Influence of PPh3 moiety in the anticancer activity of new organometallic ruthenium complexes

Article abstract of DOI:10.1016/j.jinorgbio.2014.03.002

The effect of the PPh₃ group in the antitumor activity of some new organometallic ruthenium(II) complexes has been investigated. Several complexes of the type [Ru^(II)(Cl)(PPh₃)(Lig-N)], [Ru ^(II)(Cl)₂(Lig-N)] (where Lig-N = pyridine derivate) and [Ru^(II)(Cl)(PPh₃)₂], have been synthesized and characterized. A noticeable increment of the antitumor activity and cytotoxicity of the complexes due to the presence of PPh₃ moiety has also been demonstrated, affording IC₅₀ values of 5.2 μM in HL-60 tumor cell lines. Atomic force microscopy, circular dichroism and electrophoresis experiments have proved that these complexes can bind DNA resulting in a distortion of both secondary and tertiary structures. Ethidium bromide displacement fluorescence spectroscopy studies and viscosity measurements support that the presence of PPh₃ group induces intercalation interactions with DNA. Indeed, crystallographic analysis, suggest that intra-molecular π-π interactions could be involved in the intercalation within DNA base pairs. Furthermore, high performance liquid chromatography mass spectrometry (HPLC-MS) studies have confirmed a strong interaction between ruthenium complexes and proteins (ubiquitin and potato carboxypeptidase inhibitor - PCI) including slower kinetics due to the presence of PPh ₃ moiety, which could have an important role in detoxification mechanism and others. Finally, ion mobility mass spectrometry (IMMS) experiments have proved that there is no significant change in the gas phase structural conformation of the proteins owing to their bonding to ruthenium complexes.

Full text of DOI:10.1016/j.jinorgbio.2014.03.002

Journal of Inorganic Biochemistry 136 (2014) 1–12
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Influence of PPh3 moiety in the anticancer activity of new organometallic
ruthenium complexes
Rubén Sáez a, Julia Lorenzo b, Ma Jose Prieto c, Mercè Font-Bardia d, Teresa Calvet d, Nuria Omeñaca e,
Marta Vilaseca e, Virtudes Moreno a,
⁎
a
Department de Química Inorgànica, Facultat de Química, Universitat de Barcelona, Martí y Franquès 1-11, 08028 Barcelona, Spain
Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain
Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain
Departament de Cristal·lografia, Mineralogia i Dipòsits Minerals, Universitat de Barcelona, Martí i Franquès s/n., 08028 Barcelona, Spain
b
c
d
e
IRB Barcelona-Institute for Research in Biomedicine, Parc Científic de Barcelona, Baldiri Reixac, 10-12, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2013
Received in revised form 3 March 2014
Accepted 6 March 2014
Available online 15 March 2014
The effect of the PPh3 group in the antitumor activity of some new organometallic ruthenium(II) complexes has
been investigated. Several complexes of the type [Ru(II)(Cl)(PPh3)(Lig-N)], [Ru(II)(Cl)2(Lig-N)] (where Lig-N =
pyridine derivate) and [Ru(II)(Cl)(PPh3)2], have been synthesized and characterized. A noticeable increment
of the antitumor activity and cytotoxicity of the complexes due to the presence of PPh3 moiety has also
been demonstrated, affording IC50 values of 5.2 μM in HL-60 tumor cell lines. Atomic force microscopy, circular
dichroism and electrophoresis experiments have proved that these complexes can bind DNA resulting in a
distortion of both secondary and tertiary structures. Ethidium bromide displacement fluorescence spectroscopy
studies and viscosity measurements support that the presence of PPh3 group induces intercalation interactions
with DNA. Indeed, crystallographic analysis, suggest that intra-molecular π–π interactions could be involved
in the intercalation within DNA base pairs. Furthermore, high performance liquid chromatography mass
spectrometry (HPLC–MS) studies have confirmed a strong interaction between ruthenium complexes and
proteins (ubiquitin and potato carboxypeptidase inhibitor — PCI) including slower kinetics due to the presence
of PPh3 moiety, which could have an important role in detoxification mechanism and others. Finally, ion mobility
mass spectrometry (IMMS) experiments have proved that there is no significant change in the gas phase
structural conformation of the proteins owing to their bonding to ruthenium complexes.
Keywords:
Arene ruthenium(II) complexes
Ruthenium antitumor compounds
pBR322 DNA
Calf thymus DNA
DNA interaction
Protein interaction
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
These half sandwich “piano-stool” type constructs offer much scope
for design, with the potential for modifications to the arene and its
In the last few years, ruthenium complexes have attracted much
attention as building blocks for new transition-metal-based antitumor
agents, since they present some advantages over platinum complexes
currently used in cancer chemotherapy [1,2]. Ruthenium compounds
show less toxicity, a novel mechanism of action, the prospect of non-
cross-resistance [3,4] and a different spectrum of activity [5,6]. More
concretely, organometallic ruthenium(II) complexes with arene ligands
represent an important group of ruthenium compounds with anticancer
activity that is being intensively studied in the last decades [7]. The
typical structure of organometallic ruthenium complexes bearing η6-
arene ligands is shown in Fig. 1, which consist in a half-sandwich
“piano-stool” [(η6-arene)Ru(X)(Y)(Z)] complex, where X is usually a
monodentate leaving group and Y, Z can be monodentate or chelating
ligands, depending on the porpoise of the design [8,9].
substituents (R), the monodentate leaving group (X), the ligands Y
and Z, and overall charge of the complex (n+). These features provide
handles for the control of both the thermodynamics and kinetics of
these systems as well as their overall structural architecture, allowing
a more rational drug design approach compared to platinum-based
drugs [10]. They also provide an ability to fine-tune the chemical
reactivity of the complexes, potentially allowing the control of pharmaco-
logical properties including cell uptake, distribution, and interactions with
biomolecules, toxic side effects, and detoxification mechanisms [11].
With regard to their mechanism of action, the role of the arene
moiety, as well as the influence of the other ligands on the aqueous
chemistry of several complexes have been widely investigated
[12–18], resulting in a complex structure–activity relationship [7]. As
observed for other ruthenium complexes, their cytotoxicity is usually
correlated with DNA binding [19–21], although recent works point
to other biomolecules as possible biological targets. As an example,
RAPTA complexes do not show selective binding to DNA in vitro, and
proteins and RNA appear to be the main intracellular targets [22]. In
⁎
Corresponding author.
E-mail address: virtudes.moreno@qi.ub.es (V. Moreno).
http://dx.doi.org/10.1016/j.jinorgbio.2014.03.002
0162-0134/© 2014 Elsevier Inc. All rights reserved.

2
R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
dissolving the protein directly extracted from potato, in mQ water.
Protein concentration was determined in both cases through absor-
bance measurements with UV–visible (UV–Vis) spectrophotometer
CARY 100 SCAN (Varian) as predicted by Lambert–Beer theory.
Molar absorptivity coefficient estimation was made with the method
proposed by Grimsley et al. [25].
2.2. Devices and methods
2.2.1. X-ray diffraction analysis
Crystal structures were registered with an ENRAF-NONIUS CAD4.
Software for structure refining was SHELXS97 and SHELXL97. Crystals
were obtained through diethyl ether slow diffusion in saturated dichlo-
romethane solutions of the compounds.
Fig. 1. Organometallic ruthenium(II) complex structure.
the same way, in the case of the NAMI-A antimetastatic agent, it is ap-
parent that DNA is not the target, and more likely, activity is a conse-
quence of drug–protein interaction. This is especially interesting since
the antimetastatic behavior is not unique to NAMI-A, but applicable to
other classes of ruthenium complexes [23,24]. Related to these results,
here we explore the interaction of η6-arene ruthenium(II) complexes
with some specific proteins.
Lastly, previous works suggests that the addition of the hydrophobic
PPh3 ligand in RAPTA complexes results in more cytotoxic and less
selective drugs, presumably because of increased drug uptake [22].
With the aim of developing more potent anticancer drugs, we
have synthesized and characterized six new organometallic arene–
ruthenium(II) complexes, some of them including PPh3 group in its
structure. In this work we study the influence of tri-phenyl-phosphine
moiety in the antitumor activity of several η6-arene ruthenium(II)
complexes, and try to elucidate the possible reason behind this
phenomenon.
2.2.2. Elemental analysis
Elemental analysis of (C, H, N, S) was carried out with a CARLO ERBA
EA1108.
2.2.3. Infrared spectroscopy
IR spectra between 4000 and 600 cm−1 were registered with a
spectrophotometer NICOLET 5700 FT-IR, in solid phase in KBr matrix.
2.2.4. NMR spectroscopy
1H, 31P{1H} and 19F{1H} NMR were registered in a 300 MHz VARIAN
UNITY. Samples were dissolved in CDCl3.
2.2.5. Atomic force microscopy (AFM)
Atomic force microscopy images were obtained in TMAFM mode
with a NANOSCOPE III MULTIMODE AFM from Digital Instruments Inc.
Sample preparation: DNA was treated for 15 min at room tempera-
ture to obtain a homogeneous topoisomer distribution. Stock solution
1 mg/ml was prepared in a maximum rate DMSO:HEPES 6:4, for non
water soluble complexes. It was then diluted 1:1000 in HEPES until a
final volume of 2000 μl, and therefore filtered through FP030/3 0.2 nm
pore filters (Schleicher & Schuell GmbH). Each sample consists of 1 μl
of pBR-322 plasmid DNA (0.25 μg/μl), 2 μl of drug filtered solution
and then carried to a final volume of 50 μl with HEPES. Samples were
incubated during 5 and 24 h at 37 °C. 2 μl of each sample are adsorbed
over a mica disk (Ashville-Schoonmaker Mica Co., Newport News),
washed with mQ water and dried under argon or nitrogen.
2. Experimental
2.1. Materials
2.1.1. Reactives
RuCl3, pyridine derivated ligands and methyl-benzylamines, were
purchased from Fluka. KPF6, NH4PF6, salts used for buffer preparation,
mobile phases and ubiquitin were commercial products from Sigma-
Aldrich. Solvents were purchased from Sigma-Aldrich and Panreac.
Ligand dppz was synthesized from Sigma-Aldrich commercial products.
PCI was extracted directly from potato.
2.2.6. Circular dichroism
2.1.2. Solutions and buffers
Circular dichroism spectra were registered with a spectropolarime-
ter JASCO 810, equipped with a 450 W Xenon arc lamp.
TE: 10 nM tris-HCl (tris-[hydroxymethyl]aminomethane hydro-
chloride), 0,1 mM EDTA (ethylenediaminetetraacetic acid), 50 mM
NaCl; pH was adjusted to 7.4 with NaOH.
TBE: 45 mM tris-base (tris-[hydroxymethyl]aminomethane),
45 mM boric acid, 1 mM EDTA (ethylenediaminetetraacetic acid); pH
was adjusted to 8 with NaOH.
HEPES: 40 mM de HEPES (4-(2-hydroxyethyl)-1-piperazineethane
sulfonic acid), 10 mM MgCl2; pH was adjusted to 7.4 with NaOH.
Color marker: bromophenol blue (0.25%), xilencianol FF (0.25%),
glycerol 25%.
PBS: 150 mM NaCl, 3 mM KCl, 9 mM Na2PO4, 1.3 mM K2PO4; pH was
adjusted to 7.2 with NaOH.
Sample preparation: 1 mg/ml stock solutions of each compound
were prepared immediately before using in a DMSO:TE sterilized
mixture (2% DMSO maximum). 20 μg/ml calf thymus DNA solution
was prepared in TE and stored at 4 °C. DNA quantization was verified
by UV–Vis spectroscopy, checking absorbance at 260 nm in a split
double beam SHIMADZU UV-2101-PC spectrophotometer. Compound-
DNA adduct formation was carried out by addition of solution stock
aliquots of each compound to a fixed volume of DNA solution. Amount
of drug added in each case is expressed as ri (theoretical molar ratio
compound–nucleotide) and is calculated as can be seen in the addition-
al information.
2.1.3. DNA and general materials for DNA experiments
m ꢀ Mnucl ꢀ Am
ri ¼
DNA calf thymus highly polymerized sodium salt (SIGMA); Plasmid
pBR322 0.25 μg/μl (Boehringer Mannheim GmbH); Agarose AG-200
molecular biology grade (ECOGEN); ethidium bromide (MERCK).
C ꢀ Mr ꢀ V
m
mass of compound used to prepare stock solution (μg)
average molecular mass by nucleotide (g/mol)
number of metallic atoms in compound
2.1.4. Protein solutions preparation. Proteins: Ubiquitin and potato
carboxypeptidase inhibitor (PCI)
Ubiquitin 3 mM solution was prepared dissolving commercial
lyophilized ubiquitin in mQ water. PCI solution 1.86 mM was prepared
Mnucl
Am
C
DNA solution concentration (μg/ml)

R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
3
Mr
V
molecular mass of each compound (g/mol)
sample final volume (ml)
(Applied Biosystems) mass spectrometer provided with a nitrogen
laser (337 nm, 3 ns pulsed) and applying 20–25 KV as accelerating
voltages. Samples were dissolved in suitable matrixes (DHB 2,5-
dihydroxybenzoic acid, Sigma-Aldrich, 10 mg/ml acetonitrile/H2O 1:1
volume (0.1% TFA)).
Infusion high resolution electrospray ionization mass spectrometry
(ESI-MS) spectra were carried out with a LC/MSD-TOF (Agilent
Technologies) mass spectrometer provided with double nebulizer for
exact mass determination. (See Table S1 in Supplementary informa-
tion for experimental condition details).
Liquid chromatography mass spectrometry (LC–MS) experiments
were performed on a QSTAR Elite System Hybrid Quadrupole-TOF
LC/MS/MS (AB Sciex) using an Agilent 1100 G13112B pump, and
an Agilent 1200 G1367C automatic sampler provided with a column
oven.
Potato carboxypeptidase inhibitor (PCI)-complex and ubiquitin-
complex adducts (where complex = 1.7), were obtained by aqueous
solution reaction at neutral pH and temperature of HPLC autosampler
(40 °C). Aliquots of 300 μl of PCI and ubiquitin solutions were taken,
and it was added with the necessary amount (μl) of 0.01 M compound
stock solution (DMSO/mQ water, 2% maximum DMSO) to obtain 1:1
molar ratio. The system was allowed to react and its evolution was
studied by HPLC–MS with 10 μl sample injection per hour, during 24
h. A Nucleosil 120 C18 10 μm 25 × 0.45 cm column was used for
chromatographic separation using linear gradients of acetonitrile
in aqueous solution (A: ammonium acetate 0.01 M, B: acetonitrile
0–100% flux: 1 ml/min–40 min). A 1:10 split post-column was done
for on-line coupling to the mass spectrometer. Experimental mass
spectrometry conditions are described in the Supplementary informa-
tion (Table S2).
All experiments were carried out for molar ratios of 0.1, 0.3 and 0.5,
which means that in each case there are 1, 3 and 5 molecules of
compound respectively versus each ten pairs of DNA nitrogen bases.
Through this formula the μg of compound (or μl of stock solution) that
must be added to DNA solution in each case can be calculated. The
sample holder had 5 l/min nitrogen flow purge. 1 cm path length quartz
cells were used for measurements. Each sample was registered twice in
a wavelength interval of 220 and 330 nm, rate of 50 nm/min.
2.2.7. Agarose gel electrophoresis
Electrophoresis experiments were carried out in an ECOGEN
horizontal tank connected to a PHARMACIA GPS 200/400 variable
tension source. Gel images were recorded with a thermal system
FUJIFILM FTI-500.
Sample preparation: stock solution preparation for each compound
was the same to the one described for circular dichroism. Buffer solution
was TE (2% DMSO maximum). Sample final volume was 20 μl:2.8 μl of
DNA pBR322 solution 0.25 μg/μl, the volume of stock solution necessary
to obtain the desired molar ratio (ri = 0.5), and filling until 20 μl with TE
buffer solution. In this way, the final concentration of DNA plasmid was
35 μg/ml so each sample contained 0.7 μg of DNA. After incubation at
37 °C for 24 h of 20 μl compound-DNA solution samples 4 μl of color
marker was added. The mixture went through electrophoresis in 0.5%
agarose gel in TBE buffer at 1.5 V/cm for 4 h. After that DNA was stained
with ethidium bromide solution (0.5 μg/ml in TBE) during 20 min.
Negative control was a free plasmid pBR322 DNA solution, and for
positive control cisplatin–DNA samples in the same conditions of all
other complexes were prepared.
2.2.11. Ion mobility mass spectrometry (IMS–MS)
IMS–MS experiments were carried out using a SYNAPT G1 HDMS
mass spectrometer (Waters, Manchester, UK). Samples were placed
on a 384-well plate refrigerated at 15 °C and introduced by automated
chip-base nanoelectrospray using a Triversa NanoMate (Advion Bio-
Sciences) in positive ion mode. A reduction of the source pumping
speed in the backing region (5.81 mbar) of the mass spectrometer
was done for optimal ion transmission. (For detailed experimental con-
ditions see Supplementary Table S3) The instrument was calibrated
over the m/z range 500–5000 Da using a solution of cesium iodide.
MassLynx vs 4.1 SCN 704 software and Driftscope vs 2.1 software
were used for data processing. Experimental drift times were trans-
formed into collision cross sections (CCS, Ω) by constructing a
calibration curve with proteins of known collision cross-sections. The
calibrant lists are given in Table S4 and the calibration curves are
shown in Fig. S2. Experimental drift times for these calibrants were
recorded using identical instrument conditions than the studied
complexes.
They were taken 10 μl of 3 mM ubiquitin solution and 20 μl of 1.86
mM PCI solution, and it was added with the necessary volume of
complex E1 and E2 0.01 M stock solutions to obtain 1:1 molar ratio.
Samples were incubated for 24 h at 37 °C. Afterwards they were diluted
with 600 μl of ammonium acetate buffer, and 10 μl of this diluted
solution was poured in the sample plate of the Advion Triversa
Nanomate. DMSO concentration never exceeded 2%.
2.2.8. Molecular fluorescence
Fluorescence molecular emission measurements were registered
with a spectrofluorimeter Kontron SFM-25 (Bio-Tek Instruments).
Sample preparation: several 3 ml aliquots from a calf thymus DNA
50 μM standard stocking solution were taken, adding to them necessary
amount (30 μl) of ethidium bromide 5 mM to get 1:1 molar ratio,
and they were incubated for 30 min at 37 °C. Afterwards, growing
amounts (0, 20, 40, 60, 80 y 100 μl) of compound stock solution
(1.5 mM DMSO/mQ water) were added to different samples, to obtain
different complex concentrations in each one (0, 10, 20, 30, 40 y 50
μM respectively). Emission spectra were registered between 530 and
670 nm and excitation wavelength was established in 502 nm. DMSO
concentration in final samples was always below 2%.
2.2.9. Viscosity measurements
DNA solutions viscosity measurements were carried out with a Vibro
Viscometer SV-1ª (AND A&N Company Limited).
Sample preparation: 1 ml stock solution 5 mM of each compound in
DMSO/water (4:1), and 1 mM calf thymus DNA solution were prepared.
Afterwards, several aliquots of 1 ml from this last were transferred
to different sterilized tubes, adding then 3 ml of TE buffer, which
corresponds with DNA control solution. For each compound, increasing
amounts of stock solution (20, 60 and 100 μl) were added to reach
molar ratios of 0.1, 0.3 and 0.5 DNA: complex, respectively. In all of
them viscosity at 25 °C was measured before and after mixing, and
along the time as well (0, 4, 14, 32, 44 and 56 h), keeping constant
temperature with a termostatized water bath for the samples and
isobuthyl alcohol bath for viscometer devices. Again, DMSO con-
centration in biological samples did not exceed 2%.
2.2.12. In vitro cytotoxicity and apoptosis assays on HL-60 cells
2.2.12.1. Tumor cell lines and culture conditions. The cell line used was the
human acute promyelocytic leukemia cell line HL-60 (American Type
Culture Collection (ATCC)). Cells were routinely maintained in RPMI-
1640 medium supplemented with 10% (v/v) heat inactivated fetal
bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 lg/ml
streptomycin (Gibco BRL, Invitrogen Corporation, Netherlands) in a
highly humidified atmosphere of 95% air with 5% CO2 at 37 C.
2.2.10. Mass spectrometry
Matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) mass spectra were obtained with a VOYAGER DE-RP

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R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
2.2.12.2. Cytotoxicity assays. Growth inhibitory effect of the ruthenium
complexes on the leukemia HL-60 cell line was measured by
the microculture tetrazolium, [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, MTT] assay [26]. Briefly, cells growing
in the logarithmic phase were seeded in 96-well plates (104 cells per
well), and then were treated with varying doses of the ruthenium
complex and the reference drug cisplatin at 37 C for 24 h. For each of
the variants tested, four wells were used. Aliquots of 20 μl of MTT
solution were then added to each well. After 3 h, the color formed
was quantified by a spectrophotometric plate reader at 490 nm wave-
length. The percentage of cell viability was calculated by dividing the
average absorbance of the cells treated with the complex by that of
the control; IC50 values (drug concentration at which 50% of the cells
are viable relative to the control) were obtained by GraphPad Prism
software, version 4.0.
2.3.2.2. Synthesis of [RuIICl(p-cymene)(3-picoline)PPh3][PF6] (4).
A suspension of (3) (0.1 g, 0.18 mmol), KPF6 (0.04 g, 0.2 mmol) and
3-methylpyridine (3-picoline, 400 μl, 4.0 mmol) in methanol (30 ml)
was stirred during 24 h at room temperature. Solvent was removed
under reduced pressure until a yellow oil was obtained. With the addi-
tion of diethyl ether a yellow precipitate was obtained, which was
filtered off, washed with diethyl ether and dried under reduced
pressure.
Yield: 78%; M.S.[ESI]: m/z 626.13 {M+}; Anal. Calc. C34H36ClF6NP2Ru:
52.96% C, 4.71% H, 1.82% N; Anal. Exp.: 53.13% C, 4.83, 1.82% N; 1H NMR
[CDCl3]: δ 8.76 (d, J(HH) ≈ 5.0 Hz, H, C–H{3-picoline}), δ 8.53 (s, H, C–H
{3-picoline}), δ 7.27–7.57 (m, 16H, PPh3, 3-picoline), δ 7.04 (d, J(HH)
≈ 2.0 Hz, H, C–H{3-picoline}), δ 5.95–5.30 (4d, J(HH) ≈ 5.0 Hz, 4H,
C–H{ring}), δ 2.18 (sep, J(HH) ≈ 6.4 Hz, H, CH(Me)2), δ 2.12 (s, 3H,
CH3{3-picoline}),
δ
1.65 (s, 3H, CH3{ring}),
δ
1.10 (2d,
J(HH) ≈ 7.0 Hz, 6H, CH(Me)2); 31P{1H}NMR [CDCl3]: δ 37.3 (s, PPh3),
δ −144.1 (sep, J(PF) ≈ 713 Hz, PF−6 ), 19F{1H}NMR [CDCl3]: δ −73
(d, J(FP) ≈ 713 Hz, PF−6 ); IR: 1093.02 (νP–C), 840.39 (νRu–N), 700.75
(νP–F).
2.3. Synthesis
2.3.1. Synthesis of complexes without PPh3 moiety (see Fig. S1)
2.3.2.3. Synthesis of [RuIICl(p-cymene)(3,4-lutidine)PPh3][PF6] (5). A
suspension of (3) (0.1 g, 0.18 mmol), KPF6 (0.04 g, 0.2 mmol) and 3,4-
dimethylpyridine (3,4-lutidine, 200 μl, 1.8 mmol) in methanol (30 ml)
was heated under reflux during 7 h, keeping the stirring afterwards
during 12 h more at room temperature. Solvent was removed under
reduced pressure until an orange oil was obtained. With the addition
of diethyl ether an orange precipitate was obtained, which was filtered
off, washed with diethyl ether and dried under reduced pressure.
Yield: 84%; M.S.[ESI]: m/z 640.15 {M+}; Anal. Calc. C35H38ClF6NP2Ru-
H2O: 52.34 %C, 5.02% H, 1.74% N; Anal. Exp.: 52.39% C, 4.60% H, 1.84% N;
1H NMR [CDCl3]: δ 8.60 (d, J(HH) ≈ 5.3 Hz, H, C–H{3,5-lutidine}), δ 8.35
(s, H, C–H{3,5-lutidine}), δ 7.26–7.5 (m, 15H, PPh3), δ 6.90 (d, J(HH)
≈ 5.3 Hz, H, C–H{3,5-lutidine}), δ 5.98–5.28 (4d, J(HH) ≈ 5.7 Hz, 4H,
C–H{ring}), δ 2.21 (sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2), δ 2.15 (s, 3H,
CH3{3,5-lutidine}), δ 1.99 (s, 3H, CH3{3,5-lutidine}), δ 1.64 (s, 3H, CH3
{ring}), δ 1.11 (2d, J(HH) ≈ 5.0 Hz, 6H, CH(Me)2); 31P{1H}NMR
[CDCl3]: δ 37.7 (s, PPh3), δ −144.1 (sep, J(PF) ≈ 713 Hz, PF−6 ), 19F{1H}
NMR [CDCl3]: δ −73 (d, J(FP) ≈ 713 Hz, PF−6 ); IR: 1092.35 (νP–C),
840.39 (νRu–N), 700.30 (νP–F).
2.3.1.1. Synthesis of [RuIICl2(p-cymene)]2 (1). A suspension of RuCl3
(0.1 g, 0.36 mmol) in ethanol (40 ml) was heated under reflux during
8 h with 6 equivalents (2 ml, 18 mmol) of R-α-phellandrene, keeping
the stirring afterwards during 12 h more at room temperature. Solvent
was removed under reduced pressure until an orange precipitate was
observed, which was filtered off, washed with cold methanol and
dried under reduced pressure.
Yield: 65%; M.S.[ESI]: m/z 576.9 {M-Cl}+; Anal. Calc. C20H28Cl4Ru2:
39.23% C, 4.61% H; Anal. Exp.: 39.39% C, 4.51% H; 1H NMR [CDCl3]: δa
5.48, δb 5.35 (dd, J(HH) ≈ 6.0 Hz, 4H, C2,3,5,6-H{ring}), δ 2.93
(sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2), δ 2.16 (s, 3H, CH3{ring}), δ 1.28
(d, JHH ≈ 7.0 Hz, 6H, CH(Me)2); IR: 3052.68 (νCsp2–H), 2961.22
(νCsp3–H), 1468–1386 (νC_C).
2.3.1.2. Synthesis of [RuIICl2(p-cymene)(4-(2-EtOH)Py)] (2). A suspension
of (1) (0.1 g, 0.16 mmol) and 4-(2-hydroxyethyl)pyridine (300 μl,
2.7 mmol) in methanol (30 ml) was heated under reflux during 7 h,
keeping the stirring afterwards during 12 h more at room temperature.
Solvent was removed under reduced pressure until an orange oil was
obtained. With the addition of diethyl ether an orange precipitate was
obtained, which was filtered off, washed with diethyl ether and dried
under reduced pressure.
2.3.2.4. Synthesis of [RuIICl(p-cymene)(3,5-lutidine)PPh3][PF6] (6). A sus-
pension of (3) (0.1 g, 0.18 mmol), KPF6 (0.04 g, 0.2 mmol) and 3,5-
dimethylpyridine (3,5-lutidine, 200 μl, 1.8 mmol) in methanol (30 ml)
was heated under reflux during 7 h, keeping the stirring afterwards
during 12 h more at room temperature. Solvent was removed under
reduced pressure until an orange oil was obtained. With the addition
of diethyl ether an orange precipitate was obtained, which was filtered
off, washed with diethyl ether and dried under reduced pressure.
Yield: 89%; M.S.[ESI]: m/z 640.15 {M+}; Anal. Calc. C35H38ClF6NP2-
Ru-H2O: 52.34% C, 5.02% H, 1.74% N; Anal. Exp.: 52.61% C, 4.72% H,
1.89% N; 1H NMR [CDCl3]: δ 8.43 (s, 2H, C-H{3,5-lutidine}), δ 7.27–7.50
(m, 15H, PPh3), δ 7.11 (s, H, C–H{3,5-lutidine}), δ 5.99–5.35 (4d, J(HH)
≈ 5.4 Hz, 4H, C–H{ring}), δ 2.20 (sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2),
δ 2.11 (s, 6H, 2CH3{3,5-lutidine}), δ 1.64 (s, 3H, CH3{ring}), δ 1.11
(2d, J(HH) ≈ 7.5 Hz, 6H, CH(Me)2); 31P{1H}NMR [CDCl3]: δ 38.1
(s, PPh3), δ −144.1 (sep, J(PF) ≈ 713 Hz, PF−6 ), 19F{1H}NMR [CDCl3]:
δ −73 (d, J(FP) ≈ 713 Hz, PF−6 ); IR: 1092.65 (νP–C), 836.70 (νRu–N),
700.41 (νP–F).
Yield: 83%; M.S.[ESI]: m/z 394.05 {M+-Cl}; Anal. Calc. C17H22Cl2-
NORu: 47.56% C, 5.40% H, 3.26% N; Anal. Exp.: 47.57 %C, 5.35% H, 3.35%
N; 1H NMR [CDCl3]: δ 8.88 (d, J(HH) ≈ 6.2 Hz, 2H, C–H{4-(2-EtOH)
Py}), δ 7.18 (d, J(HH) ≈ 6.2 Hz, 2H, C–H{4-(2-EtOH)Py}), δ 5.44–5.21
(2d, J(HH) ≈ 6.0 Hz, 4H, C–H{ring}), δ 3.80 (m, J(HH) ≈ 6.0 Hz, 2H,
CH2{4-(2-EtOH)Py}), δ 3.00 (sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2), δ 2.85
(t, J(HH) ≈ 6.0 Hz, 2H, CH2{4-(2-EtOH)Py}), δ 2.11 (s, 3H, CH3{ring}),
δ 1.31 (d, J(HH) ≈ 7.0 Hz, 6H, CH(Me)2); IR: 3463.70 (νOH), 816.35
(νRu–N).
2.3.2. Synthesis of complexes including PPh3 moiety (see Fig. S1)
2.3.2.1. Synthesis of [RuIICl2(p-cymene)PPh3] (3). A suspension of (1)
(0.55 g, 0.9 mmol) and PPh3 (0.6 g, 2.25 mmol) in hexane (30 ml)
was heated under reflux during 5 h, keeping the stirring until it reached
room temperature. The red precipitate result was filtered off, washed
with hexane and dried under reduced pressure.
2.3.2.5. Synthesis of [RuIICl(p-cymene)(4-(2-EtOH))PPh3][PF6] (7). A
suspension of (3) (0.1 g, 0.18 mmol), NH4PF6 (0.03 g, 0.2 mmol) and
4-(2-hydroxyethyl)pyridine (300 μl, 2.7 mmol) in methanol (30 ml)
was heated under reflux during 7 h, keeping the stirring afterwards
during 12 h more at room temperature. Solvent was removed under
reduced pressure until a yellow oil was obtained. After the addition
of some drops of DMSO, a brown precipitate was obtained with the
Yield: 82%; M.S.[ESI]: m/z 586.1 {M-Cl}+; Anal. Calc. C28H29Cl2Ru:
59.16% C, 5.14% H; Anal. Exp.: 58.83% C, 5.04% H; 1H NMR [CDCl3]:
δ 7.37–7.83 (m, 15H, PPh3), δa 5.18, δb 4.99 (2d, J(HH) ≈ 6.0 Hz, 4H,
C–H{ring}), δ 2.85 (sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2), δ 1.87 (s, 3H,
CH3{ring}), δ 1.11 (d, J(HH) ≈ 7.0 Hz, 6H, CH(Me)2); 31P{1H}NMR
[CDCl3]: δ 24.16 (s, PPh3); IR: 1091.21 (νP–C), 520.95 (πC–P–C).

R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
5
Table 1
Summary of crystallographic data.
Unit cell dimensions
Crystal system
Space group
(2)
(4)
(5)
(6)
(7)
Monoclinic
P21/c
Orthorhombic
Pbca
Orthorhombic
Pbca
Monoclinic
P21/c
Monoclinic
Cc
a
b
c
α
β
γ
10.595 (4) Å
8.411 (2) Å
19.960 (7) Å
90°
92.31 (2)°
90°
18.158 (6) Å
18.521 (6) Å
21.986 (3) Å
90°
90°
90°
18.494 (2) Å
21.875 (6) Å
21.875 (6) Å
90°
90°
90°
10.543 (6) Å
18.391 (8) Å
19.901 (8) Å
90°
94.62 (2)°
90°
10.202 (5) Å
19.636 (5) Å
17.481 (7) Å
90°
3.00 (2)°
90°
addition of H2O. The precipitate obtained was filtered off, washed with
deionized water and dried under reduced pressure.
2.4. Crystallographic analysis
Yield: 69%; M.S.[ESI]: m/z 656.14 {M+}; Anal. Calc. C35H38ClF6NOP2-
Ru-NH4: 48.67% C, 4.74% H, 3.66% N; Anal. Exp.: 49.16% C, 4.66%, 3.57%
N; 1H NMR [CDCl3]: δ 8.73 (d, J(HH) ≈ 6.3 Hz, 2H, C–H{4-(2-EtOH)Py}),
δ 7.27–7.50 (m, 15H, PPh3), δ 7.02 (d, J(HH) ≈ 6.3 Hz, 2H, C–H{4-(2-
EtOH)Py}), δ 5.93–5.25 (4d, J(HH) ≈ 6.0 Hz, 4H, C–H{ring}), δ 3.77
(m, J(HH) ≈ 7.0 Hz, 2H, CH2{4-(2-EtOH)Py}), δ 2.76 (t, J(HH) ≈ 6.0 Hz,
2H, CH2{4-(2-EtOH)Py}), δ 2.24 (sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2),
δ 1.66 (s, 3H, CH3{ring}), δ 1.10 (d, J(HH) ≈ 7.0 Hz, 6H, CH(Me)2);
31P{1H}NMR [CDCl3]: δ 37.1 (s, PPh3), δ −144.1 (sep, J(PF)
≈ 713 Hz, PF−6 ), 19F{1H}NMR [CDCl3]: δ −73 (d, J(FP) ≈ 713 Hz,
PF−6 ); IR: 3533.84 (νOH), 1093.69 (νP–C), 840.42 (νRu–N), 700.86
(νP–F).
Single crystal X-ray diffraction experiments were carried out with
suitable selected crystals of (2),(4),(5),(6) and (7), mounted at the tip
of a glass fiber on an ENRAF-NONIUS CAD4 producing graphite mono-
chromatic Mo Kα radiation (λ = 0.71073 Å). The structures were
solved using the WINGX package. A summary of the crystal data can
be seen in Table 1. Core length and refinements parameters are included
in the Supplementary information (Tables S5). Images of each one of the
complexes analyzed can be seen in Figs. 2–6. CCDC 857319–857323
contain the Supplementary crystallographic data for this paper. These
data can be obtained free of charge from the Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
3. Results and discussions
2.3.2.6. Synthesis of [RuIICl(p-cymene)(PPh3)2][PF6] (8). A suspension of
(3) (0.1 g, 0.18 mmol), KPF6 (0.04 g, 0.2 mmol) and PPh3 (0.1 g,
0.4 mmol) in methanol (30 ml) was stirred during 2 h at 35 °C. Solvent
was removed at room temperature under reduced pressure until a yel-
low oil was obtained. With the addition of hexane a yellow precipitate
was obtained, which was filtered off, washed with ethanol/hexane 1:2
and dried under reduced pressure.
3.1. DNA interaction studies
3.1.1. Circular dichroism
The circular dichroism spectrum of calf-thymus DNA in TE buffer
shows a negative band with λmax = 46 nm and a positive band with
λmax = 275 nm, characteristics of right-handed B-form DNA [27].
Although most of them induced changes in CD spectrum not very signif-
icant (with notable negative and positive bands intensity decrease),
complex (2) and complex (8) caused important changes in ellipticity
of calf thymus DNA and so distortion of its secondary structure (Fig. 7).
Yield: 78%; M.S.[ESI]: m/z 795.16 {M+}; Anal. Calc. C46H44ClF6P3Ru:
58.76% C, 4.72% H; Anal. Exp.: 58.56% C, 4.79% H; 1H NMR [CDCl3]: δ
7.45–7.22 (m, 30H, 2PPh3), δ 5.60–5.00 (2d, J(HH) ≈ 6.4 Hz, 4H, C–H
{ring}), δ 2.70 (sep, J(HH) ≈ 7.0 Hz, H, CH(Me)2), δ 1.22 (d, J(HH)
≈ 7.0 Hz, 6H, CH(Me)2), δ 1.10 (s, 3H, CH3{ring}); 31P{1H}NMR
[CDCl3]: δ 20.67 (s, PPh3), δ −144.1 (sep, J(PF) ≈ 713 Hz, PF−6 ), 19F
{1H}NMR [CDCl3]: δ −73 (d, J(FP) ≈ 713 Hz, PF−6 ); IR: 1089.04
(νP–C), 831.44 (νRu–N), 699.03 (νP–F), 516.46 (πC–P–C).
3.1.2. Agarose gel electrophoresis
Calf thymus DNA contains two main conformational topoisomers,
open circular (OC) and covalently closed circular (CCC). Agarose gel
electrophoresis studies can show the distortion of tertiary structure
due to the interaction between drugs and DNA. The image in Fig. 8
shows calf thymus DNA migration through agarose gel for untreated
DNA and several DNA-metallic complex adducts.
As seen below any of these complexes distorts DNA tertiary struc-
ture in a way able to change the topoisomer's distribution pattern.
Only DNA–cisplatin adducts show the typical coalescence of both CC
and CCC signals due to the formation of cis covalent bonding adducts.
These results suggest that the interaction between DNA and current
ruthenium complexes could be different from the one established be-
tween DNA and cisplatin. It is well known that DNA–cisplatin adducts
are preferently intrastand cis covalent binding, so it can be concluded
that current ruthenium complexes must bind DNA in a different way,
since its effect in DNA agarose gel electrophoresis migration is
completely different.
3.1.3. Molecular fluorescence
Based on previous results, ethidium bromide quenching studies
were carried out to elucidate whether π-stacking bonding could
have any contribution to DNA ruthenium complex interaction or not.
Ethidium bromide is a typical intercalator that can bond DNA nitrogen
Fig. 2. ORTEP representation of crystallographic structure of complex 2.

6
R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
Fig. 3. ORTEP representation of crystallographic structure of complex 4.
Fig. 5. ORTEP representation of crystallographic structure of complex 6.
bases intercalating between them. Ethidium bromide displacement
studies are one of the most simple and potent tools to find out if any
compound can bind DNA nitrogen bases through π-stacking interaction
[28].
binding (capable of EtBr quenching in some occasions) has no influence
on DNA viscosity [30]. Fig. 10 shows the change in viscosity of several
calf thymus DNA solutions in TE when treated with increasing ratios
of ruthenium complexes.
As seen in Fig. 10 all complexes cause an important increase in DNA
solutions viscosity when increasing its concentration, except the only
one lacking PPh3 moiety in its structure. This phenomenon confirms
the intercalative model induced by PPh3 plane rings, probably in combi-
nation with pyridine derivate ring as a π-stacking sandwich system. In
addition, higher increase in viscosity takes place for complex 8, again,
the one with more PPh3 moieties included in its structure.
Images on Fig. 9 show the molecular fluorescence spectra of DNA–
cisplatin (negative control), DNA-9-acridine (positive control) and
DNA-ruthenium complex 2 and 8 adducts. As seen below, for positive
control decrease intensity of molecular fluorescence occurs when
increasing drug ratio due to the consequent higher ethidium bromide
displacement, and so increasing fluorescence quenching. On the other
hand, negative control, shows no molecular fluorescence signal varia-
tion, as expected for compounds that are not able to stand π-stacking
interactions with DNA nitrogen bases. See additional spectra for all
compounds in the Supplementary information (Fig. S4).
3.1.5. Atomic force microscopy
These plots showed intensity decrease pattern for all the ruthenium
complexes except for complex 2, the only one lacking PPh3 moiety in its
structure. Different amounts of decrease was found for each one of
them, achieving highest quenching values for complex 8, since it
includes two PPh3 groups in its structure. These results suggest that
PPh3 presence could induce intercalation between nitrogen bases
through π-stacking based interactions.
Ruthenium complex interaction with pBR322 DNA in HEPES buffer
solution was studied by atomic force microscopy (AFM). The results
obtained are depicted in Fig. 11. As can be seen, ruthenium complex
binding causes DNA chain aggregation (complex 2), DNA chain opening
(complex 6), kinks (complexes 6, 7), cross-linking and supercoiling
(complexes 4–7, remarkably complex 5), and even chain fracture
(complexes 5, 6), showing very different DNA morphologies related to
untreated DNA.
3.1.4. Viscosity measurements
Once more, pBR322-complex 2 system shows different topoisomer
morphologies compared to the rest of ruthenium complexes, which is
consistent with the intercalation binding model proposed for all of
them except for complex 2.
Optical photophysical probes provide necessary, but not sufficient
clues to support a binding model. Hydrodynamic measurements
(i.e., viscosity and sedimentation) that are sensitive to length change
are regarded as the least ambiguous and the most critical tests of a bind-
ing model in solution in absence of crystallographic structural data [29].
A classical intercalation model results in lengthening the DNA helix as
base pairs are separated to accommodate the bound ligand, leading to
the increase of DNA viscosity. In contrast, non-intercalative model,
could bend or kink the DNA helix, reduce its effective length, and
concomitantly, its viscosity. In addition, electrostatic or minor groove
3.2. Protein interaction studies
Although DNA is considered as the primary target for most of the
metallo-drugs studied so far [31], this belief is based mainly on studies
carried out for platinum based anticancer compounds [32]. However,
mechanism of action of ruthenium-based anticancer compounds
is comparatively unexplored, although it is clear that ruthenium
Fig. 4. ORTEP representation of crystallographic structure of complex 5.
Fig. 6. ORTEP representation of crystallographic structure of complex 7.

R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
7
Fig. 7. CD spectra of DNA–cisplatin and DNA–complex 8 adducts.
compounds interact more weakly with DNA relative to platinum
complexes [33]. There is evidence suggesting that ruthenium
compounds might directly interfere with specific proteins involved in
signal transduction pathways and/or alter cell adhesion and trans-
duction processes [34–36]. With this frame, ruthenium complex
reactivity studies in the presence of model and specific proteins
(ubiquitin and potato carboxypeptidase inhibitor-PCI respectively)
have been carried out.
Ubiquitin is a model protein that plays many different rolls in metab-
olism, and it is ubiquitous in the organism. On the other hand, PCI is a
specific protein that can act as an antagonist of human epidermal
grow factors (EGF) which are over expressed in tumor cells [37,38]. In
fact, PCI is considered as a cytostatic agent, able to block the cell cycle
between G0 and G1 phases selectively in cancer cells, without directly
inducing apoptosis [39]. All these phenomena suggest the capability of
PCI to vehiculize ruthenium metallo-drugs in a selective way to tumor
cells (see structures of both proteins in Fig. S5 in the Supplementary
information).
3.2.1. HPLC–MS ruthenium complex–protein interaction study
High-resolution ESI MS has been known as a potent tool to study
covalent and non-covalent ligand–biomolecule interactions [40–42]
and to screen complex mixtures of metabolites, often without the
need for chromatographic separation of the adducts prior to analysis
[43–45]. In this case, HPLC–MS studies allowed to evaluate the inter-
action of ruthenium complexes with both model and specific proteins,
as well as to elucidate the implications of the presence of PPh3 moiety
in this interaction.
Graphics on Figs. 12 and 13 show a summary of the decrease of free
protein signal while increasing ruthenium complex–protein adduct
solution content within the time (see all complete mass spectra in the
Supporting information, Fig. S6).
In the case of PCI protein (Fig. 12), when PPh3 ligand is present the
kinetics of the reactions is very influenced, taking more time to detect
the PCI-ruthenium complex adduct and in smaller quantities. On the
other hand, when no PPh3 moiety is present, almost all the free protein
content disappears in very short period of time, to be mainly in PCI-
ruthenium complex adduct form.
Added to that, for ubiquitin protein (Fig. 13), it was not possible to
detect the presence of ubi-ruthenium complex adduct when PPh3
ligand was present. All that data suggest that the PPh3 presence affects
in a very important manner to the adduct formation process kinetics,
which could have very important consequences in the detoxification
processes and/or in the delivery of these drugs and cell uptake, allowing
slower pharmacokinetics (which usually means less secondary effects)
and higher resistance to drug removal in natural detoxification
processes.
3.2.2. IMMS — Ion mobility mass spectrometry studies
Ion mobility mass spectrometry can provide information on the
physical size and shape of ionized molecules [46] and previous works
on related Ru-based complexes have demonstrated the use of this
Fig. 8. Agarose gel electrophoresis image of untreated DNA (1), DNA-ruthenium complex
2, 4–8 (2, 3–7), and DNA–cisplatin (8) adducts.

8
R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
Fig. 9. Fluorescence emission spectra of DNA-EtBr system treated with some compounds showing different performances depending on the presence of PPh3 moiety.
technique for the separation of geometrical isomers and the calculation
of their collision cross-sections (CCSs) [47].
their mass-to-charge (m/z) ratio. Therefore, integrated IMS–MS has
the capability of separating ions not only by their mass but also by
their size, shape and charge state. IM–MS offers an extra degree of
analytical opportunity whereby conformational ensembles of species
of equivalent mass, or the same m/z, can be separated on account
of their physical shape and then mass analyzed in a single, rapid
experiment. The experimental drift times (arrival times) can be corre-
lated to collision cross sections by performing calibration curves with
protein standards of known cross sections analyzed under identical
In this technique, basically, a liquid sample is ionized and injected
into a drift chamber containing neutral gas at a controlled pressure
(e.g., 0.5 mbar of nitrogen gas). Under the influence of an electric field,
gaseous ions undergo IM separation according to the resistance they
experience through their collision with neutrals, which depends on
their collision cross section-to-charge ratio (Ω/z). After separation,
ions are sampled by a mass spectrometer and analyzed according to

R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
9
Fig. 10. Viscosity evolution for calf thymus DNA solutions treated with synthesized ruthenium complexes.
instrumental conditions. Significant changes in CCS should be evaluated
as they reflect conformational changes that could affect some functions
of the protein. More detailed information about IMS–MS can be found in
the literature [48].
As mentioned before, PCI protein can act as a vehicle for anticancer
drugs towards specific cancer cells in case the binding of the metal
complexes doesn't distort the protein structure, since it is an antagonist
of EGFs. With the aim of studying the conformational changes of PCI and
ubiquitin due to the ruthenium complexes binding IMS–MS experi-
ments were carried out.
reinforce previous conclusions out of HPLC–MS studies. As can be
seen, again, the presence of PPh3 moiety affords slower kinetics and
smaller yields of protein–metal complex binding, which should have
important consequences in terms of drug distribution and detoxifica-
tion mechanisms.
Table 2 shows the CCSs obtained when relating the drift times out of
the IMS–MS experiments for each molecule reaching the detector,
with the cross section calibration curve made with protein standards
(see also Fig. S2 in the Supplementary information).
As shown in the table, the differences in CCS upon ligand interaction
with UBI and PCI lay between 0.02 and 11.7% which suggests no signifi-
cant conformational changes in the three-dimensional gas-phase protein
MS spectra shown in Fig. 14 demonstrate the binding of different
fragments of ruthenium complexes to both PCI and ubiquitin, and
Fig. 11. Atomic force microscopy (AFM) images of pBR322-DNA plasmid solutions treated with synthesized ruthenium complexes.

10
R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
Fig. 12. HPLC–MS tuned spectra of PCI-complex 2, 7 solution. Go to Supplementary information Fig. S6 to see all complete mass spectra.
Fig. 13. HPLC–MS tuned spectra of Ubi-complex 2 solution. Go to Supplementary information Fig. S6 to see all complete mass spectra.

R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
11
Fig. 14. MS spectra of PCI, UBI, complex 2 and complex 7 combination solutions.
structure. That would support both the possibility of drug delivery by
model proteins as ubiquitin and the possibility of specific vehiculization
towards cancer cells by specific proteins as PCI.
The cytotoxic properties of the complexes including PPh3 ligand
in its structure correspond to values comparable to the cytotoxicity
obtained for cis-platin and ruthenium complexes active against cancer
cell lines in similar experiments [49] (notice that ruthenium complexes
undergo some special processes, such as hydrolysis and different bind-
ings compared to cis-platin), while the only one that lack this moiety
raises over 200 μM, so it cannot be considered an active drug towards
this type of tumor cell line.
These results added to the fact that previous investigations carried
out in our group [50] in which most of the complexes studied in the
present work, but lacking PPh3 moiety, were evaluated as antitumor
drugs showing poor antiproliferative properties, strongly suggest
an important increment of the antitumor properties of ruthenium
complexes due to PPh3 presence.
3.3. Cytotoxicity studies
Cytotoxicity studies were carried out for complexes 2–7 in HL60
Human Leukemia Tumor Cell Line, affording IC50 values shown in
Table 3.
Table 2
Cross-section variations of different detected adducts. (a) No DMSO in solution. (b) 2%
DMSO in solution.
Molecule
Adduct
CCS (Å2)
ΔCCS %
PCI (a)
PCI (b)
–
545
569
–
Table 3
–
–
IC50 values at 24 h for HL60 leukemia tumor cell line.
PCI (b)-2
PCI (b)-2
PCI (a)-7
UBI (a)
PCI (b)-[Ru(p-cymene)]
PCI (b)-[Ru(p-cymene)PPh3]
PCI (a)-[Ru(p-cymene)]
–
584
596
566
1015
1017
1028
1015
908
2.5
4.5
3.7
Complex
IC50 (μM)
Complex 2
Complex 4
Complex 5
Complex 6
Complex 7
202
10.1
5.2
5.2
15.4
UBI (b)
–
UBI (b)-2
UBI (b)-2
UBI (a)-7
UBI (b)-[Ru(p-cymene)]
UBI (b)-[Ru(p-cymene)PPh3]
UBI (b)-[Ru(p-cymene)]
1.1
0.02
11.7

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R. Sáez et al. / Journal of Inorganic Biochemistry 136 (2014) 1–12
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4. Conclusions
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Several new organometallic ruthenium complexes, some of them
including PPh3 ligands, have been synthesized and characterized. DNA
interaction studies have demonstrated the capability of these
complexes to bind DNA and distort its secondary and tertiary structure
notably. Ethidium bromide displacement experiments and viscosity
measurements prove that those complexes including PPh3 moiety in
its structure are able to intercalate into DNA base pairs, whereas those
without PPh3 ligand bind DNA only in a covalent manner. Protein inter-
action studies have shown the capability of these complexes to bind as
well as to model and specific proteins, demonstrating slower kinetics
and smaller binding yields when PPh3 group is present, presumably
due to steric impediments. These effects could have important
consequences in drug cell up-taken and/or detoxification mechanisms.
Finally, cytotoxicity studies show that IC50 values in the range of the
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PPh3 ligand. That result proves definitively the increment of ruthenium
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Acknowledgments
Financial support from the Spanish Ministry of “Ciencia e Innovación”,
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Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2014.03.002.
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