Ru(II)/N-N/PPh3 complexes as potential anticancer agents against MDA-MB-231 cancer cells (N-N?=?diimine or diamine)

Article abstract of DOI:10.1016/j.jinorgbio.2019.01.006

The rational design of anticancer agents that acts in specific biological targets is one of the most effective strategies for developing chemotherapeutic agents. Aiming at obtaining new ruthenium (II) compounds with good cytotoxicity against tumor cells, a series of new complexes of general formula [RuCl(PPh₃)(Hdpa)(N–N)]Cl [PPh₃ = triphenylphosphine, N-N = 2,2′-dipyridylamine (Hdpa) (1), 1,2-diaminoethane (en) (2), 2,2′-bipyridine (bipy) (3), 5,5′-dimethyl-2,2′-bipyridine (dmbipy) (4), 1,10-phenanthroline (phen) (5) and 4,7-diphenyl-1,10-phenanthroline (dphphen) (6)] were synthesized. The complexes were characterized by elemental analysis and spectroscopic techniques (IR, UV/Visible, and 1D and 2D NMR) and three of their X-ray structures were determined: [RuCl(PPh₃)(Hdpa)₂]Cl, [RuCl(PPh₃)(Hdpa)(en)]Cl and [RuCl(PPh₃)(Hdpa)(dmbipy)]Cl. All the complexes are more cytotoxic against the cancer cell line than against the non-tumor cell line, highlighting complexes 1 and 5, which have an index selectivity of 18 and 15, respectively. The binding constants of compounds 1–6 with human serum albumin (HSA) were determined by tryptophan fluorescence quenching, indicating moderate to strong interactions. The binding mode of the complexes to calf thymus (CT) DNA was explored by several techniques, which reveal that only the dphphen compound 6 causes distortions in the secondary and tertiary structures of DNA. The studies demonstrated that the nature of the N–N co-ligand and the presence of the PPh₃ and Hdpa ligands are features that can influence the binding affinity of the complexes by the biomolecules and in the cytotoxic activity of the complexes. Overall, the complexes with diimine co-ligand are much more cytotoxic than compound 2 with the aliphatic diamine.

Full text of DOI:10.1016/j.jinorgbio.2019.01.006

Accepted Manuscript
Ru(II)/N-N/PPh3 complexes as potential anticancer agents against
MDA-MB-231 cancer cells (N-N=diimine or diamine)
Gabriel H. Ribeiro, Legna C. Vegas, Juan C.T. Clavijo, Javier
Ellena, Marcia R. Cominetti, Alzir A. Batista
PII:
S0162-0134(18)30473-2
DOI:
Reference:
https://doi.org/10.1016/j.jinorgbio.2019.01.006
JIB 10630
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
9 August 2018
10 January 2019
12 January 2019
Please cite this article as: Gabriel H. Ribeiro, Legna C. Vegas, Juan C.T. Clavijo, Javier
Ellena, Marcia R. Cominetti, Alzir A. Batista , Ru(II)/N-N/PPh3 complexes as potential
anticancer agents against MDA-MB-231 cancer cells (N-N=diimine or diamine). Jib
(2019), https://doi.org/10.1016/j.jinorgbio.2019.01.006
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ACCEPTED MANUSCRIPT
Ru(II)/N-N/PPh3 complexes as potential anticancer agents against
MDA-MB-231 cancer cells (N-N = diimine or diamine)
Gabriel H. Ribeiroa,*, Legna C. Vegasa, Juan C. T. Clavijob, Javier Ellenab, Marcia R. Cominettic,
Alzir A. Batistaa*
a Universidade Federal de São Carlos, Departamento de Química, São Carlos-SP, Brazil
b Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil
c Universidade Federal de São Carlos, Departamento de Gerontologia, São Carlos-SP, Brazil
*Corresponding authors: Alzir A. Batista. daab@ufscar.br; Gabriel Henrique Ribeiro.
gabrielhenri10@hotmail.com
Fax: +55 (16) 3351-8350
Phone: +55 (16) 3351-8285

ACCEPTED MANUSCRIPT
Abstract
The rational design of anticancer agents that acts in specific biological targets is one of the most
effective strategies for developing chemotherapeutic agents. Aiming at obtaining new ruthenium
(II) compounds with good cytotoxicity against tumor cells, a series of new complexes of general
formula [RuCl(PPh3)(Hdpa)(N-N)]Cl [PPh3 = triphenylphosphine, N-N = 2,2’-dipyridylamine
(Hdpa) (1), 1,2-diaminoethane (en) (2), 2,2’-bipyridine (bipy) (3), 5,5’-dimethyl-2,2’-bipyridine
(dmbipy) (4), 1,10-phenanthroline (phen) (5) and 4,7-diphenyl-1,10-phenanthroline (dphphen) (6)]
were synthesized. The complexes were characterized by elemental analysis and spectroscopic
techniques (IR, UV/Visible, and 1D and 2D NMR) and three of their X-ray structures were
determined:
[RuCl(PPh3)(Hdpa)2]Cl,
[RuCl(PPh3)(Hdpa)(en)]Cl
and
[RuCl(PPh3)(Hdpa)(dmbipy)]Cl. All the complexes are more cytotoxic against the cancer cell line
than against the non-tumor cell line, highlighting complexes 1 and 5, which have an index
selectivity of 18 and 15, respectively. The binding constants of compounds 1 - 6 with human serum
albumin (HSA) were determined by tryptophan fluorescence quenching, indicating moderate to
strong interactions. The binding mode of the complexes to calf thymus (CT) DNA was explored by
several techniques, which reveal that only the dphphen compound 6 causes distortions in the
secondary and tertiary structures of DNA. The studies demonstrated that the nature of the N-N co-
ligand and the presence of the PPh3 and Hdpa ligands are features that can influence the binding
affinity of the complexes by the biomolecules and in the cytotoxic activity of the complexes.
Overall, the complexes with diimine co-ligand are much more cytotoxic than compound 2 with the
aliphatic diamine.
Introduction
Currently, ruthenium-based complexes represent promising alternative candidates to
platinum drugs, with less toxic side effects and more selectivity against cancer cells than against
non-tumor cells [1-6]. Moreover, ruthenium complexes can act by a myriad of biological targets,
such as proteins, membranes, DNA, etc [7-12]. In this context, the synthesis of new ruthenium

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polypyridyl compounds capable of targeting DNA has been widely investigated in recent years [13-
17]. These kinds of complexes have been shown to bind to DNA by various modes [10, 13], e.g.,
through intercalation of the polypyridyl ligand in the base pairs of DNA [18, 19] or via association
with the minor or major grooves [20]. Binding compounds to DNA grooves can exhibit high levels
of DNA sequence-specific recognition [13] due to many base pairs found in the biomolecule, and
also by the possible combination of Van der Waals interactions, hydrogen bonding, hydrophobic
contacts and electrostatic interactions [13, 20, 21]. Ruthenium complexes such as [Ru(bipy)3]2+ and
[Ru(Me4phen)3]2+ (bipy = 2,2’-bipyridine; Me4phen = 3,4,7,8-tetramethyl-phenanthroline) associate
electrostatically within the grooves of the DNA, without disrupting its double helix, despite the
presence of ligands that usually intercalate DNA [10].
In recent years, our research group has been attracted by the possibilities of multifunctional
chemotherapy. In this context, we have synthesized new ruthenium(II)/phosphine complexes
containing diimine ligands and various other classes of ligands, aiming at identifying synergisms
between the metallic center and functional ligands [22-25]. Thus, many compounds were
synthesized, and some of them showed high cytotoxicity [25]. Recently, we reported on the
compounds
[Ru(pyS)(bipy)(dppb)]PF6,
[Ru(Spym)(bipy)(dppb)]PF6
and
2-
[Ru(SpymMe2)(bipy)(dppb)]PF6 [dppb
=
1,4-Bis(diphenylphosphino)butane; (pyS)
=
mercaptopyridine; (Spym) = 2-mercaptopyrimidine and (SpymMe2) = 4,6-dimethyl-2-
mercaptopyrimidine], which showed, in vitro, higher cytotoxicity against the HepG2 cell line than
the cisplatin [24]. Furthermore, these compounds showed the property of inhibiting the DNA
supercoiled relaxation mediated by the human topoisomerase IB and additional assays indicate that
they inhibit the cleavage reaction, impeding the binding of the enzyme to DNA and slowing down
the relegation reaction. Thus, results suggest that the topoisomerase I is one possible target for the
synthesized complexes [24].
In this study, based on the knowledge gained by our research group with ruthenium (II)
complexes, a series of [RuCl(PPh3)(Hdpa)(N-N)]Cl complexes [ PPh3 = triphenylphosphine; N-N =

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2,2’-dipyridylamine (Hdpa), 1,2-diaminoethane (en), 2,2’-bipyridine (bipy), 5,5’-dimethyl-2,2’-
bipyridine (dmbipy), 1,10-phenanthroline (phen) and 4,7-diphenyl-1,10-phenanthroline (dphphen)]
were synthesized and characterized. The inspiration for the design of these complexes was: (a) the
fact that using hydrophobic PPh3 ligand results in complexes with good cytotoxicity, presumably
because of increasing vehiculation property of the complexes [23, 26]; (b) the ligand Hdpa can
provide a good solubility to the complexes in polar solvents, due to the presence of the NH group in
its structure [14, 27-29]; (c) we focused mainly on assessing whether design modification of the N-
N co-ligands (comparison between the diimine ligands and aliphatic bidentate diamine ligand and
with different numbers of aromatic rings) can lead to modulating the binding mode to biomolecules
and biological activity of the compounds; (d) using chloride as a labile ligand and as a hydrogen-
bond acceptor might be able to form additional hydrogen bonds, such as O—H.…Cl, N—H.…Cl, and
C—H.…Cl, with biomolecules facilitating the interaction of complex/DNA [30].
Herein the binding mode of the new complexes with DNA was also investigated by using a
variety of methods, such as absorption spectroscopy (UV/Vis and circular dichroism), viscosity
measurements and gel electrophoresis of DNA plasmid. Interaction of the compounds with other
relevant biomolecules, human serum albumin (HSA) by fluorescence quenching, was also evaluated
with the aim of initially understanding the pharmacological properties of the compounds. The
cytotoxicity of the compounds in vitro was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay, against the tumor cell line (MDA-MB-231) and the non-tumor
cell line (V79-4).
Experimental section
RuCl3.3H2O, 2,2’-dipyridylamine, 1,10-phenanthroline, 2,2’-bipyridine, 4,7-diphenyl-1,10-
phenanthroline, 4,4’-dimethyl-2,2’-bipyridine, 1,2-diaminoethane and triphenylphosphine were
obtained from Sigma-Aldrich. All chemicals were used as purchased, without further purification.
Monodimensional [1H, 13C{1H}, 31P{1H}] and bidimensional [1H—1H correlation spectroscopy
1
1
(COSY), H - 13C {1H} heteronuclear single-quantum correlation (HSQC), H - 13C {1H}

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heteronuclear multiple-bond correlation (HMBC)] nuclear magnetic resonance (NMR) experiments
were recorded on a Bruker DRX-400 spectrometer (9.4 T) equipped with an inverse 5 mm probe
1
head with an actively shielded z-gradient coil. H and 13C {1H} chemical shifts in dimethyl
sulfoxide (DMSO-d6) were referenced to the peak of a residual non-deuterated solvent [(1H) δ 2.50,
and (13C{1H}) δ 39.52 for DMSO-d6]. The 31P{1H} NMR spectra were carried out in dimethyl
sulfoxide (DMSO-d6) (using a capillary containing H3PO4 85% in D2O) and the chemical shifts
were referenced to an external 85% H3PO4 standard at 0.00 ppm.
Elemental analyses were performed on a FISIONS Instrument EA 1108 CHNS elemental analyzer
at the Microanalytical Laboratory at the Federal University of São Carlos, São Carlos (SP).
Conductivity measurements in water and dimethyl sulfoxide solutions (1.0 mmol L-1) of the
complexes were carried out on a Meter Lab CDM2300 conductivity meter using a cell of constant
0.089 cm-1. FTIR spectra were recorded on a Bomem-Michelson 102 spectrometer in the range of
4000 - 200 cm-1. The samples were examined in CsI cells. UV-visible absorption spectra were
recorded using a Varian model Cary 500 NIR spectrophotometer with 1.0 cm quartz cell in the
range of 240 - 800 nm. Electrochemical measurements were carried out using a 100 B/W
electrochemical workstation from Bioanalytical Systems with a conventional three-electrode
system. Scans were recorded on samples dissolved in dichloromethane containing 0.1 mol L-1
tetrabutylammonium perchlorate (PTBA Fluka Purum) solution. The working and auxiliary
electrodes were of platinum and Ag/AgCl, 0.10 mol L-1 PTBA in dichloromethane as the reference
electrode. Under these conditions, the ferrocene/ferrocenium oxidation occurs at 0.43 V.
Single crystal X-ray structure data analysis
Crystals of complex 1 were grown by slow evaporation of dichloromethane/methanol
solution. The crystals of complexes 2 and 4 were grown in a solution of dichloromethane/methanol,
but in these cases KPF6 (1:1, complex/KPF6) was added to the solution in order to facilitate the
crystallization. The single crystals exhibited a prism form and were mounted on an Enraf-Nonius
Kappa-CCD diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). The

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dimensions and the symmetry of the unit cell were measured based on all reflections. Data
collection was performed at room temperature (293 K) after the unit cell dimensions were
determined using the COLLECT program [31, 32]. Integration and scaling of the reflections were
carried out using the HKL Denzo-Scalepack software package [33]. The structures were solved
through direct methods of phase retrieval with SHELXS-2013 [34] and the refinement by the full-
matrix least-squares on F2 with SHELXL-20135 [34] within the WinGX-v.2013.3 [35] program
package. Absorption correction was performed by the Gaussian method [35]. Non-hydrogen atoms
were refined anisotropically and hydrogen atoms were fixed at calculated positions and refined
using the riding mode. The constrained positions and fixed isotropic thermal parameters for C–H
hydrogen atoms were the bond lengths of 0.93 and 0.97 Å for Csp2 –H (aromatic rings) and Csp3 –
H (methylene groups), respectively, considering Uiso(H) = 1.2Ueq(C). Structure analysis and the
preparation of artwork were performed using MERCURY and ORTEP-3 software [36]. WinGX
was used to prepare the material for publication (CIF file). The crystallographic data are given in
Table 1.
Table 1. Crystal data and structure refinement parameters obtained for complexes 1, 2 and 4.
Complex
Empirical formula
Formula weight (g/mol)
(1)
C39H35Cl4N6PRuO
861.57
(2)
C31H34Cl3N5P2RuF6
859.99
(4)
C40H36ClN5P2RuO2
931.20
Crystal system
Space group
Monoclínic
P 21/c
Triclinic
P -1
Triclinic
P -1
a (Å)
b (Å)
c (Å)
Α
11.11400(10)
20.8490(2)
16.4720(3)
90°
90.5830 (10)°
90 (10)°
3816.63(9)
4
1.499
10.1746(2)
10.4747(2)
18.2349(4)
104.7940(10)°
105.4150(10)°
91.8940(10)°
1800.73(12)
2
10.661(2)
11.3252(2)
20.6240(5)
74.4130(10)°
85.6900(10)°
64.132(2)°
2155.74(8)
2
Β
Γ
Volume (Å3)
Z
Density calculated (Mg/m3)
1.586
0.808
1.435
0.565
Absorption coefficient (mm-1) 0.770
F (000)
1752
868
944
Crystal size (mm3)
Θ range
0.31x 0.27 x 0.05
5.918 to 52.73°
-13≤h≤13, -26≤k≤23, -
19≤l≤20
0.03 x 0.3 x 0.19
5.968 to 51.55°
-12≤h≤12, -12≤k≤12,
21≤l≤22
0.46 x 0.44 x 0.15
6.052 to 51.362°
-13≤h≤12, -13≤k≤13, -
25≤l≤25
Index ranges
-
Reflections collected
76409
13041
38649
Independent reflections (Rint) 7789 (0.0365)
6864 (0.0283)
99.8 %
6864 / 0 / 434
1.000
R1=0.0484, wR2=0.1270
R1=0.0560, wR2=0.1316
8161 (0.0257)
99.8 %
8161 / 0 / 519
1.001
R1=0.0428, wR2=0.1250
R1=0.0496, wR2=0.1293
Completeness to θ(%)
Data/restraints/parameters
Goodness-of-fit on F2
Final R índices [I>2σ(I)]
R indices (all data)
99.8 %
7789 / 0 / 460
1.116
R1=0.0373, wR2=0.0955
R1=0.0493, wR2=0.1018

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Δρmax. and Δρmin.(e. Å-3)
1.06 and -0.58
1.30 and -1.22
0.84 and -0.70
Synthesis
All synthetic procedures were carried out under argon atmosphere using standard Schlenk
techniques. The precursors [RuCl2(PPh3)3)], cis-[RuCl2(PPh3)2(bipy)], cis-[RuCl2(PPh3)2(dmbipy)],
cis-[RuCl2(PPh3)2(phen)], cis-[RuCl2(PPh3)2(dphphen)] and trans-[RuCl2(PPh3)2(en)] were
synthesized as described in the literature [37-39].
[RuCl(PPh3)(Hdpa)2]Cl (1)
The Hdpa ligand (54 mg; 0.313 mmol) was added to a solution of [RuCl2(PPh3)3] (150 mg;
0.156 mmol) in dichloromethane (15 mL). The resulting solution was stirred for 24 h at room
temperature and an orange solid was precipitated. The solid was filtered off, washed with diethyl
ether and dried in vacuo. Yield 94 mg (77%). Elemental analysis (%) Calc. for
C38H33Cl2N6PRu.1/3CH2Cl2: C 57.20; H 4.19; N 10.21 Found: C 57.19; H 3.91; N 10.44. NMR-31P
{1H} (DMSO-d6) [δ/ppm (multiplicity, assignment)] 44.1 (s, PPh3). NMR-1H (DMSO-d6) {[δ/ppm
(multiplicity, integration, assignment, J/Hz, coordination-induced shifts (c.i.s), δcomplex - δligand]}
3
11.09 (s, 1H, NH, 1.46), 10.51 (s, 1H, NH, 0.85), 8.70 (d, 1H, H6’, J = 5.5, 0.49), 7.82 (d; 1H;
3
H10’; J = 4.8, 0.39), 7.68 (p, 2H, H8”/H12’), 7.61 – 7. 54 (m, 2H, H10” e H12”), 7.38 (t, 1H,
3
H8’, J = 7.3, -0.25), 7.32 – 7.19 (m, 5H, H9”/H13’/H3), 7.18 – 7.11 (m, 2H, H6”/H13’), 7.09 –
6.94 (m, 12H, H2/H1), 6.73 (d, 1H, H9’, 3J = 8.1, -1.01), 6.64 (t, 1H, H7”, 3J = 6.3, -0.21), 6.56 (t,
3
3
1H, H7’,3J = 6.4, -0.26), 6.49 (t, 1H, H11”, J = 6.3; -0.36), 6.38 (t, 1H, H11’, J = 6.3, -0.47).
NMR-13C {1H} (DMSO-d6) [δ/ppm (multiplicity, assignment)] 157.95 (s, C6’), 148.60 (s, C6”),
117.75 (s, C7’), 118.57 (s, C7”), 138.46 (s, C8’), 138.87 (s, C8”), 126.47 (s, C9’), 7.17 (s, C9”),
154.50 (dd, C10’/C10”), 115.51 (s, C11’), 113.50 (s, C11”), 136.42 (d, C12’/C12”), 123.86 (d,
C13’/C13”), 133.58 (d, C1), 128.70 (d, C2), 130.34 (s, C3). Selected IR (CsI, cm-1): v (N—H) 3438
cm-1 v (C=N) 1632 cm-1 v (C=C) 1469 cm-1 vas (P—CH) 1087 cm-1 v (Ru—N) 528 cm-1 v (Ru—
,
,
,
,
,
P) 511 cm-1, v (Ru—Cl) 230 cm-1. UV/visible spectrum [CH2Cl2; λ max, nm (ε, M-1 cm-1)]: 298
(55905), 368 (16261), 430 (7951).

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[RuCl(PPh3)(Hdpa)(en)]Cl (2)
The Hdpa ligand (34 mg; 0.198 mmol) was added to a solution of [RuCl2(PPh3)2(en)] (150
mg; 0.198 mmol) in dichloromethane (20 mL). The resulting solution was stirred for 48 h at room
temperature, the solvent was removed under reduced pressure to ca. 2 mL and acetone was added
for the precipitation of a yellow solid, which was filtered off, rinsed with acetone (5 x 5 mL) and
dried in vacuo. Yield 91 mg (69%). Elemental analysis (%) Calc. for C30H32Cl2N5PRu.1/10CH2Cl2:
C 53.72; H 4.81; N 10.37 found: C 53.69; H 5.09; N 10.21. NMR-31P {1H} (DMSO-d6) [δ/ppm
(multiplicity, assignment)] 53.9 (s, PPh3). NMR-1H (DMSO-d6) [δ/ppm (multiplicity, integration,
3
assignment, J/Hz, c.i.s)] 10.15 (s, 1H, NH, 0.51), 8. 86 (d, 1H, H6’, J = 6.2, 0.65), 7.68 (d, 1H,
H6”, 3J = 5.7, -0.53), 7.51 (t, 1H, H8’, 3J = 7.8; -0.12), 7.33 – 7.15 (m, 16H, H1/H2/H3/H8”), 6.98
3
3
3
(d, 1H, H9’, J = 8.3, -0.76), 6.68 (d, 1H, H9”, J = 8.2, -1.06), 6.46 (t, 1H, H7’, J = 6.6, -0.39),
6.30 (t, 1H, H7”, 3J = 6.4, -0.54), 4.85 [m, 1H, H10’ (NH2)], 4.66 [m, 1H, H10” (NH2)], 4.40 [(m,
1H, H13’ (NH2)], 3.17 [m, 1H, H13” (NH2)], 3.03 [m, 1H, H11’ (CH2)], 2.85 [m, 1H, H12’ (CH2)],
2.70 [m, 1H, H11” (CH2)], 2.25 [m, 1H, H12” (CH2)]. NMR-13C {1H} (DMSO-d6) [δ/ppm
(multiplicity, assignment)] 155.65 (s, C6’), 154.46 (s, C6”), 116.18 (s, C7’), 116.92 (s, C7”), 136.88
(s, C8’), 135.69 (s, C8”), 112.20 (s, C9’), 113.82 (s, C9”), 42.43 (s, C11’/C11”), 45.75 (s,
C12’/C12”), 133.49 (d, C1), 128.00 (d, C2), 129.00 (s, C3). Selected IR (CsI, cm-1): v (N—H) 3437
cm-1 v (C=N) 1641 cm-1 v (C=C) 1481 cm-1 vas (P—CH) 1088 cm-1 v (Ru—N) 529 cm-1 v (Ru—
,
,
,
,
,
P) 503 cm-1, v (Ru—Cl) 243 cm-1. UV/visible spectrum [CH2Cl2; λ max, nm (ε, M-1 cm-1)]: 301
(52980), 353 (9788).
[RuCl(PPh3)(Hdpa)(bipy)]Cl (3)
The Hdpa ligand (45 mg; 0.264 mmol) was added to a yellow suspension of cis-
[RuCl2(PPh3)2(bipy)] (150 mg; 0.176 mmol) in dichloromethane/methanol (1/1, 30 mL). The
mixture was refluxed for ca. 48 h under vigorous stirring, the solvent was removed under a reduced
pressure to ca. 2 mL volume and diethyl ether was added for the precipitation of a red solid, which
was filtered off, rinsed with diethyl ether (5 x 5 mL) to remove the excess of ligand and dried in

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vacuo. Yield 106 mg (79%). Elemental analysis (%) Calc. for C38H32Cl2N5PRu.1/5CH2Cl2: C 58.68;
H 4.16; N 8.95 Found: C 58.77; H 4.16; N 8.81. NMR-31P {1H} (DMSO-d6) [δ/ppm (multiplicity,
assignment)] 41.5 (s, PPh3). NMR-1H (DMSO-d6) [δ/ppm (multiplicity, integration, assignment,
J/Hz, c.i.s)] 11.05 (s, 1H, NH, 1.42), 9.45 (d, 1H, H10’, 3J = 5.5, 0.86), 8.55 (d, 1H, H10”, 3J = 5.5,
-0.03), 8.28 (d, 1H, H13’, 3J = 8.0, -0.22), 8.19 (m, 2H, H13”/6”), 7.91 (t, 1H, H12’, 3J = 7.7, 0.26),
3
7.80 (m, 2H, H8”/H12”), 7.59 – 7.51 (m, 2H, H8’/9”), 7.43 (t, 1H, H11’, J = 6.5, 0.30), 7.32 –
7.27 (m, 6H, H1/H11”), 7.16 (m, 7H, H2, H6”/H9”), 6.97 (m, 5H, H3), 6.65 (t, 1H, H7”, 3J = 6.5, -
3
0.20), 6.6 (t, 1H, H7’, J = 6.5, -0.27). NMR-13C {1H} (DMSO-d6) [δ/ppm (multiplicity,
assignment)] 157.95 (s, C6’), 148.60 (s, C6”), 117.75 (s, C7’), 118.57 (s, C7”), 138.46 (s, C8’),
138.87 (s, C8”), 115.51 (s. C9’), 113.50 (s, C9”), 154.50 (dd, C10’/C10”), 126.47 (s, C11’), 125.46
(s, C11”), 136.42 (d, C12’/C12”), 123.86 (d, C13’/C13”), 133.58 (d, C1), 128.70 (d, C2), 130.34 (s,
C3). Selected IR (CsI, cm-1): v (N—H) 3423 cm-1 v (C=N) 1627 cm-1 v (C=C) 1465 cm-1 vas (P—
,
,
,
CH) 1085 cm-1 v (Ru—N) 529 cm-1 v (Ru—P) 514 cm-1, v (Ru—Cl) 258 cm-1. UV/visible
,
,
spectrum [CH2Cl2; λ max, nm (ε, M-1 cm-1)]: 298 (55828), 366 (23695), 480 (8615).
[RuCl(PPh3)(Hdpa)(dmbipy)]Cl (4)
This compound was prepared by refluxing cis-[RuCl2(PPh3)2(dmbipy)] (150 mg; 0.170
mmol) and Hdpa ligand (44 mg; 0.255 mmol) adopting the procedure used for 3. Yield 124 mg
(92%). Elemental analysis (%) Calc. for C40H36Cl2N5PRu: C 60.84; H 4.59; N 8.87 Found: C 61.05;
H 4.34; N 8.86. NMR-31P {1H} (DMSO-d6) [δ/ppm (multiplicity, assignment)] 41.7 (s, PPh3).
NMR-1H (DMSO-d6) [δ/ppm (multiplicity, integration, assignment, J/Hz, c.i.s)] 11.14 (s, 1H, NH,
3
3
1.51), 8.91 (s, 1H, H10’), 8.32 (d, 1H, H6’, J = 5.6, 0.10), 8.20 (t, 2H, H13’/H13”, J = 9.3), 8.00
3
(s, 1H, H10”), 7.86 (t, 1H, H8”, J = 7.6, 0.22), 7.68 (t, 2H, H12’/H12”), 7.61 – 7,49 (m, 2H,
3
H8’/H9”), 7.33 (t, 3H, H3, J = 7.2), 7.23 – 7.10 (m, 7H, H2/H9’), 7.04 – 6.90 (m, 7H, H1/H6’),
6.64 (p, 2H, H7’/H7”, -0.22), 2.12 (s, 3H, CH3”), 2.08 (s, 3H, CH3’). NMR-13C {1H} (DMSO-d6)
[δ/ppm (multiplicity, assignment)] 157.23 (s, C6’), 148.59 (s, C6”), 117.86 (s, C7’), 118.40 (s,
C7”), 138.61 (s, C8’), 139.00 (s, C8”), 115.59 (s, C9’), 113.73 (s, C9”), 154.18 (d, C10’/C10”),

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18.09 (s, CH3’), 18.51 (s, CH3”), 136.87 (d, C12’/C12”), 122.64 (d, C13’/C13”), 133.41 (d, C1),
128.35 (d, C2), 129.96 (s, C3). Selected IR (CsI, cm-1): v (N—H) 3433 cm-1 v (C=N) 1624 cm-1 v
,
,
(C=C) 1466 cm-1 vas (P—CH) 1089 cm-1 v (Ru—N) 526 cm-1 v (Ru—P) 516 cm-1, v (Ru—Cl) 252
,
,
,
cm-1. UV/visible spectrum [CH2Cl2; λ max, nm (ε, M-1 cm-1)]: 304 (55348), 342 (shoulder).
[RuCl(PPh3)(Hdpa)(phen)]Cl (5)
The complex was prepared by refluxing cis-[RuCl2(PPh3)2(phen)] (150 mg; 0.171 mmol)
and Hdpa ligand (44 mg; 0.257 mmol) following the procedure used for 3. Yield 113 mg (84%).
Elemental analysis (%) Calc. for C40H32Cl2N5PRu.1/2.5CH2Cl2: C 59.20; H 4.03; N 8.54 found: C
59.09; H 4.04; N 8.24. NMR-31P {1H} (DMSO-d6) [δ/ppm (multiplicity, assignment)] 43.1 (s,
PPh3). NMR-1H (DMSO-d6) [δ/ppm (multiplicity, integration, assignment, J/Hz, c.i.s)] 10.89 (s,
3
1H, NH, 1.26), 9.84 (d, 1H, H10’, J = 5.3, 0.59), 9.11 (d, 1H, H10”, 3J = 5.3, -0.14), 8.51 (d, 1H,
H12’, 3J = 8.2, -0.05), 8.40 (d, 1H, H12”, 3J = 8.2, -0.17), 8.22 (d, 1H, H6’, 3J = 5.8, 0.01), 8.01 (dd,
2H, H13’/H13’’, 3J = 8.9, -0.04), 7.89 – 7.80 (m, 2H, H8’/H11’), 7.70 (t, 1H, H11”, 3J = 5.7, -0.16),
3
3
7.53 (d, 1H, H9’, J = 8.1, -0.20), 7.43 (t, 1H, H8”, J = 7.6, -0.21), 7.23 – 7.15 (m, 4H, H3/H6”),
7.02 (m, 7H, H2/H9”), 6.83 (t, 6H, H1), 6.61 (t, 1H, H7’, 3J = 6.5, -0.24), 6.47 (t, 1H, H7”, 3J = 6.5;
-0.37). NMR-13C {1H} (DMSO-d6) [δ/ppm (multiplicity, assignment)]. 157.96 (s, C6’), 148.72 (s,
C6”), 117.89 (s, C7’), 118.42 (s, C7”), 138.59 (s, C8’), 138.98 (s, C8”), 115.38 (s, C9’), 113.42 (s,
C9”), 155.06 (d, C10’), 156.51 (d, C10”), 125.65 (d, C11’), 124.61 (d, C11”), 135.42 (d, C12’),
125.69 (d, C12”), 127.60 (d, C13’/C13”), 133.18 (d, C1), 128.04 (d, C2), 129.95 (s, C3) Selected IR
(CsI, cm-1): v (N—H) 3410 cm-1 v (C=N) 1625 cm-1 v (C=C) 1468 cm-1 vas (P—CH) 1089 cm-1 v
,
,
,
,
(Ru—N) 527 cm-1 v (Ru—P) 510 cm-1, v (Ru—Cl) 256 cm-1. UV/visible spectrum [CH2Cl2; λ max
,
,
nm (ε, M-1 cm-1)]: 272 (56713), 293 (shoulder), 434 (10105), 476 (shoulder).
[RuCl(PPh3)(Hdpa)(dphphen)]Cl (6)
This compound was prepared by refluxing cis-[RuCl2(PPh3)2(dphphen)] (150 mg; 0.146
mmol) and Hdpa ligand (38 mg; 0.219 mmol) adopting the procedure used for 3. Yield 121 mg
(88%). Elemental analysis (%) Calc. for C52H40Cl2N5PRu: C 65.43; H 4.26; N 7.30 found: C 65.33;

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H 3.93; N 7.49. NMR-31P {1H} (DMSO-d6) [δ/ppm (multiplicity, assignment)] 42.5 (s, PPh3).
NMR-1H (DMSO-d6) [δ/ppm (multiplicity, integration, assignment, J/Hz, c.i.s)] 11.03 (s, 1H, NH,
3
3
3
1.40), 9.93 (d, 1H, H10’, J = 5.5), 9.15 (d, 1H, H10”, J = 5.5), 8.22 (d, 1H, H6’, J = 5.5, 0.01),
7.93 – 7.82 (m, 4H, H8’/H13’/H13”/ H11’), 7.72 – 7.48 (m, 13H, H11”/H14’/H14”/H9’/H8”), 7.30
3
3
– 7.21 (m, 4H, H3/H6”), 7.19 (d, 1H, H9”, J = 8.2, -0.55), 7.06 (t, 6H, H2, J = 7.1), 6.89 (t, 6H,
3
H1, J = 8.5), 6.62 (p, 2H, H7’/H7”, -0.23). NMR-13C {1H} (DMSO-d6) [δ/ppm (multiplicity,
assignment)] 157.98 (s, C6’), 148.89 (s, C6”), 118.06 (s, C7’), 118.71 (s, C7”), 138.47 (s, C8’),
139.00 (s, C8”), 115.42 (s, C9’), 113.97 (s, C9”), 154.96 (d, C10’), 155.61 (d, C10”), 125.70 (d,
C11’), 125.17 (d, C11”), 129.86 (d, C12’/C12”), 125.80 (d, C13’/C13”), 133.45 (d, C1), 128.03 (d,
C2), 129.90 (s, C3). Selected IR (CsI, cm-1): v (N—H) 3427 cm-1 v (C=N) 1623 cm-1 v (C=C) 1468
,
,
cm-1 vas (P—CH) 1088 cm-1 v (Ru—N) 530 cm-1 v (Ru—P) 509 cm-1, v (Ru—Cl) 232 cm-1.
,
,
,
UV/visible spectrum [CH2Cl2; λmax, nm (ε, M-1 cm-1)]: 287 (49500), 328 (shoulder), 454 (8542),
493 (shoulder).
DNA interaction studies
A standard solution of calf thymus DNA (CT DNA) was prepared in the Tris–HCl buffer (5
mM Tris–HCl and 50 mM NaCl, pH 7.4, Tris = tris(hydroxymethyl)aminomethane). CT DNA
solutions in the Tris-HCl buffer gave a ratio of UV absorbance at 260 and 280 nm of 1.8, indicating
that the DNA was sufficiently free of protein. The DNA concentration was determined
spectrophotometrically using the molar absorption coefficient of 6600 mol-1 L cm-1 at 260 nm [26].
Initially, compounds 1 - 6 were solubilized in DMSO and diluted with Tris-HCl buffer afterwards.
All the solutions of the compounds used in the experiments were prepared in the Tris–HCl buffer
containing 5% DMSO.
Circular Dichroism (CD)
CD spectra were registered on a Jasco J-810 spectropolarimetrer, equipped with a 450 W
Xenon arc lamp. All experiments were done using a standard quartz cell of 10 mm path length. The
CD measurements were performed from complex-DNA solutions in the Tris-HCl buffer (5%

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DMSO) at different molar ratios, [Complex]/[CT DNA] = 0.1, 0.2, 0.4, 0.6. The DNA concentration
in the Tris-HCl buffer was kept constant (100 μM) in all samples. Complex-DNA solutions were
incubated at 298 K for 18 h. After the incubation period, all CD spectra were recorded in the range
of 240 – 300 nm. The spectra were expressed in terms of molar ellipticity.
Spectroscopic titrations
Absorption spectral titration experiments were performed by maintaining the constant
concentration of the complexes and varying the CT DNA concentration. This was done by
successive additions of CT DNA solution to solution of the compound in a quartz cell and recording
the UV–Vis spectra after each addition of CT DNA. The binding constants were obtained using
Equation 1 [40]:
[DNA]/ (εa – εf) = [DNA]/ (εb – εf) + 1/ [Kb(εb – εf)] (Eq. 1)
Where [DNA] is the concentration of DNA in base pairs, εa, εf and εb are apparent-, free-, and
bound-complex extinction coefficients, respectively. Kb is the equilibrium binding constant of the
complex binding to DNA in M-1.
Viscosity measurements
The viscosity assays were carried out using an Ostwald viscometer maintained at a constant
temperature of 298 K in a thermostatic bath. First, 4 mL of complex-DNA solutions at different
molar ratios, [Complex]/[CT DNA] = 0.08, 0.17, 0.25, 0.33, 0.42, 0.50, 0.58, 0.67, 0.75 were
freshly prepared in the Tris-HCl buffer (5% DMSO) prior to use. The DNA concentration in the
Tris-HCl buffer was kept constant (98 μM) in all samples. Afterwards, the flow times of the
solutions on the Ostwald viscometer were measured using a digital stopwatch 5 times, taking the
average flow time into consideration. The relative viscosity of DNA in the absence (ηo) and
presence of complexes (η) was calculated from Equation 2: η/ηo = (t – to)/(tDNA - to), where to and
tDNA are the flow time of the buffer and DNA solution alone, respectively, while t is the flow time
of DNA solution in the presence of the ruthenium compounds [41a]. Data are presented as (η/η0)1/3
versus the ratio [complex]/[DNA].

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Agarose gel electrophoresis studies
pTZ57RT plasmid (100 mM) in buffer Tris-HCl buffer was treated with each compound at
different molar ratios. The different ratios of complex/plasmid were 0 (control); 0.5; 1.0; 2.0. The
solutions were incubated at 310 K for 18 h, and then 5 μL of each sample were analyzed by
electrophoresis for 90 min at 100 V on a 1% agarose gel in the TAE buffer [0.45 M Tris–HCl, 0.45
M acetic acid, 10 mM ethylenediaminetetraacetic acid (EDTA)]. The gels were stained with 1 μg
mL-1 ethidium bromide under UV light and photographed using a ChemiDoc MP. Samples of free
DNA and DNA/DMSO were used as controls.
Partition coefficient determination
Lipophilicity, commonly expressed log P (the partition coefficient of a compound in two
immiscible phases, as water and n-octanol). Water-octanol partition coefficients were determined
using the shake flask method [41b]. The determination was carried out at in a mixture of equal
volumes of water and n-octanol, and continuously shaking for 18h at room temperature. The
concentrations of complexes in n-octanol and water were measured spectrophotometrically in order
to determine values of P = [compound]n-octanol / [compound]water
.
Fluorescence quenching experiments
The protein binding study was performed by a tryptophan fluorescence quenching
experiment using human serum albumin (HSA). The extinction of emission intensity of the
tryptophan residue at 305 nm was monitored using the compounds as suppressors. The fluorescence
measurements were performed using compound-HSA solutions in the Tris-HCl buffer (5% DMSO)
at different molar ratios, [Compound]/[HSA] = 0 (control), 1, 2, 3, 4, 5, 6, 7. The HSA
concentration in the Tris-HCl buffer was kept constant (5 μM) in all samples. Emission spectra
were recorded between 300 and 500 nm upon excitation at 270 nm. Fluorescence spectra were
registered on a SpectraMax M3 at different temperatures (295 and 310 K) and in triplicate. All
experiments were conducted using an opaque 96-well plate. The fluorescence data were analyzed
using the Stern-Volmer equation (2) [42]:

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F0/F = 1 + kqt0[Q] = 1 + Ksv[Q] (Eq. 2)
Where F0 and F are fluorescence intensities in the absence and presence of the quencher,
respectively. Ksv is the Stern-Volmer quenching constant. kq is the biomolecular quenching constant
and t0 is the average lifetime of fluorophore in the absence of the quencher; [Q] is the concentration
of the quencher. The Ksv constant was obtained from the slope of the linear regression of F0/F
versus [compound] plot. The kq constant was calculated by the ratio between Ksv and t0 (kq = Ksv/t0).
The binding constant (Kb) and the number of binding sites (n) for the interactions of HSA
and compounds were determined by Equation 3.
log [(F0 – F)/F] = log Kb + n log[Q] (Eq. 3)
The thermodynamic parameters of the intermolecular forces involved in the interactions
between HSA and compounds were calculated using the modified van’t Hoff equation, Equation 4
[43]:
ln K = -∆H/RT + ∆S/R (Eq. 4)
where K is analogous to the Stern-Volmer quenching constant at the corresponding temperature (T);
the temperatures used were 295 and 310 K; R = gas constant; ∆H = enthalpy change and ∆S =
entropy change . The values were obtained from the slope of the linear regression of ln K versus 1/T
plot. Furthermore, the free energy change (∆G) was calculated using Equation 5 [43].
∆G = -RT ln K = ∆H - T∆S (Eq. 5)
Cell proliferation
In vitro cytotoxicity assays on cultured human tumor cell lines represent the standard
method for the initial screening of antitumor agents. Thus, as a first step to assess their
pharmacological properties, the ruthenium complexes were assayed using the human breast tumor
cell line MDA-MB-231 (ATCC HTB-26) and the Chinese hamster lung fibroblast non-tumor cell
line V79-4 (ATCC CCL-93). The cells were routinely maintained in Dulbecco’s Modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM),

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penicillin (100 UI mL-1) and streptomycin (100 mg mL-1) at 310 K in a humidified 5% CO2
atmosphere.
Briefly, all cell lines were prepared at a concentration of 1.5×104 cells/150 μL, in complete
medium (with 10% FBS), and plated on sterile 96 well plates for 24 h at 310 K in a humidified 5%
CO2 atmosphere. The complexes were added to the wells at different concentrations and incubated
for 48 h under the same conditions as described above. The cell proliferation assay was performed
compared to the wells where the vehicle (0.5% DMSO) was added instead of the tested compounds.
After incubation, the culture medium of each well was removed and a solution containing MTT (0.5
mg ml-1) was added (100 μL/well) [44]. The plates were then kept at 310 K for 4 h and the formed
crystals were dissolved in isopropyl alcohol. The absorbance was read on an ELISA plate reader at
a wavelength of 595 nm and the IC50 (concentration of compound that induced 50% of cell death)
value were determined.
Cell morphology
MDA-MB-231 growing cells were harvested, counted and seeded at 8 × 104 cells/well in 12-
well plates. The cells were allowed to grow at 310 K in a humidified 5% CO2 atmosphere overnight
and then, treated or not (control) with 0.2, 2.0 and 20.0 μM of compound 6 for 0, 12, 24, 36 and 48
h. Cell morphology was examined under an inverted microscope at 100x magnification.
Results and discussion
Synthesis and characterization of the complexes
The [RuCl(PPh3)(Hdpa)2]Cl (1) complex was prepared from the reaction of [RuCl2(PPh3)3]
precursor with 2,2’-dipyridylamine ligand, in dichloromethane. The cationic ruthenium (II)
complexes [RuCl(PPh3)(Hdpa)(N-N)]Cl [N-N: en (2), bipy (3), dmbipy (4), phen (5) and dphphen
(6)] were synthesized by treating the respective [RuCl2(PPh3)2(N-N)] precursor with the 2,2’-
dipyridylamine, in 1:1 dichloromethane/methanol solution, as shown in Scheme 1.
Scheme 1. Synthetic pathways for the preparation of complexes 1 – 6.

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Reagents and conditions: (a) PPh3 (5 equivalents), methanol, reflux; (b) Hdpa (2 equivalents),
CH2Cl2, agitation; (c) en (1 equivalent), CH2Cl2, agitation; (d) Hdpa (1 equivalent), CH2Cl2,
agitation; (e) N-N (1 equivalent; N-N = bipy, dmbipy, phen or dphphen), CH2Cl2, agitation; (f)
Hdpa (1.5 equivalent), dichloromethane/methanol (1:1), reflux.
All complexes were characterized by 1D (31P {1H}, 1H, 13C {1H}) and 2D (1H — 1H COSY;
1H — 13C {1H} HSQC) NMR, UV and IR spectroscopy, cyclic voltammetry, CHN analyses, molar
conductance and X-ray crystallography, for complexes 1, 2 and 4. The CHN analyses of the
complexes are according to the proposed formula. The molar conductivity values of 1 mmol L-1

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solutions of complexes 1 - 6 in dimethyl sulfoxide are in the range of 38 – 46 S cm2 mol-1,
indicating that the complexes are 1:1 electrolytes [45].
NMR spectroscopy
The 31P{1H} NMR spectra (in the Supporting Information) of complexes in DMSO-d6
present a singlet (A spin system). The chemical shifts are consistent with a structure in which the
phosphorus atom is trans to Ru—Npy bond for 1 and 3 – 6, whereas for 2 it is trans to Ru—NH2
[37].
1
The assignments of H spectra (see Figures in the Supporting Information) of the
compounds are summarized in the Experimental section. The resonances of the compounds show
inequivalent aromatic rings for the Hdpa ligand, as well as from the diimines. In all complexes, the
NH group (Hdpa ligand) resonance shows a great variation in the chemical shift toward the region
of higher frequencies of the 1H NMR spectrum after the ligand coordination to the metal center.
X-ray Crystallography
The crystal structures of complexes 1, 2 and 4 were determined by X-ray crystallography.
The ORTEP diagrams are shown in Figure 1, while the selected bond lengths and angles are
reported in Table 2. In all three structures, the cationic complexes show a distorted octahedral
coordination geometry around the Ru(II) center.

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Figure 1. ORTEP view of compounds [RuCl(PPh3)(Hdpa)2]Cl (A),
[RuCl(PPh3)(Hdpa)(dmbipy)]PF6 (B) and [RuCl(PPh3)(Hdpa)(en)]PF6 (C), showing the atom
labels and the 30% probability ellipsoids. The anions are omitted for clarity.
Table 2. Selected bond lengths (Å) and angles (°) for complexes 1, 2 and 4.
Compound
Bond Lengths (Å)
Compound
Bond Angles (°)
1
2
4
1
2
4
Ru(1)–Cl(1)
Ru(1)–P(1)
Ru(1)–N(3)
Ru(1)–N(2)
Ru(1)–N(4)
Ru(1)–N(1)
2.362(7) 2.299(1) 2.344(1)
2.434(8) 2.430(1) 2.425(1)
2.123(2) 2.157(3) 2.132(3)
2.077(2) 2.083(3) 2.061(4)
2.098(2) 2.156(3) 2.110(3)
2.097(2) 2.107(4) 2.047(3)
P(1)–Ru(1)–Cl(1) 88.89(3)
89.18(4)
99.32(3)
P(1)–Ru(1)–N(3) 173.12(7) 173.68(9) 175.97(9)
Cl(1)–Ru(1)–N(2) 172.88(9) 174.7(1) 168.20(9)
N(1)–Ru(1)–N(4) 175.35(9) 165.7(1)
171.6(1)
78.8(1)
84.1(1)
N(1)–Ru(1)–N(2)
N(3)–Ru(1)–N(4)
87.11(9)
85.28(9)
90.5(1)
80.3(1)
In all structures, the dipyridylamine ligand is chelated to the Ru(II) by pyridinic-N atoms,
with the free group NH pointed out from the rings. In compound 4, the dmbipy ligand is
coordinated in the equatorial plane, whereas the Hdpa ligand occupies a position trans to the PPh3
ligand, and another position in the equatorial plane, trans to N1(dmbipy).
The Ru—N bond lengths [2.157 (3) and 2.156 (3) Å] are observed in the complex with the
1,2-diaminoethane ligand, since it is typical for Ru—Nsp3 distances. The Ru—Nen distances are
longer than those of the Ru—N (diimine, Hdpa) bond lengths. This trend is consistent with the
characteristic moderate π acceptor of the diimine ligand, while the en ligand is only a pure σ-donor.
In complex 4, the Ru—N coordination bond lengths of the dmbipy ligand are shorter than those
Ru—N(Hdpa) distances. This finding is consistent with the better π acceptor ability of the dmbipy
ligand, compared to the Hdpa ligand [27]. The Ru—P bond lengths, in all compounds, are
influenced by the nature of the ligand in the position trans to it: in complex 2, the Ru—P bond
length trans to NH2 (en ligand) is significantly the shortest value compared to those trans to the
Hdpa ligand, in complexes 1 and 4.

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The hydrogen bonds in the crystal structures of the complexes involve the –NH– groups of
the Hdpa ligand acting as hydrogen bonds where the chlorido ligand acts as a hydrogen bond
acceptor. The intermolecular distance of 2.25 Å between H….Cl is indicative of strong hydrogen
bonds [46]. Other hydrogen bonds, defined by a hydrogen acceptor distance shorter than the sum of
van der Waals radii, are formed between C—H donors and the chlorido ligand. Relatively long
intermolecular distances suggest that they may be classified as weak interactions [46]. Considering
these observations in the crystal self-assembly, it can be expected that the –NH– group in the Hdpa
ligand may also perform hydrogen bonds and interactions in aqueous medium with biomolecules.
UV/Vis Spectroscopy Studies. In the UV region, the spectrum of each complex shows a strong
absorption band centered at about 300 nm, assigned to the intraligand-centered π—π* type
transition, also present in the spectra of the free ligands. The metal to ligand charge transfer
(MLCT) transitions from Ru (dπ) to the ligand (π) for complexes 1 – 6, which appear in the region
of the typical spectra of ruthenium compounds, where the metal is coordinated to polypyridyl
ligands. In the region of 350 - 470 nm, the complexes exhibit lower-energy absorption bands
attributed to MLCT transitions π (Hdpa) ← dπ(Ru) and π (diimine) ← dπ(Ru) [47a, 48].
Electrochemistry. The electrochemical behavior of compounds 1 – 6, in dichloromethane
solutions, was studied by cyclic voltammetry (in the Supporting Information). All the complexes
showed similar electrochemical behavior, where the CVs of the complexes exhibit a redox couple
(I) with half-wave potential (E ) values in the range of 0.7 – 1.1 V vs. Ag/AgCl (Table 3), attributed
½
to the transfer of one electron of the Ru(II)/Ru(III) couple. In addition, the peak potentials (Epa and
Epa) are nearly scan-rate independent and the ratios of the cathodic (Ipc) to anodic (Ipa) peak currents
are close to one, suggesting that the redox processes are quasi-reversible type.

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Table 3. Electrochemical data of phosphine ruthenium complexes. TBAP (0.1 M); CH2Cl2;
Ag/AgCl; scan rate of 100 mVs−1.
Compound
Epa1 / mV
813
767
984
954
1031
1010
Epc2 / mV
727
∆Ep / mV
86
E1/2 / mV
770
Ipa1/Ipc2
1.03
1.01
0.99
1.12
1.03
1.05
Epa3 / mV
1327
1226
1336
1290
pKa*
6.48
9.98
4.33
4.58
4.27
4.29
1
2
3
4
5
6
688
821
852
906
79
728
902
903
969
163
102
125
98
1318
1292
912
961
* pKa of the co-ligands: Hdpa [47b], en [47c], bipy [47d], dmbipy [47d], phen [47b], dphphen [47b].
The Ru(II) oxidation state is stabilized when the PPh3 and chlorido ligand are replaced by
the Hdpa ligand (also π-acceptor), which is consistent with enhanced π delocalization. The
oxidation potential of compound 1 is low when compared to those possessing the diimine co-
ligands, compounds 3 – 6. This is an effect of its higher energy π* orbitals and the stronger σ-
donating and less π-accepting ability of Hdpa ligand, when compared to other diimine ligands [27,
47a]. Similarly, complex 2 showed the lowest E1/2 among all the complexes, which is attributed to
the influence pure σ-donor character of the diaminoethane ligand, which results in a more electro-
rich metal center.
The difference of the oxidation potentials of the complexes, with the same composition and
structure, can be analyzed using the pKa values of the co-ligands, as shown in Figure S7
(Supporting Information). It can be observed that the compounds with the diimines (bipy, dmbipy,
phen and dphphen), which have lower pKa than the en ligand, have higher oxidation potentials than
the [RuCl(PPh3)(Hdpa)(en)]Cl (2) complex.
In addition, all complexes also exhibit one irreversible oxidation wave (Epa3) with values
falling in the range of 1.29 – 1.34 V vs. Ag/AgCl, which is attributed to oxidation of the -NH- of
Hdpa ligands. The irreversible oxidation process is also observed in the free Hdpa ligand.
Chemical behavior of the complexes in aqueous solution
Contrary to that commonly observed for ruthenium/phosphine complexes, the new
compound 2 is well soluble in water, while compound 1 is only slightly soluble in this solvent.

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Complexes 3 – 6 are insoluble in water. All the complexes are soluble and stable in solvents such as
DMSO, N,N-dimethylformamide and chlorinated solvents, and are also soluble in mixture of
1%:99% DMSO: H2O, at micromolar concentrations (relevant for biological studies).
The stability of the complexes 1 - 6 in D2O/DMSO (50:50) mixture was investigated by
31P{1H} NMR spectroscopy. Compounds 1 and 3 - 6 are stable in the tested solutions, where their
NMR spectra remained unchanged for at least 96 hours. Contrary, immediately after the
solubilization of compound 2 in D2O/DMSO mixture, a new singlet at 53 ppm was observed in its
31P{1H} spectrum, Figure S28 (Supporting Information), which increased at the expense of the
signal at 64 ppm. The new singlet can attribute to the formation of the aqua species
[Ru(OH2)(PPh3)(Hdpa)(en)]2+ (2aq) by replacing the chlorido ligand from the coordination sphere
of the ruthenium center. The species, in solution, reached an equilibrium, within about 45 min after
dissolution of the complex, with a 40:60 ratio between 2 and 2aq. The lability of the Cl- ligand in
complex 2 is attributed to the influence of the σ-donor en ligand, compared to the σ-donor and π-
acceptor diimine ligands in compounds 1 and 3 – 6, which do not undergo hydrolysis of the
complex [30].
HSA binding study by fluorescence quenching
HSA is the most abundant protein in plasma and its principal function is to transport
metabolites, playing an important role in the drug distribution and efficacy since it increases the
solubility of hydrophobic drugs in the plasma. Currently, an important study in the development of
novel drug candidates is to investigate their binding affinity with human serum albumin, which is
an important step in the pharmacological characterization [42, 49]. The interaction of ruthenium
phosphine compounds 1 - 6 with HSA was studied by tryptophan fluorescence quenching.
Figure 2A shows significant fluorescence quenching of HSA in the presence of compound 6
(Supporting Information). A substantial decrease in HSA fluorescence intensity was observed with
the increasing amounts of the complexes in the solution, and in the higher molar ratio of
compound/protein (r = 7) the fluorescence intensity of HSA was approximately 49 - 33 % of the

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initial value. The quenching of HSA fluorescence clearly indicates that the interaction of
compounds causes conformational change in the microenvironment of the Trp21p residue, located
in the subdomain IIA.

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Figure 2. (A) Fluorescence quenching spectra of HSA in the absence and presence of compound 6
at different [compound]/[HSA] ratios (a=0.5; b=1; c=2; d=3; e=4; f=5; g=6; h=7), with the
excitation wavelength at 270 nm, at 310 K in Tris-HCl buffer. Arrow indicates the increase of
quencher concentration. (B) Stern-Volmer plots for HSA fluorescence quenching observed with
compounds 1 – 6.
The fluorescence quenching of HSA, in the presence of the complexes, was measured at
different temperatures, in order to ascertain the fluorescence quenching mechanism. The
mechanisms of quenching are usually classified as dynamic quenching and static quenching, which
can be distinguished examining their temperature dependence, or by the lifetime of fluorophore
measurements [43]. The Ksv constants (Table 4) were determined using the Stern-Volmer equation.
The linearity of the Stern-Volmer plots (Figure 2B) suggests the involvement of only one type of
quenching [42]. The decrease in the Ksv values with an increase in temperature indicates the
existence of static quenching due to the formation of the fluorophore-quencher intermediate in the
ground state.

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Table 4. Stern-Volmer quenching constant (KSV), biomolecular quenching rate constant (kq),
binding constant (Kb), number of binding sites (n) and the thermodynamic parameters for the
compound-HSA system at different temperatures.
Temperature
Ksv
kq
Kb
N
∆Hº
∆Sº
∆Gº
(× 104 M-1) (× 1012 M-1 s-1)
(× 105 M-1)
(KJ mol-1) (KJ mol-1 K-1) (J mol-1 K-1)
Compound 1
1.54 ± 0.80
295
310
4.80 ± 0.06
4.73 ± 0.04
8.01 ± 0.15
7.88 ± 0.08
1.12
1.26
1.10
1.22
1.33
1.47
55.72
77.34
59.66
49.08
65.12
51.14
291.44
374.86
310.48
277.70
342.42
309.50
-31.12
-34.62
4.75 ± 1.10
Compound 2
04.44 ± 0.31
21.73 ± 4.27
Compound 3
02.94 ± 0.15
10.07 ± 0.11
Compound 4
04.67 ± 0.47
12.46 ± 0.29
Compound 5
14.31 ± 0.27
54.53 ± 15.13
Compound 6
90.35 ± 0.46
255.8 ± 25.28
295
310
3.38 ± 0.08
3.28 ± 0.07
5.63 ± 0.13
5.47 ± 0.16
-34.36
-38.86
295
310
4.28 ± 0.03
4.26 ± 0.12
7.13 ± 0.07
7.10 ± 0.20
-32.86
-36.59
295
310
4.81 ± 0.02
4.47 ± 0.11
8.01 ± 0.05
7.44 ± 0.18
-33.68
-37.01
295
310
5.66 ± 0.01
5.14 ± 0.07
9.44 ± 0.03
8.57 ± 0.11
-36.92
-41.03
295
310
9.46 ± 0.15
8.85 ± 0.22
15.76 ± 0.36
14.75 ± 0.37
-41.09
-44.80
The bimolecular quenching constants (kq) were also determined using the Stern-Volmer
equation and assuming t0 = 6 ns for HSA [50]. The maximal value resulting for dynamic quenching
of biopolymers is 2.0 x 1010 M-1 s-1 [51]. The kq constants were higher than 2.0 x 1010 M-1 s-1 for all
complexes. The kq values decrease when the temperature is increased due to the stability of the
fluorophore, while the quencher complex is lowered at higher temperatures. Therefore, the results

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suggest that the fluorescence quenching mechanism of HSA in the interaction with the compounds
is static.
The fluorescence data were treated using the modified Stern-Volmer equation to determine
the binding constants (Kb) and number of binding sites (n) for the interaction of HSA with the
complexes (Table 4). From the binding constants (Kb), the thermodynamic parameters (Table 4)
were calculated using the Van’t Hoff thermodynamic equation [43].
The signs and magnitudes of thermodynamic parameters were analyzed to evaluate the main
intermolecular forces involved in the interaction between the compounds and HSA. According to
Ross and Subramanian [43], when ∆H> 0 and ∆S> 0 it implies that the main force is due to
hydrophobic interactions; ∆H< 0 and ∆S< 0 reflect van der Waals force or hydrogen bond
formation; ∆H< 0 and ∆S> 0 imply that the main force is due to electrostatic interactions. The
negative values of ∆G reveals that the interaction processes between all compounds and HSA are
spontaneous. All complexes present positive ∆H and ∆S values, which is indicative that their
insertions in the protein framework are determined by hydrophobic interactions.
The number of binding sites between HSA and the ruthenium complexes is approximately
equal to 1. The relatively high Kb values for the complexes are reflected moderate to strong
interactions with the HSA. The preference of Ru-phosphine-compounds to bind the subdomain IIA
of the HSA [23c, 52] was shown. The new complexes 1 – 6 have a hydrophobic moiety, mainly by
the presence of PPh3 moiety. Thus, the high binding affinity of the compounds to HSA can be
explained by interactions of the compounds by hydrophobic cavity of subdomain IIA of the
biomolecule. Hence, transporting compounds 1 - 6 in human plasma would be performed by HSA,
and thus the absorption and distribution to various tissues can be feasible [49]
The compounds containing phen (5) and dphphen (6) ligands presented the strongest
interactions with HSA among the 1 - 6 complexes. Even though the hydrophobic interaction plays a
major role in the binding forces, other types of interactions, such as π-π interactions through the

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aromatic rings and size of diimine ligands, may play a further role in the binding affinity between
HSA and compounds 5 and 6 [52, 56].
DNA binding studies
Viscosity Studies
The CT DNA viscosity studies are regarded as one of the most effective methods and least
ambiguous to evaluate the binding mode between DNA and complexes, in solution [41a, 57, 58].
The viscosity measurements are sensitive to length changes of the DNA, and it is known that the
DNA viscosity enhances significantly due to complete or partial intercalation of drugs into its base
pairs [58]. In contrast, the electrostatic or groove interaction between the compounds and DNA
cause a less significant change in the viscosity of the biomolecule [21]. The covalent binding of the
complex/DNA can cause kinks or bends in the DNA double helix, decreasing its viscosity [23e].
The effects of complexes 1 - 6 on the relative CT-DNA viscosity are shown in Figure 3. The
relative viscosity of DNA solutions did not show any significant changes upon adding complexes 1
– 5. These results suggest that compounds 1 – 5 do not cause conformational changes in the DNA
double helix, which is consistent with weak DNA reversible binding. In contrast, when increasing
the concentration of compound [RuCl(PPh3)(Hdpa)(dphphen)]Cl (6), the relative viscosity of the
DNA solutions gradually decreased. This behavior can be associated with two types of interactions:
(a) Covalent binding between the complex-DNA. Indeed, studies on reactivity followed by NMR
showed that compound 6 does not react with guanosine-5’-monophosphate (5’-GMP) and its ability
to bind DNA is unlikely. (b) Cooperative surface interaction complex-DNA by the grooves. Thus,
the interaction of complex 6, by covalent binding with the DNA is not probable because this
compound is very stable in solution, but its interaction through the DNA grooves is more viable to
happen, since the dphphen ligand has phenyl rings, with torsional freedom allowing them to twist
and become isohelical with the DNA groove. As reported by GILL et al. [13], the DNA groove
binding is also dependent on a combination of van der Waals and electrostatic interactions and
hydrogen bonding that has a key role of stabilizing the complex-DNA groove interaction. Hence,

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the possible hydrogen bonding of the NH-group of Hdpa ligand with the DNA may play a further
role in stabilizing the interaction of complex 6 with the DNA groove binding [13, 27, 57].
Figure 3. Effect of increasing concentration of phosphine Ru(II) complexes 1 – 6 on the relative
viscosity of CT DNA; [DNA] = 98 µM. The lines are guides for the lecture of the points.
Electrophoretic mobility of the complexes 1-6 in gel agarose
The ability of compounds 1 – 6 to modify the tertiary structure of DNA was also examined
by monitoring changes in the electrophoretic mobility of pTZ57RT plasmid in gel agarose [59]. The
electrophoretograms of the pTZ57RT plasmid treated with different concentrations of the
compounds are given in Figure 4. The degree of folding the forms of plasmid DNA treated with
compounds 1 – 5 showed no significant changes, which is very similar to the migration pattern of
the untreated DNA. In contrast, compound 6 induced significant changes in the electrophoretic
mobility of the plasmid forms. The amount of plasmid forms decreases when the concentration of
compound 6 reaches the higher molar ratio of compound/pTZ57RT (r = 2), containing no band
corresponding to any form of the biomolecule. These changes in the migration pattern of the DNA

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bands may be due to several factors. The two most common are the fragmentation of the DNA
duplex helix by covalent binding of the compound to DNA [23e] or by quenching ethidium bromide
(EtBr) emission caused when the EtBr is expelled out of the DNA plasmid through intercalation of
the compounds between the DNA base pairs. However, on some occasions, as already described in
the literature, compounds that are very closely associated with the DNA groove structure, leading to
substantial conformational changes, are capable of expelling the EtBr from the DNA plasmid. This
is in accordance with other reported complexes with similar behavior [23d].
Figure 4. Electrophoresis mobility shift assays of plasmid pTZ57RT for compounds 1 – 6
DNA/water refers to untreated plasmid pTZRT57 in water; DNA/buffer refers to untreated plasmid

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in Tris-HCl buffer; A, B, and C correspond to [compound]/[plasmid] ratios of 0.5; 1.0; 1.5,
respectively (D corresponds to 1.25).
Circular Dichroism Studies
The possible effects of compounds 1 – 6 in the DNA secondary structures during the binding
process were investigated by the circular dichroism (CD) technique. The CD spectrum of the CT-
DNA in Trizma-HCl buffer shows a positive band with a maximum at 275 nm due to the base
stacking, and a negative band with a minimum at 240 nm due to the right-handed ellipticity,
characteristic of the B-DNA conformation [58, 60]. Alterations in the CD signals can be assigned to
modifications in the secondary structures of DNA from the DNA-complex interactions [61]. In the
CD spectra of CT-DNA (Figure 5, CD spectra of compounds 5 and 6; see Supporting Information,
CD spectra of 1 – 4) in the presence of complexes 1 – 5, minor changes are observed of the bands
compared with untreated CT-DNA, with a slight decrease in the intensity of the negative band and
no alteration in the positive region, and without a shift in the maximum of the absorption bands.
These observations indicate that the DNA binding to complexes 1 – 5 does not induce
conformational changes in the DNA secondary structures, suggesting the existence of weak
interaction between DNA/complexes [60, 62]. In contrast, the bigger change in CT-DNA was
observed for compound 6. In this case, the intensities of both the positive and negative bands
decreased, indicating that the interaction of compound 6 with the DNA molecule causes
disturbances in the wavelength and ellipticity of the DNA (compared to free DNA). This type of
alteration in the profile of the CD spectrum of CT-DNA is indicative of conformational changes
caused by a non-intercalative mode of binding of the compound to the biomolecule and offers
support that the complex is binding to the groove of the DNA [62].