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A Bioactive l-Phenylalanine-Derived Arene in Multitargeted Organoruthenium Compounds: Impact on the Antiproliferative Activity and Mode of Action.
Article
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/IC
A Bioactive L‑Phenylalanine-Derived Arene in Multitargeted
Organoruthenium Compounds: Impact on the Antiproliferative
Activity and Mode of Action
Sanam Movassaghi,† Euphemia Leung,‡ Muhammad Hanif,† Betty Y. T. Lee,† Hannah U. Holtkamp,†
Jason K. Y. Tu,† Tilo Söhnel,† Stephen M. F. Jamieson,‡ and Christian G. Hartinger*,†
†
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
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‡
S Supporting Information
*
ABSTRACT: RuII(η6-arene) compounds carrying bioactive
flavonol ligands have shown promising anticancer activity
against tumor cells via a multitargeting mode of action, i.e.,
through interaction with DNA and inhibition of topoisomerase IIα. By introducing a novel arene ligand based on the
amino acid L-phenylalanine (Phe), we aimed to alter the
pharmacological properties of the complexes. We report here
a series of novel RuII(η6-arene)Cl complexes with different
substituents on the phenyl ring of the flavonol which should
maintain the multitargeting capability of the parent η6-pcymene (cym) complexes. Studies with selected examples
revealed stability in aqueous solution after quickly forming
aqua complexes but rapid decomposition in pure DMSO. The reactions with protein and DNA models proceeded quickly and
resulted in cleavage of the flavonol or adduct formation, respectively. The compounds were found to be cytotoxic with
significant antiproliferative activity in cancer cells with IC50 values in the low μM range, while not following the same trends as
observed for the cym analogues. Notably, the cellular accumulation of the new derivatives was significantly higher than for their
respective cym complexes, and they induced DNA damage in a manner similar to that of cisplatin but to a lesser extent.
■
action.9−11 These properties are related to unique structural
features of such half-sandwich “piano-stool” compounds which
direct their specific interactions with target biomolecules.12−19
The RAPTA family with the general formula [Ru(arene)(PTA)X2] (PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane; X = halido or dicarboxylato ligands) and the RAED
compound class [Ru(arene)Cl(en)]+ (en = 1,2-ethylenediamine) are the most advanced representatives of the Ru(arene)
derivatives with their modes of action dependent on the
ligands.11,20
A recent approach in anticancer metallodrug design is to use
nature-inspired, bioactive ligands such as 8-oxyquinoline,21
quinolones,22 curcumin derivatives,23 chlorambucil,17 nonsteroidal anti-inflammatory drugs,19,24 ethacrynic acid,25,26
and flavonols27 and coordinate them to biologically active
metal centers. Flavonoids are secondary metabolites of plants
with interesting biological properties such as antioxidant, antiinflammatory, estrogenic, antimicrobial, and anticarcinogenic
activity.28,29 The 3-hydroxy-4-keto structural motif found in 3hydroxyflavones coordinates bidentately to many metal ions
and forms stable metal complexes with interesting applications
INTRODUCTION
Discovery of the anticancer activity of cisplatin has led to the
development of metallopharmaceuticals based on a variety of
metal centers.1−3 Ruthenium complexes are considered the
most promising next generation anticancer metallodrugs, and
the RuIII drug IT-139 (Chart 1, also known as NKP-1339), is
undergoing clinical trials.4−8 In the past decade, the field of
organometallic anticancer agents has received considerable
attention, and particularly RuII(η6-arene) complexes have been
widely investigated for their tunability and novel modes of
Chart 1. Chemical Structures of Anticancer Ru Complexes
Received: May 2, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b01187
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
and properties, such as intrinsic fluorescence.30−33 One
example for an application is the use as ligands in metalbased anticancer agents.34−39 We reported a series of
multitargeted anticancer Ru(arene) compounds carrying
flavonol ligands with the metal center able to form covalent
bonds to DNA, while the complex inhibits topoisomerase
IIα.27,33,40−42 The potent antiproliferative activity of the
organoruthenium compounds was driven by the cytotoxicity
of the flavonol ligands, and the complexes were more potent
topoisomerase inhibitors than the flavonols which was also
dependent on the substituents found at their phenyl ring.27,40
However, the aqueous solubility of flavonols and their
Ru(arene) complexes was limited. In the case of 8-oxyquinolinato complexes, we overcame this issue by replacing the
apolar arene with an L-phenylalanine (Phe)-derived arene
ligand.43 Phe is bioactive and inhibits alkaline phosphatase, an
enzyme which is overexpressed in many tumors.44 Herein, we
borrow the concept to study the impact of substituting η6-pcymene (cym) with the Phe-derived arene in 3-hydroxyflavonol Ru complexes on their biological activity and chemical
properties in comparison to the analogous cym complexes.
■
previously determined IC50 values. The cells were incubated with
metal complexes for 4 and 24 h, after which the medium was
removed, and the cells were washed twice with 1 mL of ice-cold PBS
buffer.
To determine the cell uptake of 1a and 1acym at the same
concentration as the DNA-damaging ability was measured, stock
solutions of 300 μM in DMSO were prepared and diluted with media
to a concentration of 1% DMSO. The cells were incubated with the
metal complexes for 6 h, after which the medium was removed, and
the cells were washed twice with 1 mL of ice-cold PBS buffer.
The cells from all the experiments were lysed with 2 mL of
concentrated nitric acid (containing 0.1 μL of a 1000 ± 3 μg/mL
thulium as an internal standard) and digested with an Ethos Up
microwave digestion system (Milestone). After the solutions were
diluted with 10 mL of H2O, the ruthenium content was determined
by ICP-MS (Agilent 7700) with an ASX-500 autosampler (CETAC
Technologies) in a Serie SuSi laminar flow hood (SPECTEC). The
ICP-MS was equipped with a MicroMist nebulizer and a Scott double
pass spray chamber. The carrier gas flow rate was 1 mL min−1. The
instrument was tuned for cerium, cobalt, lithium, magnesium,
thallium, and yttrium. The reported values are the mean of at least
three independent experiments conducted with blank wells for each
substance to account for unspecific binding to the plastic of the well
plates.
Cell Proliferation Assays. Sulforhodamine B Cytotoxicity
Assay. HCT116, SW480, and NCI-H460 cells were supplied by
ATCC, while SiHa cells were from Dr. David Cowan, Ontario Cancer
Institute, Canada. The cells were grown in αMEM (Life
Technologies) supplemented with 5% fetal calf serum (Moregate
Biotech) at 37 °C in a humidified incubator with 5% CO2.
The cells were seeded at 750 (HCT116, NCI-H460), 4000 (SiHa),
or 5000 (SW480) cells/well in 96-well plates and left to settle for 24
h. The compounds were added to the plates in a series of 3-fold
dilutions, containing a maximum of 0.5% DMSO at the highest
concentration. The assay was terminated after 72 h by addition of
10% trichloroacetic acid (Merck Millipore) at 4 °C for 1 h. The cells
were stained with 0.4% sulforhodamine B (Sigma-Aldrich) in 1%
acetic acid for 30 min in the dark at room temperature and then
washed with 1% acetic acid to remove unbound dye. The stain was
dissolved in unbuffered Tris base (10 mM; Serva) for 30 min on a
plate shaker in the dark and quantified on a BioTek EL808 microplate
reader at an absorbance wavelength of 490 nm with 450 nm as the
reference wavelength to determine the percentage of cell growth
inhibition by determining the absorbance of each sample relative to a
negative (no inhibitor) and a no-growth control (day 0). The IC50
values were calculated with SigmaPlot 12.5 using a three-parameter
logistic sigmoidal dose−response curve between the calculated growth
inhibition and the compound concentration. The presented IC50
values are the mean of at least three independent experiments, where
10 concentrations were tested in duplicate for each compound.
Thymidine Incorporation Assay. HCT116 cells were seeded at
750 per well in 96 well plates. The compounds were added to the
plates in a series of 3-fold dilutions, containing a maximum of 0.5%
DMSO at the highest concentration for 3 days. 3H-Thymidine (0.04
μCi per well) was added to each well and incubated for 6 h. The cells
were harvested on glass fiber filters using an automated TomTec
harvester. The filters were incubated with Betaplate Scint and 3Hthymidine incorporation was measured in a Trilux/Betaplate counter.
The inhibition of 3H-thymidine incorporation by the metal complexes
was determined relative to the incorporation of 3H-thymidine into
DNA of control cells. The presented IC50 values are the means of two
independent experiments, where 10 concentrations were tested in
duplicate for each compound.
Flow Cytometry. HCT116 cells (7.2 × 105 cells per well) were
plated in 6-well plates overnight and incubated with 1a, 1acym,
cisplatin (30 μM each), doxorubicin (1 μM), and camptothecin (1
μM) for 6 h. Cells were harvested, fixed with 80% ethanol for 10 min,
washed and resuspended in 1 mL of blocking buffer (1% FCS/PBS),
and incubated with antibody to γH2AX (phosphorylated Ser139;
Millipore, United States) in blocking buffer (1:500 dilution) at room
EXPERIMENTAL SECTION
All reactions were performed in Schlenk flasks with dry solvents under
nitrogen atmosphere. Chemicals acquired from commercial suppliers
were used without any prior purification. Sodium methoxide was
purchased from Fluka. Dry solvents were prepared according to
standard methods.45 [(η6-Ethyl 2-acetamido-3-phenylpropanoate)RuIICl2]243 (A), 1acym,27 and ligands 1−533,40 were synthesized
following literature procedures.
Elemental analyses were conducted on a vario EL cube (Elementar
Analysensysteme GmbH, Hanau, Germany). 1D and 2D (1H and 13C
HSQC and HMBC) NMR spectra were recorded on a Bruker Avance
AVIII 400 MHz NMR spectrometer at ambient temperature at 400.13
MHz (1H) or 100.57 MHz (13C{1H}). CDCl3, D2O, or DMSO-d6
were used as NMR solvents. High resolution mass spectra were
recorded on a Bruker micrOTOF-Q II ESI-MS in positive ion mode.
X-ray diffraction measurements of single crystals of 1a were
performed on a Rigaku Oxford Diffraction XtaLAB-Synergy-S singlecrystal diffractometer with a PILATUS 200 K hybrid pixel array
detector using Cu Kα radiation (λ = 1.54184 Å; Table S2). Single
crystals of 1acym were grown from a MeOH solution and analyzed on
a Siemens/Bruker SMART APEX II Single Crystal Diffractometer
with a CCD area detector using graphite monochromated Mo Kα
radiation (λ = 0.71073 Å; Table S2). The data were processed with
the SHELX2016 and Olex2 software packages.46,47 All non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were inserted at
calculated positions and refined with a riding model or without
restrictions. Mercury 3.10 was used to visualize the molecular
structure.
Biomolecule Interaction. The biomolecule interactions of 1a
were studied by 1H NMR spectroscopy. It was dissolved in DMSO-d6
and diluted with D2O to obtain a 10% DMSO-d6/D2O solution.
Equimolar amounts of the biomolecules L-methionine (Met), Lcysteine (Cys), L-histidine (His), or 9-ethylguanine (EtG) were added
to 1a, and NMR spectra were collected over periods of 48 h. In case
of the reaction with EtG, 1a was incubated with the DNA model at
ratios of 1:1 and 2:1 (EtG:1a), and 1H NMR spectra were recorded
after 3 h of incubation.
Cellular Uptake. The cellular uptake experiments were carried
out as described previously.21,48 HCT116 cells were grown in αMEM
(Life Technologies) supplemented with 5% fetal calf serum
(Moregate Biotech) at 37 °C in a humidified incubator with 5%
CO2. HCT116 cells (4 × 105/well) were seeded into 6-well plates and
allowed to settle for 24 h time at 37 °C and 5% CO2. Compounds 1a
and 1acym were dissolved in DMSO (379 and 443 μM, respectively)
and diluted with media to a concentration of 1% DMSO to reach their
B
DOI: 10.1021/acs.inorgchem.8b01187
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
(C3d2), 83.0 (C2d1), 83.3 (C2d2), 85.4 (C5), 95.0 (C1d1), 95.3
(C1d2), 117.9 (C21), 119.9 (C22), 120.1 (C26), 124.4 (C20), 124.5
(C18), 128.7 (C24), 128.9 (C28), 130.8 (C14), 131.6 (C25), 131.9
(C27), 133.8 (C19d1), 133.4 (C19d2), 148.8 (C23), 153.6 (C17),
153.9 (C15), 171.2 (C12), 171.4 (C9), 183.7 (C16) ppm.
Chlorido[3-(oxo-κO)-2-(4-methylphenyl)-chromen-4(1H)-onatoκO](η6-ethyl-2-acetamido-3-phenylpropanoate)ruthenium(II) (3a).
The reaction was performed according to the general procedure using
3 (100 mg, 0.40 mmol), NaOMe (20 mg, 0.44 mmol), and A (146
mg, 0.18 mmol) to afford 3a as a deep red solid (170 mg, 68%). MS
(ESI+): m/z 588.0956 [M − Cl]+ (mtheor = 588.0968). Elemental
analysis calculated for C29H28ClNO6Ru·0.1CH2Cl2: C 55.34, H 4.50,
N 2.22%. Found: C 55.30, H 4.12, N 1.82%. 1H NMR (400.13 MHz,
CDCl3): δ 1.25−1.37 (m, 6H, H11), 1.75 (s, 3H, H13d1), 1.89 (s, 3H,
H13d2), 2.39−2.46 (m, 6H, H29), 3.22−3.32 (m, 4H, H7), 4.22−4.34
(m, 4H, H10), 4.85−4.98 (m, 2H, H8), 5.37−5.42 (m, 2H, Harom),
5.45−5.51 (m, 2H, Harom), 5.66−5.73 (m, 2H, Harom), 5.76−5.88 (m,
4H, Harom), 7.27−7.38 (m, 6H, H25, H27, H20), 7.55 (d, 3J = 8 Hz,
2H, H21), 7.58−7.64 (m, 2H, H19), 7.93 (d, 3J = 8 Hz, 2H, NH),
8.13−8.21 (m, 2H, H18), 8.37 (d, 3J = 8 Hz, 2H, H24d1, H28d1), 8.42
(d, 3J = 8 Hz, 2H, H24d2, H28d2) ppm. 13C{1H} NMR (100.57 MHz,
CDCl3): δ 14.2 (C11d1), 14.3 (C11d1), 22.8 (C13d1), 23.0 (C13d2),
33.5 (C7d1), 34.3 (C7d2), 50.9 (C8d1) 51.4 (C8d2), 61.9 (C10d1), 62.1
(C10d2), 75.8 (C4), 77.6 (C6d1), 77.8 (C6d2), 78.9 (C3d1), 79.2
(C3d2), 82.9 (C2d1), 83.3 (C2d2), 84.9 (C5), 95.1 (C1d1), 95.3
(C1d2), 117.9 (C21), 120.1 (C22), 124.4 (C20), 124.5 (C18), 127.
(C24) 127.6 (C28), 129.1(C14), 129.8 (C25), 129.4 (C27), 132.8
(C19d1), 132.9 (C19d2), 140.4 (C26), 150.7 (C23), 153.0 (C17),
154.0 (15), 170.8 (C12), 171.5 (C9), 183.1 (C16) ppm.
Chlorido[3-(oxo-κO)-2-(4-trifluoromethylphenyl)-chromen-4onato-κO](η6-ethyl-2-acetamido-3-phenylpropanoate)ruthenium(II) (4a). The reaction was performed according to the general
procedure using 4 (100 mg, 0.33 mmol), NaOMe (19 mg, 0.36
mmol), and A (120 mg, 0.15 mmol) to afford 4a as an orange solid
(167 mg, 75%). MS (ESI+): m/z 642.0692 [M − Cl]+ (mtheor =
642.0680). Elemental analysis calculated for C29H25ClF3NO6Ru·
2.5H2O: C 48.25, H 4.19, N 1.94%. Found: C 48.39, H 3.99, N
1.68%. 1H NMR (400.13 MHz, CDCl3): δ 1.27−1.36 (m, 6H, H11),
1.86 (s, 3H, H13d1), 1.93 (s, 3H, H13d2), 3.20−3.33 (m, 4H, H7),
4.22−4.33 (m, 4H, H10), 4.90−4.98 (m, 2H, H8), 5.45 (d, 3J = 6 H,
2H, Harom), 5.48−5.54 (m, 2H, Harom), 5.71 (t, 3J = 5 H, 2H, Harom),
5.79−5.89 (m, 4H, Harom), 7.35−7.41 (m, 2H, H20), 7.51 (d, 3J = 8
Hz, 2H, NH), 7.58 (d, 3J = 8 Hz, 2H, H2), 7.64−7.70 (m, 2H, H19),
7.72 (d, 3J = 8 Hz, 4H, H25d1, H27d1), 7.77 (d, 3J = 8 Hz, 4H, H25d2,
H27d2), 8.16−8.24 (m, 2H, H18d1+d2), 8.59−8.65 (m, 4H, H24, H28)
ppm. 13C{1H} NMR (100.57 MHz, CDCl3): δ 14.1 (C11), 22.8
(C13d1), 23.0 (C13d2), 33.8 (C7d1), 34.5 (C7d2), 50.9 (C8d1) 51.4
(C8d2), 62.0 (C10d1), 62.1 (C10d2), 76.1(C4), 77.6 (C6d1), 77.7
(C6d2), 77.9, 79.1 (C3d1), 79.2 (C3d2), 82.7 (C2d1), 83.3 (C2d2), 83.3,
84.6 (C5d1, C5d2), 95.3 (C1d1), 95.5 (C1d2), 118.0 (C21), 119.9
(C17), 124.6 (C21), 124.7 (C18), 125.4 (C29), 127.2 (C25), 127.4
(C27), 130.0 (C20), 130.8 (C23), 133.6 (C24), 133.7 (C19), 147.8
(C15), 154.2 (C14), 155.8 (C22), 165.5 (C12), 181.1 (C9), 184.7
(C16) ppm.
Chlorido[3-(oxo-κO)-2-(3,4,5-trimethoxyphenyl)-chromen4onato-κO](η6-ethyl-2-acetamido-3-phenylpropanoate)ruthenium(II) (5a). The reaction was performed according to the general
procedure using 5 (100 mg, 0.30 mmol), NaOMe (18 mg, 0.33
mmol), and A (112 mg, 0.14 mmol) to afford 5a as an orange solid
(136 mg, 65%); MS (ESI+): m/z 664.1131 [M − Cl]+ (mtheor =
664.1124). Elemental analysis calculated for C31H32ClNO9Ru: C
53.26, H 4.61, N 2.00%. Found: C 53.00, H 4.89, N 1.88%. 1H NMR
(400.13 MHz, CDCl3): δ 1.26−1.35 (m, 6H, H11), 1.91 (s, 3H,
H13d1), 1.97 (s, 3H, H13d2), 3.14−3.35 (m, 4H, H7), 3.93 (s, 3H,
OMe), 3.99 (s, 6H, OMe), 4.19−4.30 (m, 4H, H10), 4.86−4.99 (m,
2H, H8), 5.42−5.53 (m, 4H, Harom), 5.64−5.70 (m, 2H, Harom),
5.72−5.87 (m, 4H, Harom), 7.15−7.22 (m, 1H, NH), 7.33−7.41 (m,
2H, H20), 7.53−7.67 (m, 4H, H21, H19), 7.84 (s, 2H, H24d1,
H28d1), 7.87 (s, 2H, H24d2, H28d2), 8.13−8.23 (m, 1H, H18) ppm.
13
C{1H} NMR (100.57 MHz, CDCl3): δ 14.1 (C11), 22.8 (C13),
temperature for 2 h. Cells were washed, incubated with goat
antimouse Alexa Fluor 488 Fab fragment secondary antibody
(Invitrogen, New Zealand; 1:400 in blocking buffer for 1 h, at
room temperature; dark), washed, and resuspended in 1 mL of
blocking buffer containing RNase (1 μg/mL) and propidium iodide
(PI; 10 μg/mL) for 10 min at room temperature. AntiphosphoHistone H2A.X (Ser139) Antibody, clone JBW301 (Millipore) and
Alexa Fluor 488 (Life Technologies) were used according to the
manufacturer instructions. Briefly, cells were harvested, fixed with 2%
paraformaldehyde, permeabilized with 0.1% Triton-X, and incubated
with antibody (5 μL in 300 μL blocking buffer for 2 h, at room
temperature, dark), washed and resuspended in 1 mL of blocking
buffer. Cells were analyzed in Becton Dickinson BD Accuri C6 flow
cytometer.
General Procedure for the Synthesis of the Ru(η6-ethyl-2acetamido-3-phenylpropanoate) Complexes 1a−5a. A solution
of [(η6-N-acetyl-L-phenylalanine ethyl ester)RuIICl2]2 A (0.45 equiv)
in dry methanol (5 mL) was added to a solution of 3-hydroxyflavone
1−5 (1.00 equiv) and sodium methoxide (1.10 equiv) in a mixture of
methanol (15 mL) and dichloromethane (5 mL). The reaction
mixture was stirred at room temperature and under nitrogen
atmosphere for 18 h. The solvent was evaporated in vacuo, and the
residue was dissolved in dichloromethane and filtered. The complexes
were precipitated from dichloromethane by addition of n-hexane.
Chlorido[3-(oxo-κO)-2-(4-fluorophenyl)-chromen-4(1H)-onatoκO](η6-ethyl-2-acetamido-3-phenylpropanoate)ruthenium(II) (1a).
The reaction was performed according to the general procedure using
1 (100 mg, 0.39 mmol), NaOMe (23 mg, 0.43 mmol), and A (143
mg, 0.18 mmol) to afford 1a as a deep red solid (193 mg, 72%).
Single crystals suitable for X-ray diffraction analysis were grown from
MeOH and CH2Cl2/diethyl ether. MS (ESI+): m/z 592.0695 [M −
Cl]+ (mtheor = 592.0712). Elemental analysis calculated for
C28H25ClFNO6Ru·0.3CH2Cl2: C 52.09, H 3.95, N 2.15%. Found: C
52.15, H 3.64, N 1.98%. 1H NMR (400.13 MHz, CDCl3): δ 1.20−
1.37 (m, 6H, H11), 1.83 (s, 3H, H13d1), 1.90 (s, 3H, H13d2), 2.19−
3.32 (m, 4H, H7), 4.21−4.34 (m, 4H, H10), 4.85−4.97 (m, 2H, H8),
5.41 (d, 3J = 5 Hz, 2H, Harom), 5.48 (t, 3J = 6 Hz, 2H, Harom), 5.38−
5.51 (m, 4H, Harom), 5.66−5.72 (m, 2H, Harom), 5.77−5.88 (m, 4H,
Harom), 7.12−7.28 (m, 4H, H25/H27), 7.32−7.39 (m, 2H, H20), 7.54
(d, 3J = 8 Hz, 2H, H21), 7.59−7.66 (m, 2H, H19), 7.77 (d, 3J = 8 Hz,
2H, NH), 8.11−8.22 (m, 2H, H18), 8.47−8.58 (m, 4H, H24/H28)
ppm. 13C{1H} NMR (100.57 MHz, CDCl3): δ 14.3 (C11), 22.7
(C13d1), 23.0 (C13d2), 33.4 (C7d1), 34.3 (C7d2), 50.8 (C8d1) 51.3
(C8d2), 61.9 (C10d1), 62.0 (C10d2), 75.6 (C4d2/C4d2), 77.5 (C6d1),
77.7 (C6d2), 78.8 (C3d1), 79.1 (C3d2), 82.9 (C2d1), 83.3 (C2d2), 85.1
(C5d1, C5d2), 95.0 (C1d1), 95.3 (C1d2), 115.6 (d, 2JC−F = 21 Hz,
C25d1/C27d1), 115.8 (d, 2JC−F = 21 Hz, C25d2/C27d2), 117.9 (C21),
119.9 (C22), 124.5 (C20), 124.6 (C18), 128.1 (C14), 129.6 (d, 3JC−F
= 8 Hz, C24d2/C28d2), 129.7 (d, 3JC−F = 8 Hz, C24d1/C28d1), 132.1
(C19d1), 132.2 (C19d2), 146.7 (C23), 154.0 (C17), 155.1 (C15),
164.4 (C26), 170.6 (C12d1), 171.2 (C12d2), 171.5 (C9), 183.5 (C16)
ppm.
Chlorido[3-(oxo-κO)-2-(4-bromophenyl)-chromen-4-onato-κO](η6-ethyl-2-acetamido-3-phenylpropanoate)ruthenium(II) (2a).
The reaction was performed according to the general procedure
using 2 (100 mg, 0.32 mmol), NaOMe (19 mg, 0.35 mmol), and A
(116 mg, 0.14 mmol) to afford 2a as a deep red solid (155 mg, 78%);
MS (ESI+): m/z 653.9889 [M − Cl]+ (mtheor = 653.9901). Elemental
analysis calculated for C28H25ClBrNO6Ru·0.3CH2Cl2: C 47.65, H
3.62, N 1.96%. Found: C 47.89, H 3.25, N 1.60%. 1H NMR (400.13
MHz, CDCl3): δ 1.22−1.37 (m, 6H, H11), 1.82 (s, 3H, H13d1), 1.91
(s, 3H, H13d2), 3.07−3.31 (m, 4H, H7), 4.21−4.33 (m, 4H, H10),
4.86−4.98 (m, 2H, H8), 5.42 (d, 3J = 5 Hz, 2H, Harom), 5.49 (t, 3J = 6
Hz, 2H, Harom), 5.65−5.73 (m, 2H, Harom), 5.77−5.87 (m, 4H, Harom),
7.32−7.39 (m, 2H, H20), 7.53 (d, 3J = 8 Hz, 2H, H2), 7.57−7.72 (m,
8H, H25, H27, H19, NH), 8.13−8.22 (m, 2H, H18), 8.33−8.43 (m,
4H, H24/H28) ppm. 13C{1H} NMR (100.57 MHz, CDCl3): δ 14.2
(C11d1), 14.3 (C11d1), 21.7 (C29), 22.6 (C13d1), 22.9 (C13d2), 33.2
(C7d1), 33.9 (C7d2), 50.8 (C8d1) 51.2 (C8d2), 61.8 (C10d1), 61.9
(C10d2), 75.8(C4), 77.6 (C6d1), 77.7 (C6d2), 78.8 (C3d1), 79.1
C
DOI: 10.1021/acs.inorgchem.8b01187
Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Scheme 1. Preparation of Complexes 1a−5a
[M − Cl]+ ions detected for complexes 1a−5a in positive ion
mode.
Single crystals of 1a were grown by slow diffusion of diethyl
ether into a mixture of methanol/dichloromethane (approximately 1:1) and analyzed by X-ray diffraction analysis (Figure
1). A diastereomer of the complex crystallized in the
34.6 (C7d1), 34.7 (C7d2), 51.2 (C8), 56.3 (OMe), 61.0 (OMe), 62.0
(C10), 77.8 (C4), 78.7 (C6), 81.6 (C3), 82.9 (C2), 83.5 (C5), 94.9
(C1), 105.1 (C24d1/C28d1), 105.1 (C24d2/C28d2), 114.7 (C17),
117.7 (C21), 124.3 (C20), 124.4 (C18d1), 124.5 (C18 d2), 132.7
(C23), 132.8 (C19), 138.7 (C14), 146.7 (C15), 153.0 (C25, C27),
161.8 (C22), 170.5 (C12), 171.1 (C9), 185.8 (C16) ppm.
■
RESULTS AND DISCUSSION
We recently reported a series of [Ru(arene)(8oxyquinolinato)Cl] complexes in which we replaced the
routinely used arene p-cymene with a protected Phe derivative.
This modification maintained the in vitro anticancer activity of
the complexes in the low μM range but improved the aqueous
solubility significantly and may contribute to interaction of the
complexes with other targets.43 To expand this work on highly
potent anticancer organometallics, we translate here this
concept from N,O-chelating 8-oxyquinolinato complexes to
O,O-donor systems as in 3-oxyflavonato ligands.40,41 A series of
substituted 3-hydroxyflavones 1−5 was used that had shown
promising anticancer activity in the past upon coordination to
metal centers, in particular 1.27,33,40−42 They were converted
with the dimeric Ru precursor A into the respective
organometallic compounds [Ru(η6-N-acetyl-L-phenylalanine
ethyl ester)(3-oxyflavonato)Cl] 1a−5a (Scheme 1). The 3hydroxyflavone derivatives were deprotonated using NaOMe,
and A was added in dry MeOH. The reaction mixture was
stirred at room temperature under nitrogen atmosphere for 18
h. The solvent was evaporated in vacuo, and the residue was
dissolved in dichloromethane and filtered. Complexes 1a−5a
were obtained by precipitation with n-hexane in high yields of
65−78% (Scheme 1). The reaction results in the formation of
diastereomers given the chiral nature of the Phe-derived arene
ligand and another chiral center sitting at the Ru ion.
All synthesized compounds were characterized by standard
methods including NMR spectroscopy, ESI-mass spectrometry, and elemental analysis. The 1H NMR spectra show signals
for the ethyl ester CH3 in the range 1.22−1.37 ppm and
around 1.90 ppm for the acetyl group. In both cases, we saw
more than one set of signals owing to the diastereomeric
nature of the complexes. Protons H7 and H8 of the Phederived arene ligand were found at ca. 3.3 and 4.2 ppm,
respectively, while the 5 arene protons resonated in the range
of 5.4−5.9 ppm. As expected, the signals assigned to the
flavone ligand along with the amide NH were found between
7.1 and 8.7 ppm. These signals are in similar ranges as
observed for structurally related 8-oxyquinolinato complexes.43
The ESI-MS results confirmed the nature of compounds with
Figure 1. Molecular structures of a crystallized diastereomer of 1a and
of an enantiomer of 1acym drawn at 50% probability level.
Cocrystallized solvent molecules were omitted for clarity.
monoclinic space group P21. The compound features the
pseudo-octahedral piano-stool configuration around the metal
center10,22 and is involved in a network of hydrogen bonds
through cocrystallized water molecules. The amide carbonyl
oxygen atom forms a hydrogen bond with a water molecule,
which bridges it to another molecule of 1a through an
interaction with the amide NH proton. Another H bond was
found for 1a from the deprotonated flavone O atom to another
water molecule. The flavone backbone is involved in
intermolecular π-stacking interactions, and the shortest
distance is 3.425 Å (Figure S1). The Ru−arenecentroid distance
is found at 1.638 Å (Table 1). The 3-oxyflavonato ligand forms
a virtually planar five-membered metallocycle with the Ru
center, while the phenyl substituent of the ligands are twisted
with torsion angles of 29.1°. The two Ru−O bond lengths are
very similar at 2.068(5) and 2.117(6) Å, with the longer bonds
formed between the Ru and flavone-carbonyl O atoms. The
C15−O1 bond is significantly longer (1.374(8) Å) than the
C16−O2 distance (1.259(8) Å), indicating higher single bond
character. The Ru−Cl bond length is 2.423(2) Å. Comparison
of these structural parameters with those of the analogous pcymene complex 1acym (crystals of a mixture of enantiomers
were grown from MeOH; Figure 1) shows that the Ru−Cl
bond is slightly longer than in 1acym (2.4326(10) Å). The Ru−
O1 distance is slightly shorter, while the other distances
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biomolecules L-cysteine (Cys), L-methionine (Met), Lhistidine, and 9-ethylguanine (EtG) in 10% DMSO-d6/D2O,
and the progress of the reactions was monitored by 1H NMR
spectroscopy. The spectra recorded for the reaction mixtures
with the amino acids revealed that the signals assigned to the
flavone protons vanished very rapidly (Figures 3 and S3), while
Table 1. Key Bond Lengths (Å) and Angles (°) for Complex
1a in Comparison to the Analogous p-Cymene Complex
1acym
1a
bond lengths (Å) Ru−arenecentroid
1.638
Ru−Cl
2.423(2)
Ru−O1
2.068(4)
Ru−O2
2.117(6)
torsion angles (deg)
C15−C14−C23−C24
29(1)
bond angles (deg)
C1−Ru−O1
84.8(2)
C1−Ru−O2
83.98(13)
O1−Ru−O2
79.4(2)
1acym
1.639
2.4186(6)
2.078(2)
2.112(2)
14.1(4)
85.41(5)
86.00(5)
78.39(7)
around the Ru center are similar (Table 1).27,40 The most
notable difference is the more planar nature of the 3oxyflavonato ligand in 1acym as compared to 1a.
Stability in DMSO and Aqueous Solution. The stability
in DMSO and water was determined for 1a as a representative
example for the compound class with 1H NMR spectroscopy
over a time span of two days. The complex was stable in
DMSO-d6 for less than 1 h, after which additional sets of
signals appeared in the 1H NMR spectra, probably due to a
combination of cleavage of the arene ligand and additional
ligand exchange reactions at the metal center (Figure S2).
When stability studies were carried out at low concentrations
of DMSO-d6 (10% in D2O), an immediate chlorido/aqua
ligand exchange was observed (Figure 2). This was confirmed
Figure 3. 1H NMR spectroscopy study on the reaction of 1a with His
(1:1) in 10% DMSO-d6/D2O over a period of 48 h.
in the region of the spectra characteristic for the arene ligand,
additional signals formed. These are most likely from the
formed amino acid complexes upon coordination to the Ru
center. During the experiments, in particular with Cys,
precipitation was observed, which is not unsurprising given
the low aqueous solubility of 3-hydroxyflavones when cleaved
from the metal center.
DNA has been suggested as the target for the RAED
anticancer agents.49 The reaction of 1a with the DNA model
EtG resulted in the formation of EtG complexes after ligand
exchange with Cl−. The reaction between 1a and EtG in a
molar ratio of 1:1 proceeded very quickly, and already 1 h after
the start of the incubation, the complex was transformed
quantitatively into its EtG adduct (Figure S4). This is
supported by an experiment in which a second equivalent of
EtG was added to the reaction mixture, and the peak assigned
to unreacted EtG at 7.88 ppm grew while the rest of the
spectrum remained unchanged (Figure 4). Both reaction
mixtures were also analyzed by ESI-MS in positive ion mode.
For example, the mass spectrum recorded from the 1:1
reaction mixture showed a peak at m/z 773.1609, which was
Figure 2. 1H NMR spectroscopy study on the stability of 1a in 10%
DMSO-d6 in D2O over a period of 48 h.
by the addition of 1 equiv of AgNO3, which resulted in the
same spectrum as recorded over the 48 h period. These
experiments demonstrate that the compounds are sufficiently
stable to carry out cell viability studies under conditions
routinely used in biological assays.
Biomolecule Interaction. Metal complexes are prone to
undergo ligand exchange reactions, especially in biological
systems with many binding partners available. These
interactions may be beneficial and support the accumulation
of the pharmacophore in the desired tissue or allow interaction
with the target. On the other hand, these reactions may
deactivate drugs. To understand the nature of interactions of
the novel organoruthenium compounds with biomolecules
such as proteins and DNA, we reacted 1a with the
Figure 4. 1H NMR spectroscopy study on the reaction of 1a with 1 or
2 equiv of EtG in 10% DMSO-d6/D2O after an incubation period of 3
h.
E
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assigned to the ion [1a + EtG − Cl]+ (mtheor = 773.1661),
while the base peak was identified as [1a − Cl]+ (Figure S5).
The low abundance of the adduct peak relative to the latter
species may be explained by the low stability of the EtG adduct
in the gas phase. As the samples were prepared in deuterated
solvents, species were detected in which protons had
exchanged with deuterium.
In Vitro Cytotoxicity against Human Cancer Cells and
Cellular Accumulation. The in vitro antiproliferative activity
of complexes 1a−5a was studied in HCT116 human
colorectal, NCI-H460 non-small cell lung, SiHa cervical
carcinoma, and SW480 colon adenocarcinoma cells using the
sulforhodamine B assay (SRB; Table 2) and compared to the
1acym was negligible. Increasing the incubation time to 24 h
resulted in significantly higher Ru levels for both compounds
(Figure 5); however, overall the Ru content for 1a was about 5
Table 2. In Vitro Cytotoxic Activity of Compounds 1a−5a
in the Human Cancer Cell Lines HCT116 (Colon), NCIH460 (Nonsmall Cell Lung), SiHa (Cervix), and SW480
(Colon)a
Figure 5. Cellular accumulation of 1a and 1acym determined by ICPMS after treatment of HCT116 cells for 4 and 24 h.
IC50 values (μM)
compound
HCT116
NCI-H460
SiHa
SW480
1a
2a
3a
4a
5a
1acym
2acym
3acym
4acym
5acym
4.4 ± 0.4
2.1 ± 0.1
2.8 ± 0.2
1.3 ± 0.1
8.3 ± 1.9
3.8 ± 0.1
3.4 ± 0.2
5.8 ± 0.8
8.7 ± 0.1
14 ± 1
2.4 ± 0.1
1.5 ± 0.1
2.0 ± 0.2
1.0 ± 0.3
5.9 ± 0.8
2.7 ± 0.5
2.9 ± 0.6
5.4 ± 0.3
9.2 ± 1.1
14 ± 1
22 ± 1
15 ± 1
19 ± 2
13 ± 2
20 ± 1
23 ± 1
29 ± 1
22 ± 4
64 ± 6
41 ± 2
9.5 ± 0.4
5.7 ± 0.1
7.2 ± 0.3
2.5 ± 0.1
8.1 ± 0.1
7.2 ± 0.4
8.5 ± 0.7
13 ± 4
21 ± 0.3
30 ± 1
times higher than that found for 1acym, despite similar in vitro
cytotoxicity which was, however, determined over 72 h. Note
that 1acym was strongly interacting with the well plates,
resulting in significant blanks which were considered when
determining the cellular accumulation.
In addition, the in vitro antiproliferative activity of
complexes 1a and 1acym in HCT116 cells was compared to
that of cisplatin in SRB and 3H-thymidine incorporation (TI)
assays (Figures 6 and S6, Table S2). Overall, the two assays
The IC50 values are given as means ± standard deviation.
a
cytotoxic activity of the analogous cym complexes 1acym−
5acym. All RuII(arene)−flavonoid complexes were potent
antiproliferative agents with IC50 values in the low micromolar
range, especially in HCT116 and NCI-H460 cells. Compound
4a with its trifluoromethyl substituent was the most potent
derivative, while the trismethoxy (5a) and the fluoro (1a)
derivatives were the least active, depending on the cell line
used. This trend is in contrast to that found for the analogous
cym complexes 1acym−5acym, for which the CF3 (4acym)
derivative was among the least active compounds, while the pfluoro (1acym) complex was the most potent compounds. The
introduction of the protected Phe group as an arene had a
significant impact on the cytotoxicity of complexes with ligands
2−5, which all became more cytotoxic, while 1a was similarly
potent to its cym analogue 1acym. We found a similar effect for
hydroxyquinoline complexes, where the introduction of the
Phe-based arene ligand also resulted in complexes with limited
correlation of their cytotoxic activity to those of the parent cym
complexes.43 These results suggest that organoruthenium
compounds with the Phe-derived arene may have different
modes of action and that the biological activity is not
necessarily a function of the lipophilicity and associated
increased cell penetration ability. This conclusion is also
supported by cellular accumulation data for 1a and 1acym. The
cellular Ru content after treatment of HCT116 cells with these
complexes at concentrations similar to their IC50 values (3.79
μM for 1acym and 4.43 μM for 1a) was measured after 4 and 24
h by inductively coupled plasma mass spectrometry. After
incubation of the cells with the compounds for 4 h, only 1a
was found to enter the cells, while the detected amount of
Figure 6. Relative growth (%) of HCT116 cells measured by the SRB
and TI assays, respectively, after incubation with 1a for 72 h.
gave similar IC50 values for all compounds, although the values
were slightly higher by SRB assay, especially for cisplatin. This
supports that the organometallics are cytotoxic rather than
cytostatic.
Induction of DNA Damage by 1a and 1acym. As pointed
out earlier, metal complexes are prone to undergo ligand
exchange reactions with biomolecules, and the flavone
complexes were designed to act as enzyme inhibitors while
still being able to coordinate to DNA. Therefore, we examined
whether the antiproliferative effect of 1a and 1acym is related to
DNA damage. For this purpose, HCT116 cells were exposed
to 1a, 1acym, cisplatin (30 μM each), doxorubicin (1 μM), and
camptothecin (1 μM). The DNA damage response was
measured by the level of γH2AX phosphorylation using flow
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DOI: 10.1021/acs.inorgchem.8b01187
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■
CONCLUSIONS
The design concept of coordinating bioactive ligands to metal
centers resulted in the preparation of [Ru(η6-p-cymene)(flavonolato)Cl] compounds with interesting biological
activity defined by its flavonolato ligand and the properties
of the metal center. We have borrowed here a concept in which
we replace the commonly used arene cym with a recently
introduced novel arene derived from the bioactive amino acid
L-phenylalanine, which may add to the biological activity of the
complex. The synthesis and characterization of the series of
organoruthenium compounds was complemented with studies
on the stability in water and DMSO, as well as the reactivity
with biomolecules. High stability in aqueous solution after
dissolution in DMSO was demonstrated for a representative
example of the complexes, after rapidly forming aqua
complexes. In contrast, the complex quickly decomposed in
DMSO, as determined by NMR spectroscopy. The reactions
with the amino acids His, Met, and Cys showed quick cleavage
of the flavonolato ligand from the Ru center, while incubation
with EtG resulted in substitution of the chlorido ligand with
the DNA model compound. All new compounds were potent
anticancer agents in a panel of cancer cells with IC50 values as
low as 1 μM in NCI-H460 non-small cell lung cancer cells.
The IC50 values of the Phe-derived compounds did not follow
the same trends as observed for their cym analogues, but the
values were in a similar range. This is surprising given that the
cellular accumulation of a representative Phe-derived complex
was found to be much higher than of its cym analogue,
independent of the concentration used and the incubation
times. Comparison of the IC50 values determined by 3Hthymidine incorporation and sulforhodamine B assays revealed
cytotoxic rather than cytostatic activity. As the compounds
were designed to be able to coordinate to DNA, which was
confirmed in the reactivity studies with EtG, it is important to
note that they damage DNA, which may contribute to their
cytotoxic activity. The DNA damage profile was similar to that
of cisplatin; however, the amount of DNA damage detected
was lower for the tested compounds than for cisplatin. These
data support the hypothesized DNA binding ability of the
compounds while not contradicting the topoisomerase
inhibitory activity, which was demonstrated for the parent
compounds, as another contributor to the biological activity.
cytometry as compared to control (Figure 7). Camptothecin
specifically induces DNA damage in the S phase, while the
Figure 7. DNA damaging ability in HCT116 cells as determined for
γH2AX by flow cytometry after treatment with 1a, 1acym, cisplatin (30
μM each), doxorubicin (1 μM), and camptothecin (1 μM) for 6 h.
DNA damage caused by doxorubicin is cell phase independent.
Both 1a and 1acym showed very similar DNA damaging ability,
which is surprising given the differing cellular accumulation.
The amount of Ru in the cells at 6 h was found to be about 4
times higher for 1a over 1acym when they were treated with the
compounds at the same concentration as used for the flow
cytometry sample preparation (30 μM; data not shown). The
DNA damage profile of 1a and 1acym after 6 h treatment is
unlike that of both camptothecin and doxorubicin but
resembles to some extent that of cisplatin (Figure 7). Cisplatin
was reported to induce an S/G2 cell cycle arrest and apoptosis
in V79 cells,50 which supports a similar conclusion for the
compounds studied here. However, the induction level of
γH2AX phosphorylation was significantly lower with averages
of 27% for 1a and 24% for 1acym as compared to 45% for
cisplatin. This may be a result of the higher lability of the
DNA−metal bonds for RuII as compared to those for PtII. Also,
cisplatin forms bifunctional adducts, whereas DNA can
coordinate to the organoruthenium compounds only monodentately.
Exposure of cells to 1a and 1acym for 24 h at concentrations
of 30 and 60 μM revealed that both compounds induced
higher dose-dependent DNA damage than the damage
response found after 6 h, as was indicated by a higher number
of cells expressing γH2AX (Figure S7). In all cases, 1a caused
slightly more DNA damage than its cym counterpart, which
may explain the slightly differing antiproliferative activity of the
compounds. This was especially pronounced in the samples
containing only 30 μM of the compounds.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01187.
X-ray crystallographic data and measurement parameters, NMR and mass spectrometric analysis of stability
and reactivity with biomolecules, and additional data
collected in in vitro anticancer activity and mode of
action studies (PDF)
Accession Codes
CCDC 1840317 and 1840404 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
G
DOI: 10.1021/acs.inorgchem.8b01187
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AUTHOR INFORMATION
Corresponding Author
*E-mail: c.hartinger@auckland.ac.nz; Tel.: +64 9 3737 599 ext.
83220; Website: http://www.hartinger.auckland.ac.nz.
ORCID
Muhammad Hanif: 0000-0002-2256-2317
Christian G. Hartinger: 0000-0001-9806-0893
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank the University of Auckland (University of Auckland
Doctoral Scholarships to H.H. and B.L.) for the financial
support. We are grateful to Tanya Groutso for collecting the
single crystal X-ray diffraction data, to Tony Chen for ESI-MS
analyses, and to Marjan Askarian-Amiri for useful discussions.
■
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