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Expanding on the Structural Diversity of Flavone- Derived RutheniumII(ƞ6-arene) Anticancer Agents
Metallodrugs 2015; 1, 24–35
Research Article
Open Access
Mario Kubanik, Jason K. Y. Tu, Tilo Söhnel, Michaela Hejl, Michael A. Jakupec,
Wolfgang Kandioller, Bernhard K. Keppler, Christian G. Hartinger*
Expanding on the Structural Diversity of FlavoneDerived RutheniumII(ƞ6-arene) Anticancer Agents
ruthenium(II) 2b exhibits the highest cytotoxicity with
IC50 values in the low µM range in all tested cell lines.
DOI 10.1515/medr-2015-0001
Received June 30, 2015; accepted August 8, 2015
Abstract: 3-Hydroxyflavones belong to the naturally
occurring class of flavonoids and have been extensively
studied with regard to medicinal application. Moreover,
it has been demonstrated that these compounds
act as bioactive chelates to the ruthenium(II)–arene
moiety. Such organometallic complexes have shown
promising anticancer activity against tumor cells via a
multitargeting mode of action, interacting with DNA and
inhibiting topoisomerase IIα. In this paper, we present
the synthesis and characterization of an extended series
of 3-hydroxyflavone ligands and their corresponding
ruthenium-p-cymene complexes to study the impact of
substitution pattern as well as of electron-withdrawing
and –donating substituents at the flavonol-phenyl
group. The ligands and complexes were characterized
by elemental analysis, ESI-MS, 1D as well as 2D NMR
spectroscopy. The structures of four Ru(η6-p-cymene)
complexes were determined in solid state by single-crystal
X-ray diffraction, and the impact of the substitution
pattern with regard to in vitro anticancer activity in human
cancer cell lines is discussed. Structural differences,
calculated octanol-water partition coefficients (clogP)
of the flavonols and aqueous solubility were used to
rationalize the finding that chlorido[3-(oxo-κO)-2-(3,5dimethoxyphenyl)-chromen-4-onato-κO](η6-p-cymene)
*Corresponding author: Christian Hartinger: School of Chemical
Sciences, University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand, E-mail: c.hartinger@auckland.ac.nz
Mario Kubanik, Jason K. Y. Tu, Tilo Söhnel: School of Chemical
Sciences, University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand
Michaela Hejl, Michael A. Jakupec,Wolfgang Kandioller, Bernhard
K. Keppler: University of Vienna, Faculty of Chemistry, Institute of
Inorganic Chemistry, Waehringer Str. 42, 1090 Vienna, Austria
Michael A. Jakupec, Wolfgang Kandioller, Bernhard K. Keppler:
University of Vienna, Research Platform “Translational Cancer
Therapy Research”, Waehringer Str. 42, A-1090 Vienna, Austria
Keywords:
Bioorganometallic
chemistry,
Cancer
chemotherapy, Flavonols, Ruthenium complexes, X-ray
diffraction analysis
1 Introduction
Conventional cytotoxic chemotherapeutics effectively kill
tumor cells but their low selectivity often results in damage
to healthy organs and tissues [1]. Therefore, strategies that
improve the specificity of chemotherapies in the human
body (e.g. targeted delivery, molecular targeted therapies)
maximize the drugs’ anticancer activity while they reduce
the systemic toxicity to non-tumor tissue. In recent years,
such strategies have become popular in the development
of cancer chemotherapeutics, including also metalbased drugs. For, example, the established Pt(ammine)2
pharmacophore of cisplatin has been modified to improve
the selectivity by using ligands which can specifically
bind to certain transporters, enzymes and receptors, or by
using platinum(IV) compounds as prodrug [2].
Within the course of developing non-platinum
anticancer agents, Ru compounds with different activity
profiles have moved into the focus of interest. They were
often described as less toxic than platinum complexes,
which have been attributed to a higher selectivity for tumor
cells. Both more selective delivery, tumor (cell)-dependent
activation and targeted activity have been suggested to
be responsible for these properties. Promising examples
feature ligands such as paullones [3], N-heterocyclic
carbenes [4], quinones [5] and protein-targeting
approaches (e.g. ethacrynic acid [6,7], maleimide [8,9],
acetal [10], biotin [11,12] and aldehyde [13]), or resemble
biological substrates of kinases [14,15].
We have focused in the last years on developing
organometallic compounds based on biologically active
© 2015 Mario Kubanik, et al., published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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Expanding on the Structural Diversity of Flavone-Derived RutheniumII(ƞ6-arene) Anticancer Agents
ligand systems. One of the ligand types used is the class
of flavonols which have been coordinated to different
metal centers [16-25]. Flavonols are derived from flavones
which are naturally occurring constituents of plants, have
demonstrated promising tumor-inhibiting activity and
are known to be enzyme inhibitors. Flavonoids are a large
group of secondary metabolites ubiquitously produced
in plants and consist of phenolic fragments possessing
two benzene rings linked by heterocyclic systems with
one or more hydroxyl groups [26,27]. They are the most
abundant polyphenols found in plants and are thought
to perform a variety of functions including protection
from UV radiation, defense against pathogens, pollinator
attraction, pigmentation, and play an essential role in
reproduction [28]. Flavonoids have been well-known
to exert a wide range of biological properties, including
antioxidant, antiradical, antibacterial, estrogenic,
antiviral, and also anticancer effects [29-31]. Several in
vitro studies have shown that phenolics extracted from
plants have potential in chemoprevention of hepatoma
and melanoma. They may act as cancer blocking agents
with their ability to scavenge free radicals, and/or cancer
suppressing agents, due to their capacity to target different
signal transduction pathways and preventing tumor
development by inducing tumor cell apoptosis [32-34].
Flavonols can act as bidentate ligand systems to metal
complexes, as has been shown for Ru(II) [20], Zn(II) [25],
Cu(II) [21], Pb(II) [22], and Al(III) [23,24] as the metal center.
We got interested in the ligand systems as an extension to
our early work on hydroxypyrones and -pyridones [35-37],
and reported organoruthenium, -osmium and -rhodium
[16-19] compounds (Figure 1) with the p-fluoro Ru complex
A (Figure 1, M = Ru, Ar = p-cymene, X = Cl, Y = Z = O, R
= p-F) being among the most potent in in vitro assays
[16-19]. Complexes of flavonols were found to inhibit
topoisomerase IIα while maintaining their coordination
ability to DNA model compounds. Topoisomerases
are crucial enzymes required for the resolution of
topological problems that occur during DNA replication
and transcription. This observation supports that the
compounds have multitargeted properties. Notably, the
complexes were more potent inhibitors of the enzyme
than the ligand systems, while the anticancer activity was
determined by the flavonoid [16].
Herein, we report the synthesis of 3-hydroxyflavones and
their corresponding organometallic Ru(II) complexes,
containing electron-withdrawing as well as electrondonating R groups at the phenyl ring (Figure 1), with the
aim to get a better understanding of the electronic and
structural impact of such substitution on the cytotoxic
activity of flavone-derived anticancer agents.
25
Ar
Y
M
X
O
Z
R
Figure 1. General structure of 3-hydroxypyr(id)one-derived organometallic compounds (compound A: M = Ru, Ar = p-cymene, X = Cl, Y
= Z = O, R = p-F).
2 Experimental
2.1 Materials and Methods
All air- and moisture-sensitive reactions were carried
out under nitrogen atmosphere using standard Schlenk
techniques. Chemicals and solvents were obtained from
commercial suppliers and used as received. Tetrahydrofuran (THF) and diethyl ether (Et2O) were first dried
through a solvent purification system (LC Technology
Solutions Inc., SP-1 solvent purifier) and degassed under
a N2 flow, and stored in a Schlenk flask until use. Ethanol
(EtOH) and methanol (MeOH) were degassed under N2
and stored for at least a couple of days over activated
molecular sieves (3 Å) following a standard procedure
[38]. Thin layer chromatography (TLC) was performed
with aluminum sheets pre-coated with Merck silica gel
60 F254. Detection was achieved by visualization under
UV light. Flash column chromatography was employed
on silica gel 60, 0.04–0.06 mm (Scharlau). Solvents were
evaporated under reduced pressure using a rotary evaporator. 2’-Hydroxyacetophenone (98%) was obtained from
AK Scientific, α-terpinene and 3,4,5-trimethoxybenzaldehyde (98%) from Sigma-Aldrich, 3,4-dimethoxybenzaldehyde (99%), 4-acetamidobenzaldehyde (98%),
3,5-dimethoxybenzaldehyde (98%), 2,6-difluorobenzaldehyde (98%), 4-trifluoromethylbenzaldehyde (98%),
and sodium (99.8%) from Acros Organics. Sodium
methoxide (≥97%) was purchased from Fluka, sodium
hydroxide (mini pearls AR), sodium sulfate (anhydrous
granular AR), ammonium chloride (AR), molecular sieve
3 Å, acetic acid and sodium acetate (AR) were from ECP,
hydrochloric acid (37%) from RCI, ruthenium(III) chloride hydrate (99%) from Precious Metals Online, TLC
silica gel 60 F254 from Merck, flash chromatography silica
gel 60, 0.04–0.06 mm, and sodium chloride (reagent
grade) from Scharlau. Bis[(ƞ6-p-cymene)dichloridoru- 10.1515/medr-2015-0001
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26
M. Kubanik, et al.
thenium(II)] was synthesized as described in the literature [39].
2.2 Physical Measurements
NMR spectra were recorded on Bruker DRX 400 MHz NMR
spectrometers at ambient temperature, unless otherwise
indicated. 1H and 13C{1H} chemical shifts are reported vs
SiMe4 and were determined by reference to the residual 1H
and 13C{1H} solvent peaks. In addition to 1H and 13C{1H} NMR
data, some compounds were analyzed via multinuclear 2D
(1H−1H COSY, 1H−13C HSQC, and HMBC) NMR spectroscopic
experiments, allowing unambiguous assignments of
characteristic resonances. Melting points were measured
in capillary tubes using a SMP30 Stuart Scientific Melting
Point Apparatus. Elemental analyses for all compounds
were performed at the Campbell Microanalytical
Laboratory, The University of Otago. High resolution mass
spectra were recorded on a Bruker microOTOF-Q mass
spectrometer in positive ion electrospray ionization (ESI)
mode. X-ray diffraction measurements of single crystals
were carried out on a Siemens SMART diffractometer with
a CCD area detector using graphite monochromated Mo-Kα
radiation (λ = 0.71073 A). The data was processed using
the SHELX2013 software packages [40]. All non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were
inserted at calculated positions and refined with a riding
model or without restrictions. Molecular structures were
visualized using Mercury 3.5.1.
2.3 Synthesis
General procedure for 3-hydroxyflavone synthesis 1a–1f
2’-Hydroxyacetophenone (1.0 eq) was added to a
suspension of the respective aldehyde (1.0 eq) in
ethanol and aqueous NaOH (5 M, 4.3 eq). The mixture
was stirred overnight at room temperature. Afterwards,
the reaction mixture was cooled on ice, and aqueous
acetic acid (30%) was added until the mixture was
acidic. The mixture was stirred for an additional 30 min
at 0°C, and the 2’-hydroxychalcone was collected by
filtration. Hydrogen peroxide (30%, 2.2 eq) was then
added to an ice-cold suspension of the chalcone in
ethanol (approx. 100 mL) and NaOH (5 M, 2.0 eq). The
mixture was allowed to warm to room temperature and
was stirred overnight. The mixture was acidified with
1 M HCl until acidic, and the formed precipitate was
collected by filtration. Recrystallization from methanol
afforded the flavonols.
3-Hydroxy-2-(3,4-dimethoxyphenyl)-4H-chromen-4-one
(1a). The reaction was performed according to the general
procedure by using 3,4-dimethoxybenzaldehyde (2.44 g) to
afford 1a as yellow powder. (1.84 g, 42%); m.p. 182–186°C.
1
H NMR (400.13 MHz, d6-DMSO): δ = 3.86 (s, 6H, -OCH3),
7.17 (d, 3J(H5’,H6’) = 9 Hz, 1H, H5’), 7.47–7.51 (m, 1H, H6),
7.78–7.80 (m, 2H, H2’/H6’), 7.83 (d, 3J(H7,H8) = 2 Hz, 1H,
H8), 7.89 (dd, 3J(H6/H8,H7) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H,
H7), 8.11 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5),
9.46 (s, 1H, OH) ppm. 13C{1H} NMR (100.6 MHz, d6-DMSO):
δ = 55.7 (-OMe), 55.6 (-OMe), 111 (C5’), 111.5 (C2’),
118.4 (C8), 121.3 (C4a), 121.5 (C6’), 123.6 (C1’), 124.4 (C5),
124.7 (C6), 133.4 (C7), 138.3 (C3), 145.4 (C2), 148.4 (C3’),
150.3 (C4’), 154.4 (C8a), 172.6 (C4) ppm. MS (ESI+): m/z
321.0738 [M + Na]+ (mex = 321.0722). Elemental Analysis
Calculated for C17H14O5·0.125 H2O: C 67.94, H 4.78%. Found:
C 67.98, H 4.78%.
3-Hydroxy-2-(3,5-dimethoxyphenyl)-4H-chromen-4-one
(1b). The reaction was performed according to the general
procedure by using 3,5-dimethoxybenzaldehyde (1.67 g)
to afford 1b as yellow powder. (1.60 g, 53%); m.p. 160–
163°C. 1H NMR (400.13 MHz, d6-DMSO): δ = 3.84 (s, 6H,
-OCH3), 6.69 (dd, 3J(H2’/H6’,H4’) = 8 Hz, 1H, H4’), 7.41 (d,
4
J(H2’/H6’,H4’) = 2 Hz, 2H, H2’/H6’), 7.48–7.53 (m, 1H, H6),
7.81 (d, 3J(H7,H8) = 8 Hz, 1H, H8), 7.82–7.86 (m, 1H, H7),
8.12 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5), 9.46
(s, 1H, OH) ppm. 13C{1H} NMR (100.6 MHz, d6-DMSO):
δ = 55.4 (-OMe), 101.5 (C4’), 106 (C2’/C6’), 118.4 (C8),
121.1 (C4a), 124.5 (C6), 124.7 (C5), 132.9 (C1’), 133.7 (C7),
139.3 (C3), 144.6 (C2), 154.5 (C3’/C5’), 160.4 (8a), 173
(C4) ppm. MS (ESI+): m/z 321.0732 [M + Na]+ (mex = 321.0739).
Elemental Analysis Calculated for C17H14O5·0.2 H2O:
C 67.63, H 4.81%. Found: C 67.99, H 5.31%.
3-Hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
(1c). The reaction was performed according to the general
procedure by using 3,4,5-trimethoxybenzaldehyde (2.88 g)
to afford 1c as yellow powder. (3.14 g, 65%); m.p. 179–181°C.
1
H NMR (400.13 MHz, d6-DMSO): δ = 3.77 (s, 3H, CH3), 3.88
(s, 6H, CH3), 7.48–7.53 (m, 1H, H6), 7.57–7.63 (m, 2H, H7/
H8), 7.79–7.84 (m, 2H, H2’/H6’), 8.12 (dd, 3J(H5,H6) = 8 Hz,
4
J(H5,H7) = 2 Hz, 1H, H5), 9.59 (s, 1H, OH) ppm. 13C{1H}
NMR (100.6 MHz, d6-DMSO): δ = 56.0 (CH3), 60.2 (CH3),
105.6 (C2’/C6’), 118.5 (C5), 121.2 (C8), 124.5 (C4a), 124.7 (C6),
126.5 (C7), 133.5 (C1’), 138.8 (C2), 139.2 (C4’), 144.9 (C8a),
152.7 (C3), 154.4 (C3’/C5’), 172.8 (C4) ppm. MS (ESI+): m/z
351.0844 [M + Na]+ (mex = 351.0846). Elemental Analysis
Calculated for C18H16O6·0.125 H2O: C 65.40, H 4.95%. Found:
C 65.38, H 5.15%.
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Expanding on the Structural Diversity of Flavone-Derived RutheniumII(ƞ6-arene) Anticancer Agents
3-Hydroxy-2-(2,6-difluorophenyl)-4H-chromen-4-one (1d).
The reaction was performed according to the general
procedure by using 2,6-difluorobenzaldehyde (2.09 g) to
afford 1d as milky powder (2.09 g, 55%); m.p. 210–213°C.
1
H NMR (400.13 MHz, d6-DMSO): δ = 7.33 (dd, 3J(H3’/
H5’,H4’) = 8 Hz, 2H, H3’/H5’), 7.52 (dd, 3J(H5/H7,H6) =
8 Hz, 1H, H6), 7.70–7.76 (m, 2H, H4’, H8), 7.83–7.86 (m, 1H,
H7), 8.18 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5),
9.69 (s, 1H, OH) ppm. 13C{1H} NMR (100.6 MHz, d6-DMSO):
δ = 111.9 (C3’/C5’), 112.2 (C8), 118.4 (C4a), 121.5 (C6), 124.9
(C5), 125.1 (C1’), 134.1 (C4’), 138.2 (C7), 140.9 (C3), 155.2 (C2’/
C6’), 158.4 (C8a), 160.9 (C2), 172.6 (C4) ppm. MS (ESI+): m/z
297.0329 [M + Na]+ (mex = 297.0339). Elemental Analysis
Calculated for C15H8F2O3·0.5 H2O: C 63.61, H 3.20%. Found:
C 63.69, H 2.95%.
3 - H y d r o x y -2 - ( 4 - ( t r i f l u o r o m e t h y l ) p h e n y l ) - 4 H chromen-4-one (1e). The reaction was performed
according to the general procedure by using
4-trifluoromethylbenzaldehyde (2.58 g) to afford 1e
as yellow powder (3.60 g, 80%); m.p. 181–184°C. 1H
NMR (400.13 MHz, d6-DMSO): δ = 7.48–7.52 (m, 1H, H6),
7.82–7.86 (m, 2H, H3/H5), 7.95 (d, 3J(H2’/H6’,H3’/H5’) =
8 Hz, 2H, H3’/H5’), 8.15 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7)
= 2 Hz, 1H, H5), 8.45 (d, 3J(H2’/H6’,H3’/H5’)= 8 Hz, 2H,
H2’/H6’), 10.04 (s, 1H, OH) ppm. 13C{1H} NMR (100.6 MHz,
d6-DMSO): δ = 118.5 (C8), 121.3 (C4a), 124.7 (CF3),
124.8 (C6), 125.4 (C5), 128.2 (C3’/C5’), 129.2 (C4’),
129.5 (C1’), 134.1 (C2’/C6’), 135.3 (C7), 140.1 (C3),
143.3 (C2), 154.6 (C8a), 173.2 (C4) ppm. MS (ESI+): m/z
329.0397 [M + Na]+ (mex = 329.0401). Elemental Analysis
Calculated for C16H9F3O3: C 62.75, H 2.96%. Found: C
62.91, H 2.95%.
3-Hydroxy-2-(4-acetomidophenyl)-4H-chromen-4-one (1f).
The reaction was performed according to the general
procedure by using 4-acetomidobenzaldehyde (1.63 g) to
afford 1f as dark yellow crystals. (2.18 g, 74%); m.p. 260–
262°C. 1H NMR (400.13 MHz, d6-DMSO): δ = 2.09 (s, 3H,
CH3), 7.47–7.53 (m, 1H, H7), 7.74–7.82 (m, 4H, H6, H8, H3’/
H5’), 8.12 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5),
8.20 (d, 3J(H2’/H6’,H3’/H5’) = 8 Hz, 2H, H2’/H6’), 9.51 (s,
1H, OH), 10.22 (s, 1H, NH) ppm. 13C{1H} NMR (100.6 MHz,
d6-DMSO): δ = 24.1 (CH3), 118.3 (C8), 118.5 (C3’/C5’), 121.3
(C8a), 124.5 (C6), 124.7 (C5), 125.6 (C1’), 128.4 (C2’/C6’),
133.4 (C7), 138.5 (C3), 140.7 (C4’), 145.3 (C2), 154.5 (C4a),
168.7 (C=O), 172.7 (C4) ppm. MS (ESI+): m/z 318.0747 [M +
Na]+ (mex = 318.0742). Elemental Analysis Calculated for
C17H13NO4·0.4 H2O: C 67.50, H 4.60, N 4.63%. Found: C 67.63,
H 4.52, N 4.70%.
27
General procedure for the synthesis of [(η6-p-cymene)RuII]
complexes 2a–2f
[(η6-p-cymene)RuCl2]2 (50 mg, 0.90 eq) was added to a
solution of the respective 3-hydroxyflavone 1a–1f (1.0
eq) and sodium methoxide (11 mg, 1.1 eq) in dry methanol
(10 mL). The reaction mixture was stirred at room
temperature under nitrogen atmosphere for 2–8hours.
Afterwards the reaction mixture was concentrated
under reduced pressure, the residue dissolved in
dichloromethane, and filtered and precipitated
with n-hexane. Pure compound was obtained by
recrystallization from methanol. Single crystals for X-ray
diffraction analysis were obtained by crystallization from
chloroform/n-hexane.
Chlorido[3-(oxo-κO)-2-(3,4-dimethoxyphenyl)-chromen-4onato-κO](η6-p-cymene)ruthenium(II) (2a). The synthesis
was performed according to the general procedure using
1a (54 mg) to afford a red solid (86 mg, 84%). m.p. 197°C
(decomp.). 1H NMR (400.13 MHz, CDCl3): δ = 1.41 (dd,
3
J(He,Hf) = 7 Hz, 3J(He,Hf) = 7 Hz, 6H, Hf), 2.40 (s, 3H,
Hg), 2.89–2.99 (m, 1H, He), 3.96 (s, 3H, -OMe), 4.00 (s, 6H,
-OMe), 5.33–5.38 (m, 2H, Hb), 5.62–5.66 (m, 2H, Hc), 6.97
(d, 3J(H5’,H6’) = 8 Hz, 1H, H5’), 7.30−7.34 (m, 1H, H6), 7.53
(d, 3J(H7,H8) = 8 Hz, 1H, H8), 7.55−7.60 (m, 1H, H7), 8.19
(dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5), 8.19–8.23
(m, 2H, H2’/H6’) ppm. 13C{1H} NMR (100.6 MHz, CDCl3):
δ = 18.7 (Cg), 22.5 (Cf), 31.3 (Ce), 55.9 (-OMe), 78.1 (Cb), 80.7
(Cc), 95.5 (Ca), 98.8 (Cd), 110.5 (C5’), 110.8 (C2’), 117.7 (C8),
120.1 (C4a), 121.3 (C6’), 123.9 (C1’), 124.5 (C5), 125.5 (C6),
132.2 (C7), 148.5 (C3), 149.7 (C2), 150.3 (C3’), 153.6 (C4’),
153.8 (C8a), 182.5 (C4) ppm. MS (ESI+): m/z 533.0925 [M –
Cl]+ (mex = 533.0902). Elemental Analysis Calculated for
C27H27ClO5Ru·1.3 CHCl3: C 47.00, H 3.94%. Found: C 46.73,
H 3.97%.
Chlorido[3-(oxo-κO)-2-(3,5-dimethoxyphenyl)-chromen-4onato-κO](η6-p-cymene)ruthenium(II) (2b). The synthesis
was performed according to the general procedure using
1b (54 mg) to afford a red solid (75 mg, 73%). m.p. 201°C
(decomp.). 1H NMR (400.13 MHz, CDCl3): δ = 1.42 (dd,
3
J(He,Hf) = 7 Hz, 3J(He,Hf) = 7 Hz, 6H, Hf), 2.40 (s, 3H, Hg),
2.90–3.00 (m, 1H, He), 3.88 (s, 6H, -OMe), 5.33–5.38 (m, 2H,
Hb), 5.62–5.68 (d, 2H, Hc), 6.53 (dd, 3J(H2’/H6’,H4’) = 8 Hz,
1H, H4’), 7.28−7.35 (m, 1H, H6), 7.53 (d, 3J(H7,H8) = 8 Hz, 1H,
H8), 7.56−7.63 (m, 1H, H7), 7.80 (d, 4J(H2’/H6’,H4’) = 2 Hz,
2H, H2’/H6’), 8.20 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz,
1H, H5) ppm. 13C{1H} NMR (100.6 MHz, CDCl3): δ = 18.6
(Cg), 22.5 (Cf), 31.3 (Ce), 55.5 (d, -OMe), 78.2 (Cb), 80.8 (Cc),
95.5 (Ca), 98.8 (Cd), 102.0 (C4’), 105.5 (C2’/C6’), 117.9 (C8),
119.9 (C4a), 124.0 (C6), 124.6 (C5), 132.7 (C1’), 134.2 (C7),
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M. Kubanik, et al.
153.4 (C2), 154.8 (C3’/C5’), 160.5 (C8a), 183.5 (C4) ppm. MS
(ESI+): m/z 533.0902 [M – Cl]+ (mex = 533.0902). Elemental
Analysis Calculated for C27H27ClO5Ru·0.25 H2O: C 56.64, H
4.84%. Found: C 56.38, H 4.69%.
Chlorido[3-(oxo-κO)-2-(3,4,5-trimethoxyphenyl)-chromen4-onato-κO](η6-p-cymene) ruthenium(II) (2c). The
synthesis was performed according to the general
procedure using 1c (60 mg) to afford a deep red solid
(70 mg, 70%); m.p. 220°C (decomp.). 1H NMR (400.13 MHz,
CDCl3): δ = 1.42 (dd, 3J(He,Hf) = 7 Hz, 3J(He,Hf) = 7 Hz, 6H,
Hf), 2.40 (s, 3H, Hg), 2.90–3.01 (m, 1H, He), 3.92 (s, 3H,
-OMe), 3.98 (s, 6H, -OMe), 5.33–5.38 (m, 2H, Hb), 5.60–
5.66 (m, 2H, Hc), 7.30−7.35 (m, 1H, H7), 7.54 (d, 3J(H7,H8)
= 8 Hz, 1H, H8), 7.56−7.61 (m, 1H, H6), 7.89 (s, 2H, H2’/H6’),
8.19 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5) ppm.
13
C{1H} NMR (100.6 MHz, CDCl3): δ = 18.6 (Cg), 22.5 (Cf),
31.4 (Ce), 56.2 (-OMe), 61.0 (-OMe), 78.3 (Cb), 80.7 (Cc), 95.2
(Ca), 98.7 (Cd), 105.1 (C2’/C6’), 117.7 (C5), 120.0 (C8), 124.1
(C4a), 124.6 (C6), 127.9 (C7), 132.5 (C1’), 139.6 (C2), 149.1
(C8a), 152.9 (C3), 153.7 (C3’), 154.3 (C5’), 183.1 (C4) ppm. MS
(ESI+): m/z 563.1032 [M – Cl]+ (mex = 563.1007). Elemental
Analysis Calculated for C28H29ClO6Ru·CHCl3: C 48.55,
H 4.21%. Found: C 48.37, H 4.39%.
Chlorido[3-(oxo-κO)-2-(2,6-difluorophenyl)-chromen-4onato-κO](η6-p-cymene)ruthenium(II) (2d). The synthesis
was performed according to the general procedure using
1d (50 mg) to afford a red solid (74 mg, 75%). m.p. 237°C
(decomp.). 1H NMR (400.13 MHz, CDCl3): δ = 1.35 (dd,
3
J(He,Hf) = 7 Hz, 3J(He,Hf) = 7 Hz, 6H, Hf), 2.35 (s, 3H, Hg),
2.85–2.96 (m, 1H, He), 5.33–5.38 (m, 2H, Hb), 5.59–5.63 (m,
2H, Hc), 6.92–6.98 (m, 2H, H3’/H5’), 7.31–7.34 (m, 1H, H6),
7.37–7.43 (m, 1H, H4’), 7.45–7.50 (m, 1H, H8), 7.57–7.63 (m,
1H, H7), 8.24 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H,
H5) ppm. 13C{1H} NMR (100.6 MHz, CDCl3): δ = 18.6 (Cg),
22.4 (Cf), 31.0 (Ce), 77.5 (Cb), 80.5 (Cc), 96.7 (Ca), 99.7 (Cd),
111.6 (C3’/C5’), 111.8 (C8), 120.2 (C4a), 124.1 (C6), 124.8 (C5),
131.4 (C4’), 133.9 (C7), 141.5 (C3), 155.0 (C8a), 155.1 (C1’),
159.8 (C6’), 162.4 (C2’), 183.8 (C4) ppm. MS (ESI+): m/z
509.0515 [M – Cl]+ (mex = 509.0502). Elemental Analysis
Calculated for C25H21ClF2O3Ru·0.9CHCl3: C 47.76, H 3.39%.
Found: C 47.96, H 3.44%.
Chlorido[3-(oxo -κO)-2-(4-trifluorometh ylphen yl)chromen-4-onato-κO](η6-p-cymene)ruthenium(II)
(2e).
The synthesis was performed according to the general
procedure using 1e (56 mg) to afford a red solid (81 mg,
75%). m.p. 240°C (decomp.). 1H NMR (400.13 MHz, CDCl3):
δ = 1.37–1.46 (m, 6H, Hf), 2.41 (s, 3H, Hg), 2.95–3.05 (m,
1H, He), 5.35–5.43 (m, 2H, Hb), 5.62–5.71 (m, 2H, Hc),
7.31–7.36 (m, 1H, H6), 7.53–7.57 (m, 1H, H8), 7.60–7.66 (m,
1H, H7), 7.70 (d, 3J(H2’/H6’,H3’/H5’) = 8 Hz, 2H, H3’/H5’),
8.21 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5), 8.69
(d, 3J(H2’/H6’,H3’/H5’) = 8 Hz, 2H, H2’/H6’) ppm. 13C{1H}
NMR (100.6 MHz, CDCl3): δ = 18.7 (Cg), 22.4 (Cf), 31.3 (Ce),
78.0 (Cb), 81.0 (Cc), 96.0 (Ca), 99.1 (Cd), 117.9 (C8), 120.0
(C4a), 124.3 (CF3), 124.8 (C6), 125.1 (C5), 127.1 (C3’/C5’), 129.7
(C4’), 130.0 (C1’), 130.3 (C2’), 133.2 (C6’), 135.9 (C7), 147.0
(C3), 154.2 (C2), 155.3 (C8a), 184.2 (C4) ppm. MS (ESI+): m/z
541.0588 [M – Cl]+ (mex = 541.0564). Elemental Analysis
Calculated for C26H22ClF3O3Ru·0.75 H2O: C 52.98, H 4.02%.
Found: C 52.84, H 3.71%.
Chlorido[3-(oxo-κO)-2-(4-acetomidophenyl)-chromen-4onato-κO](η6-p-cymene)ruthenium(II) (2f). The synthesis
was performed according to the general procedure using
1f (54 mg) to afford a red solid (56 mg, 55%). m.p. 270°C
(decomp.). 1H NMR (400.13 MHz, CDCl3): δ = 1.41 (dd,
3
J(He,Hf) = 7 Hz, 3J(He,Hf) = 7 Hz, 6H, Hf), 1.97 (s, 3H,
CH3), 2.41 (s, 3H, Hg), 2.88–2.98 (m, 1H, He), 5.34–5.38 (m,
2H, Hb), 5.62–5.66 (m, 2H, Hc), 7.30–7.37 (m, 2H, H3’/H5’),
7.50–7.63 (m, 3H, H6,H7,H8), 7.72 (brs, 1H, NH), 8.18 (dd,
3J(H5,H6) = 8 Hz, 4J(H5,H7) = 2 Hz, 1H, H5), 8.44 (d, 3J(H2’/
H6’,H3’/H5’) = 8 Hz, 2H, H2’/H6’) ppm. MS (ESI+): m/z
530.0916 [M – Cl]+ (mex = 530.0905). Elemental Analysis
Calculated for C27H26ClNO4Ru·1.75 H2O: C 54.36, H 4.98,
N 2.35 %. Found: C 54.23, H 4.61%, N 2.40%.
2.4 In vitro anticancer activity
Cell lines and culture conditions. CH1 cells (identified via
STR profiling as PA-1 ovarian teratocarcinoma cells by
Multiplexion, Heidelberg, Germany; see also [41]) were
obtained 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, Department
of Medicine I, Medical University of Vienna, Austria).
Cells were grown in 75 cm² culture flasks as adherent
monolayer cultures in Minimal Essential Medium (MEM)
supplemented with 10% heat-inactivated fetal calf serum,
1 mM sodium pyruvate, 4 mM l-glutamine and 1% nonessential amino acids (100×). Cultures were maintained at
37°C in a humidified atmosphere containing 95% air and
5% CO2.
MTT assay conditions. Cytotoxicity was determined
by the colorimetric MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) microculture assay. For
this purpose, cells were harvested from culture flasks by
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Expanding on the Structural Diversity of Flavone-Derived RutheniumII(ƞ6-arene) Anticancer Agents
trypsinization and seeded in 100 µL aliquots into 96–well
microculture plates. 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 untreated controls throughout the experiment. Cells
were allowed to settle and resume exponential growth in
drug-free complete culture medium for 24 h. Stocks of the
test compounds in DMSO were appropriately diluted in
complete culture medium such that the maximum DMSO
content did not exceed 1%. The dilution was added in
100 µL aliquots to the microcultures and cells were exposed
to the test compounds for 96 hours. At the end of exposure,
all media were replaced by 100 µL/well RPMI1640 culture
medium (supplemented with 10% heat-inactivated fetal
bovine serum) plus 20 µL/well MTT solution in phosphatebuffered 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 DMSO
per well. Optical densities at 550 nm were measured with
a microplate reader, using a reference wavelength of
690 nm to correct for unspecific absorption. The quantity
of viable cells was expressed relative to untreated control
microcultures, 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.
29
of the compound in DMSO, for example by diluting 2.5 µL
of the DMSO stock with another 2.5 µL of DMSO, and
this solution was then added to 495 µL of medium. This
process was repeated until no precipitation was observed.
3 Results and Discussion
3-Hydroxyflavones (3-HFs) have shown remarkable
activity in medicinal applications, and the modification
of 3-hydroxyflavones has widened the bioavailability.
In order to make them suitable to coordinate to metal
centers, a series of substituted 3-hydroxyflavones was
synthesized. A Claisen-Schmidt condensation of various
substituted benzaldehydes and 2’-hydroxyacetophenone
in the presence of aqueous sodium hydroxide gave the
corresponding chalcones (Scheme 1). A subsequent AlgarFlynn-Oyamada reaction with alkaline hydrogen peroxide
produced the substituted 3-HFs 1a–f in yields of 25–80%
[43].
2.5 Lipophilicity
ChemBioDrawUltra 12.0 and software tools from
Molinspiration (http://www.molinspiration.com) and
VCCLAB (Virtual Computational Chemistry Laboratory,
http://www.vcclab.org, 2005) were used to estimate the
compounds’ lipophilicity based on calculated logarithmic
octanol−water partition coefficients (clogP) for the ligands
1a–1f [42]. The ligands were chosen for the calculations
since the Ru(η6-p-cymene)Cl moiety is present in all
complexes.
2.6 Solubility
DMSO stock solutions of the compounds were used to
determine the solubility in α-MEM medium, supplemented
with 5% fetal calf serum (FCS), by adding 1 vol% of the
stock solution to an appropriate amount of medium. In a
standard experiment, 2.5 µL of the DMSO stock was added
to 247.5 µL of medium. If precipitation was observed, the
experiment was repeated with a decreased concentration
Scheme 1. Synthesis of 3-hydroxyflavonol ligands and the respective
Ru(cym)Cl complexes.
To
obtain
the
ruthenium
complexes
2a–2f,
3-hydroxyflavones 1a–1f (1.0 eq) and sodium methoxide
(1.1 eq) were dissolved in dry methanol and stirred for
10 min (Scheme 1). This results in deprotonation of the
3-HF and allows reaction with dimeric [(η6-p-cymene)
RuCl2]2 (0.90 eq) dimer, under cleavage of the chloride
bridge to yield neutral [Ru(cym)Cl] (cym = η6-p-cymene)
complexes (yield: 55–85%).
All ligands were characterized by melting point, 1H
NMR and 13C{1H} NMR spectroscopy in d6-DMSO. New
ligands were further analyzed by ESI-MS, and elemental
analysis.
The 1H NMR spectra featured the hydroxyl protons
generally around 9.10–9.40 ppm [16]. The signals of the
phenolic protons from 3-HFs are slightly shifted, due to
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30
M. Kubanik, et al.
the different strength electronic effects of the substituents
on the phenyl rings. The protons of the methyl group
of ligand 1f appear at 2.09 ppm (singlet), whereas the
protons from the methoxy group of ligands 1a–c give
a singlet in the range of 3.80–3.90 ppm. This is due to
the presence of the electronegative oxygen atom which
results in a downfield shift of the methyl protons. 13C{1H}
NMR spectroscopy shows the C=O carbon signal generally
around 173 ppm for all ligands. The methyl group of 1f
appears at 24.1 ppm, which is in the typical range of alkyl
groups. Signals for the methoxy carbon atoms of 1a–c
are typically located around 56 ppm. Fluoro-substituted
ligands (1d, 1e) showed additional coupling to the C1’,
C2’/C6, C3’/C5’ and C4’ carbon atoms.
The complexes were characterized by melting point,
1
H and 13C{1H} NMR spectroscopy, mass spectrometry,
elemental analysis and single crystal X-ray diffraction
analysis. Complexes 2a, 2d and 2e contained CHCl3 as
determined by elemental analysis which may contribute
to their cytotoxicity. However, 2b was the most cytotoxic
compound in the series (see below). Some characteristic
features for 3-HF ligands and their complexes were
unequivocally identified by 1D NMR spectroscopy in CDCl3.
The chemical shifts of the complexes are very close to that
of the corresponding ligand. However, due to the electronwithdrawing effect induced by the ruthenium center, the
signals appear slightly down-field in the spectra of the
complexes as compared to free ligands. The protons of the
two methyl groups of cymene (Hf) give rise to two doublets
in the 1H NMR spectra at ca. 1.4 ppm, each integrating for
3 protons, whereas the protons of the methyl group Hg are
found at ca. 2.4 ppm (singlet). The proton He resonates at
ca. 3.0 ppm. The proton signals of Hb and Hc of the arene
region are observed in two regions (ca. 5.4 and 5.6 ppm)
as multiplets, with a total integral of four protons. In
the 13C{1H} NMR spectra, the two methyl carbons Cf
resonate at ca. 23 ppm, and Cg is found at 19 ppm. Both
of these signals are in the typical range of primary alkyl
groups. Ce is detected at ca. 31 ppm, while both aromatic
cymene carbons Cb and Cc appear at ca. 78 and 81 ppm,
respectively. The signals for the two quaternary carbon
atoms Ca and Cd were assigned in the 13C{1H} NMR spectra
with the aid of HMBC spectra.
Single crystals of 2b–2e suitable for X-ray diffraction
analysis were obtained by crystallization from chloroform/
n-hexane (Figure 2). The enantiomeric mixtures of all the
Ru(II) compounds crystallized in the pseudo-tetrahedral
“piano-stool” configuration with a π-bound p-cymene
Figure 2. ORTEP diagram of one of two enantiomers found in the molecular structures of 2b–2e. The thermal ellipsoids were set at 50%
probability and solvent molecules in the structures of 2c and 2d were deleted for clarity.
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Expanding on the Structural Diversity of Flavone-Derived RutheniumII(ƞ6-arene) Anticancer Agents
ring forming the seat through coordination in η6 fashion to
the Ru center and the ligands act as the legs. Complex 2b
crystallized in the triclinic centrosymmetric space group
P-1, whereas the other three compounds crystallized in
monoclinic cells with the space groups P21/c (2c), C2/c
(2d) and P21/n (2e) (Table 1).
The 3-hydroxyflavone ligand binds bidentately to the
ruthenium center, forming a non-planar, envelope-like fivemembered cycle. The meta and para substituted phenyl rings
of the flavone ligands show in the X-ray structures small
torsion angles, whereas the ortho substitution of complex 2d
results in a twisted phenyl ring with a torsion angle of 60.22°
Table 1. Details of collected X-ray data for complexes 2b–2e
2b
2c
C27H27ClO5Ru
C28H29ClO6Ru ∙ 3CHCl3 C25H21ClF2O3Ru ∙ 0.5CHCl3
C26H22ClF3O3Ru
1402549
1402550
1402551
1402552
Molecular weight (g mol )
568.01
598.05
543.95
575.96
Temperature (K)
100(2)
99(2)
101(2)
100(2)
Wavelength (Å)
0.71073
0.71073
0.71073
0.71073
Crystal system
Triclinic
Monoclinic
Monoclinic
Monoclinic
Space group
P-1
P21/c
C2/c
P21/n
a (Å)
8.9186(5)
15.4715(10)
22.9901(9)
9.7631(4)
b (Å)
12.0214(7)
17.6062(11)
11.9033(5)
10.3130(5)
c (Å)
13.1440(8)
15.0190(9)
19.5172(8)
22.9814(10)
α (°)
99.220(4)
90
90
90
β (°)
108.583(3)
111.494(3)
113.802(2)
101.528(2)
γ (°)
110.975(3)
90
90
90
Volume (Å3)
1185.82(12)
3806.6(4)
4886.8(4)
2267.25(18)
Z
2
4
8
4
Calculated density (g cm-3)
1.591
1.668
1.479
1.687
Absorption coefficient (mm-1)
0.811
1.155
0.789
0.861
F(000)
580
1920
2192
1160
Crystal size (mm × mm × mm)
0.38 × 0.28 × 0.20
0.4 × 0.4 × 0.3
0.38 × 0.10 × 0.05
0.22 × 0.20 × 0.18
2θ (min, max) (°)
1.72, 27.94
1.83, 28.11
1.94, 27.88
1.81, 27.88
Limiting indices
-11 ≤ h ≤ 11,
-15 ≤ k ≤ 15,
-17 ≤ l ≤ 17
-20 ≤ h ≤ 20,
-23 ≤ k ≤ 23,
-19 ≤ l ≤ 19
-30 ≤ h ≤ 30,
-15 ≤ k ≤ 15,
-19 ≤ l ≤ 25
-12 ≤ h ≤ 12,
-13 ≤ k ≤ 13,
-30 ≤ l ≤ 30
Reflections collected / unique
Completeness to theta = 25.24
27231 / 5614 [R(int) = 83035 / 9183 [R(int) = 29504 / 5804 [R(int) =
0.0559]
0.0800]
0.0627]
98.8%
99.9%
99.8%
28277 / 5376 [R(int) =
0.0404]
100.0%
Data / restraints / parameters
5614 / 0 / 328
9183 / 0 / 471
5804 / 0 / 324
5376 / 0 / 330
Goodness-of-fit on F2 a)
1.083
1.061
1.068
1.030
R1 = 0.0356,
wR2 = 0.1047
R1 = 0.0470,
wR2 = 0.1097
1.539 and -0.621
R1 = 0.0548,
wR2 = 0.1509
R1 = 0.0711,
wR2 = 0.1677
3.677 and -1.766
R1 = 0.0374,
wR2 = 0.0823
R1 = 0.0553,
wR2 = 0.0908
0.890 and -0.997
R1 = 0.0251,
wR2 = 0.0609
R1 = 0.0298,
wR2 = 0.0637
0.579 and -0.343
Formula
CCDC Nr.
-1
Final R indices [I>2σ(I)]
b)
R indices (all data)
Largest diff. peak and hole (eÅ-3)
31
2d
2e
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32
M. Kubanik, et al.
(Table 2). Due to the small torsion angles of the meta and para
substituted flavones, π-stacking of the aromatic ligands was
observed with the other enantiomer present in the crystal
structure (Figure 3) All four compounds show Ru–O bond
lengths in the ranges of 2.101(3)–2.1197(19) and 2.067(2)–
2.093(2) Å for the Ru–O2 and Ru–O3 bonds, respectively,
which is in good agreement with the literature. Table 2
summarizes selected features of the Ru complexes 2b–2e in
comparison to reported Ru(arene)(flavonol) complexes [16].
The majority of the bond lengths and angles involving the
ruthenium center and oxygen atoms and chlorido ligands are
very similar as well as the distance between the ruthenium
center to centroidcymene. The most significant differences were
observed for the torsion angles at C(3)–C(2)–C(1’)–C(2’),
where different substituents induce rotation of the phenyl
ring around the C(2)–C(1’) bond. This results in almost
co-planar configuration for phenyl rings with substituents
in meta and para positions, while substituents in ortho
position induce a twist towards an orthogonal position to the
flavonoid backbone of the ligand.
Lipophilicity is regarded as a supportive factor for
high anticancer activity at least in an in vitro setting, as it
influences the ability of a drug to penetrate through the cell
membrane and accumulate in cells. In order to compare the
lipophilic properties of the organoruthenium compounds
developed, we compared the clogP values of the flavonol
ligands, as the Ru(cym)Cl component is present in all studied
compounds and the coordination geometries around the Ru
center, as apparent from the X-ray diffraction studies, are
very similar. We used three widely used software solutions,
i.e., ChemDraw, Molinspiration and ALOGPS 2.1, for the
calculation of clogP values of 1a–1f and compared the
values to that of the p-fluoro-substituted 3-hydroxyflavone
(Aflavone) as the ligand in the most cytotoxic Ru(cym)(HF)Cl
complex A (Figure 1). While the values obtained from the
different software solutions are varying, the trends are very
similar in each set of calculations with 1e being the most
lipophilic compound (and the least soluble in 1% DMSO/
medium mixture; Table 4) and 1f being the least lipophilic
in the series while Aflavone shows intermediate clogP values.
Table 2. Selected bond lengths (Å) and angles (°) for complexes 2b–2e as compared to the reported ruthenium-p-cymene complex with
2-chloro substituted flavone ligand [16].
Compound
Bond Lengths (Å)
Ru–O(2)
Ru–O(3)
Ru–Cl
Ru–centroidarene
Torsion Angles (°)
C(3)–C(2)–C(1’)–C(2’)
Bond Angles (°)
O(2)–Ru–O(3)
O(2)–Ru–Cl
O(3)–Ru–Cl
2b
2c
2d
2e
2-chloro
2.103(2)
2.067(2)
2.4161(10)
1.634
2.101(3)
2.077(3)
2.4202(9)
1.646
2.1197(19)
2.093(2)
2.4159(8)
1.643
2.1193(13)
2.0730(12)
2.4196(5)
1.643
2.134(2)
2.087(2)
2.410(7)
1.647
-6.46(9)
4.42(10)
60.52(8)
4.82(7)
48.41(4)
79.16(10)
86.85(7)
83.73(7)
78.53(10)
85.59(8)
83.71(8)
78.81(8)
84.98(6)
84.80(6)
78.10(5)
85.13(4)
84.06(4)
78.08(6)
84.77(5)
84.31(5)
Figure 3. π stacking interaction between the planar 3-hydroxyflavone backbones of the two enantiomers of 2d.
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Expanding on the Structural Diversity of Flavone-Derived RutheniumII(ƞ6-arene) Anticancer Agents
Obviously, this is not a quantitative comparison but rather
a relative measure to estimate the compounds’ potential
to accumulate in cells, which in turn has an impact on the
cytotoxicity (Table 4).
Table 3. Comparison of the clogP values obtained from ChemDraw,
Molinspiration and ALOGPS 2.1.
Compound
clogP
ChemDraw
Molinspiration
ALOGPS 2.1
1a
2.71
3.092
2.20
1b
3.06
3.487
2.19
1c
2.35
3.077
2.08
1d
1e
1f
Aflavone a
3.36
3.96
2.09
2.35
3.677
4.341
2.664
3.609
2.32
3.13
2.04
2.38
In order to estimate the tumor-inhibiting potential of the
ligands 1a–1f and their complexes 2a–2f, the cytotoxicity
in human ovarian teratocarcinoma [CH1(PA-1)], colon
adenocarcinoma (SW480) and non-small cell lung (A549)
cancer cells was determined and compared to A and the
respective HF ligand Aflavone (Table 4). All complexes, except
2e, show activity in the low to intermediate µM range and
33
most of them are nearly as active as A. With the exception
of 1e, the free ligands are not active in A549 and SW480
cells, but very potent against the ovarian teratocarcinoma
CH1(PA-1) cells, which are generally more chemosensitive
than the ABC-transporter-overexpressing SW480 and
A549 cells. Compound 2e was not sufficiently soluble to be
included in the cytotoxicity assays. This can be explained
by the lipophilic nature of its HF ligand 1e, which however,
was the most potent ligand in the in vitro anticancer
activity assays. The activity of the methoxy-substituted
flavones seems to be determined by the position of the
OMe groups rather than by the lipophilicity (compare
Tables 3 and 4). This is suggested by the fact that 2a with
a methoxy-substituted phenyl ring in positions 3’ and 4’
is slightly less active than the 3’,5’-dimethoxy derivative
2b. Also, increasing the lipophilicity by addition of a third
methoxy group in position 4’ led to decreased cytotoxicity.
Addition of a second fluoro substituent in para position as
in 2d seems to result in even higher IC50 values compared
with monosubstituted examples [16,17]. In general, the
cytotoxicity of these compounds seems to be increased by
ortho and meta substitution, but decreased by introducing
halogens in ortho position. Considering the data obtained
in X-ray diffraction studies, this may be related to the
ortho-substituent inducing a twist of the phenyl ring
around the C(2)–C(1’) bond (Figure 2).
Table 4. IC50 values ± standard deviation for 1a–1e and 2a–2e in A549, SW480 and CH1(PA-1) cells with an exposure of 96 h and the experimentally determined solubility in 1%DMSO/medium.
Compound
methoxy
acetamide
a
b
IC50 / µM
1% DMSO/medium
A549
SW480
CH1(PA-1)
1a
330
> 25
> 25
2.1 ± 0.2
2a
423
18 ± 2
8.7 ± 0.8
2.2 ± 0.5
1b
122
> 25
> 25
1.4 ± 0.2
2b
358
9.0 ± 0.5
4.5 ± 0.2
1.5 ± 0.2
a
111
> 25
8.6 ± 1.5
2.0 ± 0.2
2c a
142
23 ± 5
9.7 ± 1.9
2.5 ± 0.3
1d
92
> 25
> 25
18 ± 1
2d
353
55 ± 15
20 ± 4
5.1 ± 0.8
1e
31
6.5 ± 1.5
2.6 ± 0.2
1.0 ± 0.1
2e
n.s.
–
–
–
Aflavone b
217
7.9 ± 1.2
3.7 ± 0.4
0.60 ± 0.10
A
b
257
9.5 ± 0.5
3.8 ± 0.5
0.86 ± 0.06
1f
2f
103
187
> 25
21 ± 1
> 25
18 ± 1
7.2 ± 0.3
5.3 ± 0.5
1c
fluoro
Solubility /µM
Values should be taken with caution because precipitation was observed within 24 h at concentrations > 3 µM (1c) and > 0.8 µM (2c);
taken from ref. [17], n.s. = not sufficiently soluble in 1%DMSO/medium.
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34
M. Kubanik, et al.
4 Conclusions
[2]
Half-sandwich
ruthenium-arene
compounds
of
3-hydroxyflavones have demonstrated potential in the
development as anticancer agents. In order to expand our
knowledge about their structure-activity relationships,
we have introduced substituents at the phenyl ring
of the HF ligand and prepared the respective air and
light stable Ru(cym)Cl complexes in good yields. As
the lipophilicity is an important parameter in drug
development, we calculated the clogP values for the
3-hydroxyflavones, and also determined the solubility
of the compounds. While the ligands show low aqueous
solubility, complexation increases the solubility up to
4-times, with the exception of 2e. While the stability is
not reported for the prepared compounds, the aquation
of structurally-related compounds has been reported to be
rapid which is, however, not expected to impact behavior
in aqueous solution significantly. The cytotoxicity of
the new compounds in human ovarian teratocarcinoma
[CH1(PA-1)], colon adenocarcinoma (SW480) and nonsmall cell lung (A549) cancer cells was compared to that
of one of the most active compounds in the series so far,
i.e., the p-fluoro derivative A. The compounds inhibited
cell proliferation with IC50 values in the low µM range
but were slightly less active than A. Both the ligands
and the complexes were particularly potent in CH1(PA-1)
cells, while in the other cell lines the complexes were
significantly more active than the ligands. There was
however, no clear-cut relationship between lipophilicity
and cytotoxicity. By analysing the molecular structures of
complexes 2b–e and comparing them to other compounds
reported earlier, it appears that the substitution pattern at
the phenyl ring has a substantial impact on cytotoxicity.
This may be related to twisting of the phenyl ring out of
the plane which may cause different interactions with
biological targets while factors such as lipophilicity and
electronic effects seem to play a minor role.
Acknowledgements: We thank the University of
Auckland, Genesis Oncology Trust (GOT-1263-RPG), and
COST CM1105 for financial support. The authors are
grateful to Tanya Groutso for collecting the single crystal
X-ray diffraction data and Nick Lloyd for ESI-MS analyses.
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