Polypyridyl ruthenium complexes: Novel DNA-intercalating agents against human breast tumor

Article abstract of DOI:10.21577/0103-5053.20170019

This paper describes a new series of four DNA-intercalating agents with promising anticancer activities, based on ruthenium(II) with the planar ligand dpqQX (dpqQX = dipyrido[3,2-a:2',3'-c] quinoxaline[2,3-b]quinoxaline). The complexes identified as trans-[RuCl₂(dppb)(dpqQX)], cis-[RuCl₂(dppb)(dpqQX)], ct-[RuCl(CO)(dppb)(dpqQX)]PF₆and ct-[RuCl₂(PPh₃)₂(dpqQX)] (dppb = 1,4-bis(diphenylphosphine)butane and PPh₃ = triphenylphosphine) were characterized by ³¹P{¹H} nuclear magnetic resonance (NMR) and infrared spectroscopies, cyclic voltammetry,molar conductance measurements, elemental analysis, mass spectrometry and X-ray diffraction analysis for complex ct-[RuCl₂(PPh₃)₂(dpqQX)]. Their in vitro cytotoxic activities against MDAMB-213 and MCF-7 breast cancer cells were evaluated and compared with normal L-929 cells. Low drug concentration at which 50percent of the cells are viable relative to the control (IC₅₀) values were obtained for all four complexes compared with a reference metallodrug, cisplatin. In addition, DNA affinity studies from titrations, as well as the images obtained by atomic force microscopy (AFM)involving pBR322 plasmid DNA, suggest interactions between the metal complexes and the DNA macromolecule, in which they act as intercalating agents. The intercalation of the complexes with DNA was confirmed by viscosity measurements.

Full text of DOI:10.21577/0103-5053.20170019

http://dx.doi.org/10.21577/0103-5053.20170019
J. Braz. Chem. Soc., Vol. 28, No. 10, 1879-1889, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Polypyridyl Ruthenium Complexes: Novel DNA-Intercalating Agents against
Human Breast Tumor
João P. Barolli,a Rodrigo S. Corrêa,b Fabio S. Miranda,c Juliana U. Ribeiro,a Carlos Bloch Jr.,d
Javier Ellena,e Virtudes Moreno,f Márcia R. Cominettig and Alzir A. Batista*,a
aDepartamento de Química, Universidade Federal de São Carlos, CP 676, 13561-901 São Carlos-SP, Brazil
bDepartamento de Química, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto-MG, Brazil
cInstituto de Química, Universidade Federal Fluminense, 24020-141 Niterói-RJ, Brazil
dLaboratório de Espectrometria de Massa (LEM), EMBRAPA-Recursos Genéticos e Biotecnologia,
70770-917 Brasília-DF, Brazil
eInstituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos-SP, Brazil
fDepartment de Química Inorgànica, Universitat de Barcelona, Martí y Franquès 1-11,
08028 Barcelona, Spain
gDepartamento de Gerontologia, Universidade Federal de São Carlos, 13565-905 São Carlos-SP, Brazil
This paper describes a new series of four DNA-intercalating agents with promising anticancer
activities, based on ruthenium(II) with the planar ligand dpqQX (dpqQX = dipyrido[3,2-a:2’,3’-c]
quinoxaline[2,3-b]quinoxaline). The complexes identified as trans-[RuCl2(dppb)(dpqQX)],
cis-[RuCl2(dppb)(dpqQX)], ct-[RuCl(CO)(dppb)(dpqQX)]PF6 and ct-[RuCl2(PPh3)2(dpqQX)]
(dppb = 1,4-bis(diphenylphosphine)butane and PPh3 = triphenylphosphine) were characterized
by 31P{1H} nuclear magnetic resonance (NMR) and infrared spectroscopies, cyclic voltammetry,
molar conductance measurements, elemental analysis, mass spectrometry and X-ray diffraction
analysis for complex ct-[RuCl2(PPh3)2(dpqQX)]. Their in vitro cytotoxic activities against MDA-
MB-213 and MCF-7 breast cancer cells were evaluated and compared with normal L-929 cells. Low
drug concentration at which 50% of the cells are viable relative to the control (IC50) values were
obtained for all four complexes compared with a reference metallodrug, cisplatin. In addition, DNA
affinity studies from titrations, as well as the images obtained by atomic force microscopy (AFM)
involving pBR322 plasmid DNA, suggest interactions between the metal complexes and the DNA
macromolecule, in which they act as intercalating agents. The intercalation of the complexes with
DNA was confirmed by viscosity measurements.
Keywords: ruthenium complex, metallo-intercalator, DNA interaction, tumor cells, cytotoxicity
Introduction
π-stacking interactions and grooving, leading to hindrance
of vital biological functions due to alterations in the tertiary
structure of the DNA.4,5
Metal complexes containing a planar polypyridyl
ligand obtained from 1,10-phenanthroline have been
extensively studied in the last decades due to their DNA
binding ability.1-3 Specifically, ruthenium(II) complexes
coordinated with phenazine and quinoxaline derivatives
can bind to DNA through intermolecular forces. The
important interactions between the complexes and DNA
are electrostatic binding, non-covalent intercalation via
Ligands derived from 1,10-phenanthroline present a
rigid and planar structure with highly conjugated electron
clouds. Many ruthenium(II) complexes with phenazine
derivatives have shown promising results as DNA-linkers,
interacting with nucleobase pairs of this biomolecule6,7
and leading to the death of cancer cells. Furthermore,
ruthenium complexes with 1,10-phenanthroline derivatives
exhibit high redox potential, photochemical and interesting
photophysical properties, due to the high energy involved in
*e-mail: daab@ufscar.br

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Polypyridyl Ruthenium Complexes: Novel DNA-Intercalating Agents against Human Breast Tumor
J. Braz. Chem. Soc.
metal-to-ligand charge transfer (MLCT) and, consequently,
possible effects upon DNA systems.8,9
synthesized by exchange of a chorine trans to phosphorus
to one carbonyl ligand (Scheme 1). All complexes were
characterized and their biological properties such as
cytotoxicity against invasive MDA-MB 231 and non-invasive
MCF-7 tumor cells lines were evaluated, including their
interaction with the DNA molecule.
Previously, Miranda et al.10 synthesized three new
α,α’-diimine ligands based on 1,10-phenanthroline-5,6-dione
condensation with 1,2-phenylenediamine derivatives,
using different approaches, including the first example of
a dipyrido[3,2-f:2’,3’-h]quinoxalino[2,3-b]quinoxaline
(dpqQX) heterocyclic system. Recently, we have synthesized
several classes of phosphine ruthenium(II) complexes
with N-heterocyclic ligands and we have evaluated their
cytotoxicity against tumor cancer cells, in which promising
results have been obtained.11,12 Therefore, as part of our
ongoing research to obtain new compounds with potential
cytotoxic effects, three neutral complexes were obtained
with the general formula [RuCl2(P-P)(dpqQX)], where
P-P is dppb (1,4-bis(diphenylphosphino)butane) or (PPh3)2
(triphenylphosphine) and dpqQX = dipyrido[3,2-a:2’,3’-c]
quinoxaline[2,3-b]quinoxaline, a planar heteroaromatic
ligand with high electronic conjugation. Also, an ionic
compound ct-[RuCl(CO)(dppb)(dpqQX)]PF6 was
Experimental
Materials for synthesis
All manipulations were carried out under purified
argon with standard Schlenk techniques. The solvents were
degassed and distilled according to standard procedures,
where reagent grade solvents were appropriately distilled
and dried before use.All chemicals were reagent grade and
were used as received from commercial suppliers unless
otherwise stated. RuCl3⋅3H2O, 1,4-bis(diphenylphosphino)
butane and triphenylphosphine were used as supplied by
Sigma-Aldrich. The phosphinic ruthenium precursors
Scheme 1. Synthetic route of the complexes 1-4.

Vol. 28, No. 10, 2017
Barolli et al.
1881
[RuCl2(dppb)(PPh3)]13 and [RuCl2(PPh3)3]14 and the dpqQX
ligand10 were prepared as reported elsewhere.
were carried out at room temperature in CH2Cl2 containing
0.1 mol L-1 tetrabutylammonium perchlorate (TBAP Fluka
Purum) using a BAS-100B/W Bioanalytical Systems
Instrument. The working and auxiliary electrodes were
stationary Pt foils; the reference electrode was Ag/AgCl,
in a Luggin capillary probe, a medium in which
ferrocene (Fc) is oxidized at 0.43 V (Fc+/Fc). The
voltammogram was performed at a scan rate of 0.10 V s-1,
X-ray diffraction data
Brown crystals of ct-[RuCl2(PPh3)2(dpqQX)]
(compound 4) were grown by slow evaporation of a mixed
dichloromethane/ether solution at room temperature. The
X-ray diffraction experiment was carried out at 298 K
on an Enraf-Nonius Kappa-CCD diffractometer (95 mm
CCD camera on goniostat) using graphite monochromated
Mo-Kα radiation (0.71073Å). The structure was solved by
direct methods with SHELXS-97.15 The model was refined
by full-matrix least squares on F2 with SHELXL-97.15
Hydrogen atoms in the aromatic rings of the PPh3 and
dpqQX ligands were set as isotropic with a thermal
parameter 20% greater than the equivalent isotropic
displacement parameter of the atom to which each one was
bonded. Structural analysis and figures were made using
the MERCURY16 and ORTEP-3 programs.17 Although
the X-ray experiment afforded the molecular connectivity
of compound 4, the low quality of the crystals and the
disorder associated with the solvent molecules and PPh3
groups prevented complete refinement of the model. Crystal
data: Ru2C130H120Cl4N12O0.50P4, MW = 2326.20, monoclinic,
a = 28.859(1) Å, b = 35.335(3) Å, c = 25.476(2) Å,
β = 122.209(3)°, V = 21980.6(5) Å3, T = 298(2) K, space
group C2/c, Z = 8, Dc = 1.406 g cm-3, μ(Mo Kα) = 0.490 mm-1,
θ-range for data collection = 2.98-25.00, 0 ≤ h ≤ 34,
−0 ≤ k ≤ 42, −30 ≤ l ≤ 25, 17822 reflections collected,
completeness to θ = 25° of 92.0%, F000 = 9632, 1192
parameters refined.
o
at 298 C. The electronic spectra were obtained with
scanning on a Hewlett-Packard diode array model 8452A
spectrophotometer. The microanalyses were performed at
the Microanalytical Laboratory at the Federal University
of São Carlos, São Carlos city, São Paulo, using a FISONS
CHNS, EA 1108 microanalyser.
Synthesis
Inthiswork, complexestrans-[RuCl2(dppb)(dpqQX)](1)
and cis-[RuCl2(dppb)(dpqQX)] (2) differ in respect to the
position of the chlorine ligands to each other. For complex
ct-[RuCl(CO)(dppb)(dpqQX)]PF6 (3), the first letter of
the prefix ct is related to the position of the chlorine to
the carbonyl ligand and the second letter refers to the
position of the carbonyl relative to the phosphorous atom.
For complex ct-[RuCl2(PPh3)2(dpqQX)] (4), the first letter
refers to the relative positions of the chlorine atoms with
respect to each other and the second refers to the geometric
positions of the two PPh3 ligands relative to each other.
The complexes trans-[RuCl2(dppb)(dpqQX)] (1)
and ct-[RuCl2(PPh3)2(dpqQX)] (4) were obtained from
the precursors [RuCl2(dppb)(PPh3)] and [RuCl2(PPh3)3],
respectively. The complex cis-[RuCl2(dppb)(dpqQX)] (2)
was obtained from isomerization of compound 1.
Compound ct-[RuCl(CO)(dppb)(dpqQX)]PF6 (3) was
obtained from compound 2 by coordination of carbon
monoxide, which was generated from the dehydration of
formic acid by sulfuric acid.
Physical measurements
The IR spectra of the complexes were recorded on a
FT-IR Bomem-Michelson 102 spectrometer in the range
4000-200 cm-1 using solid samples pressed in CsI pellets.
31P{1H} nuclear magnetic resonance (NMR) spectra were
recorded at 293 K using a Bruker Avance III spectrometer,
(400 MHz for hydrogen frequency) at 161.98 MHz, with
CH2Cl2 as solvent (external reference 85% H3PO4) and with
a capillary containing D2O. The splitting resonances are
defined as s = singlet or d = doublet. The molar conductance
measurements (Λ) were carried out in CH2Cl2 at 25 °C
using concentrations of 1.0 × 10-3 mol L-1 in a Micronal,
model B-330, equipped with a Pt electrode. Mass spectra
were obtained by direct injection in a MicroTof-Q II Bruker
Daltonics Mass Spectrometer (Le) in the positive ion mode,
utilizing CH3OH (LC-MS grade from Honeywell-B&J
Brand) as solvent. Cyclic voltammetry (CV) experiments
trans-[RuCl2(dppb)(dpqQX)] (1)
The trans-[RuCl2(dppb)(dpqQX)] (1) was prepared by
reacting the precursor [RuCl2(dppb)(PPh3)] (0.116 mmol;
100 mg) with dpqQX ligand (0.116 mmol; 38.9 mg) in
dichloromethane (50.0 mL) under an Ar atmosphere for
20 min. The final brown solution was concentrated to
ca. 3.0 mL and diethyl ether was added for the precipitation
of a brown solid, which was filtered off and washed well
with diethyl ether (3 × 5.0 mL), and hexane (3 × 5.0 mL)
and dried under vacuum for 24 hours. Yield: 91%
(98.1 mg); anal. calcd. for C48H38N6P2Cl2Ru.0.33CH2Cl2:
exptl. (calcd.)%:C60.50(60.40), H4.27(4.06), N8.80(8.74);
IR (CsI) n / cm-1 3051 (m) n(C−H)arom; 2920 (w), 2989 (w)

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Polypyridyl Ruthenium Complexes: Novel DNA-Intercalating Agents against Human Breast Tumor
J. Braz. Chem. Soc.
n(C−H)aliph; 1587 (w), 1542 (w), 1494 (m), 1434 (s), 1417 (m),
1383 (s), 1349 (m), 1315 (w) n(C=N + C=C) + δ(C−H);
1114 (m) n(P−C); 1090 (m) δ(C=N); 699 (s) γ(aromatic
ring); 508 (m) n(Ru-P); 425 (w) n(Ru-N); 317 (w)
n(Ru−Cl); 31P{1H} NMR (161.98 MHz, CH2Cl2): d 32.6 (s);
HR TOF-MS-ES: m/z [M – Cl]+ calcd.: 897.137 Da; found:
897.138 Da; [M – H – 2Cl]2+ calcd.: 430.076 Da; found:
430.073 Da; UV-Vis (CH2Cl2, 1.0 × 10-5 mol L-1): λ / nm
(ε / L mol-1 cm-1) 302 (2.61 × 104), 426 (4.49 × 103). The
solid is soluble in dichloromethane, chloroform, methanol,
dimethylformamide and dimethylsulfoxide.
N 7.30 (7.27); IR (CsI) n / cm-1 3060 (m) n(C−H)arom
;
2927 (w), 2851 (w) n(C−H)aliph; 1996 (s) n(CO); 1587 (w),
1542 (w), 1500 (s), 1471 (s), 1436 (s), 1422 (s), 1397 (s),
1386 (s), 1351 (w), 1314 (w) n(C=N + C=C) + δ(C−H);
1116 (m) n(P−C); 1090 (m) δ(C=N); 844 (s) n(P−F),
697 (s) γ(aromatic ring); 558 (s) δ(P−F), 507 (m) n(Ru−P);
427 (w) n(Ru−N); 320 (w) n(Ru−Cl); 31P{1H} NMR
(161.98 MHz, CH2Cl2): d 36.9 (d), 7.9 (d), 2Jpp 30.0 Hz;
HR-TOF-MS-ES m/z [M – PF6]+ calcd.: 925.131 Da;
found: 925.130 Da; [M – CO – PF6]+ 897.137 Da; found:
897.138 Da; UV-Vis (CH2Cl2, 1.0 × 10-5 mol L-1) λ / nm
(ε / M-1 cm-1) 298 (3.14 × 104), 432 (1.79 × 104). The
complex is soluble in dichloromethane, chloroform,
methanol, dimethylformamide and dimethylsulfoxide.
cis-[RuCl2(dppb)(dpqQX)] (2)
A CH2Cl2 (100 mL) solution of 1 (150 mg, 0.052 mmol)
was refluxed for 60 h under Ar. The resulting brown
solution was concentrated to ca. 2 mL and diethyl
ether was added for the precipitation of a brown solid,
which was filtered off, washed well with diethyl ether
(3 × 5.0 mL) and hexane (3 × 5.0 mL), and dried under
vacuum for 24 hours. Yield: 81% (43.4 mg); anal. calcd.
for C48H38N6P2Cl2Ru.0.33CH2Cl2 exptl. (calcd.) %:
C 60.50 (60.40), H 4.33 (4.06), N 8.33 (8.74); IR (CsI)
ct-[RuCl2(PPh3)2(dpqQX)] (4)
The complex ct-[RuCl2(PPh3)2(dpqQX)] (4) was
prepared by stirring the precursor [RuCl2(PPh3)3]
(0.104 mmol; 100 mg) with the dpqQX ligand (0.104 mmol;
34.9 mg) in dichloromethane (50.0 mL) for 20 min. The
final brown solution was concentrated to ca. 2.0 mL and
diethyl ether was added for the precipitation of a brown
solid, which was filtered off, washed well with diethyl
ether (3 × 5.0 mL) and hexane (3 × 5.0 mL), and dried
under vacuum for 24 hours. Yield: 87% (93.4 mg); anal.
calcd. for C56H40N6P2Cl2Ru: 0.67CH2Cl2: exptl (calc) %:
C 62.39 (62.58), H 3.71 (3.83), N 7.65 (7.73); IR (CsI)
n / cm-1: 3053 (m) n(C−H)arom; 1587 (w), 1539 (w),
1481 (m), 1434 (m), 1419 (m), 1384 (F), 1346 (w),
1309 (w) n(C=N + C=C); 1112 (m) n(P−C); 1090 (m)
δ(C=N); 697 (s) γ(aromatic ring); 518 (m) n(Ru-P); 430 (w)
n(Ru-N); 309 (w), 291 (w) n(Ru−Cl); 31P{1H} NMR
(161.98 MHz, CH2Cl2): d 22.6 (s); HR-TOF-MS-ES:
m/z [M – Cl-]+ calcd.: 995.152 Da; found: 995.158 Da;
[M – 2Cl-]2+ calcd.: 480.092 Da; found: 480.090 Da; UV-Vis
(CH2Cl2, 1.0 vacuum 10-5 mol L-1): λ / nm (ε / M-1 cm-1) 302
(2.61 vacuum 104), 436 (4.49 vacuum 103). The complex is
soluble in dichloromethane, chloroform, methanol, ethanol,
dimethylformamide and dimethylsulfoxide.
n/cm-1:3053(m)n(C−H)arom;2923(w), 2853(w)n(C−H)aliph
;
1588 (w), 1542 (w), 1496 (m), 1469 (s), 1434 (s), 1418 (m),
1384 (s), 1350 (w), 1312 (w) n(C=N + C=C) + δ(C−H);
1116 (m) n(P−C); 1092 (m) δ(C=N); 697 (s) γ(aromatic
ring); 507 (m) n(Ru−P); 419 (w) n(Ru−N); 302 (w),
280 (w) n(Ru−Cl); 31P{1H} NMR (161.98 MHz, CH2Cl2):
2
d 43.5 (d); 30.8 (d), Jpp 33.8 Hz; HR-TOF-MS-ES:
m/z [M – Cl]+ calcd.: 897.137 Da; found: 897.136 Da;
[M – H – 2Cl]2+ calcd: 430.076 Da; found: 430.074 Da;
UV-Vis (CH2Cl2, l.0 × 10-5 mol L-1) λ / nm (ε / M-1 cm-1)
288 (4.54 × 104), 424 (2.97 × 104). The complex 2 is
soluble in dichloromethane, chloroform, methanol,
dimethylformamide and dimethylsulfoxide.
ct-[RuCl(CO)(dppb)(dpqQX)]PF6 (3)
The ct-[RuCl(CO)(dppb)(dpqQX)]PF6 complex was
synthesized from compound cis-[RuCl2(dppb)(dpqQX)] (2)
(50 mg, 0.053 mmol) dissolved in dichloromethane (5 mL).
The brown solution was exposed to CO gas generated from
a mixture of H2SO4/HCOOH, forming a yellow suspension.
The mixture was stirred at room temperature and, after the
addition of 8.7 mg; (0.053 mmol) of NH4PF6 dissolved
in 1.0 mL of methanol, the resulting yellow solution was
concentrated to ca. 2 mL and diethyl ether was added for
the precipitation of a yellow solid, which was filtered off,
washed well with diethyl ether (3 × 5.0 mL), and hexane
(3 × 5.0 mL), and dried under vacuum for 24 hours.Yield:
88% (51.6 mg); anal. calcd. for C48H38ClON6P3F6Ru.CH2Cl2
exptl. (calcd.) %: C 51.86 (51.98), H 3.74 (3.49),
DNA titration
All the measurements with calf thymus DNA
(CT-DNA) were carried out in Tris-HCl buffer (5 mM
Tris-HCl and 50 mM NaCl, pH 7.4). The CT-DNA
concentration per nucleotide was determined by absorption
spectrophotometric analysis using the molar absorption
coefficient 6.600 M-1 cm-1 at 260 nm.18,19 The spectroscopic
titrations were carried out by adding increasing amounts of
CT-DNA (15 μL, ca. 3 mM) to a solution of the complex
at a fixed concentration (2 mL, 75 μM) in a quartz cell,

Vol. 28, No. 10, 2017
Barolli et al.
1883
and recording the UV-Vis spectra after each addition.
While measuring the absorption spectra, an equal amount
of CT-DNA was added to the complex solution and the
reference solution to eliminate the absorbance of the
CT-DNA itself. The complex-DNA affinity was obtained
by using the Scatchard equation:19,20
cell lines were performed using a standard method for
initial screening of antitumor agents. The complexes were
tested against the invasive human breast tumor cell line
MDA-MB-231 (ATCC No. HTB-26), the non-invasive
breast tumor cell line MCF-7 (ATCC No. HTB-22), and
also on a non-tumor line of mouse fibroblasts, L929
(ATCC No. CCL-1). The MDA-MB-231 and L929 cell
lines were routinely maintained in Dulbecco’s Modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), and the MCF-7 cell line was cultured
in Roswell Park Memorial Institute (RPMI) 1640 medium
supplemented with 20% FBS. All cell lines were kept at
37 ºC in a humidified 5% CO2 atmosphere. After reaching
confluence, cells were detached by trypsinization and
1 × 104 cells well-1 were seeded in 200 μL of complete
medium in 96-well assay microplates (TPP). The plates
were incubated at 37 ºC in 5% CO2 for at least 12 h to allow
cell adhesion prior to drug testing. All tested compounds
were dissolved in sterile dimethylsulfoxide (DMSO) (a
stock solution with a maximum concentration of 20 mM)
and diluted to 0.05 to 200 μM (final concentration in each
well), after which 2 μL aliquots were added to 200 μL of
medium (final concentration 1% DMSO per well). Cells
were incubated with compounds for 48 h at 37 ºC in 5%
CO2.
r/Cf = nK(n−1)
(1)
where r is the number of mol of Ru complex bound to 1 mol
of CT-DNA (Cb/CDNA), n is the number of equivalent binding
sites, and K is the affinity of the complex for those sites.
Concentrations of free (Cf) and bound (Cb) complexes were
calculated from Cf = C(1–α) and Cb = C − Cf, respectively,
where C is the total concentration of the ruthenium(II)
complex. The fraction of bound complex (α) was calculated
from:
α = (Af − A)/(Af − Ab)
(2)
where Af and Ab are the absorbance of the free and fully
bound complex at the selected wavelengths, and A is the
absorbance at any given point during the titration. The plot
of r/Cf vs. r gives the binding constant Kb as the slope of
the graph.
To verify the potential cytotoxic effects of the
complexes, cell viability was measured by the MTT method
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide], a colorimetric assay wherein the mitochondria
of viable cells reduce the soluble yellow tetrazolium
salt to blue formazan crystals.21 After incubation with
the complexes, cells were washed twice with phosphate
buffer saline (PBS) and MTT solution (0.5 mg mL-1,
50 μL per well) was added to the cells and incubated during
4 hours, after which 100 μL of isopropanol was added to
dissolve the precipitated formazan crystals. The conversion
of MTT to formazan by metabolically viable cells was
measured in an automated microplate reader at 595 nm. To
analyze the effects of the complexes on cell viability, the
viability in the control wells (cells receiving only DMSO)
was taken as a reference (100%). The viability rates of
treated cultures were then expressed as percentages of the
control value, and % cell viability was plotted against drug
concentration (logarithmic scale) to determine the IC50
(drug concentration at which 50% of the cells are viable
relative to the control), with the error estimated from the
average of three trials. IC50 values were calculated using
GraphPad Prism 4.02 software (GraphPad Software, San
Diego, CA, USA).22
AFM imaging
Atomic force microscopy (AFM) samples were
prepared by casting a 3-μL drop of test solution onto freshly
cleaved Muscovite green mica disks as support. The drop
was allowed to stand undisturbed for 3 min to favor the
adsorbate-substrate interaction. Each DNA-laden disk
was rinsed with Milli-Q water and blown dry with clean
compressed argon gas directed normal to the disk surface.
Samples were stored over silica prior to AFM imaging.
All AFM observations were made with a Nanoscope III
Multimode AFM (Digital Instrumentals, Santa Barbara,
CA). Nano-crystalline Si cantilevers of 125 nm length
with a spring constant of 50 N m-1 average ended with
conical-shaped Si probe tips of 10-nm apical radius
and cone angle of 35° were utilized. High-resolution
topographic AFM images were collected in air at room
temperature (relative humidity 40%) on different specimen
areas of 2 × 2 μm operating in intermittent contact mode
at a rate of 1-3 Hz.
Cell culture assay
To evaluate the effects of ruthenium complexes on
tumor cells, in vitro cytotoxicity assays on human tumor
Viscosity measurements were carried out using an
Ostwald viscometer immersed in a water bath maintained at

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Polypyridyl Ruthenium Complexes: Novel DNA-Intercalating Agents against Human Breast Tumor
J. Braz. Chem. Soc.
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 (η/η0)1/3
versus the ratio [complex]/[DNA], where η and η0 are the
specific viscosity of DNA in the presence and absence of
the complex, respectively. The values of η and η0 were
calculated by use of the expression (t − tb)/tb, where t is the
observed flow time and tb is the flow time of buffer alone.
phosphorous trans phosphorous, such as previously reported
for ruthenium/phosphine/diimine complexes.24
Cyclic voltammograms of the complexes showed
quasi-reversible processes for compounds 1, 2 and 4
and an irreversible process for complex 3 involving one-
electron, corresponding to the oxidation of ruthenium(II)
to ruthenium(III) (Table 1).
Table 1. Electrochemical and molar conductivity data for the ruthenium
complexes
Λ (25 °C) /
Complex
Epa / V
Epc / V
E1/2a / V
(S cm2 mol−1)
Results and Discussion
1
2
3
4
0.72
0.82
1.86b
0.65
0.56
0.69
–
0.64
0.76
–
2.1
2.7
Synthesis and characterization of the complexes
21.6
1.8
Compounds 1-4 are stable under ambient conditions.
They are insoluble in water, hexane, diethyl ether and ethanol,
being soluble in methanol, chloroform, dichloromethane,
dimethylsulfoxide and dimethylformamide. The purity
of the complexes was confirmed by elemental analysis
and 31P{1H} NMR spectra suggesting the formation
of trans-[RuCl2(dppb)(dpqQX)] (1), cis-[RuCl2(dppb)
(dpqQX)] (2), ct-[RuCl(CO)(dppb)(dpqQX)]PF6 (3) and
ct-[RuCl2(PPh3)2(dpqQX)] (4) compounds. ESI-MS(+)
spectra showed the isotopic pattern and molecular parent
of all the complexes in the study, in agreement with the
assigned formulations for mononuclear ruthenium(II) species
(Figure S1 in Supplementary Information section). The molar
conductivity data in CH2Cl2 at 25 °C indicated that only
complex 3 is a 1:1 electrolyte, while the other compounds
are neutral, in accordance with their proposed formulations.
The 31P{1H} NMR spectra of complexes 2 and 3
presented typical AX spin systems, two doublets with
chemical shifts at 43.5 (d); 30.8 (d), and 36.9 (d);
0.44
0.55
aScan rate: 100 mV s-1, in CH2Cl2 vs. Ag/AgCl; Epa: irreversible, in
CH3CN vs. Ag/AgCl; Epa: anodic peak potential; Epc: cathodic peak
potential; Λ: molar conductance (dichloromethane).
b
The electrochemical oxidation of 3 forms a d5
configuration for the ruthenium(III) ion, leading to
the dissociation of the CO ligand from the oxidized
complex, which explains the irreversibility of the cyclic
voltammogram of this compound. As can be seen from
Table 1 data the cis-[RuCl2(dppb)(dpqQX)] (2) isomer
is electrochemically more stable than the trans isomer,
complex 1. This was also previously observed for the
[RuCl2(dppb)(N-N)] (N-N =bipy = 2,2’-bipyridine and
1,10-phenanthroline) complexes.25,26 Additionally, the lower
oxidation potential for complex 4 can be explained by the
competitive effect between the two PPh3 ligands, which are
in trans position to each other.24
The FTIR band assignments are represented in Table S1.
The most characteristic vibrational mode of the free dpqQX
ligand is nC=N of the phenanthroline and quinoxaline
moieties at 1412 and 1384 cm-1, respectively.10 The C=C
skeletal vibrations of the aromatic rings can be observed
at 1550 and 1540 cm-1. For all four complexes n(C=N)
stretching appears in the 1500 cm-1 region, indicating that
the dpqQX ligand is coordinated. The infrared spectrum
of complex 3 shows a typical nCO band at 1996 cm-1,
which is in agreement with a relatively strong interaction
(metal-carbonyl back-bonding). The absorptions at
419-430 cm-1 can be assignable to ruthenium-nitrogen
bonding, in agreement with the coordination of the dpqQX
to ruthenium(II). Additional peaks were observed for
complex 3 at 844 and 558 cm-1 due to the n(P–F) and δ(P–F)
vibration modes of the anion.
2
7.9 (d) ppm, with JP-P 33.8 and 30.0 Hz, respectively,
indicating the formation of asymmetrical structures in which
dpqQX is not in the same plane as the dppb ligand. The
large difference in the chemical shifts of the phosphorus
atoms in the ct-[RuCl(CO)(dppb)(dpqQX)]PF6 (3)
complex compared with that observed in its precursor,
cis-[RuCl2(dppb)(dpqQX)] (2), shows that one phosphorus
atom is in the trans-position with respect to the carbonyl
group. The shielding observed for one phosphorus atom,
which is trans to the carbonyl group in the 31P{1H} NMR
spectra, is due to the trans-weakening caused by the carbonyl
to the Ru-P bond, in which this aspect was previously
reported.23 For complexes 1 and 4, their 31P{1H} spectra
present a singlet at 32.6 and 22.6 ppm, respectively, indicating
the formation of symmetrical structures in which the dpqQX
ligand is trans to the dppb in (1) or trans to the chlorine in
complex 4. In complex 4 the chemical shift is typical of
The complex 4 crystallizes in the monoclinic system,
with space group C2/c with two molecules in the

Vol. 28, No. 10, 2017
Barolli et al.
1885
asymmetric unit. The geometry observed for the complex
is a distorted octahedron, as can be seen by the bond angles
and metal-ligands distances around the ruthenium(II)
metal center (Table 2) and its crystal structure is shown in
Figure 1. The N–Ru–N bond angle is far from ideal value
(90o) due to the five member chelate ring tension. The
distances for the Ru–Cl, Ru–P and Ru–N bonds lengths and
bond angles found for the compound is within the normal
range for similar ruthenium(II) complexes.12,25,27
Figure 2. Representation of π-π interactions occurring in the ligand
dpqQX, contributing for crystal packing stabilization of the complex 4.
(A) Ring d involved in the π-stacking contacts with adjacent molecule;
(B) interplanar distance between the dpqQX ligands.
Figure 1. Crystal structure of the complex 4 with the main atoms labeled.
assigned to π → π* ligand charge (LC) transitions, also
observed in the free ligands (dppb and dpqQX). The metal-
to-ligand charge transfer transition is a shoulder band on the
ligand centered absorption at ca. 470 nm, and it is assigned
to charge transfer from ruthenium(II) to the dpqQX ligand.
The influence of CT-DNA on the LC bands of the ruthenium
complexes was investigated using UV-Vis spectroscopic
titrations and compared to the previously reported data for
ruthenium(II)-dppz complexes.28 Strong DNA binding by
intercalation is suggested for the ruthenium complexes that
contain the planar ligand with the largest aromatic surface
area,28 which is expected to the complexes studied here.
The addition of small amounts of CT-DNA into
the complex solution, in buffer, resulted in large
hypochromism, MLCT or π → π* absorption bands
in compounds (1-4) (see spectra in Supplementary
Information section). Table 3 shows that significant
levels of hypochromism occur upon addition of DNA to
In Figure 2A the six rings of the dpqQX ligand labeled
(a-f) are depicted. Rings a and b are connected with the
ruthenium(II), while rings c-f (tetraaza center) are in the
outer-coordination sphere responsible for intermolecular
interactions. As can be seen, the d ring is involved in an
intermolecular π-π interaction with the d ring of an adjacent
molecule. This interaction in the solid state shows the
possibility of this complex binding to DNA by π-stacking
interactions through the tetraaza moiety of the dpqQX
(Figure 2B).
DNA binding studies by UV-Vis titration
UV-Vis experiments were carried out to investigate
the ability of the ruthenium(II) complexes (1-4) to bind to
calf-thymus DNA (CT-DNA). Electronic spectra showed
two bands in the UV region (280-300 nm) and (ca. 430 nm)
Table 2. Selected bond lengths and angles for complex ct-[RuCl2(PPh3)2(dpqQX)] (4) with estimated standard deviations in parentheses
Bond length / Å
(Angle / degree)
Bond length / Å
(Angle / degree)
Bond length / Å
(Angle / degree)
Ru1–N1
Ru1–N2
Ru1−Cl1
Ru1–Cl2
Ru1–P1
Ru1–P2
2.018(19)
2.034(17)
2.443(7)
2.432(6)
2.382(7)
2.390(7)
N1–Ru1–N(2)
N1–Ru1–Cl(1)
N1–Ru1–Cl(2)
N1–Ru1–P(1)
N1–Ru1–P(2)
P1–Ru1–P(2)
78.8(7)
170.0(5)
93.4(5)
95.0(5)
91.5(5)
173.5(3)
P1–Ru1–Cl1
P1–Ru1–Cl2
P1–Ru1–N2
Cl1–Ru1–P2
N2–Ru1–P2
N2–Ru1–Cl2
88.1(2)
88.9(2)
91.7(5)
85.6(2)
89.7(5)
172.2(5)

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Polypyridyl Ruthenium Complexes: Novel DNA-Intercalating Agents against Human Breast Tumor
J. Braz. Chem. Soc.
Table 3. Electronic absorption data and interaction constants upon addition
of CT-DNA to the solutions of complexes 1-4
the complexes in study. These types of perturbations are
generally considered to arise from the intercalation of the
bidentate ligand into the DNA duplex.29 By increasing
the concentration of the CT-DNA, the intensity of the LC
band decreased along with a red shift (Figures 3 and S2
in Supplementary Information section). The complexes
1 and 3 showed high hypochromicity in both electronic
transitions (300 and 432 nm).
Complex
λ / nm
432
Kb / (L mol-1) Hypochromism / %
1
2
3
4
7.0 × 106
1.4 × 105
5.4 × 105
2.0 × 106
38.4
35.2
37.0
14.3
432
432
434
observed changes in supercoil forms of DNA after 3 hours
of incubation at 37 °C.As can be seen, ruthenium complex
binding causes DNA chain aggregation (complexes 1, 3
and 4) and supercoiling (complexes 2-4), showing very
different DNA morphologies related to untreated DNA. In
these complexes, the interaction on the DNA is observed
with some formation of agglomerates, also observed
previously in palladium(II) and platinum(II) complexes.33
The mode of interaction observed for complexes 1-4 can
be compared to other ruthenium compounds that intercalate
into DNA and also to intercalating organic compounds such
as doxorubicin, ethidium bromide and netropsin.34,35 The
images obtained byAFM show changes in DNA, which are
in good agreement with the results of CT-DNA/complex b
inding constants. Thus, complex 2 with lower Kb value
shows a behavior less aggressive against pBR-322 plasmid
DNA. The complex 3, presenting moderate Kb value, is
more aggressive against pBR-322 plasmid DNA compared
with complex 2. In addition, the complexes 1 and 4 with
higher Kb constants show the typical images of intercalating
agents.
Figure 3. UV-Vis spectra of 2 with increasing amounts (15 μL, ca. 3 mM)
of CT-DNA in Tris-HCl buffer (5 mM Tris-HCl and 50 mM NaCl, pH 7.4),
to a solution of the complex at a fixed concentration (2 mL, 75 μM), in
a quartz cell.
The intrinsic binding constants (Kb) of complexes were
between 105 and 106 L mol-1 are consistent with intercalation
of the complexes with the CT-DNA.30-32 The lower values of
binding constants for complexes 2 and 3 may be explained
by the positions of the dppb and dpqQX ligands related
to each other, which can difficult the intercalation of the
complexes to DNA, mainly through the dpqQX ligand. The
close values of binding constants between these complexes
suggest that the charge of complex 3 does not play important
role regarding interaction of the complex with DNA. In the
same way, the complexes 1 and 4 present similar binding
constants, showing that in this case the different ancillary
phosphine ligands do not play important role concerning
binding to DNA, probably due to the equatorial position
of the dpqQX ligand, allowing better intercalation of these
complexes with DNA, when compared with the other two,
complexes 2 and 3.
Figure 4. AFM images showing the modifications in pBR322 DNA due
Atomic force microscopy (AFM) studies
to interaction with the ruthenium dpqQX complexes.
AFM images were obtained of the DNA plasmid
pBR322 as well as pBR322 DNA incubated with each
complex or with cisplatin that was used as a reference for
the in vitro tests (Figure 4). In these experiments we have
Viscosity is very sensitive to the change in the length
of the DNA double helix, and it is considered one of the
most unambiguous methods to determine intercalation
or non-intercalation binding modes of complexes to

Vol. 28, No. 10, 2017
Barolli et al.
1887
DNA, in solution.36 The effect of increasing amounts of
complexes on the relative viscosities of CT-DNA is shown
in Figure 5, together with thiazole orange, for comparison
purpose. The observed increase in the viscosity of the
DNA with the increasing of the complex concentration
suggests an intercalation of the complexes with the
DNA. This behavior is similar to that reported for the
complex [Ru(bpy)2(dppz)]2+, which acts like the classical
intercalators ethidium bromide or thiazole orange.37
the cytotoxicity of cisplatin was evaluated under the same
experimental conditions. The IC50 values calculated from
the dose-survival curves are listed in Table 4.
The new dpqQX ruthenium(II) complexes 1-4
exhibited good activity against the human tumor cell
lines MCF-7 and MDA-MB-231. As can be observed
from the data represented in Table 4, complex 3 induced
more significant cell death than cisplatin (reference
metallodrug) in both MDA-MB-231 and MCF-7 tumor
cells. Complexes 1 and 2 are also active, presenting IC50
values comparable with other ruthenium(II)/phosphine
complexes.11,12,27 The isomer cis-[RuCl2(dppb)(dpqQX)] (2)
is more active than the trans-[RuCl2(dppb)(dpqQX)] (1)
one. Probably, due to the lability of the chlorine ligand
trans to a phosphorus atom, forming the cationic species
[RuCl(L)(dppb)(dpqQX)]+ (where L = solvent), such as
occur in analogous complexes.36 Therefore, it is relevant to
mention that the cis-[RuCl2(dppb)(dpqQX)] complex can
be considered pro-drug due to this rapid exchange of one
chloride ligand. The complex ct-[RuCl2(PPh3)2(dpqQX)] (4)
is stable in the biological solution, as showed by
31P {1H} NMR experiments, and it shows better activity
than complexes 1 and 2.
Figure 5. Effect of increasing concentration of the complexes
trans-[RuCl2(dppb)(dpqQX)] (1), cis-[RuCl2(dppb)(dpqQX)] (2),
ct-[RuCl(CO)(dppb)(dpqQX)]PF6 (3) and ct-[RuCl2(PPh3)2(dpqQX)] (4),
on the relative viscosity of CT-DNA at 25 ºC.
By comparing the cell viability of complexes and free
ligands, there was a significant reduction in the cellular
viability of complexes than compared the dppb and dpqQX
ligands. The cytotoxicity assays showed that complexes 3
and 4 are the most promising for use against MDA-MB
231 tumor cells. Compared with cisplatin, complex 3
was twenty-four and twenty-two fold more active in
the MDA-MB-231 and MCF-7 cell lines, respectively,
indicating its potential as an antitumor agent. The increased
activity of complex 3 could be related to the presence of
the carbonyl ligand. We do not exclude the possibility of a
parallel mechanism of action involving the release of CO
from the metal center, in which this molecule could play a
Antitumoral activity
The new ruthenium complexes were submitted to
cytotoxic assays to study the effects of the complexes on the
viability of invasive and non-invasive human breast tumor
cells, MDA-MB-231 and MCF-7, respectively, in vitro, by
the MTT method. The cells were exposed to each compound
for a period of 48 hours in order to allow them to reach
the DNA or any other biological target. For comparison,
Table 4. IC50 values for cytotoxic assays against human breast cancer cells MDA-MB-231, MCF-7 and mouse fibroblasts L929 (non-tumoral cells) for the
ruthenium complexes 1-4, free bases and cisplatin
IC50 / µM
Complex
ISa
ISb
MDA-MB-231
29.09 3.56
6.02 0.46
0.10 0.22
3.14 0.66
> 200
MCF-7
23.75 2.41
17.05 0.69
0.41 0.02
26.11 0.81
> 200
L929
1
12.65 0.17
8.22 0.56
0.32 0.01
12.26 1.02
0.27 0.06
N.R.
0.43
1.36
0.53
0.48
2
3
3.90
0.78
4
4.02
0.47
dpqQX
dppb
PPh3
cisplatin
< 0.01
N.R.
N.R.
6.80
< 0.02
N.R.
N.R.
1.86
> 200
> 200
> 200
> 200
N.R.
2.43 0.20
8.91 2.60
16.53 2.38
aIS = IC50 L-929/IC50 MDA-MB-231; bIS = IC50 L929/IC50 MCF-7; N.R.: not realized.

1888
Polypyridyl Ruthenium Complexes: Novel DNA-Intercalating Agents against Human Breast Tumor
J. Braz. Chem. Soc.
synergistic role.38 Such as the selectivity index corresponds
to the ratio between the IC50 value for each compound
tested on fibroblasts (normal cell line) and the IC50 value
on neoplastic cells, this index can be an initial step toward
potential use in subsequent clinical trials.
The fact that CO is able of interacting with specific
biomacromolecules, such as mitochondrial enzymes,
cellular membranes and ion channels, showed that the
CO can be a useful molecule for drug design.39,40 Thus,
metal-complexes containing carbonyl bonds to metals
can be a good model for the design of complexes with
good anticancer activity. Moreover, it has been shown that
the metal-carbonyl complexes, in which the CO does not
dissociate easily in solution, can be pharmacologically
active as anticancer agents, indicating that stable metal-
carbonyl complexes can be attractive candidates for drug
development.41,42
Finally, it is worth mentioning that for the evaluation of
the complexes on tumor cells the complexes are dissolved
in DMSO. Thus, in this case the complex 2 can have its
chlorido ligand trans to the phosphorus atoms, dissociated,
due to the strong trans effect of the phosphorus atoms, as
observed previously for the cis-[RuCl2(dppb)(bipy)].36 In
this case the species cis-[RuCl(DMSO)(dppb)(bipy)]+ will
be formed, which can be detected by 31P NMR experiment.
To avoid this process the dissolution of the complex in
DMSO and its transference to the biological medium has
to be done quickly. Thus, in this medium the complex is
stable, as confirmed by NMR experiment, for at least 48 h.
Acknowledgments
The authors gratefully acknowledge the financial
support provided by CAPES/PROEX, CNPq, FAPEMIG,
FAPERJ (Process No. E-26-111.177/2011) and FAPESP
(Process No. 2012/06013-4). J. P. Barolli thanks FAPESP
for a Pos-Doc fellowship (Process No. 2013/21611-8).
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