Cl ligand by the CH CN
3
molecule. The CVs in acetonitrile (Figure 3c) show a quasi-reversible process (III/IV) at a potential
close to 1.0 V, which correspond to the redox pair Ru(II)/Ru(III), and an irreversible oxidation
process (V) at around 1.65 V, studies developed by Bernhard Gollas and coworkers [33,34] indicate
that this irreversible process at a potential around +1.65 V is due to arene ligand oxidation and it
depends on the substituent groups of this ligand [35]. Complex 3 shows oxidation (I) and reduction
6
(
II) processes at negative potentials that also belong to the coordinated η -C H ligand, as described
10
14
for similar complexes [36]. Complexes 6–9, containing CQ, revealed cyclic voltammograms similar
to those represented in Figure 3d. The CQ exhibited two oxidation processes in the anodic region
(Figure 3S), where both processes are irreversible and may be attributed to irreversible oxidation of
the N-heterocyclic nitrogen of the aminoquinoline moiety (first peak) and the nitrogen of the
alkylamino side chain group of the chloroquine molecule (second peak) [37]. In complexes 6–9, the
first oxidation process was not observed, confirming its coordination to the Ru metal center. This
process was observed only after the first sweep, indicating that the CQ was dissociated from the
metal center. These data are in accordance with reports in the literature for Ru(II) arene complexes
with phosphine [38] and pyrazolyl [39].
1
5

ACCEPTED MANUSCRIPT
Crystals of compounds of 1–5 were grown by slowly diffusing diethyl ether into a
concentrated solution of the complexes in CH Cl or acetone, yielding yellow or orange crystals,
2
2
which were investigated by X-ray crystallography. All complexes adopted the familiar half-sandwich
three-legged piano-stool, in which the immediate coordination sphere around the Ru(II) consists of
two nitrogen atoms of the N-N heterocyclic chelating ligand, where the chloride ligand is the legs of
6
the stool, and the six carbon atoms of the η -C H ligand form the seat. Figure 4 (a-f) shows the
1
0
14
structures of these ruthenium complexes. The most important interatomic distances and bond angles
6
are collected and can be seen in Table 4. Ru(II) to η -C H distances did not vary significantly
1
0
14
between all the derivatives, ranging from [2.139(14) to 2.153(10) Å]. The Ru−Cl bond lengths in the
complexes were almost the same (~2.400 Å), comparable to the Ru-Cl bond length observed in
6
+
6
10 14
+
6
10 14
+
[
RuCl(η -tetralin)(en)] [11], [RuCl(η -C H )(mimid) ] and [RuCl(η -C H ) (bimid) ]
2 2
4
0
[
].Ru(1)−N(1) and Ru(1)−N(2) bond lengths were ~2.080 Å. The N(1)−Ru(1)−N(2) bond angles in
these complexes did not differ significantly from each other. An interesting feature in the crystal
structure of these complexes is that, depending on the N-N heterocyclic chelating ligand used, the
complex may adopt different configurations. In the first configuration, adopted by complex 1 and
6
complex 3, the isopropyl part of the η -C H ligand is on the same side of the chloride ligand, while
1
0
14
in the second configuration (adopted by the other complexes) the isopropyl moiety is inverted,
occupying the side opposite the chloride ligand. This fact is easily 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
complexes. In the crystalline state of 3, the arene adopts two different orientations, presenting two
different molecules in the asymmetric unit.
6
In the crystal structure of complex 2, the η -C H group is disordered in two positions (Figure 4S).
1
0
14
6
In this cation complex, the η -C H group can adopt two different positions; however, the Ru-C
1
0
14
bond lengths are very similar for the two positions.
.2 Interactions with DNA
3
1
6

ACCEPTED MANUSCRIPT
The Ru-arene complexes interact with various targets including DNA and proteins, which is
probably the explanation for their anticancer activity [41]. It is known that free CQ intercalates into
DNA, while its Au-CQ and Ru-CQ complexes bind to DNA through intercalation and electrostatic
interactions [6-42]. Experiments of absorption titration, viscosity and electrophoresis in agarose were
performed to study the mode of interactions of the new compounds with DNA. In the spectroscopic
titrations, in the presence of ctDNA, all complexes showed decreases in absorption (hypochromism-
%
H) thus determining the constant of interaction (Kb , Kb ) by a nonlinear Scatchard curve (Figure
2 1
1
4S). The Kb and Kb (Table 5) are within the accepted range for compounds considered to interact
1 2
with DNA [6]. Overall, complexes 1-5 present low hypochromism and also low Kb and Kb values
1
2
compared with their analogous with CQ, and the overall positive charge could offer an interacts
electrostatics with the phosphate groups. The constants exhibited by complexes 6, 7, 8 and 9 were
similar to those found for free CQ, and the same order of magnitude for analogous ruthenium-CQ
and platinum-CQ complexes [43].
Viscosity measurements were used successfully to determine intercalation or non-intercalation
binding modes of the complexes to DNA [44]. The observed increase in the viscosity of this
biomolecule in the presence of complexes 6 to 9 (Figure 15S), suggests intercalation of the
complexes with the DNA probably is solely due to the CQ moiety. This behavior is similar to that
2
+
reported for the complex [Ru(bpy) (dppz)] , which acts like the classical intercalator ethidium
2
bromide [45]. While the viscosity of DNA remains unchanged upon addition of the ruthenium
complexes (1-5), suggesting no interaction between these species.
It is well known that plasmid DNA exists in two main forms, a supercoiled form, which
migrates faster, and an open circular form, which migrates more slowly. Changes in the mobilities of
these bands are generally interpreted as evidence of DNA binding with another molecule [46]. This
prompted a study aiming to evaluate the electrophoretic mobility of plasmid pBR322 DNA in
1
7

ACCEPTED MANUSCRIPT
agarose after incubation of ruthenium complexes for 18 h at 37 °C (compound: DNA r 0.5 and 2.0)
i
compared to free pBR322 and pBR322 treated with CQ (Figure 5).
The results showed that exposure of pBR322 to increasing complex concentrations did not induce a
noticeable alteration in the tertiary structure of the plasmid under these conditions, similar to that
obtained for CQ and Ru–CQ complexes and commonly associated with the strength of the DNA
interaction. Analyzing all the results of our DNA binding studies together, we suggest that
compounds 6-9 interact with DNA primarily through intercalation.
3
.3 Interactions with BSA
Fluorescence quenching of BSA. Quenching can occur by different mechanisms, usually
classified as either dynamic or static quenching, which can be distinguished by their differing
dependence on temperature and viscosity, or preferably by lifetime measurements [47]. Higher
temperatures result in faster diffusion and hence a larger degree of collisional quenching. Higher
temperatures will also result in the dissociation of weakly bound complexes, and hence lead to less
static quenching. As shown in Figure 6a, BSA showed strong fluorescence emission, while the
ruthenium-arene complexes displayed almost no intrinsic fluorescence under the experimental
conditions used for the measurements. The fluorescence intensity of BSA decreased with increasing
ruthenium-arene complex concentrations, accompanied by a red shift in the maximum emission
wavelengths in the fluorescence spectra. In order to ascertain the fluorescence quenching
mechanism, the fluorescence quenching data were measured at different temperatures (295, 305 and
3
10 K). These results show that in all complexes the KSV values increased when the
temperature is increased (see Figure 6 and Table 6), except to 2, 5 and CQ. Moreover, the
1
2
12
-1
values of Kq for all complexes were in the range of 1.45x10 to 6.35×10 mol·L , far higher than
1
0
-1
2
.0×10 mol·L , the maximum possible value for dynamic quenching [48], indicating the
existence of static quenching mechanism.
1
8

ACCEPTED MANUSCRIPT
3
.3.1 Determinations of binding constants, numbers of binding sites and thermodynamic parameters
The quenching data for the static quenching process were analyzed according to the modified
Stern-Volmer equation Eq. (2). The binding constants K and binding sites, n, were calculated from
b
the slopes of the static quenching equation: log(F -F)/F vs. log[Q]. The results are summarized in
o
Table 6. The number of binding sites between BSA and the Ru/arene complexes is approximately
4
-1
equal to 1. The interaction constant for all complexes is between 0.34 x10 and 5.67 M , the same
range reported for Ru [49-50], V [51] and Zn [52] complexes, showing hydrophobic interactions
with BSA. The interaction forces between drugs and biomolecules may include electrostatic
interactions, multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric contacts
within the antibody-binding site, etc. [53]. The thermodynamic parameters, enthalpy change (ΔH),
entropy change (ΔS) and free energy change (ΔG), are the main means used to confirm the binding
modes. From the thermodynamic standpoint, ΔH> 0 and ΔS> 0 imply a hydrophobic interaction;
ΔH< 0 and ΔS< 0 reflects van der Waals force or hydrogen bond formation; and ΔH< 0 and ΔS> 0
suggests an electrostatic force.
The homologous series of ruthenium-containing aromatic dinitrogen chelating ligands utilized in this
study provides an opportunity to relate these substituents to protein association process. To elucidate
the protein association process between these ruthenium-arene complexes and BSA, the
thermodynamic parameters (ΔH and ΔS) were calculated from the van’t Hoff plots, Eq. (3), ΔG was
estimated from Eq. (4) and the results are presented in Table 6. The negative value of ΔG reveals
that the interaction process is spontaneous. Complexes 1, 3 and all complexes containing
chloroquine presents positive ΔH and ΔS values indicated hydrophobic association with BSA, while
complex 2 and 5 show negative ΔH and positive ΔS values indicated an electrostatic association, this
behavior can be related to the size and electronic density of the dinitrogen ligand, where ligands with
larger substituents and more activators like phenyl and tercbuthyl, promotes localization of
electronic density, which can be related to electrostatic affinity for BSA, in the case of complex 7 the
1
9

ACCEPTED MANUSCRIPT
inclusion of chloroquine in the coordination sphere favors a delocalization of the electronic density,
which is evidenced by an increase of the oxidation potential, leading to a hydrophobic association
with the BSA. On the other hand, although hydrophobic interactions may play a key role in their
interactions with BSA, electrostatic interactions could not be excluded because it was not possible to
account for the thermodynamic parameters of the Ru/arene-BSA compounds 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-octanol is the most widely used measure
of chemical compound lipophilicity [54] as it is a major structural factor governing both the
pharmacokinetics and pharmacodynamics of drugs, because up to a certain limit, compounds with
higher lipophilicity have higher permeation across biological membranes (but lower aqueous
solubility). In general terms, for an effective anticancer drug to initiate cell death, it must reach
viable cells in a tumor, and be retained at a sufficient concentration on a relevant time scale. The
partition coefficient (log P-Table 7) values for complexes 1-9 are in the range of 0.21 to 1.20, which
do not necessarily correlate with cytotoxicity, and it can be observed that, in general, the substituents
in bipy and phen ligands, as well as the coordination of chloroquine, increase lipophilicity of the
ruthenium complexes. This combination of structural effects and enhanced drug lipophilicity result
in increasing accumulation in biological membranes.
3
.5 Cytotoxicity assay and morphological observations
Cell respiration as an indicator of cell viability was determined by a mitochondrial dependent
reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan [34].
The results obtained using this assay are listed in Table 7. Only the complexes 2, 5 and 7 show
cytostatic effects at doses appreciably lower in comparison with cisplatin, chloroquine and
doxorubicin under the same conditions. Interestingly, the complex 7 was the most active, with an
2
0

ACCEPTED MANUSCRIPT
IC of 0.95, 2.30 and 0.80 µM in A549, MDA-MB-231 and MCF-7 cells, respectively, showing that
5
0
the coordination of CQ enhances the biological activity of the metal complexes, as reported
previously [6, 8]. Under an inverted microscope, cell shape and changes in it can be observed
clearly. As shown in Figure 8, MDA-MB-231 breast cancer cells appeared phenotypically as spindle
shaped in the control group and there were very few round cells. Cells treated with 2, 5 and 7 showed
obvious morphological changes after the first 6 h; cells treated for 48 h showed, in addition to
morphological changes, a loss of adhesion, a spindle shaped form and confluence, and there was a
clear concentration-response tendency, indicating the possibility of apoptosis.
Complexes 2 and 5 not only showed greater lipophilicity, but also presented higher interaction
constants with BSA (K ), DNA (Kb ), in comparison with those ones obtained for complexes 1, 3
sv
1
and 4. These desirable biological properties are reflected in the anticancer activity shown by
complexes 2 and 5 at lower concentrations. Likewise, the Ru-CQ derivatives showed the same
tendency 7> 6> 9 ≈ 8 for Kb (DNA), K (BSA) and log P. In accordance with this trend, complex
1
sv
7
showed the best IC values compared to all tumor cell lines tested. Its potential cytotoxicity can
50
be associated to the combination of its affinity to BSA, an interaction with DNA and lipophilic
properties which increases cellular uptake. Multi-target properties of complex 7 make this a
promising candidate for the next stages of anticancer evaluation.
4
. CONCLUSIONS
Piano-stool type complexes of the formulae [RuCl(η -C H )(N-N)]PF [N-N= 1,10-
6
10
14
6
phenanthroline 4,7-diphenil-1,10-phenanthroline, 2,2’-bipyridine, 5,5’-dimethyl-2,2’-bipyridine,
,4’-di-t-buthyl-2,2’-bipyridine and their CQ derivates were synthesized and fully characterized,
4
both in solution and in the solid state. All complexes were air stable and presented distorted
octahedral coordination spheres around the ruthenium center in the solid state, as shown in their X-
II
III
ray crystal structures. All complexes showed quasi-reversible metal-based Ru /Ru electrochemical
processes. Complexes 6–9 interact in an intercalative manner with DNA, to an extent comparable to
2
1

ACCEPTED MANUSCRIPT
that of free chloroquine. The experimental results indicated that all ruthenium complexes under study
bind to BSA with moderate affinity and the intrinsic fluorescence of BSA was quenched through a
static quenching mechanism. The thermodynamic parameters, negative or positive for ΔH, positive
for ΔS and negative for ΔG, indicate that the hydrophobic properties of the complexes and hydrogen
bonding interactions between the complexes and BSA play a major role in the complex/BSA binding
process.
Complexes 2, 5 and 7 showed greater lipophilicity and also presented higher interaction constants
with BSA (K ) and DNA (Kb ) in comparison with the rest of the analogous compounds evaluated
sv
1
in this work. These desirable biological properties are reflected in the growth inhibition on human
tumor cell lines at lower concentrations, with complex 7 showing the most promising results.
Acknowledgments
The authors gratefully acknowledge the support provided by FAPESP, CNPq, CAPES and
CYTED.
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FIGURE CAPTIONS
Figure 1. Synthetic pathway of ruthenium complexes.
1
15
Figure 2. H- N HMBC spectra in dmso-d for (A) Chloroquine, (B) Complex 2 and (C) Complex
6
7
. Bruker standard pulse sequences; spectral width of 20.02 ppm (F2) and 301.10 ppm (F1); 4K (F2)
and 256 (F1) data points; 32 scans and the nitromethane signal at 380.2 ppm as an external reference.
Figure 3. Typical cyclic voltammograms with a Pt disc electrode, 100 mV/s and a scan rate of
complex 3 in (a) dichloromethane, (b) dichloromethane with a few drops of acetonitrile and (c)
acetonitrile, (d) solution of 8 representing the electrochemical behavior of 6–9 in acetonitrile.
Figure 4. ORTEP view of complexes 1–5 with the thermal ellipsoids at the 50 % probability level.
The solvent molecules, PF anions and hydrogens have been omitted for clarity.
6
Figure 5. Electrophoresis of plasmid pBR322 DNA incubated in 5 mM Tris/HCl buffer for 18 h at
3
7 °C treated with 0.5, 1.0 and 2.0 eq. of CQ and complexes 6, 7, 8 and 9. MM: molecular markers,
lanes 2 and 14: pBR322 DNA; lanes 3 and 15: DNA treated with DMSO.
Figure 6. (a) Fluorescence quenching spectra of BSA with different concentrations of complex 7
with the excitation wavelength at 280 nm at 310 K in a Trizma buffer, pH 7.4. The arrow shows the
intensity changes upon increasing the concentration of the quencher. (b) Stern–Volmer plots showing
tryptophan quenching in BSA at 310 K.
Figure 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.
Figure 8. Morphological study under an inverted microscope (100x) of MDA-MB-231 control cells
and cells treated with the IC concentrations of complexes 2, 5 and 7. In all panels, the images are
5
0
representative of many pictures taken in n = 3 experiments.
2
6

ACCEPTED MANUSCRIPT
TABLE CAPTIONS
Table 1.Electronic spectra data for complexes in dimethylsulfoxide.
1
5
Table 2. N NMR chemical shifts (δ) for Chloroquine (CQ), free ligands and metal-CQ complexes
in DMSO-d6.
Table 3. Electrochemical potentials of ruthenium complexes in acetonitrile.
6
6
Table 4. Selected bond lengths (Å) and angles (°) in [Ru(η -C H )(phen)Cl]∙PF (1), [Ru(η -
10
14
6
6
C H )(dphphen)Cl]∙PF ∙CH CH OCH CH (2). [Ru(η -C H )(bipy)Cl]∙PF (3, two molecules in
1
0
14
6
3
2
2
3
10 14
6
6
6
the
assymetric
unit),
[Ru(η -C H )(dmebipy)Cl]∙PF
(4),
and
[Ru(η -
10
14
6
C H )(dbutbipy)Cl]∙PF ∙0.5CH Cl (5).
10
14
6
2
2
Table 5. Binding constants for the interactions between the ruthenium complexes and ctDNA.
-
1
Table 6. Stern-Volmer quenching constant (K L mol ), biomolecular quenching rate constant (Kq,
sv,
-1 -1
-1
0
-1
0
L mol s ), Binding constant (K , M ), the number of binding sites (n), ΔG (KJ.mol ), ΔH
KJ.mol ) and ΔS (J.mol K) values for the complex-BSA system at different temperatures.
b
-1
0
-1
(
Table 7. In vitro anticancer activity in A549, MDA-MB-231, MCF-7 and L929 cells compared to
chloroquine, cisplatin and doxorubicin after 48 h.
2
7

ACCEPTED MANUSCRIPT
Figure 1
Figure 2
2
8

ACCEPTED MANUSCRIPT
Figure 3
2
9