Structure-Activity relationships of targeted RuII(η 6- P -Cymene) anticancer complexes with flavonol-Derived ligands

Article abstract of DOI:10.1021/jm301376a

Ru^II(arene) complexes have been shown to be promising anticancer agents, capable of overcoming major drawbacks of currently used chemotherapeutics. We have synthesized Ru^II(η⁶-arene) compounds carrying bioactive flavonol ligands with the aim to obtain multitargeted anticancer agents. To validate this concept, studies on the mode of action of the complexes were conducted which indicated that they form covalent bonds to DNA, have only minor impact on the cell cycle, but inhibit CDK2 and topoisomerase IIα in vitro. The cytotoxic activity was determined in human cancer cell lines, resulting in very low IC₅₀ values as compared to other Ru^II(arene) complexes and showing a structure-activity relationship dependent on the substitution pattern of the flavonol ligand. Furthermore, the inhibition of cell growth correlates well with the topoisomerase inhibitory activity. Compared to the flavonol ligands, the Ru^II(η⁶-p-cymene) complexes are more potent antiproliferative agents, which can be explained by potential multitargeted properties.

Full text of DOI:10.1021/jm301376a

Article
pubs.acs.org/jmc
Structure−Activity Relationships of Targeted RuII(η6‑p‑Cymene)
Anticancer Complexes with Flavonol-Derived Ligands
Andrea Kurzwernhart,† Wolfgang Kandioller,†,‡ Simone Bachler,§ Caroline Bartel,† Sanela Martic,∥
̈
Magdalena Buczkowska,⊥ Gerhard Muhlgassner,† Michael A. Jakupec,†,‡ Heinz-Bernhard Kraatz,∥
̈
Patrick J. Bednarski,⊥ Vladimir B. Arion,† Doris Marko,§ Bernhard K. Keppler,†,‡
and Christian G. Hartinger*,†,‡,#
†Institute of Inorganic Chemistry, University of Vienna, Waehringer Strasse 42, 1090 Vienna, Austria
‡Research Platform “Translational Cancer Therapy Research”, University of Vienna, Waehringer Strasse 42, 1090 Vienna, Austria
§Institute of Food Chemistry and Toxicology, University of Vienna, Waehringer Strasse 38, 1090 Vienna, Austria
∥Department of Physical and Environmental Sciences, University of Toronto, Scarborough, Canada
⊥Department of Pharmaceutical and Medicinal Chemistry, University of Greifswald, 17487 Greifswald, Germany
#The University of Auckland, School of Chemical Sciences, Private Bag 92019, Auckland 1142, New Zealand
S
* Supporting Information
ABSTRACT: RuII(arene) complexes have been shown to be promising
anticancer agents, capable of overcoming major drawbacks of currently
used chemotherapeutics. We have synthesized RuII(η6-arene) compounds
carrying bioactive flavonol ligands with the aim to obtain multitargeted
anticancer agents. To validate this concept, studies on the mode of action
of the complexes were conducted which indicated that they form covalent
bonds to DNA, have only minor impact on the cell cycle, but inhibit CDK2
and topoisomerase IIα in vitro. The cytotoxic activity was determined in
human cancer cell lines, resulting in very low IC50 values as compared to
other RuII(arene) complexes and showing a structure−activity relationship
dependent on the substitution pattern of the flavonol ligand. Furthermore,
the inhibition of cell growth correlates well with the topoisomerase
inhibitory activity. Compared to the flavonol ligands, the RuII(η6-p-
cymene) complexes are more potent antiproliferative agents, which can be explained by potential multitargeted properties.
Chart 1. Chemical Structures of KP1019, NAMI-A, and
RAPTA-C
INTRODUCTION
■
Metallodrugs have become important compounds in cancer
therapy, and, in particular, platinum complexes are used
worldwide against many tumor types.1 To overcome severe
side effects and drug resistance during treatment, which are
their major drawbacks,2 complexes with metal ions other than
platinum have become the focus of research.1 Especially,
ruthenium complexes offer a number of interesting properties
such as lower toxicity and a range of physiologically accessible
oxidation states.3 The RuIII complexes NAMI-A and KP1019
have shown the most promising results in preclinical and
clinical studies (Chart 1).4−6 The more selective activity of
these compounds due to an efficient uptake as protein adducts
and activation by reduction inside the tumor is thought to be
responsible for the low general toxicity.4 During the last years,
organometallic RuII complexes, especially half-sandwich
RuII(arene) compounds, moved into the focus of interest
because biological activity and pharmacological properties can
easily be modulated by ligand selection. Important examples of
this compound type comprise the RAPTA family7 (Chart 1)
containing the pta ligand (1,3,5-triaza-7-phosphatricyclo-
[3.3.1.1]decane) and RuII(arene) complexes of bidentate
ethylenediamine, which are at an advanced preclinical develop-
ment stage.8
The RAPTA complexes are a good example of organo-
metallics which can be equipped with ligands to obtain desired
Received: July 30, 2012
Published: November 7, 2012
© 2012 American Chemical Society
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properties. Whereas the parent compound RAPTA-C is a
metastasis inhibitor, tethering the organometallic fragment to
ethacrynic acid resulted in compounds with glutathione-S-
transferase inhibitory activity, accompanied by a cleavage of the
enzyme inhibiting moiety from the metal fragment which can
target a second biomolecule, e.g., DNA. This concept of
multitargeted anticancer agents, i.e., drugs designed to act
against several individual molecular targets, offers a number of
advantages over classic chemotherapeutics, including tunable
pharmacological properties and anticancer activity and altered
metabolism and resistance development.9 Such drugs can be
prepared by linking metal fragments to biologically active ligand
systems and, as shown in the RAPTA case, by functionalization
of an arene ligand or, as previously reported by us, by direct
coordination of 3-hydroxyflavones to a RuII(cym) moiety (cym
= η6-p-cymene).10
Scheme 1. Synthesis of 3-Hydroxyflavone Ligands and their
RuII(η6-p-Cymene) Complexes
Flavonoids are known as natural components of plants, fruits,
and vegetables and exhibit interesting biological properties such
as antioxidant, anti-inflammatory, estrogenic, antimicrobial, and
anticarcinogenic activity.11−13 The 3-hydroxy-4-keto motif of 3-
hydroxyflavones offers facile coordination to many metal ions.
The resulting metal complexes exhibit high stability constants,
high molar absorbances, and fluorescence properties and are
biocompatible.14,15 Therefore, such compounds have been
investigated as new fluorochromic indicators for ion chelation
and biomembrane structure studies in the human body.16 In
addition, a few examples of tumor-inhibiting metal−flavonoid
complexes are known.17
pseudo-octahedral “piano-stool” configuration similar to related
RuII(cym) complexes.10,22 The 3-hydroxyflavone acts as a
bidentate ligand, forming a nonplanar, envelope-like five-
membered metallocycle. The phenyl substituent of the ligand
is twisted with a torsion angle of 48.4(4)° in 3h, but only
5.1(3)° in 3f, 0.2(3)° in 3d, and 15.3(6)° in 3b. The two Ru−
O bonds were found to be slightly different as in 3b with
2.0664(14) and 2.1154(14) Å in 3d, 2.0747(11) and
2.1098(11) in 3f, and 2.0870(16) and 2.1339(16) Å in 3h,
which is in accordance to Ru−O bonds in similar
compounds.23,24 The Ru−Cl bonds [2.4105(5) Å (3d),
2.4132(4) (3f), and 2.4100(7) Å (3h)] are in the range of
recently published RuII(cym) complexes [2.4200(11)−
2.4273(8) Å]4−7,10 but shorter than in 3b with 2.4326(10).
The Ru−cymcentroid distances were found at 1.638 for both 3d
and 3f and at 1.647 Å for 3h.
Herein, we report a series of RuII(cym) complexes with 3-
hydroxyflavone ligands with the aim to study the influence of
the substituent on the phenyl ring of the ligand on the
anticancer activity. To support the hypothesis of multitargeted
compounds, their behavior in aqueous solution and in the
presence of nucleotides was studied. Furthermore, the
inhibition of cyclin-dependent kinases (CDKs) and their effect
on the cell cycle has been assayed, as well as their human
topoisomerase IIα inhibitory activity.
RESULTS AND DISCUSSION
■
We have extensively studied the application of O,O-chelating
hydroxypyrone complexes as potential anticancer agents and
recently extended these investigations to 3-hydroxyflavones
with the same binding motif.10 To establish structure−activity
relationships (SAR), a series of derivatives was synthesized in
two steps, starting with a Claisen−Schmidt condensation of 2-
hydroxyacetophenone 1 and benzaldehydes a−j in alkaline
solution. In the second step, the obtained 2′-hydroxychalcones
were converted into the respective 3-hydroxyflavones 2a−j
under Algar−Flynn−Oyamada reaction conditions with hydro-
gen peroxide and NaOH (Scheme 1).18−20 The RuII(cym)
complexes 3a−j were synthesized by deprotonation of 2a−j
with sodium methoxide and subsequent reaction with bis-
[dichlorido(η6-p-cymene)ruthenium(II)] (Scheme 1).21 All
complexes were obtained in moderate to good yields (30−
68%) and are stable for more than one year, even if exposed to
sunlight and air, which was confirmed by NMR spectroscopy
and elemental analysis.
1
The aqueous stability of the complexes was studied by H
NMR spectroscopy (Figure 2). Dissolution of organometallic
Ru(arene) compounds featuring an O,O-chelating motif and a
halido ligand often results in rapid exchange of the chlorido
ligand by an aqua moiety.25 The same behavior was
demonstrated for such complexes which aquated within
seconds, leading to charged 4a−c, 4f, and 4i (Scheme 1).26,27
The hydrolyzed compounds are relatively stable in aqueous
solution for about 24 h, however, after 6 days, the formation of
the dimeric hydrolysis side product [Ru2(cym)2(OH)3]+ was
clearly visible in ESI-mass and NMR spectra. This species does
not affect the cytotoxic activity as it is thermodynamically stable
and unreactive toward nucleophiles.25,28 Notably, 3a−j exhibit
approximately 10-fold better solubility in water than the
respective ligands, e.g., 0.03 mg/mL for 2a and 0.3 mg/mL
for 3a in 1% DMSO/H2O.
Cytotoxic Activity. The in vitro anticancer activity was
determined in the human cancer cell lines CH1 (ovarian
carcinoma), SW480 (colon carcinoma), and A549 (non-small
cell lung carcinoma) by means of the colorimetric MTT assay
and in human urinary bladder (5637), human large cell lung
(LCLC-103H), and human pancreatic carcinoma cell lines
(DAN-G) with the crystal violet assay, both after 96 h.
All synthesized compounds were characterized by standard
methods (see Experimental Section), and single crystals of 3d,
3f, and 3h were analyzed by X-ray diffraction methods (Figure
1) and are compared to 3b.10 Complex 3d crystallizes in the
centrosymmetric space group P1 and 3f and 3h in the
̅
monoclinic space group P21/n. The compounds feature the
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Figure 1. Molecular structures of the RuII(cym) complexes 3d (left), 3f (center), and 3h (right).
resistant A549 cells. In comparison to compound 3a with an
unsubstituted ligand structure, ortho substitution of the phenyl
ring (3e and 3h) appears unfavorable, whereas meta and para
substitution (3f, 3g, 3i) increases the anticancer activity. This
may be due to a structural effect, as the phenyl ring is
considerably more twisted in the ortho derivative than in the
meta- and para-substituted compounds (Figure 1), which may
influence the interaction with biological targets.31 However, the
type of substituent, whether electron-withdrawing or electron-
donating, seems to be of minor importance, whereas their
position appears to be crucial for the cytotoxic activity. We have
shown earlier that the ligands are the bioactive moiety, which is
mainly responsible for the antiproliferative activity.10 However,
they are poorly soluble in aqueous solutions and therefore
hardly suitable for pharmaceutical applications.
1
Figure 2. Hydrolysis of 3c in 10% DMSO-d6/D2O studied by H
The crystal violet assay for antiproliferative activity in the
5637, the LCLC-103H, and the DAN-G cell lines (Table 1)
revealed slightly higher IC50 values than that for the MTT assay
in the CH1 cell line but still notable for RuII(arene) complexes.
Compounds 3d and 3f exhibit the lowest IC50 values at around
5 μM in these three cell lines and also show the highest potency
in the MTT assays. Their antiproliferative activity is in the same
range as that of related dinuclear Ru(arene)(pyridone)
compounds.32 Compounds 3b and 3d show the best overall
activity in the used cell lines. However, some compounds are
more specific for one of the three cell lines used in the crystal
violet assays, e.g., 3i for DAN-G, 3f and 3j for 5637, and 3c and
3g for LCLC-103H, which is a highly desirable characteristic for
anticancer agents.32,33
NMR spectroscopy. After 6 d, the formation of the dimeric hydrolysis
side product [Ru2(cym)2(OH)3]+ was observed giving signals at 5.1
and 5.3 ppm.
In general, the biological activity as determined by the MTT
assay and given as the IC50 values (50% inhibitory
concentration; Table 1) was in the low micromolar range,
which is very unusual for RuII(arene) compounds, and only a
few more examples with similar cytotoxicity are known.4,29,30
The chemosensitive CH1 cell line was most sensitive to the test
compounds with IC50 values lower than 7.9 μM, followed by
SW480 with IC50 values ranging from 3.4−26 μM. The lowest
cytotoxic potency was found for the generally more chemo-
Table 1. In Vitro Anticancer Activity of 3a−j (96 h Exposure) in Human Ovarian, Colon, Non-Small Cell Lung, Urinary
Bladder, Large Cell Lung, and Pancreatic Carcinoma Cell Lines (Mean Values SD of Three Independent Determinations
unless Otherwise Noted)
IC50 values/μM
compd
CH1
SW480
A549
5637
11
5.7 3.2
33
LCLC-103H
13
DAN-G
12
6.6 2.5
12
3a
3b
3c
3d
3e
3f
2.1 0.2
1.8 0.2
1.7 0.4
1.5 0.1
4.0 0.8
0.86 0.06
1.0 0.1
7.9 0.6
1.2 0.2
2.3 0.7
9.6 1.5
7.2 0.5
7.9 2.0
7.0 1.0
20
2
2
1
1
1
5
6
2
17
18
15
30
5.2 0.8
5.5 5.2
4.3 1.1
nd
5
2
4.3 2.5
nd
5.3 1.6
nd
24
3
3.8 0.5
7.0 0.7
9.5 0.5
3.3 1.1
13
1
19
19
7
5
3g
3h
3i
12
51
2
5
30
nd
12
2
5.0 3.5
nd
26
1
nd
5.7 1.9
20
3.4 0.1
7.2 0.4
8.6 0.7
17
1
19
16
6
4
3j
3
5.9 1.2
5
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Furthermore, the time dependence of the cytotoxicity of 3j
was investigated and the IC50 values of 3j were determined after
1, 4, 24, and 96 h in SW480 cells (Figure 3). The calculated
IC50 values for 3j decreased from about >40 μM after 1 h to 12
μM after 24 h and finally to 7 μM after 96 h.
Figure 4. Integrated current densities (estimated from square-wave
voltammograms) observed in CDK2/cyclin A kinase-catalyzed Fc-
phosphorylation of the surface bound peptide in the presence of the
organometallic compounds (3a−3h) and compared to roscovitine (R)
(inhibitor concentration = 10 μM, 100 mV/s scan rate, 0.1 M
phosphate buffer pH 7.4 electrolyte, triplicate measurements).
Significances indicated refer to the control calculated by Student’s t
test (*p < 0.1, **p < 0.05).
Figure 3. Time-dependent anticancer activity of 3j in SW480 cells.
Significances indicated refer to the next shorter incubation time
calculated by Student’s t test (*p < 0.1, **p < 0.05).
accompanied by a decrease of the G0/G1-phase fraction,
whereas at higher concentrations these effects tend to level out
again. The impact on the S phase is less pronounced, but a
slight increase can be observed at increasing concentrations of
3b, 3c, and especially 3f (>16 μM, Figure 5). These results
indicate that CDK2 is not very likely to be an intracellular
target because this kinase is involved in the G1/S transition of
the cell cycle, which is not influenced in a dose-dependent
manner by the Ru complexes studied.
Inhibition of CDK2. The anticancer activity of the
flavonoid flavopiridol is linked to its inhibition of cyclin-
dependent kinases (CDKs),34 and therefore this group of
proteins is also a possible target for Ru−flavonoid complexes.
To estimate the compounds’ potential to inhibit kinases, the
inhibition of CDK2 was studied by an electrochemical assay
employing Fc-ATP as a cosubstrate in the kinase phosphor-
ylation reaction.35,36 The peptide HHASPRK was used as a
substrate and immobilized onto the Au surface for the study of
CDK2/cyclin A protein kinase activity. Following the kinase-
catalyzed Fc-phosphorylation reaction, the Fc group is
transferred from Fc-ATP to the immobilized peptide and
results in an electrochemical signal. Hence, the electrochemical
readout is directly related to the extent of Fc-phosphorylation
by a protein kinase. In general, the Fc-phosphorylations are
monitored by square-wave voltammetry wherein the oxidation
peak at ∼440
5 mV signals the successful Fc-phosphor-
ylation. The dependence of the integrated current density in
CDK2/cyclin A kinase assays as a function of inhibitors 3a−h
(10 μM) is shown in Figure 4. With the exception of 3c and 3f,
all compounds inhibit CDK2 in about the same range as
structurally related HOPO complexes37 and are nearly as active
as the well-known CDK2 inhibitor roscovitine (R), which was
included for comparison (Figure 4). It appears that para
substituents at the phenyl ring of the flavone ligands are less
favorable than ortho and meta substitution. However, no direct
structure−activity relationships can be derived when compared
to the in vitro anticancer activity data set. Therefore,
considering also the minor influence on the cell cycle (see
below), CDK2 and CDKs in general are not considered as the
main target of this type of compounds.
Impact on the Cell Cycle. The cell cycle distribution of
A549 cells was studied by treating exponentially growing A549
cells with 3a−c and 3e in various concentrations for 48 h. Then
cells were stained with propidium iodide and analyzed for their
DNA content by flow cytometry. The four Ru compounds have
comparable effects on the cell cycle, most probably due to their
similar structures. The effects vary in dependence of the
concentration (Figure 5). Low concentrations (<32 μM of 3a,
3b, and 3c and <16 μM of 3e) induce a slight G2/M arrest,
Figure 5. Concentration-dependent impact of 3f on the cell cycle
distribution of A549 cells after exposure for 48 h (values are means
standard deviations of two independent experiments).
Topoisomerase Inhibition. Topoisomerases are overex-
pressed in many types of cancer and thus are major targets for
antineoplastic agents such as doxorubicin, etoposide, and
mitoxantrone.38,39 Flavonoids are also known to inhibit
human topoisomerases,40,41 which are enzymes that change
the topology of DNA by introducing a transient break in the
DNA strand, allowing a second DNA region from either the
same molecule (relaxation, knotting, or unknotting) or a
different molecule (catenation or decatenation) to pass
through. During this process, the enzymes are covalently
bound to the DNA via an active tyrosine residue, termed
“cleavable complex”. After the DNA is untangled or unwound,
the strands are reannealed by the enzyme so that the overall
composition of the DNA strand does not change. DNA
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topoisomerases are participating in nearly all biological
processes involving DNA including replication, transcription,
recombination, and chromatin remodeling.42,43 They are
classified as type I and type II depending on if they induce
single- or double-strand breaks, respectively.
CONCLUSIONS
■
Flavonoids exhibit interesting biological properties, and
members of this compound class have been investigated in
clinical trials as anticancer agents. By combining such moieties
with metal fragments, we aimed to obtain anticancer agents that
can interact with more than one target, facilitating single-
molecular combination therapy. A series of light- and air-stable
RuII(η6-p-cymene) complexes with flavonol ligands were
prepared in good yields. The flavonol ligands 2a−j are hardly
soluble in aqueous solutions, which prevents their potential use
as anticancer agents. In contrast, the RuII(cym) complexes 3a−j
exhibit approximately 10-fold better solubility in water than the
ligands. However, they aquate within seconds by an exchange
of the chlorido ligand with an aqua moiety, leading to 4a−j,
which are then able to bind to DNA, as shown for the DNA
model compound 5′-GMP. The flavonol-derived compounds
exhibit IC50 values in the low μM range, which is unusual for
RuII(cym) complexes, but this indicates the central role of the
ligand system as the anticancer activity determining factor. This
is also highlighted by comparison of the IC50 values of the
coordination compounds and the respective ligand systems.10
Complexes 3f, 3g, and 3i with para- and meta-substituted
ligands exhibit lower IC50 values than their unsubstituted
analogue 3a or the ortho-substituted derivatives 3e and 3h.
To gain information on the mode of action of the complexes
and in light of the biological properties of the ligands, the
inhibitory activity of the complexes on topoisomerase IIα and
CDK2 was assayed and flow cytometry analyses of the cell cycle
were conducted. These studies revealed that 3a−c and 3e have
an influence on the cell cycle distribution, and especially at
concentrations around the IC50 values of the compounds, an
increase in the cell fraction in G0/G1 phase was observed.
Furthermore, most of the complexes were shown to inhibit
CDK2 to an extent approaching that of roscovitine, a well-
known CDK2 inhibitor. However, these results did not
resemble the activity pattern observed in the in vitro anticancer
assays. Notably, the inhibition of topoisomerase IIα correlated
well with the in vitro anticancer activity data, with the
compounds exhibiting the lowest IC50 values in the MTT
assay being also the most potent topoisomerase IIα inhibitors.
Compared to the unsubstituted flavonol ligands 2a−j, which
are considered as the topoisomerase-inhibiting moiety in the
coordination compound, the respective RuII(cym) complexes
3a−j are shown to inhibit topoisomerase IIα to a greater extent.
This may be explained by their additional ability to form bonds
to the DNA base guanine and thereby acting in a bifunctional
manner, which could be beneficial in tumor therapy.
In the case of topoisomerase inhibitors, an increase of the
proportion of cells in the S-phase and at higher concentrations
a G2/M arrest are expected. In the case of the Ru complexes
studied here, a slight increase in the S-phase fraction was
observed (Figure 5) and therefore topoisomerase inhibition
studies were conducted. The catalytic activity of topoisomerase
IIα is determined by means of the decatenation assay.
Catenated kinetoplast DNA (kDNA) consists of interlocked
DNA minicircles (mostly 2.5 kb), which can be released to
single DNA circles by catalytically active topoisomerase II. If
topoisomerase II is inhibited, kDNA stays in its catenated form,
which is not able to enter the agarose gel, whereas the single
DNA circles, released by topoisomerase II, are able to migrate
into the gel (Figure 6, compare lane 1 with lane 2). Thus,
Figure 6. Concentration-dependent effect of the Ru complexes 3a, 3c,
3d, and 3f on the catalytic activity of topoisomerase IIα, as determined
by the decatenation assay. The topoisomerase poison etoposide was
used as a control.
kDNA was incubated with topoisomerase IIα in the presence of
different concentrations of the RuII(cym) complexes 3a−3d, 3f,
3g, 3i, and 3j and the flavonols 2a−c, 2f, and 2i. All complexes
inhibit the catalytic activity of human topoisomerase IIα at
concentrations ≥10 μM. The extent of inhibition appears to be
well correlated to their in vitro anticancer potency, with 3f
being the most potent inhibitor followed by 3d (Figure 6), and
with 3a as the least cytotoxic and weakest inhibitor of
topoisomerase IIα. Compared to the flavonols, their RuII(cym)
complexes are more potent in all studied cases, demonstrating
that the inhibition of topoisomerase IIα is enhanced by the
linkage of the topoisomerase-inhibiting flavonoid scaffold to the
RuII(cym) moiety. We explain this observation by multitargeted
properties of the complexes, as they are also able to interact
with the DNA.10
EXPERIMENTAL SECTION
■
All solvents were dried and distilled prior to use. 2-Hydroxyaceto-
phenone 1 (Fluka, Acros Organics), benzaldehyde a (Fluka), 4-
tolualdehyde (Acros Organics), 4-fluorobenzaldehyde c (Fluka), 3-
fluorobenzaldehyde d (Fluka), 2-fluorobenzaldehyde e (Fluka), 4-
chlorobenzaldehyde f (Acros Organics), 3-chlorobenzaldehyde g
(Aldrich), 2-chlorobenzaldehyde h (Aldrich), 4-bromobenzaldehyde i
(Aldrich), 3-bromobenzaldehyde j (Aldrich), ruthenium(III) chloride
(Johnson Matthey), α-terpinene (Acros Organics), and sodium
methoxide (Aldrich) were purchased and used without further
purification. Bis[(η6-p-cymene)dichloridoruthenium(II)] was synthe-
sized as described elsewhere.45
Nucleotide Binding. To help estimate the reactivity to
DNA, the interaction of the aqua species 4a−c, 4f, and 4i with
1
the DNA model compound 5′-GMP was investigated by H
NMR spectroscopy. The 5′-GMP N7-adducts were formed
immediately, as indicated by an upfield shift of the H8 signal of
5′-GMP from approximately δ = 8.1 to 7.6 ppm.10,44 Therefore,
DNA represents a suitable binding partner for this class of
compounds.
Melting points were determined with a Buchi melting point B-540
̈
apparatus. Elemental analyses were carried out with a Perkin-Elmer
2400 CHN elemental analyzer by the Microanalytical Laboratory of
the University of Vienna. NMR spectra were recorded at 25 °C using a
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Bruker FT-NMR spectrometer Avance III 500 MHz. 1H NMR spectra
were measured at 500.10 MHz and 13C{1H}-NMR spectra at 125.75
MHz in DMSO-d6 or CDCl3. The 2D NMR spectra were measured in
a gradient-enhanced mode.
3-Hydroxy-2-(3-fluorophenyl)-chromen-4(1H)-one (2d). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and d (1.82 g, 14.7 mmol), affording 2d as a
1
yellow powder (0.89 g, 50%); mp 171−173 °C. H NMR (500.10
4
3
The X-ray diffraction data for 3d, 3f, and 3h were collected on a
Bruker X8 APEX II CCD diffractometer at 100 K. The single crystals
were positioned at 35, 35, and 40 mm from the detector, and 1746,
1367, and 1667 frames were measured, each for 20, 5, and 5 s over 1°
scan width. The data were processed using the SAINT software
package.46 The structures were solved by direct methods and refined
by full-matrix least-squares techniques. Non-hydrogen atoms were
refined with anisotropic displacement parameters. H atoms were
inserted at calculated positions and refined with a riding model. The
following computer programs were used: structure solution, SHELXS-
97; refinement, SHELXL-97;47 molecular diagrams, ORTEP-3.48 The
crystallographic data files for 3d, 3f, and 3h have been deposited with
the Cambridge Crystallographic Database as CCDC 886667, 886666,
and 886665, respectively.
General Procedure for the Synthesis of the 3-Hydroxy-
flavone Ligands 2a−2j. NaOH (5 M, 4.3 equiv) was added to a
solution of 2-hydroxyacetophenone 1 (1.0 equiv) and aldehydes a−i
(1.0 equiv) in ethanol, and the solution was stirred for 18 h at room
temperature. The reaction mixture was acidified to pH 6 by addition of
acetic acid (30%), and the 2′-hydroxychalcone was isolated by
filtration. The 2′-hydroxychalcone (1.0 equiv) was suspended in
ethanol, NaOH (5 M, 2.0 equiv), and H2O2 (30%, 2.2 equiv) were
added at 4 °C. The mixture was stirred for 18 h at room temperature,
afterward acidified to pH 1 with HCl (2 M) and poured onto water
(400 mL). The precipitate was collected by filtration, and the pure
product was obtained by recrystallization from methanol.
MHz, DMSO-d6): δ = 7.37 (ddd, J(H,F) = 2 Hz, J(H,H) = 8 Hz,
3J(H,H) = 8 Hz, 1H, H4′), 7.48−7.51 (m, 1H, H7), 7.64 (ddd,
3
3
4J(H,F) = 2 Hz, J(H,H) = 8 Hz, J(H,H) = 8 Hz, 1H, H5′), 7.81−
4
3
7.86 (m, 2H, H6/H8), 8.04 (dd, J(H,H) = 2 Hz, J(H,H) = 11 Hz,
1H, H5), 8.10−8.14 (m, 2H, H2′/H6′), 9.93 (br s, 1H, OH) ppm.
13C{1H} NMR (125.75 MHz, DMSO-d6): δ = 114.6 (d, 2J(C,F) = 24
2
Hz, C2′), 117.1 (d, J(C,F) = 21 Hz, C4′), 119.0 (C8), 121.7 (C8a),
4
124.1 (d, J(C,F) = 3 Hz, C6′), 125.2 (C7), 125.3 (C5), 131.1 (d,
3J(C,F) = 8 Hz, C5′), 134.0 (d, J(C,F) = 9 Hz, C1′), 134.4 (C6),
3
1
140.0 (C2), 144.0 (C3), 155.0 (C4a) 162.0 (d, J(C,F) = 243 Hz,
C3′), 173.6 (C4) ppm. Elemental Anal. Calcd for C15H9FO3: C 70.31,
H 3.54%. Found: C 69.94, H 3.39%.
3-Hydroxy-2-(2-fluorophenyl)-chromen-4(1H)-one (2e). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and e (1.82 g, 14.7 mmol), affording 2e as a
1
yellow powder (0.87 g, 49%); mp 181−182 °C. H NMR (500.10
MHz, DMSO-d6): δ = 7.37−7.42 (m, 2H, H3′/H6′), 7.48−7.51 (m,
1H, H7), 7.59−7.63 (m, 1H, H4′), 7.66 (d, 3J(H,H) = 8 Hz, 1H, H8),
4
7.76−7.82 (m, 2H, H5′/H6), 8.16 (dd, J(H,H) = 1 Hz, 3J(H,H) = 8
Hz, 1H, H5), 9.41 (br s, 1H, OH) ppm. 13C{1H} NMR (125.75 MHz,
2
DMSO-d6): δ = 116.6 (d, J(C,F) = 21 Hz, C3′), 118.9 (C8), 119.5
2
3
(d, J(C,F) = 14 Hz, C1′), 122.3 (C8a), 124.9 (d, J(C,F) = 3 Hz,
4
C6′), 125.2 (C7), 125.5 (C5), 131.7 (d, J(C,F) = 2 Hz, C5′), 133.0
3
(d, J(C,F) = 8 Hz, C4′), 134.3 (C6), 140.0 (C3), 143.9 (C2), 155.5
(C4a), 159.0 (d, 1J(C,F) = 252 Hz, C2′), 173.2 (C4) ppm. Elemental
Anal. Calcd for C15H9FO3·0.05H2O: C 70.31, H 3.54%. Found: C
69.98, H 3.42%.
3-Hydroxy-2-phenyl-chromen-4(1H)-one (2a). The synthesis was
performed according to the general procedure by using 1 (2.00 g, 14.7
mmol) and a (1.56 g, 14.7 mmol) to afford 2a as a yellow powder
(2.63 g, 75%); mp 165−168 °C. 1H NMR (500.10 MHz, DMSO-d6):
3-Hydroxy-2-(4-chlorophenyl)-chromen-4(1H)-one (2f). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and f (2.10 g, 14.7 mmo) to afford 2f as yellow
3
3
δ = 7.49−7.54 (m, 2H, H4′/H7), 7.59 (dd, J(H,H) = 7 Hz, J(H,H)
= 7 Hz, 2H, H3′/H5′), 7.79 (d, 3J(H,H) = 7 Hz, 1H, H8), 7.83−7.81
1
powder (2.31 g, 58%); mp 202−204 °C. H NMR (500.10 MHz,
DMSO-d6): δ = 7.48−7.51 (m, 1H, H7), 7.65 (d, 3J(H,H) = 8 Hz, 2H,
H3′/H5′), 7.78 (d, 3J(H,H) = 8 Hz, 1H, H8), 7.83−7.85 (m, 1H, H6),
8.13 (dd, 4J(H,H) = 1 Hz, 3J(H,H) = 8 Hz, 1H, H5), 8.26 (d, 3J(H,H)
= 9 Hz, 2H, H2′/H6′), 9.84 (br s, 1H, OH) ppm. 13C{1H} NMR
(125.75 MHz, DMSO-d6): δ = 118.9 (C8), 121.7 (C8a), 125.1 (C7),
125.3 (C5), 129.2 (C2′/C6′), 129.8 (C3′/C5′), 130.7 (C2), 134.4
(C6), 139.9 (C4′), 140.3 (C1′), 144.5 (C3), 155.0 (C4a), 173.5 (C4)
ppm. Elemental Anal. Calcd for C15H9ClO3: C 66.07, H 3.33%.
Found: C 65.80, H 3.17%.
4
3
(m, 1H, H6), 8.14 (dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5),
3
8.24 (d, J(H,H) = 7 Hz, 2H, H2′/H6′), 9.64 (br s, 1H, OH) ppm.
13C{1H} NMR (125.75 MHz, DMSO-d6): δ = 118.9 (C8), 121.8
(C8a), 125.1 (C7), 125.3 (C5), 128.1 (C2′/C6′), 129.0 (C3′/C5′),
130.4 (C4′), 131.8 (C2), 134.2 (C6), 139.6 (C1′), 145.7 (C3), 155.1
(C4a), 173.5 (C4) ppm. Elemental Anal. calcd for C15H10O3: C 75.62,
H 4.23%. Found: C 75.62, H 4.09%.
3-Hydroxy-2-(4-methylphenyl)-chromen-4(1H)-one (2b). The
synthesis was performed according to the general procedure by
using 1 (2.00 g, 14.7 mmol) and b (1.76 g, 14.7 mmol) to afford 2b as
3-Hydroxy-2-(3-chlorophenyl)-chromen-4(1H)-one (2g). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and g (2.10 g, 14.7 mmol), affording 2g as a
1
yellow crystals (1.64 g, 44%); mp 191−194 °C. H NMR (500.10
3
MHz, DMSO-d6): δ = 2.41 (s, 3H, CH3), 7.40 (d, J(H,H) = 8 Hz,
1
2H, H3′/H5′), 7.47−7.50 (m, 1H, H7), 7.79 (d, 3J(H,H) = 7 Hz, 1H,
H8), 7.79−7.82 (m, 1H, H6), 8.14 (dd, 4J(H,H) = 1 Hz, 3J(H,H) = 8
Hz, 1H, H5), 8.16 (d, 3J(H,H) = 8 Hz, 2H, H2′/H6′), 9.56 (br s, 1H,
OH) ppm. 13C{1H} NMR (125.75 MHz, DMSO-d6): δ = 21.5 (CH3),
118.9 (C8), 121.7 (C8a), 125.0 (C7), 125.3 (C5), 128.1 (C2′/C6′),
129.1 (C2), 129.6 (C3′/C5′), 134.1 (s, C6), 139.4 (C1′), 140.3 (C4′),
145.9 (C3), 155.1 (C4a), 173.4 (C4) ppm. Elemental Anal. Calcd for
C16H12O3·0.15H2O: C 75.37, H 4.86%. Found: C 75.38, H 4.47%.
3-Hydroxy-2-(4-fluorophenyl)-chromen-4(1H)-one (2c). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and c (1.82 g, 14.7 mmol) to afford 2c as yellow
yellow powder (1.15 g, 29%); mp 157−159 °C. H NMR (500.10
MHz, DMSO-d6): δ = 7.48−7.51 (m, 1H, H4′), 7.58−7.64 (m, 2H,
3
H5′/H7), 7.81−7.85 (m, 2H, H6/H8), 8.12 (d, J(H,H) = 8 Hz, 1H,
H5), 8.20 (d, 3J(H,H) = 8 Hz, 1H, H6′), 8.28 (s, 1H, H2′), 9.93 (br s,
1H, OH) ppm. 13C{1H} NMR (125.75 MHz, DMSO-d6): δ = 119.0
(C8), 121.7 (C8a), 125.2 (C7), 125.3 (C5), 126.7 (C6′), 127.5 (C2′),
130.1 (C4′), 131.0 (C5′), 133.8 (C2/C3′), 134.4 (C6), 140.1 (C1′),
143.9 (C3), 155.1 (C4a), 173.6 (C4) ppm. Elemental Anal. Calcd for
C15H9ClO3·0.1H2O: C 65.64, H 3.38%. Found: C 65.69, H 3.19%.
3-Hydroxy-2-(2-chlorophenyl)-chromen-4(1H)-one (2h). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and h (2.10 g, 14.7 mmol), affording 2h as deep-
1
needles (1.65 g, 44%); mp 151−152 °C. H NMR (500.10 MHz,
1
yellow needles (2.69 g, 67%); mp 177−179 °C. H NMR (500.10
DMSO-d6): δ = 7.42−7.45 (m, 2H, H3′/H5′), 7.47−7.50 (m, 1H,
H7), 7.79 (d, 3J(H,H) = 7 Hz, 1H, H8), 7.81−7.83 (m, 1H, H6), 8.13
MHz, DMSO-d6): δ = 7.49−7.54 (m, 2H, H5′/H7), 7.57−7.61 (m,
4
4
3
1H, H3′), 7.66−7.68 (m, 2H, H4′/H8), 7.72 (dd, J(H,H) = 2 Hz,
(dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5), 8.29−8.32 (m, 2H,
H2′/H6′), 9.72 (br s, 1H, OH) ppm. 13C{1H} NMR (125.75 MHz,
3J(H,H) = 8 Hz, 1H, H6′), 7.80−7.83 (m, 1H, H6), 8.17 (dd, 4J(H,H)
= 1 Hz, 3J(H,H) = 8 Hz, 1H, H5), 9.34 (br s, 1H, OH) ppm. 13C{1H}
NMR (125.75 MHz, DMSO-d6): δ = 118.9 (C8), 122.5 (C8a), 125.2
(C7), 125.5 (C5), 127.7 (C5′), 130.2 (C4′), 130.5 (C2′), 132.3 (C3′),
132.5 (C6′), 133.2 (C1′), 134.3 (C6), 139.7 (C2), 146.6 (C3), 155.5
(C4a), 173.4 (C4) ppm. Elemental Anal. Calcd for
C15H9ClO3·0.1H2O: C 65.64, H 3.38%. Found: C 65.69, H 3.27%.
2
DMSO-d6): δ = 116.1 (d, J(H,H) = 22 Hz, C3′/C5′), 118.9 (C8),
121.8 (C8a), 125.1 (C7), 125.3 (C5), 128.3 (d, 4J(C,F) = 3 Hz, C1′),
3
130.6 (d, J(C,F) = 8 Hz, C2′/C6′), 134.2 (C6), 139.3 (C2), 144.9
1
(C3), 155.0 (C4a), 163.0 (d, J(C,F) = 249 Hz, C4′), 173.4 (C4)
ppm. Elemental Anal. Calcd for C15H9FO3·0.15H2O: C 69.58, H
3.62%. Found: C 69.66, H 3.47%.
10517
dx.doi.org/10.1021/jm301376a | J. Med. Chem. 2012, 55, 10512−10522

Journal of Medicinal Chemistry
Article
3-Hydroxy-2-(4-bromophenyl)-chromen-4(1H)-one (2i). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and I (2.70 g, 14.7 mmol), affording 2i as a yellow
(CH3,Cym), 31.2 (CHCym), 77.9 (C3/C5Cym), 80.9 (C2/C6Cym), 95.9
(C4Cym), 98.9 (C1Cym), 117.8 (C8), 120.4 (C8a), 124.0 (C7), 124.5
(C5), 127.3 (C2′/C6′), 129.0 (C3′/C5′), 130.0 (C2), 132.6 (C6),
139.8 (C4′), 149.9 (C1′), 153.7 (C4a), 154.2 (C3), 183.1 (C4) ppm.
Elemental Anal. Calcd for C26H25ClO3Ru: C 59.82, H 4.83%. Found:
C 59.82, H 4.57%.
1
powder (2.31 g, 58%); mp 163−167 °C. H NMR (500.10 MHz,
DMSO-d6): δ = 7.47−7.50 (m, 1H, H7), 7.77−7.87 (m, H3′/H5′/
4
3
H6/H8), 8.12 (dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5), 8.19
(d, 3J(H,H) = 9 Hz, 2H, H2′/H6′), 9.85 (br s, 1H, OH) ppm.
13C{1H} NMR (125.75 MHz, DMSO-d6): δ = 118.9 (C8), 121.8
(C8a), 123.8 (C4′), 125.1 (C7), 125.3 (C5), 130.0 (C2), 132.1 (C3′/
C5′), 134.4 (C6), 139.9 (C1′), 144.5 (C3), 155.0 (C4a), 173.5 (C4)
ppm. Elemental Anal. Calcd for C15H9BrO3·0.1H2O: C 56.49, H
2.91%. Found: C 56.51, H 2.68%.
[Chlorido{3-(oxo-κO)-2-(4-fluorophenyl)-chromen-4(1H)-onato-
κO}(η6-p-cymene)ruthenium(II)] (3c). The reaction was performed
according to the general complexation procedure by using 2c (187 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3c as red needles (230 mg, 66%); mp
1
235−236 °C (decomp). H NMR (500.10 MHz, CDCl3): δ = 1.40−
1.44 (m, 6H, CH3,Cym), 2.42 (s, 3H, CH3,Cym), 2.97−3.04 (m, 1H,
3-Hydroxy-2-(3-bromophenyl)-chromen-4(1H)-one (2j). The syn-
thesis was performed according to the general procedure by using 1
(2.00 g, 14.7 mmol) and j (2.70 g, 14.7 mmol), affording 2j as orange
CHCym), 5.40 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H3/H5Cym),
3
3
5.66 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H2/H6Cym), 7.15−
7.18 (m, 2H, H3′/H5′), 7.33−7.36 (m, 1H, H7), 7.54 (d, 3J(H,H) = 8
1
crystals (0.82 g, 18%); mp 165−167 °C. H NMR (500.10 MHz,
4
3
Hz, 1H, H8), 7.60−7.62 (m, 1H, H6), 8.22 (dd, J(H,H) = 1 Hz,
DMSO-d6): δ = 7.48−7.51 (m, 1H, H7), 7.56 (dd, J(H,H) = 8 Hz,
3J(H,H) = 8 Hz, 1H, H5′), 7.72−7.74 (m, 1H, H4′), 7.82−7.86 (m,
3J(H,H) = 8 Hz, 1H, H5), 8.61−8.64 (m, 2H, H2′/H6′) ppm.
13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.7 (CH3,Cym), 22.5
(CH3,Cym), 31.3 (CHCym), 78.0 (C3/C5Cym), 81.0 (C2/C6Cym), 95.9
4
3
2H, H6/H8), 8.13 (dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5),
8.24−8.25 (m, 1H, H6′), 8.41−8.43 (m, 1H, H2′), 9.92 (br s, 1H,
OH) ppm. 13C{1H} NMR (125.75 MHz, DMSO-d6): δ = 119.0 (C8),
121.8 (C8a), 122.3 (C3′), 125.2 (C7), 125.3 (C5), 126.9 (C6′), 127.5
(C2′), 130.4 (C4′), 131.2 (C5′), 132.9 (C4′), 134.1 (C2), 134.4 (C6),
140.1 (C1′), 143.8 (C3), 155.1 (C4a), 173.6 (C4) ppm. Elemental
Anal. Calcd for C15H9BrO3: C 56.81, H 2.86%. Found: C 56.65, H
2.76%.
2
(C4Cym), 98.9 (C1Cym), 115.2 (d, J(C,F) = 21 Hz, C3′/C5′), 117.7
(C8), 120.1 (C8a), 124.1 (C7), 124.6 (C5), 128.8 (C2), 129.4 (d,
3J(C,F) = 8 Hz, C2′/C6′), 132.6 (C6), 148.4 (d, 4J(C,F) = 1 Hz, C1′),
1
153.8 (C4a), 154.1 (C3), 163.0 (d, J(C,F) = 251 Hz, C4′), 183.3
(C4) ppm. Elemental Anal. Calcd for C25H22ClFO3Ru: C 57.09, H
4.22%. Found: C 56.98, H 4.06%.
General Procedure for the Synthesis of the RuII(η6-p-
Cymene) Complexes 3a−3j. A solution of [Ru(η6-p-cymene)Cl2]2
(0.45 equiv) in dichloromethane (15 mL) was added to a solution of
3-hydroxyflavones 2a−j (1.00 equiv) and sodium methoxide (1.10
equiv) in methanol (15 mL). The reaction mixture was stirred at room
temperature and under argon atmosphere for 18 h. The solvent was
evaporated in vacuo, and the residue was extracted with dichloro-
methane, filtered, and concentrated. The pure product was obtained
by recrystallization from methanol.
[Chlorido{3-(oxo-κO)-2-(3-fluorophenyl)-chromen-4-onato-
κO}(η6-p-cymene)ruthenium(II)] (3d). The reaction was performed
according to the general complexation procedure by using 2d (187 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3d as deep-red powder (180 mg,
51%); mp 210−212 °C (decomp). Single crystals suitable for X-ray
diffraction analysis were grown from CHCl3/n-hexane. 1H NMR
(500.10 MHz, CDCl3): δ = 1.40−1.46 (m, 6H, CH3,Cym), 2.42 (s, 3H,
3
CH3,Cym), 2.99−3.06 (m, 1H, CHCym), 5.40 (d, J(H,H) = 6 Hz, 2H,
[Chlorido{3-(oxo-κO)-2-phenyl-chromen-4(1H)-onato-κO}(η6-p-
cymene)ruthenium(II)] (3a). The reaction was performed according to
the general complexation procedure by using 2a (164 mg, 0.73 mmol),
NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2 (200 mg,
0.33 mmol) affording 3a as a deep-red powder (170 mg, 51%); mp
3
3
H3/H5Cym), 5.68 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H2/
H6Cym), 7.08 (ddd, 4J(H,F) = 2 Hz, 3J(H,H) = 8 Hz, 3J(H,H) = 8 Hz,
1H, H4′), 7.33−7.36 (m, 1H, H7), 7.44 (ddd, 3J(H,F) = 6 Hz,
3J(H,H) = 8 Hz, 3J(H,H) = 8 Hz, 1H, H5′), 7.56 (d, 3J(H,H) = 8 Hz,
1H, H8), 7.61−7.65 (m, 1H, H6), 8.22 (dd, 4J(H,H) = 1 Hz, 3J(H,H)
1
229−230 °C (decomp). H NMR (500.10 MHz, CDCl3): δ = 1.41−
3
= 8 Hz, 1H, H5), 8.31 (d, J(H,H) = 8 Hz, 1H, H6′), 8.44−8.48 (m,
1.44 (m, 6H, CH3,Cym), 2.43 (s, 3H, CH3,Cym), 2.99−3.05 (m, 1H,
1H, H2′) ppm. 13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.7
CHCym), 5.39 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H3/H5Cym),
(CH3,Cym), 22.5 (2CH3,Cym), 31.3 (CHCym), 78.0 (C3/C5Cym), 80.0
3
3
5.67 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H2/H6Cym), 7.33−
2
3
3
(C2/C6Cym), 95.9 (C4Cym), 99.0 (C1Cym), 114.1 (d, J(C,F) = 25 Hz,
7.35 (m, 1H, H7), 7.41 (dd, J(H,H) = 7 Hz, J(H,H) = 7 Hz, 1H,
C2′), 115.8 (d, 2J(C,F) = 22 Hz, C4′), 117.9 (C8), 119.9 (C8a), 122.5
H4′), 7.48 (dd, 3J(H,H) = 7 Hz, 3J(H,H) = 7 Hz, 2H, H3′/H5′), 7.57
4
3
(d, J(C,F) = 3 Hz, C6′), 124.2 (C7), 124.7 (C5), 127.2 (C2), 129.6
(d, J(H,H) = 8 Hz, 1H, H8), 7.59−7.61 (m, 1H, H6), 8.22 (dd,
3
3
(d, 3J(C,F) = 8 Hz, C5′), 132.9 (C6), 125.5 (d, 3J(C,F) = 9 Hz, C1′),
4J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5), 8.61 (d, J(H,H) = 7 Hz,
1
153.9 (C4a), 154.9 (C3), 162.0 (d, J(C,F) = 243 Hz, C3′), 183.8
2H, H2′/H6′) ppm. 13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.7
(CH3,Cym), 22.5 (CH3,Cym), 31.3 (CHCym), 78.0 (C3/C5Cym), 81.0
(C2/C6Cym), 95.9 (C4Cym), 98.9 (C1Cym), 117.9 (C8), 120.0 (C8a),
124.0 (C7), 124.6 (C5), 127.3 (C2′/C6′), 128.2 (C3′/C5′), 129.3
(C4′), 132.5 (C2), 132.6 (C6), 149.2 (C1′), 153.9 (C4a), 154.0 (C3),
183.3 (C4) ppm. Elemental Anal. Calcd for C25H23ClO3Ru: C 59.11,
H 4.56%. Found: C 59.04, H 4.39%.
(C4) ppm. Elemental Analysis Calcd for C25H22ClFO3Ru·0.25H2O: C
56.61, H 4.28%. Found: C 56.51, H 4.28%.
[Chlorido{3-(oxo-κO)-2-(2-fluorophenyl)-chromen-4-onato-
κO}(η6-p-cymene)ruthenium(II)] (3e). The reaction was performed
according to the general complexation procedure, by using 2e (187
mg, 0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-
cymene)Cl2]2 (200 mg, 0.33 mmol), affording 3e as red−brownish
powder (220 mg, 63%); mp 199−202 °C (decomp). 1H NMR
(500.10 MHz, CDCl3):): δ = 1.37−1.42 (m, 6H, CH3,Cym), 2.39 (s,
3H, CH3,Cym), 2.97−3.02 (m, 1H, CHCym), 5.36 (dd, 3J(H,H) = 6 Hz,
[Chlorido{3-(oxo-κO)-2-(4-methylphenyl)-chromen-4(1H)-onato-
κO}(η6-p-cymene)ruthenium(II)] (3b). The reaction was performed
according to the general complexation procedure by using 2b (184 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3b as a red powder (240 mg, 68%).
Single crystals suitable for X-ray diffraction analysis were grown from
CHCl3/n-hexane; mp 235−236 °C (decomp). 1H NMR (500.10
MHz, CDCl3): δ = 1.40−1.43 (m, 6H, CH3,Cym), 2.42 (s, 3H,
CH3,Cym), 2.44 (s, 3H, CH3), 2.98−3.04 (m, 1H, CHCym), 5.38 (dd,
3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H3/H5Cym), 5.65 (dd, 3J(H,H)
3
3J(H,H) = 6 Hz, 2H, H3/H5Cym), 5.68 (d, J(H,H) = 6 Hz, 2H, H2/
H6Cym), 7.14−7.19 (m, 1H, H3′), 7.21 (ddd, 4J(H,F) = 1 Hz, 3J(H,H)
= 8 Hz, 3J(H,H) = 8 Hz, 1H, H6′), 7.33−7.36 (m, 1H, H7), 7.38−7.42
(m, 1H, H4′), 7.51 (d, 3J(H,H) = 8 Hz, 1H, H8), 7.58−7.62 (m, 1H,
H6), 8.23 (dd, 4J(H,H) = 1 Hz, 3J(H,H) = 8 Hz, 1H, H5), 8.31 (ddd,
3
3
4J(H,F) = 1 Hz, J(H,H) = 7 Hz, J(H,H) = 7 Hz, 1H, H5′) ppm.
13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.7 (CH3,Cym), 22.5
(2CH3,Cym), 31.2 (CHCym), 77.7 (C3/C5Cym), 81.2 (C2/C6Cym), 96.0
3
3
= 6 Hz, J(H,H) = 6 Hz, 2H, H2/H6Cym), 7.28 (d, J(H,H) = 9 Hz,
2H, H3′/H5′), 7.32−7.35 (m, 1H, H7), 7.55 (d, 3J(H,H) = 8 Hz, 1H,
H8), 7.58−7.61 (m, 1H, H6), 8.21 (dd, 4J(H,H) = 1 Hz, 3J(H,H) = 8
2
(C4cym), 98.9 (C1Cym), 116.3 (d, J(C,F) = 22 Hz, C3′), 118.3 (C8),
3
2
3
Hz, 1H, H5), 8.50 (d, J(H,H) = 8 Hz, 2H, H2′/H6′) ppm. 13C{1H}
120.1 (d, J(C,F) = 10 Hz, C1′), 120.1 (C8a), 123.6 (d, J(C,F) = 3
3
NMR (125.75 MHz, CDCl3): δ = 18.7 (CH3,Cym), 21.6 (CH3), 22.5
Hz, C6′), 124.1 (C7), 124.6 (C5), 131.1 (d, J(C,F) = 8 Hz, C4′),
10518
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4
131.6 (d, J(C,F) = 2 Hz, C5′), 132.7 (C6), 146.9 (C2/C3), 154.4
1.45 (m, 6H, CH3,Cym), 2.42 (s, 3H, CH3,Cym), 2.98−3.04 (m, 1H,
CHCym), 5.39 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H3/H5Cym),
(C4a), 159.0 (d, 1J(C,F) = 256 Hz, C2′), 183.6 (C4) ppm. Elemental
Anal. Calcd for C25H22ClFO3Ru·0.5H2O: C 56.13, H 4.33%. Found: C
56.22, H 4.60%.
3
3
5.66 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H2/H6Cym), 7.33−
3
7.36 (m, 1H, H7), 7.54 (d, J(H,H) = 8 Hz, 1H, H8), 7.59−7.64 (m,
4
3
[Chlorido{3-(oxo-κO)-2-(4-chlorophenyl)-chromen-4(1H)-onato-
κO}(η6-p-cymene)ruthenium(II)] (3f). The reaction was performed
according to the general complexation procedure by using 2f (199 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3f as deep-red powder (226 mg,
63%); mp 214−217 °C (decomp). 1H NMR (500.10 MHz, CDCl3): δ
= 1.32−1.38 (m, 6H, CH3,Cym), 2.42 (s, 3H, CH3,Cym), 2.85−2.91 (m,
3H, H3′/H5′/H6), 8.21 (dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H,
H5), 8.48 (d, J(H,H) = 9 Hz, 2H, H2′/H6′) ppm. 13C{1H} NMR
3
(125.75 MHz, CDCl3): δ = 18.7 (CH3,Cym), 22.5 (CH3,Cym), 31.3
(CHCym), 77.9 (C3/C5Cym), 80.0 (C2/C6Cym), 96.0 (C4Cym), 99.0
(C1Cym), 117.8 (C8), 120.0 (C8a), 123.4 (C4′), 124.1 (C7), 124.7
(C5), 128.6 (C2′/C6′), 131.4 (C3′/C5′), 131.7 (C2), 132.8 (C6),
148.0 (C1′), 153.9 (C4a), 154.7 (C3), 183.5 (C4) ppm. Elemental
Anal. Calcd for C25H22BrClO3Ru·0.25H2O: C 50.77, H 3.83%. Found:
C 50.74, H 3.57%.
3
3
1H, CHCym), 5.39 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H3/
H5Cym), 5.66 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H2/H6Cym),
7.32−7.35 (m, 1H, H7), 7.44 (d, 3J(H,H) = 9 Hz, 2H, H3′/H5′), 7.54
[Chlorido{3-(oxo-κO)-2-(3-bromophenyl)-chromen-4-onato-
κO}(η6-p-cymene)ruthenium(II)] (3j). The reaction was performed
according to the general complexation procedure by using 2j (231 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3j as dark-brown crystals (153 mg,
3
(d, J(H,H) = 8 Hz, 1H, H8), 7.62−7.63 (m, 1H, H6), 8.21 (dd,
4J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5), 8.55 (d, J(H,H) = 9 Hz,
3
3
2H, H2′/H6′) ppm. 13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.7
(CH3,Cym), 22.4 (CH3,Cym), 30.0 (CHCym), 77.9 (C3/C5Cym), 81.0
(C2/C6Cym), 95.9 (C4Cym), 99.0 (C1Cym), 117.8 (C8), 120.0 (C8a),
124.1 (C7), 124.7 (C5), 125.5 (C2′/C6′/C3′/C5′), 131.0 (C2), 132.8
(C6), 134.9 (C4′), 143.5 (C1′), 153.9 (C4a), 154.6 (C3), 183.5 (C4).
Elemental Anal. Calcd for C25H22Cl2O3Ru: C 55.36, H 4.09%. Found:
C 55.28, H 3.90%.
1
40%); mp 213-219 °C (decomp). H NMR (500.10 MHz, CDCl3): δ
= 1.44−1.49 (m, 6H, CH3,Cym), 2.44 (s, 3H, CH3,Cym), 3.00−3.07 (m,
3
3
1H, CHCym), 5.39 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H3/
3
3
H5Cym), 5.68 (d, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H2/H6Cym),
7.33−7.36 (m, 2H, H5′/H7), 7.40 (ddd, 4J(H,H) = 1 Hz, 4J(H,H) = 2
3
3
Hz, J(H,H) = 8 Hz, 1H, H4′), 7.56 (d, J(H,H) = 8 Hz, 1H, H8),
[Chlorido{3-(oxo-κO)-2-(3-chlorophenyl)-chromen-4-onato-
κO}(η6-p-cymene)ruthenium(II)] (3g). The reaction was performed
according to the general complexation procedure by using 2g (199 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3g as deep-red powder (244 mg,
68%); mp 213−220 °C (decomp). 1H NMR (500.10 MHz, CDCl3): δ
= 1.43−1.48 (m, 6H, CH3,Cym), 2.44 (s, 3H, CH3,Cym), 3.00−3.07 (m,
4
3
7.62−7.65 (m, 1H, H6), 8.21 (dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz,
1H, H5), 8.45−8.47 (m, 1H, H6′), 8.87−8.89 (m, 1H, H2′) ppm.
13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.7 (CH3,Cym), 22.7
(2CH3,Cym), 31.3 (CHCym), 77.9 (C3/C5Cym), 81.2 (C2/C6Cym), 95.7
(C4Cym), 98.8 (C1Cym), 117.9 (C8), 119.8 (C8a), 122.4 (C3′), 124.2
(C7), 124.8 (C5), 125.3 (C6′), 129.7 (C2′), 129.9 (C5′), 131.8 (C4′),
133.0 (C6), 134.5 (C2), 147.1 (C1′), 154.0 (C4a), 154.9 (C3), 183.8
(C4) ppm. Elemental Anal. Calcd for C25H22BrClO3Ru·0.25H2O: C
49.64, H 4.00%. Found: C 49.40, H 3.85%.
Hydrolysis and Interaction with 5′-GMP. Hydrolysis and
stability in water were investigated by 1H NMR spectroscopy. Because
of the lipophilic character of the organometallics, all experiments were
performed in 10% DMSO-d6/D2O solutions. For the 5′-GMP binding
studies, the complexes (ca. 0.1 mg/mL) were dissolved in 10%
DMSO-d6/D2O, yielding the corresponding highly reactive aqua
species. The aqua complexes were converted in situ by addition of 50
μL increments of 5′-GMP solution (10 mg/mL) into the respective 5′-
GMP adduct, and the reaction was monitored by 1H NMR
spectroscopy until unconverted 5′-GMP was observed.
3
3
1H, CHCym), 5.39 (dd, J(H,H) = 5 Hz, J(H,H) = 5 Hz, 2H, H3/
H5Cym), 5.68 (dd, 3J(H,H) = 5 Hz, 3J(H,H) = 5 Hz, 2H, H2/H6Cym),
3
3
7.33−7.36 (m, 2H, H4′/H7), 7.40 (dd, J(H,H) = 8 Hz, J(H,H) = 8
3
Hz, 1H, H5′), 7.55 (d, J(H,H) = 8 Hz, 1H, H8), 7.63−7.65 (m, 1H,
H6), 8.21 (dd, 4J(H,H) = 1 Hz, 3J(H,H) = 8 Hz, 1H, H5), 8.41 (ddd,
4J(H,H) = 1 Hz, J(H,H) = 8 Hz, J(H,H) = 8 Hz, 1H, H6′), 8.72−
3
3
8.73 (m, 1H, H2′) ppm. 13C{1H} NMR (125.75 MHz, CDCl3): δ =
18.7 (CH3,Cym), 22.5 (2CH3,Cym), 31.3 (CHCym), 77.9 (C3/C5Cym),
81.2 (C2/C6Cym), 95.8 (C4Cym), 98.9 (C1Cym), 117.9 (C8), 119.9
(C8a), 124.2 (C7), 124.7 (C5), 124.8 (C6′), 127.1 (C2′), 128.9
(C4′), 129.4 (C5′), 133.0 (C6), 134.2 (C2/C3′), 147.3 (C1′), 154.0
(C4a), 154.9 (C3), 183.8 (C4) ppm. Elemental Anal. Calcd for
C25H22Cl2O3Ru·0.15H2O: C 55.08, H 4.12%. Found: C 55.11, H
3.85%.
Cytotoxicity in Cancer Cell Lines. Cell Lines and Culture
Conditions. CH1 cells originate from an ascites sample of a patient
with a papillary cystadenocarcinoma of the ovary and were a gift from
Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute of
Cancer Research, Sutton, UK. SW480 (human adenocarcinoma of the
colon) and A549 (human non-small cell lung cancer) cells were
provided by Brigitte Marian (Institute of Cancer Research, Depart-
ment of Medicine I, Medical University of Vienna, Austria). The cell
lines 5637 (bladder cancer), LCLC-103H (lung cancer), and DAN-G
(pancreatic cancer) were obtained from the German Collection of
Microorganisms and Cell Culture (DSMZ, Braunschweig, FRG). Cells
were grown in 75 cm2 culture flasks (Iwaki) as adherent monolayer
cultures in either Minimum Essential Medium (MEM) supplemented
with 10% heat inactivated fetal calf serum (FCS), 1 mM sodium
pyruvate, 4 mM L-glutamine, and 1% nonessential amino acids (from
100× stock) (i.e., CH1, SW480, and A549) or in RPMI 1640 medium
supplemented with 10% FCS (i.e., 5637, LCLC-103H, DAN-G). All
cell culture reagents were obtained from Sigma-Aldrich. Cultures were
maintained at 37 °C in a humidified atmosphere containing 95% air
and 5% CO2.
[Chlorido{3-(oxo-κO)-2-(2-chlorophenyl)-chromen-4-onato-
κO}(η6-p-cymene)ruthenium(II)] (3h). The reaction was performed
according to the general complexation procedure by using 2h (199 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3h as brown powder (109 mg, 30%);
mp 205−208 °C (decomp). Single crystals suitable for X-ray
diffraction analysis were grown from MeOH/n-hexane. 1H NMR
(500.10 MHz, CDCl3): δ = 1.35−1.39 (m, 6H, CH33,Cym), 2.37 (s, 3H,
CH3,Cym), 2.93−3.00 (m, 1H, CHCym), 5.35 (dd, J(H,H) = 5 Hz,
3J(H,H) = 5 Hz, 2H, H3/H5Cym), 5.63 (dd, 3J(H,H) = 5 Hz, 3J(H,H)
= 5 Hz, 2H, H2/H6Cym), 7.34−7.38 (m, 3H, H3′/H5′/H7), 7.48−
7.52 (m, 2H, H4′/H8), 7.60−7.62 (m, 1H, H6), 7.99−8.02 (m, 1H,
4
3
H6′), 8.24 (dd, J(H,H) = 1 Hz, J(H,H) = 8 Hz, 1H, H5) ppm.
13C{1H} NMR (125.75 MHz, CDCl3): δ = 18.9 (CH3,Cym), 22.3
(2CH3,Cym), 31.2 (CHCym), 77.5 (C3/C5Cym), 80.5 (C2/C6Cym), 96.1
(C4Cym), 98.9 (C1Cym), 118.2 (C8), 120.2 (C8a), 124.1 (C7), 124.6
(C5), 126.3 (C5′), 130.3 (C4′), 130.5 (C3′), 130.7 (C2′), 132.7
(C6′), 132.8 (C6), 133.4 (C1′), 148.5 (C2), 154.3 (C3), 154.5 (C4a),
184.1 (C4) ppm. Elemental Anal. Calcd for C25H22Cl2O3Ru: C 55.36,
H 4.09%. Found: C 55.29, H 3.79%.
MTT Assay. Cytotoxicity was determined by the colorimetric MTT
[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide,
Fluka] microculture assay. For this purpose, cells were harvested
from culture flasks by trypsinization and seeded in 100 μL aliquots into
96-well microculture plates (Iwaki). Cell densities of 1.5 × 103 cells/
well (CH1), 2.5 × 103 cells/well (SW480), and 4 × 103 cells/well
(A549) were chosen in order to ensure exponential growth of
[Chlorido{3-(oxo-κO)-2-(4-bromophenyl)-chromen-4-onato-
κO}(η6-p-cymene)ruthenium(II) (3i). The reaction was performed
according to the general complexation procedure, by using 2i (231 mg,
0.73 mmol), NaOMe (43 mg, 0.8 mmol), and [Ru(η6-p-cymene)Cl2]2
(200 mg, 0.33 mmol), affording 3i as red powder (179 mg, 46%); mp
1
228−233 °C (decomp). H NMR (500.10 MHz, CDCl3): δ = 1.40−
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untreated controls throughout the experiment. Cells were allowed to
settle and resume exponential growth in drug-free complete culture
medium for 24 h. Stock solutions of the test compounds in DMSO
were appropriately diluted in complete culture medium so that the
maximum DMSO content did not exceed 1%. These dilutions were
added in 100 μL aliquots to the microcultures, and cells were exposed
to the test compounds for 96 h. In case of the studies on time-
dependent cytotoxicity, SW480 cells were treated for 1, 4, 24, and 96 h
with 3j followed by incubation in drug-free medium for the rest of a
total incubation time of 96 h. At the end of incubation, all media were
replaced by 100 μL/well RPMI1640 culture medium (supplemented
with 10% heat-inactivated FCS) plus 20 μL/well MTT solution in
phosphate-buffered saline (5 mg/mL). After incubation for 4 h, the
supernatants were removed and the formazan crystals formed by viable
cells were dissolved in 150 μL of DMSO per well. Optical densities at
λ = 550 nm were measured with a microplate reader (Tecan Spectra
Classic) by using a reference wavelength of 690 nm to correct for
unspecific absorption. The quantity of viable cells was expressed in
terms of T/C values by comparison to untreated controls, and 50%
inhibitory concentrations (IC50) were calculated from concentration−
effect curves by interpolation. Evaluation is based on means from at
least three independent experiments, each comprising at least three
replicates per concentration level.
Crystal Violet Assay Conditions. This assay has been described in
detail elsewhere.49 Culture conditions were the same as used in the
MTT assay. Briefly, cells were seeded into 96-well microculture plates
(Sarstedt, FRG) in cell densities of 1.0 × 103 cells/well, except for
LCLC-103H, which was seeded at 250 cells/well. After a 24 h
preincubation, cells were treated with the test substance for 96 h. Stock
solutions of the test substance were prepared to 20 mM in DMF and
diluted 1000-fold in RPMI 1640 culture medium containing 10% FCS.
Substances that showed a ≥50% growth inhibition at 20 μM were
tested at five serial dilutions in four wells/concentration to determine
the IC50 values as described.49 The staining of the cells was done for 30
min with a 0.02% crystal violet solution in water followed by washing
out the excess dye. Cell bound dye was redissolved in 70% ethanol/
water solution, and the optical densities at λ = 570 nm were measured
with a microplate reader (Anthos 2010).
Cell Cycle Analysis. One million A549 cells were seeded into Petri
dishes and allowed to recover for 24 h. Cells were then exposed for 48
h to the test compounds. Control and drug-treated cells were
collected, washed with PBS, fixed in 70% ice-cold ethanol, and stored
at −20 °C. To determine cell cycle distribution, cells were transferred
in physiological NaCl solution into PBS, incubated with 10 μg/mL
RNase A for 30 min at 37 °C, followed by 30 min treatment with 5
μg/mL propidium iodide. Fluorescence was measured by flow
cytometry by using FACS Calibur (Becton Dickinson, Palo Alto,
CA). The resulting DNA histograms were quantified by Cell Quest
Pro software (Becton Dickinson and Company, New York, USA).
Determination of Topoisomerase IIα Activity. Effects on the
catalytic activity of topoisomerase IIα were determined using a
decatenation assay. Catenated kinetoplast DNA (kDNA) was used as a
substrate. kDNA is an aggregate of interlocked DNA minicircles
(mostly 2.5 kb), which can be released by topoisomerase IIα. kDNA
(200 ng, TopoGen, OH, USA) was incubated in a final volume of 30
μL (containing 40 ng of topoisomerase IIα; 50 mM Tris, pH 7.9; 120
mM KCl; 10 mM MgCl2; 1 mM ATP; 0.5 mM DTT; 0.5 mM EDTA;
0.03 mg/mL BSA) at 37 °C for 60 min. The reaction was stopped by
the addition of 1/10 volume of 1 mg/mL proteinase K in 10% (w/v)
SDS and incubation at 37 °C for further 30 min. Gel electrophoresis
was performed in the absence of ethidium bromide at 60 V for 3 h in
1% (w/v) agarose gels with Tris acetate/EDTA buffer (40 mM Tris; 1
mM EDTA, pH 8.5; 20 mM acetic acid). Subsequently, the gel was
stained in 10 μg/mL ethidium bromide solution for 20 min. The
fluorescence of ethidium bromide was detected with the LAS-4000
system (Fujifilm, Raytest, Germany).
procedure published elsewhere.35 Gold disk electrodes (99.99%
purity) with surface area of 0.02 cm2 were obtained from
CHInstruments. All experiments were conducted in aqueous
conditions using ultrapure water (18.2 MΩ cm) from a Millipore
Milli-Q system.
Fabrication of Kinase Biosensor. The gold electrodes were cleaned
by polishing with slurry of 1 μm Al2O3 until a mirror finish was
obtained. After 5 min sonication in Milli-Q water, the gold electrodes
were rinsed with water and ethanol. The electrodes were then cleaned
electrochemically by cyclic voltammetry (CV) in the negative potential
range from −0.6 to −2.3 V in 0.5 M KOH, followed by cycling in 0.5
M H2SO4 in the 0−1.2 V potential range. Next, the gold electrodes
were incubated with 2 mM lipoic acid N-hydroxysuccinimide ester
solution in ethanol for 3 days at 273 K. After extensive washing with
freshly distilled ethanol, the gold electrodes were incubated with a 0.1
mM peptide solution in Milli-Q water for 18 h at 273 K.
Consequently, the modified electrodes were rinsed with Milli-Q
water and then incubated with 100 mM ethanolamine solution in
absolute ethanol for 1 h. Finally, the electrodes were immersed in 10
mM dodecanethiol solution in ethanol for 20 min to block any
unmodified gold surface.
Kinase-Catalyzed Fc-Phosphorylation Reaction. The peptide-
modified gold electrodes were immersed in the kinase assay buffer
based on 60 mM HEPES (pH 7.5), 3 mM MnCl2, 3 mM MgCl2, 0.5
μg/μL of PEG 20000, 3 μM sodium ortho-vanadate, 1 μg/mL CDK2/
cyclin A protein kinase and 200 μM Fc-ATP. The phosphorylation
reaction was performed for 6 h at 37 °C in a heating block (VWR
Scientific, USA). The modified gold electrodes were washed five times
using the kinase assay buffer and 0.1 M phosphate buffer (pH 7.4)
prior to the electrochemical measurement. For the inhibitor studies,
the CDK2/cyclin A protein and compounds 3a−h (1 mM, DMSO)
were added to the kinase buffer while maintaining the working
concentration of the protein and inhibitor at 1 μg/mL and 10 μM,
respectively. The DMSO concentration was maintained at a low level
(<2%). After 30 min, the kinase reaction was initiated by the addition
of Fc-ATP and was followed by the procedure outlined above.
Electrochemical Experiments. All electrochemical experiments
were carried out using a CHInstrument 660B system potentiostat
(Austin, TX) at a 100 mV/s scan rate unless otherwise specified. The
electrochemical measurements were performed in 0.1 M phosphate
buffer (pH 7.4). Typical electrochemical experimental set up included
a three-electrode system: a modified gold electrode as the working
electrode, Ag/AgCl in 3 M KCl as the reference electrode, which was
connected with the electrolyte via a salt bridge, and platinum wire as
the counter electrode. For each electrode, cyclic voltammetry was
performed at a scan rate of 100 mV/s and square-wave voltammetry
was recorded from 0.2 to 0.6 V, with an amplitude of 25 mV at 20 Hz
frequency.
ASSOCIATED CONTENT
■
S
* Supporting Information
Single crystal X-ray diffraction data for compounds 3d, 3f, and
3h, NMR spectra demonstrating the stability of 3i over two
years and the binding ability of 3a to the DNA model 5′-GMP,
concentration−effect curves for different cell lines, the cell cycle
distribution in dependence of the concentration of Ru complex,
data on the topoisomerase IIα inhibitory activity, and
electrochemical data. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +64 9 373 7599 ext 83220. Fax: +64 9 373 7599
87422. E-mail: c.hartinger@auckland.ac.nz. Web site: http://
hartinger.wordpress.fos.auckland.ac.nz/. Address: The Univer-
sity of Auckland, School of Chemical Sciences, Private Bag
92019, Auckland 1142, New Zealand.
CDK2/Cyclin A Protein Kinase Inhibition Assay. The CDK2
peptide substrate, HHASPRK, and the CDK2/Cyclin A protein
complex were purchased from Enzo Life Sciences. Adenosine 5′-[γ-
ferrocene] triphosphate (Fc-ATP) was synthesized according to the
10520
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Article
inflammation, heart disease, and cancer. Pharmacol. Rev. 2000, 52,
673−751.
Notes
The authors declare no competing financial interest.
(14) Sathish, S.; Narayan, G.; Rao, N.; Janardhana, C. A self-
organized ensemble of fluorescent 3-hydroxyflavone−Al(III) complex
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ACKNOWLEDGMENTS
■
We thank the University of Vienna, the Austrian Science Fund
(FWF), the Johanna Mahlke geb. Obermann Foundation, and
COST D39 for financial support. We gratefully acknowledge
Alexander Roller for collecting the X-ray diffraction data. V.
Dirsch and D. Schachner (Department of Pharmacognosy,
University of Vienna) are gratefully acknowledged for providing
the FACS instrument and for technical instructions, respec-
tively.
́
̌ ́ ́ ́
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ABBREVIATIONS USED
■
5′-GMP, guanosine 5′-monophosphate; CDK, cyclin depend-
ent kinase; CV, cyclic voltammetry; cym, η6-p-cymene; Fc-ATP,
adenosine 5′-[γ-ferrocene] triphosphate; IC50, 50% inhibitory
concentration; kDNA, kinetoplast DNA; pta, 1,3,5-triaza-7-
phoshatricyclo-[3.3.1.1]decane; SWV, square-wave voltamme-
try
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