Arene-ruthenium(II) acylpyrazolonato complexes: Apoptosis-promoting effects on human cancer cells

Article abstract of DOI:10.1021/jm500458c

A series of ruthenium(II) arene complexes with the 4-(biphenyl-4-carbonyl)- 3-methyl-1-phenyl-5-pyrazolonate ligand, and related 1,3,5-triaza-7- phosphaadamantane (PTA) derivatives, has been synthesized. The compounds have been characterized by NMR and IR

Full text of DOI:10.1021/jm500458c

Article
pubs.acs.org/jmc
Arene−Ruthenium(II) Acylpyrazolonato Complexes:
Apoptosis-Promoting Effects on Human Cancer Cells
Riccardo Pettinari,*,† Claudio Pettinari,† Fabio Marchetti,# Brian W. Skelton,ϕ Allan H. White,§
Laura Bonfili,‡ Massimiliano Cuccioloni,‡ Matteo Mozzicafreddo,‡ Valentina Cecarini,‡ Mauro Angeletti,‡
Massimo Nabissi,† and Anna Maria Eleuteri‡
#
‡
†School of Pharmacy, School of Science and Technology, and School of Biosciences and Biotechnology, University of Camerino,
via S. Agostino 1, 62032 Camerino, Italy
ϕ
§School of Chemistry and Biochemistry M313 and Centre for Microscopy, Characterization and Microanalysis M010,
The University of Western Australia, Crawley, WA 6009, Australia
S
* Supporting Information
ABSTRACT: A series of ruthenium(II) arene complexes with the 4-
(biphenyl-4-carbonyl)-3-methyl-1-phenyl-5-pyrazolonate ligand, and related
1,3,5-triaza-7-phosphaadamantane (PTA) derivatives, has been synthesized.
The compounds have been characterized by NMR and IR spectroscopy,
ESI mass spectrometry, elemental analysis, and X-ray crystallography. Anti-
proliferative activity in four human cancer cell lines was determined by MTT
assay, yielding dose- and cancer cell line-dependent IC50 values of 9−34 μM
for three hexamethylbenzene−ruthenium complexes, whereas the other metal
complexes were much less active. Apoptosis was the mechanism involved in
the anticancer activity of such compounds. In fact, the hexamethylbenzene−
ruthenium complexes activated caspase activity, with consequent DNA
fragmentation, accumulation of pro-apoptotic proteins (p27, p53, p89 PARP
fragments), and the concomitant down-regulation of antiapoptotic protein Bcl-2.
Biosensor-based binding studies indicated that the ancillary ligands were critical in
determining the DNA binding affinities, and competition binding experiments further characterized the nature of the interaction.
ligand has shown both p-cymene and benzene derivatives to be
slightly less cytotoxic than the complex with hexamethylbenzene.12
We have previously reported a study on the coordination
chemistry of ruthenium arene fragments with unsymmetrical
β-diketones containing a pyrazole ring fused to the O,O′-
chelating moiety.13 In this paper, we report the preparation and
characterization of some ruthenium arene complexes contain-
ing the 4-(biphenyl-4-carbonyl)-3-methyl-1-phenyl-5-pyrazolo-
nato ligand, with an associated series of biological tests,
performed for the first time using this class of metal complexes.
The cytotoxicity of arene and ancillary ligands coordinated to
ruthenium has been investigated against cervical carcinoma
(HeLa), breast adenocarcinoma (MCF-7), hepatocarcinoma
(HepG2), and colorectal carcinoma (HCT-116) human cell
lines, and the molecular mechanisms beneath the observed effects
have been evaluated and dissected.
INTRODUCTION
■
One of the major medical breakthroughs for metal-based drugs was
the discovery of the potent antitumor activity of cisplatin.1 In the
effort of fighting drug resistance and severe side effects during
treatment, which are the major drawbacks,2 complexes containing
metals other than platinum have become the focus of research.3
Among the other numerous metal complexes explored for
anticancer activity, those of ruthenium occupy a prominent
position;4 two of them, namely NAMI-A5 and KP1019,6 having
entered phase II clinical trials. Ruthenium(III) complexes are likely
to be activated by reduction to Ru(II) in the body and a range of
highly active organometallic Ru(II) anticancer complexes have
been recently developed by Dyson7 and Sadler.8 The structures of
their Ru(II) half-sandwich complexes allow for variation of their
three main building blocks, the arene, the bidentate ligand, and the
monodentate ligand, in fine-tuning their biological properties.9 The
activity of ruthenium arene complexes containing the chelating
ligand ethylenediamine, for example [(η6-arene)Ru(en)Cl]+, has
been shown to be highly dependent on the nature of the bound
arene, with increasing hydrophobicity correlating with increasing
cytotoxicity,10 although further studies have demonstrated that the
structure−activity relationship is more complex.11
RESULTS AND DISCUSSION
■
The pro-ligand HQbiph was synthesized as previously reported.14
The novel ruthenium arene complexes 1−3 were prepared
in high yield from the reaction of the appropriate dimer,
In the “RAPTA” compounds, a class of organometallic ruthenium-
(II) arene complexes containing the PTA ligand, variation of the arene
Received: October 30, 2013
© XXXX American Chemical Society
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Scheme 1
Scheme 2
[(arene)RuCl2]2 (arene = cym (1); benz (2); hmb (3)) with HQbiph
and KOH in methanol (Scheme 1). The complexes are air-stable
in solution and in the solid-state; they are highly soluble in most
organic solvents and sparingly soluble in water. The IR spectra of
1−3 show the typical shift of their ν(CO) vibrations to lower
frequency consequent on coordination of the acylpyrazolone ligand
to the metal ion.15 In the positive ESI mass spectra of 1−3, peaks
due to the cationic fragment [(arene)Ru(Qbiph)]+, generated from
loss of Cl−, are observed as the predominant species.
[(cym)Ru(Qbiph)(CH3OH)][PF6] (4), [(benz)Ru(Qbiph)-
(CH3OH)][PF6] (5), and [(hmb)Ru(Qbiph)(CH3OH)][PF6]
(6), wherein one methanol molecule supplants the chloride in
the ruthenium coordination sphere. The labile methanol mole-
cule in each of 4, 5, and 6 can also be replaced by the water-
soluble phosphine 1,3,5-triaza-7-phosphaadamantane, affording
the complexes [(cym)Ru(Qbiph)(PTA)][PF6] (7), [(benz)Ru-
(Qbiph)(PTA)][PF6] (8), and [(hmb)Ru(Qbiph)(PTA)][PF6]
(9), as depicted in Scheme 2.
Complexes 4−9 are soluble in alcohols, acetonitrile, DMSO,
and acetone, sparingly soluble in water, and poorly soluble in
chlorinated solvents. In acetonitrile, complexes 4−9 display con-
ductivity values within the range typical of 1:1 electrolytes.16 In
the IR spectra of derivatives 4−9 strong sharp absorptions are
1
The H NMR and 13C NMR spectra of 1−3 display distinct
shifts of the resonances of the acylpyrazolone protons in compar-
ison with the equivalent protons in the free pro-ligand.
The chloride ligands in 1, 2, and 3 can be easily removed by
their metathesis reaction with AgPF6 in methanol, to afford
B
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observed at 825 and 830 cm−1 arising from their PF6
The single-crystal studies of 1−3 confirm the overall
stoichiometry and the mononuclear nature and the quasioctahe-
dral (considering the arene-donor as η3) coordination environ-
ment, about each ruthenium atom, similar to those recorded in
numerous other studies.13With modification of the arene-donor
as the only difference between the three complexes, consequent
differences in dimensions are trivial. The Ru−C(0)(arene-
centroid) distances are 1.629/1.646, 1.638, 1.652 Å, with that line
having a maximum angle of 0.33(14)° between it and the normal
to the ligand plane, suggesting little or no difference in whatever
interaction there may be between the arene-ligand and the
remainder of the substituents, but with considerable conforma-
tional differences between the diverse ligands within the one
complex (1) and between them (1−3), where all interplanar
dihedral angles associated with the pendants now vary widely.
Considerable differences are found in counterpart dimensions
between the free pro-ligand HQbiph (which is in the enol form)
and those of the complexed form in 1−3, concomitant on
replacement of the protonic hydrogen atom by the ruthenium
atom, most notably in the O···O bite distance which increases
from 2.4815(13) Å in the free ligand to 2.906(6)−2.942(3) Å in
the complexes. Changes in the other distances are less notable,
but changes of 5° or more are found in those angles forming the
chelate ring.
−
counteranions.17
1
The H NMR spectra of 7, 8, and 9 show all the expected
signals due to the coordinated arene ring, acylpyrazolone, and
PTA, two types of methylene protons being detected for the
coordinated PTA ligand. The 31P NMR spectra of 4−9 contain a
−
septet ca. −143 ppm, typical of an ionic PF6 , and those of 7, 8,
and 9 exhibit single resonances at −29, −26, and −35 ppm,
respectively, due to the PTA ligand, in the region typical of
related complexes, and in accordance with the existence of only
one species in solution.17 In the positive ESI mass spectra of 7−9,
peaks arising from the cationic fragment [(arene)Ru(Qbiph)-
(PTA)]+, generated from the coordination of PTA, are observed
as the predominant species.
Structure Determinations. “Low”-temperature (ca. 100 K)
single-crystal X-ray structure determinations are recorded for
complexes (1−3) [(arene)Ru(Qbiph)Cl], and the parent pro-
ligand HQbiph, all except the latter being to some degree solvated
(Table S1, Supporting Information). In all except 1, a single
molecule (plus solvent, as appropriate), devoid of crystallo-
graphic symmetry, comprises the asymmetric unit of the struc-
ture; in 1, two such molecules. In 1, there is a single molecule of
methanol, hydrogen-bonding to the chlorine atom of molecule
1 (rather than displacing it, as in 6) (Table S2, Supporting
Information); in 2 and 3, in each case a phenyl ring (15× in 2, 1×
in 3) was modeled as rotationally disordered about the pendant
bond over a pair of sites, solvent occupancy being concerted with
that of the minor component. Figure 1b shows a projection of 3
The pro-ligand itself, HQbiph (Figure 1a), is quite closely planar
except for a twist of the “inner” ring of the biphenyl pendant of
ca. 38° (Table S2, Supporting Information), notwithstanding
intercomponent hydrogen contacts which in many cases lie
snugly at or just below their van der Waals sums; in 3, the
biphenyl interplanar dihedral angle is as small as 9.4(2)°. The
crystal packing is interesting with the molecules lying in sheets
normal to a, made up of strings of quasiherringbone dispositions
within each sheet.
Effect on Cancer Cells. All the compounds were tested (see
Experimental Section for details) for their in vitro anticancer
activity against cervical carcinoma (HeLa), breast adenocarcinoma
(MCF-7), hepatocarcinoma HepG2, and colorectal carcinoma
HCT-116 human cell lines (Table 1).
Table 1. Cytotoxicity (IC50, μM) of HQbiph and Complexes
1−9 in Human HeLa, MCF-7, HepG2, and HCT-116 Cancer
Cell Lines (n.e.: no effect)
compound
HQbiph
HeLa
MCF-7
HepG2
n.e.
HCT-116
n.e.
n.e.
n.e.
n.e.
n.e.
1
n.e.
32
30
28
38
35
32
40
30
27
1218
3
1
2
3
2
6
3
5
6
>100
2
>100
31
4
6
3
13
3
26
4
30
4
50
7
5
3
n.e.
56
>100
>100
34
5
84
8
2
9
6
25
24
3
4
7
>100
>100
14
1318
88
38
8
n.e.
13
718
>100
9
2
1
9
5219
3
cisplatin
Figure 1. (a) Projection of a molecule of the pro-ligand HQbiph normal
to its plane; (b) projection of a single molecule of 3 [(hmb)Ru(Qbiph)-
Cl] (major component; pendant phenyl ring 1 is disordered).
The HQbiph pro-ligand was always totally ineffective against
all cancer cell lines. However, the nature of the arene ring
plays a major role in establishing the anticancer potency of the
ruthenium compounds. In fact, both cancer cell lines tested were
particularly sensitive only to hmb-conjugated 3, 6, and 9 at 24 h,
with IC50 values in the range 10−30 μM, whereas benzene- and
as representative of the three complexes, and Figure 1a the pro-
ligand. Molecular dimensions and conformational descriptors are
given extensively in the summary Table S2, Supporting Information.
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Figure 2. (a) DEVDase activity; (b) DNA fragmentation assay at 24 h. Plots and 1.8% agarose gels stained with EtBr are representative of three separate
experiments.
cymene-counterpartsgenerally induced minor, cellline-dependent
effects.
to explore their ability to activate the apoptotic pathway. In detail,
cells were exposed to these compounds for 6 and 24 h and the
apoptotic pathway was explored.
Among the cell lines tested, HepG2 cells were in general the
most sensitive to all the treatments, as IC50 values in the range
27−40 μM were observed. HeLa cells were negligibly affected by
the cymene and benzene complexes, with only the CH3OH
ancillary ligand contributing to partial enhancement of the cell-
killing effect of the cymene and benzene compounds. Also MCF-7
cells were generally resistant to cymene and benzene compounds
over the range of concentration tested, only the benzene−CH3OH
combination (compound 5) inducing a significant effect. HCT-116
cell model showed the highest degree of sensitivity to compound 9,
also being significantly affected by compounds 2, 3, 6, and 7.
On the basis of these results, cells were then treated with
compounds 3, 6, and 9 (those showing the highest cytotoxicity)
Apoptotic Events. Caspase (DEVDase) activity was
measured to elucidate the basis for the observed induction of
cellular death, as it plays a central role in the execution of the
apoptotic program.20 Its activity was significantly enhanced in all
cell lines tested, being clear at 6 h for HeLa and MCF-7 cells, and
more evident at 24 h for HepG2 and HCT116 (Figure 2a).
In detail, at 6 h, caspase activity in HeLa cells was similarly up-
regulated by all the tested compounds (2- to 2.5-fold increases
were observed). Differently, caspase was strongly activated by 3
and 9 in MCF-7 cells. Compound 6 was the most effective in
HepG2 and HCT-116 cells.
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Late events of apoptosis include DNA cleavage, resulting in the
formation of a “ladder” visualized by agarose gel electrophoresis
(Figure 2b). DNA fragmentation assay confirmed the pro-
gression of apoptosis in all cell types upon 24 h treatment with
compounds 3, 6, and 9, consequent on caspase-3 activation and with
the fragmentation extent in line with the degree of caspase activation.
Caspases-3 and -7 are primarily responsible for the cleavage of
poly(ADP-ribose)polymerase protein (PARP) early during
apoptosis. PARP is a nuclear enzyme and catalyzes the
poly(ADP-ribosyl)ation of several nuclear proteins using NAD
as substrate. During apoptosis, caspase-3-mediated PARP
cleavage inactivates the enzyme by destroying its ability to
respond to DNA strand breaks.20 PARP processing by caspases
generates an 89 kDa C-terminal fragment. The results on the
induction of caspase (DEVDase) activity display a corresponding
time- and cell- dependent increase in the levels of the 89 kDa
PARP fragment in all cell lines (Figure 3).
To further confirm the activation of apoptosis, we investigated
two additional markers of this death pathway, namely proteins
p27 and p53. p27/Kip1 protein has been shown to play an
important role in human cancer cell apoptosis. MCF-7 cells
showed its highest increase in the presence of 3 and 9 after 24 h
exposure, whereas a 3- to 4-fold increase was evident at 6 h and
persisted up to 24 h for all compounds in HeLa, HepG2, and
HCT-116 cell lines.
Regarding the transcription factor p53, its activation and
accumulation occur in response to DNA damages, thus leading to
cell cycle arrest or apoptosis.21 We observed the up-regulation of
p53 protein in MCF-7 at short exposure time to 3 and 9 and to all
compounds in both HepG2 and HCT-116 cells, respectively.
Conversely, increased p53 levels were observed in HeLa cells at
24 h exposure to compound 6.
Bcl-2 is an intracellular membrane-associated oncogene
responsible for extending cellular survival.22 The down-
regulation of its expression levels upon treatment with
compounds 3, 6, and 9 in all cell models considered further
confirmed that cell death occurred via apoptosis.
Cell Cycle Analysis. The effects on cell cycle progression of
compounds 3, 6, and 9 at 8 and 16 h post-treatments, were
analyzed in all cell lines. The results showed differential effects on
cell cycle phases at 16 h. In particular, we observed a slight cell
accumulation in G2/M phase with compound 3 in HeLa and
HepG2 cell lines, with compound 6 in all cell lines, and with
compound 9 in HeLa, MCF-7, and HepG2 cell lines, compared
to vehicle-treated cells (see Supporting Information).
DNA-Binding Assay. The DNA binding ability of
compounds 1−9 was evaluated according to a biosensor-based
approach. Sensing surfaces were obtained as described in
Experimental Section. The procedure was optimized after several
experiments based upon the variation of streptavidin concen-
tration and immobilization pH value. A streptavidin concentra-
tion value of 200 μg/mL and a value of 5 for the immobilization
pH (chosen on the basis of the streptavidin isoelectric point)
were found to best suit a good immobilization of the protein
without affecting its functional properties. Readout in the range
600 arcsec were obtained: these conditions resulted in the
coupling of a “Langmuir” partial monolayer of the capturing
macromolecule. These data were adequate to minimize possible
steric hindrance or shielding effects, which could affect the
analysis. Upon saturation of the streptavidin monolayer with
biotinylated dsDNA (3′-CCACCCATACCCTGGTGGATGC-
AATGT-5′), binding experiments were carried out at increasing
ruthenium compound concentrations, each repeated in triplicate.
At any titration step, baseline achievement was assessed be-
fore adding new ruthenium compounds, and the regeneration
steps were then achieved as described in Experimental Section.
Each binding reaction was followed up to equilibrium. The
monoexponential kinetics of each ruthenium compound to
immobilized DNA were recorded (Figure 4): this behavior was
ascribed to the ability of the tested compounds to bind DNA on a
single site. To assess the validity of the monophasic model in
fitting each time course, a standard F-test procedure was used:
the biphasic model was statistically nonsignificant at 95% con-
fidence.23 All complexes displayed moderate affinity to surface-
blocked DNA, with equilibrium dissociation constants in the
range 1.7−19 μM (Table 2); 7 and 9 displayed the highest
Table 2. Kinetic and Equilibrium Parameters of DNA−Ru
Complex Binding
compound
kass (M−1 s−1
)
kdiss (s−1
)
KD (μM)
1
2
3
4
5
6
7
8
9
4300 800
3050 700
0.063 0.002
0.058 0.013
0.047 0.020
0.035 0.011
0.051 0.010
0.062 0.020
0.037 0.010
0.056 0.010
0.043 0.007
14.7 2.3
19.0 6.1
10.7 5.7
3.9 1.5
8.7 2.3
6.0 2.3
1.4 0.4
4.1 0.8
1.7 0.3
4409 1415
9047 1987
5888 1047
10340 2300
26168 1200
13539 673
24752 1520
affinity. The nature of the ancillary ligands was critical in deter-
mining the DNA binding affinities of the tested complexes, with
the compounds ranking in the following order: PTA-conjugated
> CH3OH-conjugated > chloride-conjugated. Conversely, the
nature of the arene rings affected the DNA binding ability of the
compounds only to a minor extent.
Interestingly, the difference in terms of KD was mainly
attributable to the recognition phase, as the rank of association
kinetic constants perfectly matched that of the equilibrium
parameters, whereas all dissociation kinetics were generally
comparable within experimental error (Table 2). The binding
stoichiometry was 1:1, as calculated from the extent of binding at
asymptotically high concentration of each compound (data not
shown).
DNA Binding Competition Experiments. To identify the
binding site for compounds 1−9 on the DNA 30-mer, we
performed three distinct competitive binding studies using two
established DNA binders, namely DAPI (4′,6-diamidino-2-
phenylindole dihydrochloride) and methyl green, and a DNA
intercalator, namely EtBr. In each case, the competition with a
second molecule for binding to DNA would result in the loss of
either fluorescence or absorbance of the complexes. Globally, all
compounds tested were capable of selectively forming a complex
with DNA at the minor groove, as proved by the concentration-
dependent decrease in fluorescent intensity of DNA-bound
DAPI (see Supporting Information). Conversely, no significant
decrease in the absorbance of methyl green−DNA complex was ob-
served. Moreover, four out of the nine compounds, namely com-
pounds 1, 3, 4, and 6, were able to intercalate DNA, as they signif-
icantly decreased the fluorescence intensity of EtBr bound to DNA
(although to different extents), indicating the competition with EtBr.
CONCLUSIONS
Nine novel ruthenium−arene complexes with the 4-(biphenyl-4-
carbonyl)-3-methyl-1-phenyl-5-pyrazolonate ligand have been
■
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Figure 3. Apoptotic protein markers at 6 and 24 h: autoradiographs of PARP 89 kDa fragment, p27, p53, and Bcl-2 protein levels in HeLa, MCF-7,
HepG2, and HCT-116 upon treatment with 3, 6, and 9 (representative Western blots are shown). The densitometric analyses from five separate blots
provided for quantitative analysis are presented.
synthesized, and the structures have been established by
spectroscopic methods together with X-ray diffraction studies.
Modification of these Ru(II)−arene complex structures capable
of affecting tumor-inhibitory activity in vitro toward four human
cancer cell lines (HeLa, MCF-7, HepG2, and HCT116) was
investigated. The biological activity of these complexes was
F
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(products) were obtained from commercial sources and were used
as received. IR spectra were recorded from 4000 to 600 cm−1 on a
1
PerkinElmer Spectrum 100 FT-IR instrument. H, 13C, and 31P NMR
spectra were recorded on a 400 Mercury Plus Varian instrument
operating at room temperature (400 MHz for 1H, 100 MHz for 13C, and
161 MHz for 31P). Referencing is relative to TMS (1H and 13C) and 85%
H3PO4 (31P). Positive and negative ion electrospray mass spectra were
obtained with a Series 1100 MSI detector HP spectrometer, using an
acetonitrile mobile phase. Solutions (3 mg/mL) for electrospray
ionization mass spectrometry (ESI-MS) were prepared using reagent-
grade acetonitrile. Mass and intensities were compared to those
calculated using the IsoPro Isotopic Abundance Simulator, version
2.1.28. Melting points are uncorrected and were taken on an STMP3
Stuart Scientific instrument and on a capillary apparatus. Samples for
microanalysis were dried in vacuo to constant weight (20 °C, ca.
0.1 Torr) and were analyzed on a Fisons Instruments 1108 CHNS-O
elemental analyzer. Electrical conductivity measurements (ΛM, reported
as S cm2 mol−1) of acetonitrile and dichloromethane solutions of the
complexes were recorded using a Crison CDTM 522 conductimeter at
room temperature. Cervical carcinoma (HeLa) and breast adenocarci-
noma (MCF-7) human epithelial cell lines were purchased from
American Type Culture Collection (ATCC, Manassas, VA). Compound
purity was confirmed by elemental analysis with a minimum percentage
of 95%.
Synthesis of the Pro-ligand HQbiph. To a hot 1,4-dioxane solution
of 1-phenyl-3-methylpyrazol-5-one (4.184 g, 0.024 mol) was added
calcium hydroxide (3.556 g, 0.048 mol). The resulting mixture
was stirred under reflux for 30 min. Then biphenyl-4-carbonyl chloride
(5.00 g, 0.023 mol) was added dropwise to the suspension and
the reaction mixture stirred under reflux for 24 h. The brown precipitate
formed was treated with 350 mL of 2 M HCl and then filtered off and re-
crystallized from octane (orange crystals, 80% yield). Mp 148−150 °C.
Anal. Calcd for C23H18N2O2: C, 77.95; H, 5.12; N, 7.90. Found: C,
77.90; H, 5.19; N, 7.84%. IR (cm−1): 3033w, 1606s, 1592s, 1575s, 1534s,
1506m ν(CC; CN). 1H NMR (CDCl3, 298 K): δ, 2.16 (s, 3H, C3-
CH3), 7.26−7.48 (m, 6H), 7.62−7.87 (m, 9H), 10.80 (sbr, 1H,
OH). 13C NMR (CDCl3, 298 K): δ, 16.3 (s, C3-CH3), 103.9 (s, C4),
121.0, 129.9, 127.3, 127.5, 128.4, 128.8, 129.2, 129.3, 136.4, 137.4, 140.1,
145.0, 148.1, 161.8, 191.6 (s, ligand). ESI-MS (−) CH3OH (m/z,
relative intensity %): 353 [100] [Qbiph]−.
Figure 4. Binding of the ruthenium compounds to DNA. Representative
superposition of monoexponential binding curves for compound 2 to
surface-blocked DNA 30-mer.
shown to be highly dependent on the nature of the bound arene,
the most effective complexes containing hexamethylbenzene as
the arene group. Analyzing the IC50 values of the compounds, we
observed that hexamethylbenzene compounds 3, 6, and 9 were
particularly active in inducing cell death in all cell lines, with a
general potency fully comparable to that previously reported for
cisplatin, an approved drug used in the treatment of a number of
tumors. Interestingly, upon treatment of HeLa and MCF-7 cells
with these most effective molecules, the increase in the caspase
DEVDase activity, consequent DNA fragmentation in all cell
types, increased expression levels of other downstream proteins
involved in the apoptotic pathway, and down-regulation of
antiapoptotic protein Bcl-2 were observed. The up-regulation of
apoptotic markers was clearly cell type- and compound-dependent,
with HeLa, HCT-116, and HepG2 cells being similarly responsive
to the three molecules, whereas MCF-7 cells were mainly sensitive
to 3 and 9.
Furthermore, compounds 3, 6, and 9 were capable of slightly
(but significantly) inducing cell cycle block in G2/M phase in a
cell type-dependent manner, probably as a result of DNA inter-
calation and consistently with the in vitro competitive binding
experiments. In fact, besides being all capable of selectively
targeting the minor groove of a dsDNA 30-mer with moderate
affinity, ruthenium compounds 1, 3, 4, and 6 were also shown to
intercalate DNA.
Syntheses of Ruthenium Complexes. [(cym)Ru(Qbiph)Cl] (1). To
the proligand HQbiph (231.4 mg, 0.652 mmol) dissolved in methanol
(20 mL) was added KOH (36.6 mg, 0.652 mmol). The mixture was
stirred 1 h at room temperature, and then [(cym)RuCl2]2 (200.0 mg,
0.326 mmol) was added. The resulting solution was refluxed under
stirring for 24 h. The solvent was removed under reduced pressure,
and dichloromethane (10 mL) was added. The mixture was filtered
to remove sodium chloride. The solution was concentrated to ca.
2 mL and stored at 4 °C. The red crystals obtained and collected
(362.5 mg, 0.580 mmol, yield 88%) were soluble in diethyl ether,
alcohols, acetone, acetonitrile, DMSO, and chlorinated solvents.
Mp 146−148 °C. Anal. Calcd for C33H31N2RuClO2: C, 63.50; H, 5.01;
N, 4.49. Found: C, 63.31; H, 4.89; N, 4.36%. Λm (MeCN, 298 K,
10−4 mol/L): 24 S cm2 mol−1. IR (cm−1): 3059w, 1602s, 1591s, 1577s,
1557sh, 1524m ν(CC; CN). 1H NMR (CDCl3, 298 K): δ, 1.38 (d,
3H, 3J = 7.0 Hz, CH3C6H4CH(CH3)2), 1.74 (s, 3H, C3-CH3), 2.26 (s, 3H,
CH3-C6H4CH(CH3)2), 2.97 (sept, 1H, 3J = 6.9 Hz, CH3C6H4CH(CH3)2),
5.32 (m, 2H, CH3C6H4CH(CH3)2), 5.58 (m, 2H, CH3C6H4CH(CH3)2),
7.20−7.93 (m, 14H, Qbiph). 13C NMR (CDCl3, 298 K): δ, 16.7 (s,
C3-CH3), 18.3 (s, CH3C6H4CH(CH3)2), 22.5 (s, CH3C6H4CH(CH3)2),
31.0 (s, CH3C6H4CH(CH3)2), 79.2, 79.3, 82.5, 82.6, 96.9, 99.7 (s,
CH3C6H4CH(CH3)2), 106.6 (s, C4), 121.2, 125.5, 126.9, 127.4,
128.1, 128.7, 129.1, 137.7, 138.7, 140.3, 143.6, 149.2, 163.8, 188.0 (s,
The structures of these ruthenium−arene complexes allow a
“twin-track approach”: the ancillary ligand determines the DNA
binding affinity, whereas the nature of the arene group has a
critical role for the apoptosis-based cytotoxicity on the cancer cell
types here used. In conclusion, our results demonstrate that
chemical modifications of the arene−Ru(II) compound
modulate the anticancer activity.
Q
biph). ESI-MS (+) CH3OH (m/z, relative intensity %): 589 [100]
[(cym)Ru(Qbiph)]+.
[(benz)Ru(Qbiph)Cl] (2). The synthesis was performed as for 1 using
[(benz)RuCl2]2 (163.3 mg, 0.326 mmol). 2 is soluble in alcohols, ace-
tone, acetonitrile, DMSO, and chlorinated solvents. Mp 264−266 °C.
Anal. Calcd for C29H23N2O2RuCl: C, 61.32; H, 4.08; N, 4.93. Found: C,
61.01; H,4.16; N, 4.86%. Λm (MeCN, 298 K, 10−4 mol/L): 18 S cm2 mol−1.
EXPERIMENTAL SECTION
The dimers [(arene)RuCl2]2 were purchased from Aldrich and TCI
Europe and were used as received. The acylpyrazolone pro-ligand
HQbiph was synthesized using literature methods.11 All other materials
■
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dx.doi.org/10.1021/jm500458c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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IR (cm−1): 3051w, 1592vs ν(CO), 1579s, 1556sh, 1528m ν(CC;
CN). 1H NMR (CDCl3, 298 K): δ, 1.71 (s, 3H, C3-CH3), 5.73 (s,
6H, C6H6), 7.22−7.92 (m, 14H, Qbiph). 13C NMR (CDCl3, 298 K):
δ, 16.6 (s, C3-CH3), 82.3 (s, C6H6), 106.6 (s, C4), 121.1, 125.7,
127.0, 127.3, 127.4, 128.1, 128.6, 128.9, 129.2, 137.3, 138.7, 140.3,
143.6, 149.3, 163.4, 188.4 (s, Qbiph). ESI-MS (+) CH3OH (m/z,
relative intensity %): 533 [100] [(benz)Ru(Qbiph)]+.
(s, Qbiph). 31P NMR (CDCl3, 298 K): δ = −143.2 (sept, PF6). ESI-MS (+)
CH3OH (m/z, relative intensity %): 617 [100] [(hmb)Ru(Qbiph)]+.
[(cym)Ru(Qbiph)(PTA)][PF6] (7). AgPF6 (58.3 mg, 0.231 mmol) was
added to a solution of [(cym)Ru(Qbiph)Cl] (144.2 mg, 0.231 mmol) in
acetone. The reaction mixture was stirred for 1 h at room temperature,
and PTA (36.4 mg, 0.231 mmol) was added. After 24 h, the mixture was
filtered to remove AgCl, the solution was dried under vacuum, and the
red residue was identified as compound 3 (185.2 mg, 0.208 mmol, 90%
yield). It is soluble in diethyl ether, alcohols, acetone, acetonitrile,
DMSO, and chlorinated solvents and sparingly soluble in water. Mp
189−191 °C. Anal. Calcd for C39H43F6N5O2P2Ru: C, 51.54; H,4.99;
N,7.71. Found: C, 51.05; H, 4.69; N, 7.39%. Λm (MeCN, 298 K, 10−4
mol/L): 145 S cm2 mol−1. IR (cm−1): 3062w, 1599s, 1573s, 1555sh,
1534w ν(CC; CN), 830s ν(PF6). 1H NMR (CDCl3, 298 K): δ,
1.23 (d, 6H, CH3C6H4CH(CH3)2), 1.90 (s, 3H, C3-CH3), 1.98 (s, 3H,
CH3C6H4CH(CH3)2), 2.55 (m, 1H, CH3C6H4CH(CH3)2), 4.21 (s, 6H,
PCHAHBN, PTA), 4.50 (m, 6H, PTA), 5.92 (d, 4H, CH3C6H4CH-
(CH3)2), 7.25−7.83 (m, 14H, Qbiph). 13C NMR (CDCl3, 298 K): δ, 16.9
(s, C3-CH3), 17.6 (s, CH3C6H4CH(CH3)2), 22.1 and 22.5 (s,
CH3C6H4CH(CH3)2), 31.1 (s, CH3C6H4CH(CH3)2), 51.3 (d, P-
CH2N, PTA), 73.1 (d, N-CH2N, PTA), 86.6, 88.0, 88.6, 85.5, 97.6, 103.7
(s, CH3C6H4CH(CH3)2), 108.2 (s, C4), 120.4, 126.7, 127.4, 127.6,
128.7, 129.3, 135.8, 137.8, 139.6, 145.3, 149.2, 163.1, 191.3 (s, Qbiph).
31P NMR (CDCl3, 298 K): δ, −29.1; −143.2 (sept, PF6). ESI-MS (+)
CH3OH (m/z, relative intensity %): 746 [100] [(cym)Ru(Qbiph)-
(PTA)]+.
[(hmb)Ru(Qbiph)Cl] (3). The synthesis was performed as for 1 using
[(hmb)RuCl2]2 (218.3 mg, 0.326 mmol). 3 is soluble in alcohols,
acetone, acetonitrile, DMSO, and chlorinated solvents. Mp 295−
297 °C. Anal. Calcd for C35H35N2O2RuCl: C, 64.46; H, 5.41; N, 4.30.
Found: C, 64.18; H, 5.43; N, 4.25%. Λm (MeCN, 298 K, 10−4 mol/L):
13 S cm2 mol−1 IR (cm−1): 3030w, 1602s, 1591s, 1578s, 1539m
ν(CC; CN). 1H NMR (CDCl3, 298 K): δ, 1.72 (s, 3H, C3-CH3),
2.08 (s, 18H, C6(CH3)6), 7.17−7.97 (m, 14H, Qbiph). 13C NMR
(CDCl3, 298 K): δ, 15.2 (s, C6(CH3)6), 15.8 (s, C3-CH3), 92.0 (s,
C6(CH3)6), 102.2 (s, C4), 119.5, 122.0, 124.7, 124.8, 124.9, 126.9, 127.8,
128.3, 128.4, 128.7, 129.0, 129.1, 137.5, 138.6, 140.4, 143.8, 149.1, 163.7,
188.2 (s, Qbiph). ESI-MS (+) CH3OH (m/z, relative intensity %): 617
[100][(hmb)Ru(Qbiph)]+.
[(cym)Ru(Qbiph)(CH3OH)][PF6] (4). Silver hexafluorophosphate
(78.0 mg, 0.312 mmol) was added to a solution of [(cym)Ru(Qbiph)Cl]
(1, 184.6 mg, 0.312 mmol) in methanol. The reaction mixture was
refluxed for 24 h and then allowed to cool to room temperature. The
solvent was removed under reduced pressure, and chloroform (15 mL)
was added. The mixture was filtered to remove AgCl, the solution was
dried under vacuum, and the red residue was identified as 4 (224.2 mg,
0.298 mmol, 72% yield) which is soluble in alcohols, acetone,
acetonitrile, DMSO, and sparingly soluble in chlorinated solvents. Mp
169−171 °C. Anal. Calcd for C34H36F6N2O3PRu: C, 53.26; H, 4.73; N,
3.65. Found: C, 53.03; H, 4.53; N, 3.54%. Λm (MeCN, 298 K, 10−4 mol/L):
130 S cm2 mol−1. IR (cm−1): 3078w, 1600s, 1576s, 1555hs, 1520m
ν(CC; CN), 829s ν(PF6). 1H NMR (CD2Cl2, 298 K): δ, 1.03 and
1.15 (d, 3H, 3J = 6.8 Hz, CH3C6H4CH(CH3)2), 1.26 (s, 3H, C3-CH3),
1.80 (s, 3H, CH3C6H4CH(CH3)2), 2.13 (sbr, 3H, CH3OH), 2.45 (m, 1H,
[(benz)Ru(Qbiph)(PTA)][PF6] (8). The synthesis was performed as for 7
using [(benz)RuCl2]2 (177.5 mg, 0.312 mmol). 8 is soluble in alcohols,
acetone, acetonitrile, DMSO, and chlorinated solvents and sparingly
soluble in water. Mp 240−243 °C. Anal. Calcd for C35H36F6N5O2P2Ru:
C, 50.30; H, 4.34; N, 8.38. Found: C, 50.06; H, 4.24; N, 8.31%. Λm
(MeCN, 298 K, 10−4 mol/L): 141 S cm2 mol−1. IR (cm−1): 3053w,
1598s, 1590s, 1572s, 1544s, 1519w ν(CC; CN), 827s ν(PF6). 1H
NMR (CD3CN, 298 K): δ, 1.82 (s, 3H, C3-CH3), 4.21 (s, 6H,
PCHAHBN, PTA), 4.50 (s, 6H, NCHAHBN, PTA), 5.90 (s, 6H, C6H6),
7.32−7.97 (m, 14H, Qbiph). 13C NMR (CD3CN, 298 K): δ, 16.4 (s,
C3-CH3), 50.7 (d, P-CH2N, PTA), 72.8 (d, N-CH2N, PTA), 87.4 (d,
C6H6), 119.6, 120.8, 125.3, 125.6, 127.0, 127.1, 127.3, 127.7, 127.9,
128.2, 128.3, 128.6, 129.1, 129.3, 135.4, 138.8, 140.2, 142.8, 149.1,
162.21, 190.3 (s, Qbiph). 31P NMR (CD3CN, 298 K):δ, −26.54; −143.3
(sept, PF6). ESI-MS (+) CH3OH (m/z, relative intensity %): 690 [100]
[(benz)Ru(Qbiph)(PTA)]+.
3
CH3C6H4CH(CH3)2), 4.08 (d, 1H, J = 6.0 Hz, CH3C6H4CH(CH3)2),
5.60 (d, 2H, 3J = 6.0 Hz, CH3C6H4CH(CH3)2), 5.71 (d, 2H, 3J = 6.0 Hz,
3
CH3C6H4CH(CH3)2), 5.90 (d, 1H, J = 6.0 Hz, CH3C6H4CH(CH3)2),
7.21−8.04 (m, 14H, Qbiph). 13C NMR (CDCl3, 298 K): δ, 16.7 (s,
C3-CH3), 18.3 (s, CH3C6H4CH(CH3)2), 22.6 (s, CH3C6H4CH(CH3)2),
31.1 (s, CH3C6H4CH(CH3)2), 79.2, 79.3, 82.5, 82.6, 96.9, 99.7 (s,
CH3-C6H4−CH(CH3)2), 106.5 (s, C4), 121.2, 121.3, 125.4, 125.9, 126.9,
127.0, 127.5, 128.1, 128.7, 129.1, 137.7, 138.7, 140.3, 143.6, 149.2, 163.8,
188.0 (s, Qbiph). 31P NMR (CDCl3, 298 K): δ = −143.2 (sept, PF6). ESI-MS
(+) CH3OH (m/z, relative intensity %): 590 [100] [(cym)Ru(Qbiph)]+.
[(benz)Ru(Qbiph)(CH3OH)][PF6] (5). The synthesis was performed
as for 4 using [(benz)RuCl2]2 (177.5 mg, 0.312 mmol). 5 is soluble in
alcohols, acetone, acetonitrile, and DMSO and sparingly soluble in
chlorinated solvents. Mp 230−232 °C. Anal. Calcd for C30H28F6N2O3PRu:
C, 50.71; H, 3.97; N, 3.94. Found: C, 50.43; H, 3.84; N, 3.98%. Λm (MeCN,
298 K, 10−4 mol/L): 136 S cm2 mol−1. IR (cm−1): 3051w, 1592vs ν(CO),
1580s, 1556sh, 1528m ν(CC; CN), 825s ν(PF6). 1H NMR (CD3CN,
298 K): δ, 1.69(s, 3H, C3-CH3), 2.01 (2H, H2O), 5.00(s, 6H, C6H6), 7.22−
8.03 (m, 14H, Qbiph). 13C NMR (CDCl3, 298 K): δ, 16.6 (s, C3-CH3), 82.3
(s, C6H6), 106.6 (s, C4), 121.1, 125.7, 127.0, 127.3, 127.4, 128.1, 128.6,
[(hmb)Ru(Qbiph)(PTA)][PF6] (9). The synthesis was performed as for 7
using [(hmb)RuCl2]2(208 mg, 0.312 mmol). 9 is soluble in alcohols,
acetone, acetonitrile, DMSO, and chlorinated solvents and sparingly
soluble in water. Mp 215−217 °C. Anal. Calcd for C41H47F6N5O2P2Ru:
C, 53.59; H, 5.16; N, 7.62. Found: C, 53.36; H, 5.04; N, 7.41%. Λm
(MeCN, 298 K, 10−4 mol/L): 146 S cm2 mol−1. IR (cm−1): 3050w,
1595s, 1584s, 1570s, 1540s, 1516w ν(CC; CN), 829s ν(PF6). 1H
NMR (CDCl3, 298 K): δ, 1.90 (s, 3H, C3-CH3), 2.08 (s, 18H,
C6(CH3)6), 4.10 (s, 6H, PCHAHBN, PTA), 4.48 (s, 6H, NCHAHBN,
PTA), 7.25−7.87 (m, 14H, Qbiph). 13C NMR (CDCl3, 298 K): δ, 15.6 (s,
C6(CH3)6), 16.2 (s, C3-CH3), 49.8 (d, P-CH2N, PTA), 73.2 (d, N-
CH2N, PTA), 97.7 (s, C6(CH3)6), 120.6, 120.9, 125.2, 126.8, 127.5,
127.7, 128.6, 129.1, 129.3, 129.4 (s, Qbiph). 31P NMR (CD3CN, 298 K):δ,
−35.7; −143.2 (sept, PF6). ESI-MS (+) CH3OH (m/z, relative intensity %):
774 [100] [(hmb)Ru(Qbiph)(PTA)]+.
128.9, 129.2, 137.3, 138.7, 140.3, 143.6, 149.3, 163.4, 188.4 (s, Qbiph). 31
P
NMR (CDCl3, 298 K): δ= −143.3 (sept, PF6). ESI-MS (+) CH3OH (m/z,
Structure Determinations. Full spheres of “low”-temperature
(100 K) CCD area-detector diffractometer data were measured (mono-
chromatic Mo Kα radiation (λ = 0.71073 Å), ω-scans) yielding Ntotal
reflections, these merging to N unique (Rint cited), after analytical (1−3)
or “empirical”/multiscan (HQbiph) absorption correction, which were
used in the full matrix least-squares refinements on F2, refining
anisotropic displacement parameter forms for the non-hydrogen atoms,
hydrogen atom treatment following a riding model (reflection weights:
relative intensity %): 533 [100] [(benz)Ru(Qbiph)]+.
[(hmb)Ru(Qbiph)(CH3OH)][PF6] (6). The synthesis was performed as
for 4 using [(hmb)RuCl2]2 (203.7 mg, 0.312 mmol). 6 is soluble in
alcohols, acetone, acetonitrile, and DMSO and sparingly soluble in
chlorinated solvents. Mp 197−199 °C. Anal. Calcd for C36H39F6N2O3PRu:
C, 54.47; H, 4.95; N, 3.53. Found: C, 54.18; H, 4.73; N, 3.25%. Λm (MeCN,
298 K, 10−4 mol/L): 138 S cm2 mol−1. IR (cm−1): 3030w, 1602s, 1591s,
1578s, 1539m ν(CC; CN), 827s ν(PF6). 1H NMR (CDCl3, 298 K):
δ, 1.73 (s, 3H, C3-CH3), 2.09 (s, 18H, C6(CH3)6), 7.25−7.96 (m, 14H,
2
(σ2(F0 ) + (aP)2 (+bP))−1 (P = (F02 + 2Fc2)/3)); N0 with I > 2σ(I) were
considered “observed”. Neutral atom complex scattering factors were
used within the SHELXL97 program.24 Pertinent results are given below
and in the tables and figures, the latter showing 50% probability ampli-
tude displacement envelopes for the non-hydrogen atoms, hydrogen
Q
biph). 13C NMR (CDCl3, 298 K): δ, 15.6 (s, C6(CH3)6), 16.8 (s, C3-CH3),
90.0 (s, C6(CH3)6), 100.2 (s, C4), 121.0, 125.2, 126.8, 127.1, 127.3, 127.4,
128.1, 128.6, 128.7, 128.8, 129.2, 129.3, 138.4, 140.0, 143.0, 149.1, 163.7
H
dx.doi.org/10.1021/jm500458c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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atoms having arbitrary radii of 0.1 Å. Full cif depositions (excluding
structure factors) lodged with the Cambridge Crystallographic Data
Centre [CCDC 942931 (for HQbiph), 942932 (for 1), 942933 (for 2),
942934 (for 3)] 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.
Cell Viability Assays. Cells were grown in a 5% CO2 atmosphere at
37 °C in 100 mm tissue culture dishes. Growth media were D-MEM
supplemented with 10% FBS, antibiotic, and antimycotic for HeLa cells,
and MEM supplemented with 10% FBS, 1% sodium pyruvate, antibiotic,
and antimycotic for MCF-7 and HepG2. HCT-116 were grown in RPMI
supplemented with 10% FBS. Media and reagents for cell culture were
purchased from EuroClone S.p.A. (Milan, Italy).
The effect of arene−Ru derivatives on HeLa, MCF-7, HepG2, and
HCT-116 cells viability was determined by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay.25 After individual treat-
ments with compounds 1−9, MTT was added to the culture medium at
a final concentration of 0.5 mg/mL and incubated for 2 h at 37 °C. The
medium was replaced with 100 μL of DMSO, and the optical density was
measured at 550 nm after 10 min on a microplate reader. At least six
cultures were used for each time point.
Cell Treatment. Cells were treated for 6 and 24 h with compounds 3
(30 μM), 6 (30 μM), and 9 (15 μM). Upon treatment, cells were
harvested in PBS and centrifuged, and the pellet was dispersed in a lysis
buffer (20 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA, and 5 mM
β-mercaptoethanol) and passed through a 29-gauge needle at least
10 times. Lysates were centrifuged at 12 000g for 15 min, and the
supernatants were stored at −80 °C. Protein concentration in cell lysates
was determined by the Bradford method26 using bovine serum albumin
as standard.
Caspase Activity Assay. Caspase-3 and -7 (DEVDase) activity
assays were performed in cell lysates (5 μg of total proteins in the
mixture) using the Ac-Asp-Glu-Val-Asp-AMC substrate (Sigma-Aldrich
S.r.L., Milan, Italy) in 50 mM Tris-HCl, 50 mM NaCl, 5 mM CaCl2,
1 mM EDTA, 0.1% CHAPS, 5 mM β-mercaptoethanol, pH 7.5. The
incubation was carried out at 37 °C for 60 min, and the hydrolysis
product was detected (AMC: λexc = 365 nm, λem = 449 nm) on a
SpectraMax Gemini XPS microplate reader.
for 30 min at 37 °C, stained for 30 min at room temperature with 20 μg/mL
of propidium iodide (Sigma-Aldrich), and finally analyzed by flow
cytometry using linear amplification.
DNA Binding Assay. The abilities of compounds 1−9 to bind DNA
were tested according to a biosensor-based assay. The carboxylate
surface of the biosensor cuvette was washed and equilibrated with PBS
buffer (10 mM Na2HPO4, 2.7 mM KCl, 138 mM NaCl, pH = 7.4) and
then activated by addition of an equimolar mixture of N-hydroxy-
succinimide and N-ethyl-N-(dimethylaminopropyl)carbodiimide hy-
drochloride.29 Streptavidin was dissolved in 10 mM CH3COONa buffer
(pH 5) and then anchored to the carboxylic surface. Free carboxylic sites
on the sensor surface were deactivated by injection of 1 M ethanol-
amine, pH 8.5. Finally, 5′-biotinylated dsDNA (3′-CCACCCAC-
TACCCTGGTTGGATGCTAATGT-5′) was coupled to the strepta-
vidin-coated surface. Compounds 1−9 were added to the DNA-
functionalized surface at different concentrations, and binding kinetics
were routinely followed up to equilibrium. Dissociation steps were
performed by a single 1 min wash (80 μL) with fresh PBS buffer,
whereas free DNA surface regeneration was achieved by serial PBS
washes (the number of washing cycles depending on the interaction
strength), each time assessing the recovery of free DNA baseline prior to
any further addition of the ruthenium compounds. Raw data were
globally fitted to both mono- and biexponential models, and the validity
of each model to fit a particular time course was assessed by a standard F-
test procedure.
DAPI Displacement Assay. DAPI is a DNA minor groove binder,30
whose fluorescence intensity is enhanced upon DNA complexation.
DAPI displacement was assayed by recording the emission spectra
of solutions containing different concentrations of compounds 1−9
(0−100 μM), DNA (20 μM), and DAPI (15 μM) in phosphate buffer
(10 mM, pH 7.4) at room temperature after excitation at 338 nm on a
Gemini XPS microplate reader.
Methyl Green Displacement Assay. Methyl Green is a major
groove binder,31 whose evanescent native UV−vis absorbance is re-
tained in complexes with DNA. Methyl Green was suspended in 50 mM
Tris-HCl buffer, pH 7.5, containing 7.5 mM MgSO4 and incubated with
DNA (200 μM) for 24 h at 37 °C. Next, the displacement assay was
performed by monitoring the absorbance at 630 nm upon addition of
candidate competitors (compounds 1−9 (100 μM)) with a Bio-Tek
Visible plate reader.
EtBr Competitive Binding Assay. EtBr is an established DNA
intercalator.32 EtBr binding assay was performed by monitoring the
changes in the emission spectra of solutions containing different
concentrations of compounds 1−9 (0−100 μM), DNA (20 μM), and
EtBr (10 μM) in phosphate buffer (pH 7.4) at room temperature upon
excitation at 480 nm on a Gemini XPS microplate reader.
Statistical Analysis. Results are expressed as mean values with their
standard deviations obtained from five separate experiments. Statistical
analysis was performed with one-way ANOVA, followed by the Bonferroni
test using Sigma-stat 3.1 software (SPSS, Chicago, IL). p-Values <0.05 and
<0.01 were considered to be significant.
DNA Fragmentation Assay. An amount of 1 ×106 cells was grown
in six-well microtiter plates; upon 24 h treatment with compounds 3
(30 μM), 6 (30 μM), and 9 (15 μM), the Buonnano et al. procedure27
was followed: cells were collected and the pellets were suspended in
lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 0.5% SDS, and
0.5 mg/mL proteinase K). After 1 h incubation at 50 °C, 10 mg/mL
RNase was added to the lysates and incubated for 1 h at 50 °C and for
10 min at 70 °C. DNA was precipitated with NaOAc pH 5.2 and ice-cold
100% EtOH, incubated on ice for 10 min, and centrifuged at 10 000g for
10 min. Pellets were dissolved in sterile water. Samples were resolved on
a 1.8% agarose gel stained with ethidium bromide.
Western Blotting Analyses. Western Blotting assays were per-
formed to analyze intracellular levels of the apoptotic markers
poly(ADP-ribose) polymerase (PARP), p27, p53, and Bcl-2 following
treatment with the hmb-bearing compounds 3, 6, and 9. Primary and
secondary antibodies were obtained from Santa Cruz Biotech
(Heidelberg, Germany). Cell lysates (20 μg) were loaded on 12%
SDS-PAGE (10% for PARP) and electroblotted onto PVDF membranes
Millipore (Milan, Italy). After incubation with primary antibodies, the
immunoblot detections were carried out with the Enhanced
ChemiLuminescence Western Blotting analysis system (Amersham-
Pharmacia-Biotech). Every gel was loaded with molecular weight
markers including proteins from 6.5 to 205 kDa. As a control for equal
protein loading, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was utilized: membranes were stripped and reprobed for GAPDH using
a monoclonal antibody diluted 1:500. A previously described densitometric
algorithm was used to quantitate the Western Blot results.28
ASSOCIATED CONTENT
■
S
* Supporting Information
Tables S1,2 comprising crystal/refinement data and non-hydrogen
atom molecular dimensions for HQbiph and compounds 1−3.
Fluorescence spectra and absorbance readings of competitive DNA
binding experiments. Cell cycle curves obtained by FACS analysis.
Crystallographic data in CIF format for HQbiph, 1−3. This material
is available free of charge via the Internet at http://pubs.acs.org.
Cell Cycle Analysis. For these analysis, 3 × 105 cells/mL were
incubated with compounds 3, 6, and 9 for 8 and 16 h. Upon incubation,
cells were fixed for 1 h by adding ice-cold 70% ethanol and then washed
with PBS containing 2% FBS and 0.01% NaN3. Next, the cells were
treated with 10 μg/mL ribonuclease A solution (Sigma-Aldrich), incubated
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +39 0737402338. Fax: +39 0737 402338. E-mail:
riccardo.pettinari@unicam.it.
I
dx.doi.org/10.1021/jm500458c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
design of organometallic ruthenium arene anticancer agents. Chimia
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Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the University of Camerino for financial support.
■
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