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3-Hydroxyflavones vs. 3-hydroxyquinolinones: structure–activity relationships and stability studies on RuII(arene) anticancer complexes with biologically active ligands

03 Apr 2013-Dalton Transactions (The Royal Society of Chemistry)-Vol. 42, Iss: 17, pp 6193-6202

TL;DR: To expand knowledge about the structure-activity relationships and to determine the impact of lipophilicity of the arene ligand and of the hydrolysis rate on anticancer activity, a series of novel 3-hydroxyflavone derived Ru(II)(η(6)-arene) complexes were synthesised.

AbstractRuII(η6-arene) complexes, especially with bioactive ligands, are considered to be very promising compounds for anticancer drug design. We have shown recently that RuII(η6-p-cymene) complexes with 3-hydroxyflavone ligands exhibit very high in vitro cytotoxic activities correlating with a strong inhibition of topoisomerase IIα. In order to expand our knowledge about the structure–activity relationships and to determine the impact of lipophilicity of the arene ligand and of the hydrolysis rate on anticancer activity, a series of novel 3-hydroxyflavone derived RuII(η6-arene) complexes were synthesised. Furthermore, the impact of the heteroatom in the bioactive ligand backbone was studied by comparing the cytotoxic activity of RuII(η6-p-cymene) complexes of 3-hydroxyquinolinone ligands with that of their 3-hydroxyflavone analogues. To better understand the behaviour of these RuII complexes in aqueous solution, the stability constants and pKa values for complexes and the corresponding ligands were determined. Furthermore, the interaction with the DNA model 5′-GMP and with a series of amino acids was studied in order to identify potential biological target structures.

Summary (2 min read)

Introduction

  • Ruthenium complexes represent a promising class of metal-based 20 chemotherapeutics.
  • In the course of ruthenium anticancer drug development programmes, organometallic and especially half-sandwich RuII(η6-arene) complexes have more and more demonstrated their 35 potential.
  • 4,14-16 Tethering ethacrynic acid to the arene ligand of RAPTA led to a compound capable of overcoming the glutathione transferase drug resistance mechanism of tumour cells and triggered several biological 55 pathways involving either endonuclease G, caspases or c-Jun Nterminal kinase.
  • More recently, the authors have demonstrated that RuII(cym) (cym = η6-p-cymene) complexes of 3-hydroxyflavones are potent tumour cell growth inhibitors.
  • These properties are compared with those of structurally related 3-hydroxyquinolinone complexes featuring a 15 nitrogen atom in the heterocyclic ligand.

Synthesis

  • -derived b, the hydrolysis of , Supporting corresponding complex 2 were investigated and stability and dissociation constants were studied.
  • Therefore, the pKa value spectra of the ligand species (HL, L on the basis of deconvoluted spect λmax values of both the protonated and the deprotonated forms of 90 ligand b are identical to those of the unsubstituted 3 hydroxyflavone a.25.
  • Its p due to the electron withdrawing effect of the fluoro substituent.
  • The appearance of the two emission bands indicates t pathways for deactivation of the excited state.

Solution equilibria of [RuII(cym)X3]

  • This was studied in 20% (w/w) DMSO/H2O by UVvis spectrophotometric titrations .
  • Similar but not identical speciation was found in pure aqueous solution.
  • 65 Based on the increased proton dissociation constants of ligand b and maltol (see above), higher stability constants of [RuII(cym)(L)X]n+ are expected in 20% (w/w) DMSO/H2O than in pure aqueous solution.
  • As also found for the maltolato complex, partial hydrolysis and dissociation of 2 are probable at 15 physiological pH.

Reactivity towards biomolecules

  • For 12’ a reaction with Cys was observed but the compound also decomposed partly within 24 h.
  • Two minutes after addition only traces of coordinated glycine (two doublets at approximately δ = 3.1 ppm)11 were observed in 12’ and only after 18 h in 13’, indicating again higher stability of the 3-hydroxyquinolinone 65 complexes concerning reactions with amino acids.
  • In vitro anticancer activity The cytotoxic activity of the RuII complexes was determined in the human cancer cell lines CH1 (ovarian 80 carcinoma), SW480 (colon carcinoma) and A549 (non-small cell lung carcinoma) by means of the colorimetric MTT assay (Table 1).
  • The latter compound class showed a strong dependence of 10 cytotoxicity on the coordinated arene.

Conclusions

  • The authors have extended the series of compounds by varying the arene and halido ligands to learn about their influence on the biological activity, as well as compared the 3-hydroxyflavone complexes to quinolinone analogues in terms of cytotoxicity and reactivity towards 45 biomolecules.
  • Considering stability data and in vitro anticancer activity, 3- hydroxyflavones seem to be a well-suited ligand system for anticancer RuII(cym) complexes and those represent a 70 promising compound class for further drug design.
  • The authors thank the 75 University of Vienna, the Austrian Science Fund (FWF), the Johanna Mahlke geb.
  • Obermann Foundation, and COST D39 for financial support.

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3-Hydroxyflavones vs. 3-Hydroxyquinolinones: Structure-Activity
Relationships and Stability Studies on Ru
II
(arene) Anticancer
Complexes with Biologically Active Ligands
Andrea Kurzwernhart,
a,b
Wolfgang Kandioller,
a,b
Éva A. Enyedy,
c
Maria Novak,
a
Michael A. Jakupec,
a,b
Bernhard K. Keppler
a,b
and Christian G. Hartinger
a,b,d,
*
5
Ru
II
(η
6
-arene) complexes, especially with bioactive ligands, are considered as very promising compounds
for anticancer drug design. We have shown recently that Ru
II
(η
6
-p-cymene) complexes with 3-
hydroxyflavone ligands exhibit very high in vitro cytotoxic activities correlating with a strong inhibition
of topoisomerase IIα. In order to expand the structure-activity relationships and to determine the impact
10
of lipophilicity of the arene ligand and of the hydrolysis rate on anticancer activity, a series of novel 3-
hydroxyflavone derived Ru
II
(η
6
-arene) complexes were synthesised. Furthermore, the impact of the
heteroatom in the bioactive ligand backbone was studied by comparing the cytotoxic activity of Ru
II
(η
6
-p-
cymene) complexes of 3-hydroxyquinolinone ligands with that of their 3-hydroxyflavone analogues. To
better understand the behaviour of these Ru
II
complexes in aqueous solution, the stability constants and
15
pK
a
values for complexes and corresponding ligands were determined. Furthermore, the interaction with
the DNA model 5’-GMP and with a series of amino acids was studied in order to elucidate potential
biological target structures.
Introduction
Ruthenium complexes represent a promising class of metal-based
20
chemotherapeutics. The octahedral geometry of ruthenium, its
binding ability to plasma proteins and the number of possible
oxidation states in biological environments, makes it well suitable
for drug design.
1
By now, several ruthenium complexes have
shown interesting properties in vivo and a generally lower
25
toxicity than for platinum drugs was observed.
2
Two Ru
III
compounds, namely [ImH][trans-Ru(DMSO)(Im)Cl
4
] (NAMI-A,
Im = imidazole) and [IndH][trans-Ru(Ind)
2
Cl
4
] (KP1019, Ind =
indazole) (Chart 1) are currently undergoing clinical trials with
very promising results.
3-5
30
Chart 1. Structures of Ru anticancer agents.
In the course of ruthenium anticancer drug development
programmes, organometallic and especially half-sandwich
Ru
II
(η
6
-arene) complexes have more and more demonstrated their
35
potential.
6-10
Their hydrophobic arene ligand is thought to
facilitate the diffusion through the lipophilic cell membrane.
11
The three remaining Ru coordination sites can be filled with
various mono-, bi- or tridentate ligands, which offers a number of
possibilities to modulate biological and pharmacological
40
properties by proper ligand selection.
12
Important examples for
this substance type are Ru
II
(arene) complexes of bidentate
ethylenediamine, such as RM175 (Chart 1), and the RAPTA-type
compounds containing the monodentate 1,3,5-triaza-7-
phosphatricyclo[3.3.1.1]decane (pta) ligand. RM175 binds to
45
DNA either covalently via the N7 of guanine or non-covalently
by intercalation of the arene, leading to cell death by modulation
of the p53-p21-bax pathway.
2,13
As opposed to this, the RAPTA
compounds have very different chemical and biological
properties. RAPTA-T (Chart 1) is selectively activated in the
50
hypoxic conditions of solid tumours and is capable of inhibiting
metastasis both in vitro and in vivo.
4,14-16
Tethering ethacrynic
acid to the arene ligand of RAPTA led to a compound capable of
overcoming the glutathione transferase drug resistance
mechanism of tumour cells and triggered several biological
55
pathways involving either endonuclease G, caspases or c-Jun N-
terminal kinase.
17
This is an example of linking a biological
active molecule to a metal centre and modulating thereby its
biological properties. Other related approaches involve
Ru
II
(arene) compounds with ligand systems that resemble the
60
kinase inhibitor staurosporine
18
or complexes of paullones, which
are cyclin-dependent kinase (CDK) and glycogen synthase
kinase-3 inhibitors.
19
More recently, we have demonstrated that
Ru
II
(cym) (cym = η
6
-p-cymene) complexes of 3-hydroxyflavones
are potent tumour cell growth inhibitors.
20
65
3-Hydroxyflavones belong to the naturally occurring class of
flavonoids which are polyphenols of plants, fruits and vegetables.
They are well known for their beneficial effects on health due to
their antioxidant and antiradical, antiinflammatory, antiviral and
anticarcinogenic properties. These effects are caused primarily by
70

the
scavenging of free radicals by the flavonoid structure and
interaction with a number of enzymes.
21
Flavonoids are capable
of forming stable chelate complexes with
a broad range of
ions, which have alre
ady shown biological activity in the
treatment of diseases like AIDS, diabetes
mellitus, some genetic
5
diseases and also cancer.
22
The Ru
II
(
cym
hydroxyflavones were found to exhibit
not only
anticancer activity in human cancer cell lines
human topoisomerase IIα activity,
which
cytotoxic potency.
20
10
In order to study the impact of
the nature of the
halogenido ligands on the stability and
cytotoxic activity,
of Ru
II
(arene)X complexes with 3-
hydroxyflavones has been
synthesised.
These properties are compared with those of
structurally related 3-
hydroxyquinolinone complexes featuring a
15
nitrogen atom in the heterocyclic ligand. These studies are
complemented with UV/vis and flu
orescence spectroscopy
experiments to gain information on the
stability
the hydrolysis products and ligand systems.
Results and discussion
20
Synthesis
Within the course of a project to prepare 3
complexes, we have reported
the synthesis of
cymene comple
xes with various substituted 3
and the influence of the
substitution pattern
25
substituent on the in vitro
anticancer activity
order to extend the structure-
activity relationships (SAR
series of Ru
II
(arene) complexes with 3-
hydroxyflavone
3-hydroxyquinolinones d and e was
synthesised
of the ligands with sodium methoxide and
30
with the respective bis[dihalido(η
6
-
([RuX
2
(arene)]
2
; η
6
-arene = cym, toluene,
biphen
I), yielding complexes 1-13
in good to very good yields
(Scheme 1). The compounds were
characterised
analytical methods (see experimental part
) and
35
over one year
though exposed to sunlight and air
Scheme 1. Synthesis of Ru
II
(η
6
-arene)
complexes
the hydrolysis products 1’-13’
in aqueous solution
Behaviour and stability in aqueous soluti
on
In order to study the properties of the 3-
hydroxyflavone
40
Ru
II
(cym) complexes in aqueous solution, the proton dissociation
process of the p-fluoro-
substituted ligand
[Ru
II
(cym)X
3
]
n+
(n = –1 2; X = Cl
, H
2
O
or DMSO
Information)
and the complex formation process of the
scavenging of free radicals by the flavonoid structure and
by
Flavonoids are capable
a broad range of
metal
ady shown biological activity in the
mellitus, some genetic
cym
) complexes of 3-
not only
high in vitro
anticancer activity in human cancer cell lines
but also inhibit
which
correlates to their
the nature of the
arene and
cytotoxic activity,
a series
hydroxyflavones has been
These properties are compared with those of
hydroxyquinolinone complexes featuring a
nitrogen atom in the heterocyclic ligand. These studies are
orescence spectroscopy
stability
and pK
a
values of
Within the course of a project to prepare 3
-hydroxy-4-pyrone
the synthesis of
ruthenium(II)-
xes with various substituted 3
-hydroxyflavones
substitution pattern
and the nature of the
anticancer activity
was studied.
20,23
In
activity relationships (SAR
s), a
hydroxyflavone
s ac and
synthesised
by deprotonation
of the ligands with sodium methoxide and
subsequent reaction
-
arene)ruthenium(II)]
biphen
yl; X = Cl, Br,
in good to very good yields
characterised
with standard
) and
were stable for
though exposed to sunlight and air
.
complexes
1-13 and formation of
in aqueous solution
.
a
from refs. 20,23.
on
hydroxyflavone
-derived
(cym) complexes in aqueous solution, the proton dissociation
substituted ligand
b, the hydrolysis of
or DMSO
, Supporting
and the complex formation process of the
corresponding complex 2
were investigated and stability and
dissociation constants were studi
ed.
Proton dissociation process of ligand
The proton dissociation constant (p
determined by UV-
vis spectrophotometry in 20% (w/w) dimethyl
sulfoxide (DMSO)/H
2
O because of the poor solubility of the
75
ligand and its complex in pure water. Since flavonoids may suffer
from photodegradation,
24
spectra were measured at various pH
values employing the
batch technique instead of continuous
titrations. This guarantees
minimal UV exposure and helps
avoiding
photolysis, especially at high pH
80
pH-
dependent spectra of the ligand s
increasing pH values
. The deprotonation (HL
attributed to the hydroxyl
functional group
bathochromic shift of the λ
max
and a small increase in intensity.
The isosbestic point is constant
at 366 n
85
at higher pH most probably due to the photodegradation of the
ligand. Therefore, the pK
a
value
spectra of the ligand species (HL, L
on the basis of deconvoluted
spect
λ
max
values of both the protonated and the deprotonated forms of
90
ligand b
are identical to those of the unsubstituted 3
hydroxyflavone a.
25
However,
its p
due to the electron withdrawing effect of the fluoro substituent.
The pK
a
of the structurally relate
0.01), which was also determined under the same conditions,
95
in the same range as that of b.
26
Fig. 1. UV-vis spectra of ligand b
at various pH values (a) and calculated
individual absorbance spectra of the HL and L
80
10
-5
M; T = 25˚C; I = 0.20 M
(KCl); 20% (w/w) DMSO/H
342 nm (λ
342 nm
= 10210 mol
−1
dm
3
10755 mol
1
In addition, the proton
dissociation process of
85
was monitored by
fluorimetry (Fig
concentration
. The ligand excitation maximum was found at 342
nm and the emission spectrum represents two maxima at 504 and
411 nm. The appearance of the two emission bands indicates t
pathways for deactivation of the excited state. The pH
90
were investigated and stability and
ed.
Proton dissociation process of ligand
b
The proton dissociation constant (p
K
a
) of ligand b was
vis spectrophotometry in 20% (w/w) dimethyl
O because of the poor solubility of the
ligand and its complex in pure water. Since flavonoids may suffer
spectra were measured at various pH
batch technique instead of continuous
minimal UV exposure and helps
photolysis, especially at high pH
values (Figure 1). The
dependent spectra of the ligand s
how characteristic changes at
. The deprotonation (HL
L
+ H
+
)
functional group
is accompanied by a
and a small increase in intensity.
at 366 n
m up to pH 10.4 but shifts
at higher pH most probably due to the photodegradation of the
of 8.70 ± 0.01 and the individual
spectra of the ligand species (HL, L
; Figure 1b) were calculated
spect
ra recorded at pH < 10.4. The
values of both the protonated and the deprotonated forms of
are identical to those of the unsubstituted 3
-
its p
K
a
value is significantly lower
due to the electron withdrawing effect of the fluoro substituent.
of the structurally relate
d pyrone ligand maltol (8.76 ±
0.01), which was also determined under the same conditions,
was
at various pH values (a) and calculated
individual absorbance spectra of the HL and L
species (b) {c
ligand
= 5 ×
(KCl); 20% (w/w) DMSO/H
2
O}. HL: λ
max
=
3
cm
−1
); L
: λ
max
= 402 nm (λ
402 nm
=
1
dm
3
cm
−1
).
dissociation process of
b in aqueous phase
uorimetry (Fig
ure S1a) at much lower
. The ligand excitation maximum was found at 342
nm and the emission spectrum represents two maxima at 504 and
411 nm. The appearance of the two emission bands indicates t
wo
pathways for deactivation of the excited state. The pH

dependence of the fluorescence emission spectra shows that the
emission intensity is strongly sensitive to the pH, and
deprotonation results in a significant decrease of the intensity.
From the spectral changes in water a pK
a
value of 8.30 ± 0.09
was obtained, which verifies the pK
a
determined in 20% (w/w)
5
DMSO/H
2
O and which is again in the same range as the pK
a
of
maltol in aqueous solution (8.44).
25
Solution equilibria of [Ru
II
(cym)X
3
]
n+
and complex 2
In order to understand the behaviour of the flavonoid complex in 10
aqueous solution, the hydrolysis of [Ru
II
(cym)X
3
]
n+
(n = –1 2;
X = Cl
, H
2
O or DMSO) needed to be determined under the same
conditions. This was studied in 20% (w/w) DMSO/H
2
O by UV-
vis spectrophotometric titrations (Figure S2). Based on the
spectral changes, stability constants of the minor
15
[Ru
2
(cym)
2
(OH)
2
X
m
]
n+
(m = 1, 2) and the major
[Ru
2
(cym)
2
(OH)
3
]
+
dinuclear hydrolysis products were
determined as log
[(Ru(cym))
2
H
−2
]
2+
= −9.85 ± 0.06 and logβ
[Ru
2
(cym)
2
H
−3
]
+
= −15.11 ± 0.03, respectively (Supporting
Information). As the titrations were performed in the presence of
20
0.2 M KCl, these constants are regarded as conditional stability
constants. Similar but not identical speciation was found in pure
aqueous solution.
27
The presence of DMSO can suppress the
hydrolysis of [Ru
II
(cym)X
3
]
n+
which is then shifted to higher pH
values (Figure S2b).
25
The complex formation processes of the ruthenium(II)-cym
complex 2 were studied under the same conditions as for
[Ru
II
(cym)X
3
]
n+
(Figure 2a) and is compared to the maltol-
ruthenium(II)-cym system (Figure 2b).
11
The pH-dependent
spectral changes of the ruthenium(II)-cym-containing systems
30
(Figure 2c) compared to the free ligands reveal that the complex
formation starts pH > ~4 in both cases. The complex formation
results in a significant shift of the λ
max
values and this new band is
different from the bands belonging to the protonated and
deprotonated forms of the metal-free ligands. This band is
35
especially well-separated in the case of 2 (Figure 2a) (i.e. λ
max
of
complex: 436 nm, HL: 342 nm, L
: 402 nm). Analysis of changes
in the overlapping ligand and charge transfer (CT) bands shows
the exclusive formation of mononuclear species
[Ru
II
(cym)(L)X]
n+
with a 1:1 metal-to-ligand ratio. By
40
deconvolution of the UV-vis spectra (Figure S3), a stability
constant logβ ([Ru
II
(cym)(L)X]
n+
) = 7.13 ± 0.08 for 2 was
determined, which is in about the same range as the maltolato
complex (logβ = 7.04 ± 0.05).
At neutral and alkaline pH various parallel processes take
45
place, namely the complex [Ru
II
(cym)(L)X]
n+
starts to hydrolyse
forming the mixed hydroxido species [Ru
II
(cym)(L)(OH)] and to
dissociate giving the tris-hydroxido-bridged dinuclear species
[Ru
2
(cym)
2
(OH)
3
]
+
and the metal-free ligand (Figure 2d). The
dissociation of (O,O)-pyrone ligands such as maltol of mono-
50
ligand complexes is relatively slow.
28
However, in case of
flavonoid complexes, the photodegradation of the ligand is a
possible side reaction at pH > ~10. Due to these reasons the
deconvolution of the spectra becomes more difficult and stability
data of the [Ru
II
(cym)(L)(OH)] species could only be obtained
55
with lower accuracy as logβ = 0.3 ± 0.1 for 2 and 0.1 ± 0.1 for
maltol.
Fig. 2. (a) UV-vis spectra of 2 and (b) for comparison of a maltolato
Ru
II
(cym) complex at various pH values. (c) Absorbance values at 402
60
nm (●) and at 436 nm (○) for complex 2 and at 322 nm () and at 328 nm
() for the maltolato Ru
II
(cym) complex plotted against the pH value. (d)
Concentration distribution curves of the complex 2 {c
complex
= 5 × 10
-5
M
(8 × 10
-5
M in the case of maltol); T = 25 °C; I = 0.20 M (KCl); 20%
(w/w) DMSO/H
2
O; pH = 2.5–11.5}.
65
Based on the increased proton dissociation constants of ligand
b and maltol (see above), higher stability constants of
[Ru
II
(cym)(L)X]
n+
are expected in 20% (w/w) DMSO/H
2
O than
in pure aqueous solution. However, a logβ = 9.05 was reported
for the maltolato complex in water,
29
which is actually by two
70
orders of magnitude higher than the constant obtained in 20%
(w/w) DMSO/H
2
O mixture. DMSO complexes of Ru
II
are known
and DMSO coordination can suppress the formation of

[Ru
II
(cym)(L)X]
n+
complexes. The speciation and the stability of
2 and the maltolato complex show very strong similarities due to
similar metal binding sites of the ligands. The fluorescence
spectra of ligand b (Figure S1a) and complex 2 in aqueous
solution (Figure S1b) show similar features up to pH ~4. When
5
further increasing the pH, a band with high intensity at 448 nm
develops reaching a maximum at pH ~5 and decreasing upon
increasing pH. The appearance of this strong new band is most
probably related to the formation of [Ru
II
(cym)(L)X]
n+
, while the
formation of the mixed hydroxido species [Ru
II
(cym)(L)(OH)] is
10
accompanied by a considerable loss of intensity. Therefore, this
latter species seems to be much less fluorescent than
[Ru
II
(cym)(L)X]
n+
, but somewhat more fluorescent than the
metal-free ligand. As also found for the maltolato complex,
partial hydrolysis and dissociation of 2 are probable at
15
physiological pH.
Reactivity towards biomolecules
In aqueous solution, compounds 1-3,
20
5, 7, 9 and 11 are aquated
immediately to the charged aqua species 1’-3, 5’, 7’, 9’ and 11’,
which can further react with biomolecules. The solubility of 4, 6,
20
8 and 10 in aqueous solution limited investigations, however, due
to the structural similarity similar behavior is expectable. Several
Ru
II
(arene) complexes are known to bind to the DNA model
compound 5’-GMP and therefore are also able to form adducts
with DNA, which is a possible target for metal-based anticancer
25
agents.
1,2,11,30-33
Similarly, 5, 7, 9 and 11 show interactions with
5’-GMP, as observed in
1
H NMR spectroscopy studies. However,
due to their low solubility and even lower solubility of their 5’-
GMP adducts, the binding mode and stability of the adducts are
elusive.
30
The 3-hydroxyquinolinone-derived Ru
II
(arene) complexes 12
(Figure S4) and 13 show the same aquation behaviour, but
already 5 min after addition of D
2
O the first signs of the
hydrolysis side product [Ru
2
(η
6
-arene)
2
(OH)
3
]
+
were observed in
the
1
H NMR spectrum, which increased within 24 h. This side
35
product is thermodynamically stable and unreactive towards
nucleophiles.
7
Compounds 12 and 13 bind immediately to the N7
atom of 5’-GMP as indicated by an upfield shift of the H8 signal
of 5’-GMP from approximately δ = 8.1 to 7.6 ppm (Figure S5).
To gain more insight into possible interactions with proteins
40
and pharmacokinetic pathways, the reactions of the representative
hydrolysis products 1’, 12’ and 13’ with the amino acids
L
-
methionine,
L
-histidine,
L
-cysteine and glycine were investigated
(Figures S6–S12). The reactivity was found to be similar to
pyrone-derived Ru
II
(cym) complexes. All compounds reacted
45
immediately with Met and His by replacement of the aqua ligand
with the respective amino acid, which is coordinated to the Ru
II
centre via the sulphur atom or via the N1 or N3 atoms of the
imidazole moiety, respectively.
11
In the case of 1’, the ligand was
cleaved off and precipitated completely within 24 h. The same
50
behaviour was observed for the 3-hydroxyquinolinone-derived
complexes. However, after 24 h especially for 12’ still signals of
coordinated quinolinone ligands were visible. This may be due to
a slightly higher stability of the 3-hydroxyquinolinone complexes
towards reaction with amino acids. Addition of Cys led to
55
immediate decomposition of 1’ and to a lower extent of 13’. For
12’ a reaction with Cys was observed (Figure 3) but the
compound also decomposed partly within 24 h. In the case of
glycine, also differing behaviour between 3-hydroxyflavone and
quinolinone complexes was observed. Glycine reacted
60
immediately with 1’, whereas the reaction with 12’ and especially
13’ was significantly slower. Two minutes after addition only
traces of coordinated glycine (two doublets at approximately δ =
3.1 ppm)
11
were observed in 12’ and only after 18 h in 13’,
indicating again higher stability of the 3-hydroxyquinolinone
65
complexes concerning reactions with amino acids. However, the
cytotoxicity of 3-hydroxyflavone and quinolinone Ru
II
(cym)
complexes was similar (see below), although the MTT assay to
determine the IC
50
values is carried out in amino acid-containing
medium. This indicates that the reaction with amino acids does
70
not seem to significantly alter their in vitro anticancer potency,
most probably due to their higher lipophilicity which may result
in enhanced cellular uptake.
Fig. 3. Reaction mixtures of 1’ (a) and 12’ (b) with equimolar amounts of
L
-cysteine analysed by
1
H NMR spectroscopy after 5 min show
75
immediate decomposition of 1’ after addition of Cys, whereas minor
effects on the quinolinone signals of 12’ were observed.
In vitro anticancer activity
The cytotoxic activity of the Ru
II
(arene) complexes was
determined in the human cancer cell lines CH1 (ovarian
80
carcinoma), SW480 (colon carcinoma) and A549 (non-small cell
lung carcinoma) by means of the colorimetric MTT assay
(Table 1). Recently, we have shown that the type and especially
the position of the substituent on the phenyl ring of the ligand
have a crucial impact on their biological activity.
20
Meta- and
85
para-substitution led to more cytotoxic compounds, whereas
ortho-substituted or unsubstituted ligand structures showed less
in vitro potency (Table 1, compare compounds 2 and 3 with 1).
These data correlate well with the inhibition of topoisomerase IIα
activity. All synthesised complexes exhibit promising tumour-
90
inhibiting properties with IC
50
values in the low µM range, which
is very remarkable for Ru
II
(arene) complexes. In order to

determine the effect of the lipophilicity on the anticancer activity,
complexes bearing different arene ligands were synthesised. The
toluene derivatives 8 and 9 exhibit a similar activity to their
Ru
II
(cym) analogues 1 and 3, whereas the biphenyl complexes 10
and 11 are slightly less cytotoxic. Therefore, the influence of the
5
arene ligands seems to be of minor importance for this type of
compounds. The same activity pattern was observed for pyrone
and especially thiopyrone-derived Ru
II
(arene) complexes,
19
which
is in contrast to for example ethylenediamine complexes. The
latter compound class showed a strong dependence of
10
cytotoxicity on the coordinated arene. The change from benzene
to p-cymene to biphenyl resulted in a large increase of their
growth inhibitory activity in relation to an increasing size and
hydrophobicity.
34
It may be that the change in lipophilicity by the
modification of the arene ligand is too marginal to outperform the
15
contribution of the flavonoid ligand on the lipophilicity.
Furthermore, as already shown for analogous pyrone- and
thiopyrone Ru
II
(arene) derivatives and also for ethylenediamine
complexes, different halides as leaving groups show only little or
no impact on the antiproliferative activity (compare 1, 3, 47).
20
This can be explained by the quick aquation of the Ru centre,
leading to the same aqua products.
When changing from 3-hydroxyflavones to 3-
hydroxyquinolinones as ligands, no improvement of the in vitro
anticancer activity was observed. The quinolinone complexes 12
25
and 13 exhibit cytotoxic activities in the same range as 1. Also
variation of the unsubstituted 3-hydroxyquinolinone 12 to the 1-
methylated form in 13 showed no impact on the cytotoxic
activity, indicating that the backbone of the ligand rather than the
functional group seems to be crucial for the biological activity of
30
this type of Ru
II
(arene) complexes.
Table 1. In vitro anticancer activity of 113 in ovarian (CH1), colon
(SW480) and non-small cell lung carcinoma (A549) cell lines.
a
IC
50
[µM]
R Y X arene CH1 SW480 A549
1
b
H O Cl cym 2.1 ± 0.2 9.6 ± 1.5 20 ± 2
2
b
p-F O Cl cym 1.7 ± 0.4 7.9 ± 2.1 18 ± 1
3
b
p-Cl
O Cl cym 0.9 ± 0.1 3.8 ± 0.5 9.5 ± 0.5
4
H O Br cym 2.8 ± 0.4 12 ± 1 27 ± 3
5
p-Cl
O Br cym 0.9 ± 0.1 3.4 ± 0.4 7.9 ± 0.6
6
H O I cym 1.6 ± 0.2 9.6 ± 1.5 16 ± 1
7
p-Cl
O I cym 1.2 ± 0.3 4.7 ± 0.9 8.9 ± 0.8
8
H O Cl tol 3.2 ± 0.1 12 ± 3 19 ± 1
9
p-Cl
O Cl tol 0.9 ± 0.2 4.7 ± 0.6 7.8 ± 2.5
10
H O Cl biphen 5.5 ± 1.2 9.2 ± 1.9 28.3 ± 5.0
11
p-Cl
O Cl biphen 6.3 ± 1.1 21.1 ± 4.0 59.1 ± 1.1
12
H N-H Cl cym 4.0 ± 0.2 14 ± 1 17 ± 2
13
H N-CH
3
Cl cym 5.3 ± 0.2 12 ± 2 19 ± 1
a
IC
50
= 50% inhibitory concentration, 96 h exposure.
b
taken from refs.
20,23. tol = toluene, biphen = biphenyl.
35
Conclusions
Ru
II
(arene) complexes bearing biologically active ligand systems
exhibit very interesting features and promising properties for
anticancer drug design.
12
3-Hydroxyflavone-derived Ru
II
(arene)
complexes are potent cytotoxic agents with good correlation to
40
their topoisomerase IIα inhibitory activity.
26
We have extended
the series of compounds by varying the arene and halido ligands
to learn about their influence on the biological activity, as well as
compared the 3-hydroxyflavone complexes to quinolinone
analogues in terms of cytotoxicity and reactivity towards
45
biomolecules. All compounds exhibit in vitro anticancer activity
in the low µM range and showed interaction with the DNA model
compound 5’-GMP. Substitution of the arene and halido ligands
had only a minor effect on the cytotoxic activity. The 3-
hydroxyquinolinone analogues behave similarly to the flavones in
50
aqueous solutions and in anticancer activity assays, but are more
stable in presence of amino acids. Extensive solution phase
studies by NMR, UV-vis and fluorescence spectroscopy revealed
that the para-fluoro substituted 3-hydroxyflavone b [2-(4-
fluorophenyl)-3-hydroxy-4H-chromen-4-one] exhibits a proton
55
dissociation constant (pK
a
) of 8.70 ± 0.01 in 20% (w/w)
DMSO/H
2
O and of 8.30 ± 0.09 in aqueous solution. The complex
formation processes of the corresponding ruthenium(II)-cym
complex 2 starts at pH > ~4, forming mononuclear species
[Ru
II
(cym)(L)X]
n+
with a stability constant of logβ = 7.13 ± 0.08.
60
At pH 7, hydrolysis of [Ru
II
(cym)(L)X]
n+
leads to the mixed
hydroxido species [Ru
II
(cym)(L)(OH)] (logβ = 0.3 ± 0.1) and
partial dissociation giving the tris-hydroxido-bridged dinuclear
species [Ru
2
(cym)
2
(OH)
3
]
+
and the metal-free ligand. The
stability constants of the hydroxyflavone-derived ruthenium(II)-
65
cym compounds are therefore in the range of structurally-related
maltolato complexes.
Considering stability data and in vitro anticancer activity, 3-
hydroxyflavones seem to be a well-suited ligand system for
anticancer Ru
II
(cym)(chlorido) complexes and those represent a
70
promising compound class for further drug design.
This work was supported by the Hungarian Research Foundation
OTKA 103905 and É.A. Enyedy gratefully acknowledges the
financial support of J. Bolyai research fellowship. We thank the
75
University of Vienna, the Austrian Science Fund (FWF), the
Johanna Mahlke geb. Obermann Foundation, and COST D39 for
financial support. We gratefully acknowledge Filip Groznica for
doing parts of the synthetic work.
Experimental part
80
Materials and methods
All solvents were dried and distilled prior to use. All chemicals
were purchased from commercial suppliers and used without
further purification. Bis[(
6
-p-cymene)dichloridoruthenium(II)],
bis[dichlorido(η
6
-toluene)ruthenium(II)],
35
bis[(η
6
-
85
biphenyl)dichloridoruthenium(II)], bis[dibromido(η
6
-p-
cymene)ruthenium(II)], bis[(η
6
-p-
cymene)diiodidoruthenium(II)],
36
3-hydroxy-2-phenyl-4H-
chromen-4-one (a), 2-(4-fluorophenyl)-3-hydroxy-4H-chromen-
4-one (b), 2-(4-chlorophenyl)-3-hydroxy-4H-chromen-4-one (c),
90
[chlorido{3-(oxo-κO)-2-phenyl-chromen-4(1H)-onato-κO}(η
6
-p-
cymene)ruthenium(II)] (1), [chlorido{3-(oxo-κO)-2-(4-
fluorophenyl)-chromen-4(1H)-onato-κO}(η
6
-p-
cymene)ruthenium(II)] (2), [chlorido{3-(oxo-κO)-2-(4-
chlorophenyl)-chromen-4(1H)-onato-κO}(η
6
-p-
95
cymene)ruthenium(II)] (3),
23
3-hydroxy-2-phenyl-1H-quinolin-4-
one (d) and 3-hydroxy-1-methyl-2-phenyl-1H-quinolin-4-one
(e)
37,38
were synthesised according to literature procedures.

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TL;DR: The aim of this review, a summary of the putative biological actions of flavonoids, was to obtain a further understanding of the reported beneficial health effects of these substances.
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Related Papers (5)
Frequently Asked Questions (1)
Q1. What have the authors contributed in "3-hydroxyflavones vs. 3-hydroxyquinolinones: structure-activity relationships and stability studies on ru(arene) anticancer complexes with biologically active ligands" ?

The authors have shown recently that Ru ( η-p-cymene ) complexes with 3hydroxyflavone ligands exhibit very high in vitro cytotoxic activities correlating with a strong inhibition of topoisomerase IIα.