Gold(i) complexes of water-soluble diphos-type ligands: Synthesis, anticancer activity, apoptosis and thioredoxin reductase inhibition

Article abstract of DOI:10.1039/c1dt10368g

Gold(i) complexes of imidazole and thiazole-based diphos type ligands were prepared and their potential as chemotherapeutics investigated. Depending on the ligands employed and the reaction conditions complexes [L(AuCl)₂] and [L₂Au]X

Full text of DOI:10.1039/c1dt10368g

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Dalton
Transactions
Cite this: Dalton Trans., 2011, 40, 9212
www.rsc.org/dalton
PAPER
Gold(I) complexes of water-soluble diphos-type ligands: Synthesis, anticancer
activity, apoptosis and thioredoxin reductase inhibition†
Corinna Wetzel,a Peter. C. Kunz,*a Matthias U. Kassack,b Alexandra Hamacher,b Philip Bo¨hler,c Wim Watjen,c
Ingo Ott,d Riccardo Rubbianid and Bernhard Spinglere
Received 3rd March 2011, Accepted 9th June 2011
DOI: 10.1039/c1dt10368g
Gold(I) complexes of imidazole and thiazole-based diphos type ligands were prepared and their
potential as chemotherapeutics investigated. Depending on the ligands employed and the reaction
conditions complexes [L(AuCl)2] and [L2Au]X (X = Cl, PF6) are obtained. The ligands used are
diphosphanes with azoyl substituents R2P(CH2)2PR2 {R = 1-methylimidazol-2-yl (1),
1-methylbenzimidazol-2-yl (4), thiazol-2-yl (5) and benzthiazol-2-yl (6)} as well as the novel ligands
RPhP(CH2)2PRPh {R = 1-methylimidazol-2-yl (3)} and R2P(CH2)3PR2 {R = 1-methylimidazol-2-yl
(2)}. The cytotoxic activity of the complexes was assessed against three human cancer cell lines and a
rat hepatoma cell line and correlated to the lipophilicity of the compounds. The tetrahedral gold
complexes [(3)2Au]PF6 and [(5)2Au]PF6 with intermediate lipophilicity (logD7.4 = 0.21 and 0.25) showed
significant cytotoxic activity in different cell lines. Both compounds induce apoptosis and inhibit the
enzymes thioredoxin reductase and glutathione reductase.
that mitochondria play a critical role in the regulation of apoptosis,
making them an attractive target for the design of new anticancer
Introduction
drugs.6–8 Gold complexes have been investigated for potential
anti-tumour properties9,10 and gold(I) phosphane derivatives have
shown the most reproducible and significant activity in murine
tumour models in vivo.11 Many complexes of the general for-
mula [(R3P)Au(thiolate)] related to auranofin exhibit promising
antitumor potential, some even in the case of cisplatin resistant
cell lines.12–16 More recently, also tetrahedral gold(I) complexes
of chelating bisphosphanes have shown promising activity in
murine tumour models17 and some anticancer gold(I)–phosphane
complexes exhibit significant autophagy-inducing properties in
cancer cells.18
Since the authorization of cisplatin in 1978, the interest in and
development of metal-based drugs prospers consistently. Still,
cisplatin, cis-[Pt(NH3)2Cl2], and its analogs, especially oxaliplatin
and carboplatin, are basic chemotherapeutics in combination
therapy.1,2 The therapeutic effect of cisplatin is based on DNA
metallation and the formation of cisplatin adducts with DNA
is thought to trigger apoptosis.3,4 The major draw-back for
a successful chemotherapy is acquired resistance towards the
applied drug in the course of therapy.5 Resistance to a specific
chemotherapeutic drug can by circumvented by using drugs, which
address alternative cellular targets. There is much recent evidence
Gold(I) complexes like [(dppe)2Au]+ (dppe
=
1,2-bis-
(diphenylphosphino)ethane) and analogue compounds belong to
the group of delocalised lipophilic cations (DLCs),19–21 which
can pass cellular membranes and accumulate in mitochondria.7,22
The elevated membrane potential of cancerous cells can be used
for selective drug accumulation as it leads to increased uptake
and retention of lipophilic cations.23,24 Despite the promising
cytotoxicity profile of [(dppe)2Au]+, preclinical studies showed that
[(dppe)2Au]+ has severe adverse effects, e.g. cardiac, hepatic and
vascular toxicity, all correlated to the highly lipophilic nature of
the compound.25,26
In order to circumvent the lipophilicity related side effects, more
hydrophilic diphos complexes were developed.27,28 In the group
of Berners-Price diphos-type ligands were used where the phenyl
rings of dppe were replaced by pyridinyl substituents leading to
more selective compounds.29–33 It was shown that the balance
between lipophilicity and hydrophilicity of the gold compounds
aInstitut fu¨r Anorganische Chemie und Strukturchemie, Lehrstuhl I: Bioanor-
ganische Chemie und Katalyse, Heinrich-Heine-University Du¨sseldorf,
Universita¨tsstr. 1, D-40225, Du¨sseldorf, Germany. E-mail: peter.kunz@
uni-duesseldorf.de; Fax: (+) 49 211 8112287; Tel: +49 (0)211 8112873
bInstitut fu¨r Pharmazeutische und Medizinische Chemie, Heinrich-Heine-
University Du¨sseldorf, Universita¨tsstr. 1, D-40225, Du¨sseldorf, Germany.
E-mail: matthias.kassack@uni-duesseldorf.de; Fax: +49 (0)211 81-10801;
Tel: +49 (0)211 8114587
cInstitut fu¨r Toxikologie, Heinrich-Heine University Du¨sseldorf, Uni-
versita¨tsstr. 1, D-40225, Du¨sseldorf, Germany. E-mail: wim.waetjen@
uni-duesseldorf.de; Fax: +49 (0)211 81-13013; Tel: +49 (0)211 8113003
dInstitut fu¨r Pharmazeutische Chemie, Technische Universita¨t Braunschweig,
Beethovenstr. 55, D-38106, Braunschweig, Germany. E-mail: ingo.ott@
tu-bs.de; Fax: +49 (0)5313918456; Tel: +49 (0)5313912743
eAnorganisch-Chemisches Institut, Universita¨t Zu¨rich-Irchel, Winterthur-
erstr. 190, CH-8057, Zu¨rich, Switzerland. E-mail: spingler@aci.uzh.ch;
Fax: +41 (0)44 6356803; Tel: +41 (0)44 6354656
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1dt10368g
9212 | Dalton Trans., 2011, 40, 9212–9220
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
is a very important parameter in optimizing biodistribution,
activity and selectivity of the drugs. Especially the selectivity of
gold(I)bisphosphane drugs for cancer over normal tissue could
be improved by optimizing the lipophilic–hydrophilic balance of
the complexes by ligand design. Additionally, when compared
to gold(I) complexes with monodentate phosphane ligands like
auranofin these tetrahedral bischelated gold(I) complexes are
much more stable towards ligand exchange in the presence of
serum proteins, thiols and disulfides. Therefore, the rational design
of gold complex properties is critical for their future as potential
antitumor drugs.34,35
distilled 1-methyl-2-trimethylsilylimidazole to give the two novel
imidazole-based ligands 2 and 3 in good yields (Scheme 1).
A potential cellular target for anticancer metallo-drugs alterna-
tive to DNA, are the seleno-enzymes of the thioredoxin reductase
(TrxR) family, which play a crucial role in the regulation of the
cellular redox state.36–39 A cytosolic (TrxR1) and a mitochondrial
form (TrxR2) are known and reduce the 12 kDa disulfid protein
thioredoxin (Trx) to the corresponding dithiolic form.40 Inhibition
of the active site of TrxR is a common for cytotoxic metal-based
drugs.41–44 An increase in Trx and TrxR activities is correlated to
circumvention of apoptosis and acceleration of tumour growth,
while in turn efficient inhibition of TrxR leads to apoptosis of
cancer cells.
Recently we reported on the cytotoxic potency of gold(I)
chlorido complexes with water soluble imidazolylphosphanes.45
In this work we studied structural and biological properties of
a series of linear and tetrahedral coordinated gold(I) complexes
[(L)(AuCl)2] and [(L)2Au]X with imidazolyl- and thiazolyl-based
water soluble diphos-type ligands. The ligands are used to tune the
solubility and lipophilicity of the gold complexes, which, in turn,
have marked influence on their cytotoxic activity against different
cell lines.
Scheme 1 Synthesis of ligands 1–6.
The complexes [(L)(AuCl)2] and [(L)2Au]Cl were prepared by
reaction of stoichiometric quantities of the corresponding ligand
and [(tht)AuCl] (tht = tetrahydrothiophene) in dichloromethane
or chloroform (Scheme 2). Within this series, the tetrahedral
complexes [(4)2Au]Cl and [(6)2Au]Cl could not be prepared by this
procedure. The MALDI MS spectra of all complexes [(L)(AuCl)2]
show the signal for the ion [(L)Au2Cl]+ and the spectra of
all complexes [(L)2Au]Cl the ion [(L)2Au]+ as the basis peak,
respectively.
Results and discussion
Recently we have investigated synthetic routes towards
a
series of azoyl substituted diphosphane ligands and reported
on the preparation of compounds bis(di-1-methylimidazol-
2-ylphosphino)ethane
(1),
bis(di-1-methylbenzimidazol-2-
ylphosphino)ethane (4), bis(dithiazol-2-ylphosphino)ethane
(5) and bis(dibenzthiazol-2-ylphosphino)ethane (6).46 The
synthesis of 4, 5 and 6 involves reaction of the corresponding
heteroaromatic compound with an slight excess of n-BuLi followed
by Cl2P(CH2)2PCl2. The analogous imidazole-based compound 1
was synthesised by reaction of 1-methyl-2-trimethylsilylimidazole
with Cl2P(CH2)2PCl2. This procedure was applied in this work
to synthesise the analogous imidazolyl derivative of dppp
(1,3-bis(diphenylphosphino)propane), 1,3-bis(1-methylimidazol-
2-ylphosphono)propane (2), as well as the asymmetric ligand
Scheme 2 Synthesis of the gold(I) complexes [(L)(AuCl)2] and [(L)2Au]Cl.
1
The 1H and 31P{ H} NMR chemical shifts of the ligands and the
resulting complexes are summarised in Table 1 and are very similar
to those of the pyridinyl analogs published by Berners-Price et al.33
Upon coordination the 31P NMR resonance is shifted 30–
50 ppm towards lower field. The 31P NMR resonances of com-
plexes [(L)2Au]X with the dppe-type ligands are found at about
5
2 ppm to higher field compared to the [(L)(AuCl)2] complexes.
bis(1-methylimidazol-2-ylphenylphosphino)ethane
(3).
The
The corresponding complexes of the dppp-type ligand 2 show
different resonance at -10 ppm for [(2)(AuCl)2] and -37 ppm
for [(2)2Au]Cl. In the H NMR spectra the signal of the CH2-
protons of the ethylene bridge of ligands 1, 3 and 5 are shifted in
the corresponding complexes [(L)2Au]X and [(L)(AuCl)2]. Upon
coordination this signal is shifted slightly more towards lower field
in the complexes [(L)(AuCl)2] than in the corresponding complexes
[(L)2Au]X.
starting material Cl2P(CH2)3PCl2 was synthesised according to
the procedure published by Berven et al. which involves reaction
of 1,3-dibromopropane with triethylphosphite, subsequent
reduction with LiAlH4 and chlorination using triphosgene.47
1,2-Bis(chlorophenylphosphione)ethane, ClPhP(CH2)2PPhCl,
was prepared according a modified synthesis by Long and Jones.
Dppe was treated with lithium in tetrahydrofuran. PCl3 was
added in situ to the orange solution of LiPhP(CH2)2PPhLi to give
ClPhP(CH2)2PPhCl.48 The chlorophosphanes react with freshly
1
The asymmetric ligand 3 shows two signals in dmso-d6 for
1
the rac and meso isomers in the 31P{ H} spectrum at -38.3 and
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 9212–9220 | 9213
©

View Article Online
Table 1 1H and 31P NMR data for ligands (L) 1, 2, 3, 4, 5, 6 as well as their complexes [(L)(AuCl)2] and [(L)2Au]X
d(1H)
d/ppm
NCH3(1)
H4
H5
CH2(Ha)
CH2(Hb)
d(31P)
1a
3.67
3.79
3.44
3.57
3.73
3.57
3.75
7.27
7.20
7.32
7.10
7.19
7.19
7.10
7.34
7.60
7.43
7.23
7.57
7.38
7.30
2.62
3.28
3.02
2.59
3.08
2.77
2.08
2.35
2.77
—
—
—
1.63
2.07
1.78
—
-54a
-12b
-17b
-53a
-10b
-37b
-38.3
-38.5
5.5b
6.7b
4.2b
-45c
-6c
[(1)(AuCl)2]b
[(1)2Au]Cla
2a
[(2)(AuCl)2]b
[(2)2Au]Clb
3b
[(3)(AuCl)2]c
3.81
7.13
7.26
—
[(3)2Au]Cld
4c
3.05
3.72
4.14
—
—
—
—
—
2.58
3.09
3.66
2.73
3.32
3.09
3.01
3.77
—
—
—
—
—
—
—
—
e
e
—
—
7.58
8.32
7.67
—
—
—
8.06
8.32
7.89
—
[(4)(AuCl)2]c
5c
-23c
10b
[(5)(AuCl)2]b
[(5)2Au]Clc
6c
3c
—
—
-15c
16c
[(6)(AuCl)2]c
—
—
Measured ina MeOD-d4, b dmso-d6, c CDCl3 or d D2O. e The signals for the imidazolyl- and phenyl-protons overlap to give a multiplet.
1
-38.5 ppm and in the H NMR spectrum for the protons of the
–(CH2)2– bridge at 2.08 and 2.35 ppm, respectively. Reaction of
3 with 2 equivalents of [(tht)AuCl] yielded a 1 : 1.5 equilibrium
mixture of the rac- and meso-isomers of [(3)(AuCl)2] with signals
1
in 31P{ H} NMR spectrum in dmso-d6 at 6.7 and 5.5 ppm,
respectively. Possible equilibria between these compounds and
[(3)2Au]Cl has been investigated by NMR titrations of [(tht)AuCl]
with ligand 3 in CDCl3 and dmso-d6 (see ESI†). Such solvent-
dependent equilibria have been observed for many other diphos-
phane complexes.49,50 At low L : Au ratios (L : Au £ 2) [(3)(AuCl)2]
is the predominant species, whereas at high L : Au ratios only
[(3)2Au]Cl is observed (dP = 4.2 ppm). The rac- and meso-isomers
of [(3)(AuCl)2] could be separated conveniently by their different
solubility and stability in chloroform. The chloroform soluble
Fig. 1 Molecular structure of [(3)2Au]+ in [(3)2Au]PF6. Displacement
ellipsoids are drawn at the 50% level and and atoms are
drawn as capped sticks. H-atoms, minor components of the disordered
phenyl/methylimidazole groups, the counter ion and co-crystallised sol-
compound was identified as [(3)(AuCl)2]. Suitable crystals for
X-ray crystallography of this compound were obtained by slow
diffusion of c-pentane into a chloroform solution of [(3)(AuCl)2].
The structures of the complexes [(L)(AuCl)2] (L = 1, 3, 4, 6) and
[(3)2Au]PF6 were determined by single crystal X-ray diffraction.
In the molecular structure of [(3)2Au]PF6 the gold atom is
C
N
˚
vent molecules are omitted for clarity. Selected bond lengths [A] and angles
[◦]: Au1–P1 2.4301(9), Au1–P2 2.4197(9), Au1–P31 2.4130(9), Au1–P32
2.4198(9), P1–Au1–P2 84.94(3), P1–Au1–P31 120.26(3), P1–Au1–P32
127.30(4), P2–Au1–P31 121.54(3), P2–Au1–P32 122.04(3), P31–Au1–P32
85.44(3).
2
coordinated tetrahedral by the two bidentate ligands in a k P,P
fashion (Fig. 1). The metric parameters of [(3)2Au]PF6 are within
the range reported for other monomeric bis-chelated tetrahedral
[(L)2Au]+ complexes (Table 2).49 In this structure a phenyl and 1-
methylimidazole ring connected to the same phosphorous atom
are disordered in a 6 : 4 ratio. In one case, four of the imidazole
ring atoms overlap with four of the phenyl ring carbon atoms, in
the other case, all atoms are separated. Due to this disorder, not all
atoms could be refined anisotropically and none of the hydrogen
atoms were added.
by a phosphorus and chlorine atom (Fig. 2 and 3). The metric
parameters are within the range found in other diphos complexes
of this type, e.g. [(dppe)(AuCl)2].51
The structures of these complexes vary considerably in their
PCCP torsion angles (from 180◦ to 64.4◦). In the complexes
[(1)(AuCl)2], [(4)(AuCl)2] and [(6)(AuCl)2] an anti-conformation is
found, since in all cases, the middle of the PC–CP bond is situated
on an center of inversion. On the other hand in the solid state of
The gold atoms in the solid-state structures of [(1)(AuCl)2],
[(3)(AuCl)2], [(4)(AuCl)2] and [(6)(AuCl)2] are coordinated linearly
˚
[(3)(AuCl)2] intramolecular aurophilic contacts (dAu–Au = 3.122 A)
9214 | Dalton Trans., 2011, 40, 9212–9220
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Table 2 Distribution coefficients of bisphosphane ligands and corre-
sponding gold(I) complexes
Table 3 IC50 values (mM) of [(L)2Au]Cl and [(L)(AuCl)2] against human
ovarian carcinoma cell lines sensitive (A2780 sens.) or resistant to cisplatin
(A2780 cis.)a,b
Ligand
L
[(L)(AuCl)2]
[(L)2Au]X
[(L)2Au]X
[(L)(AuCl)2]
A2780 sens.
1
2
3
5
-0.73 0.03
-0.74 0.05
1.65 0.03
0.73 0.01
-1.05 0.03
-1.51 0.09
0.79 0.08
n.d.a
-1.73 0.03
-1.38 0.01
0.25 0.03
0.21 0.02
Ligand
A2780 sens.
A2780 cis.
A2780 cis.
Cisplatin
1.32
28.9
21.6
0.398
n.a.c
0.446
n.a.c
13.4
34.6
41.2
0.812
n.a.c
2.18
n.a.c
1.32
30.1
24.0
0.575
2.34
2.63
6.55
13.4
32.9
37.8
18.8
11.8
6.76
>10
1
2
3
4
5
6
a n.d. not determined, insoluble in water/n-octanol.
a cisplatin as reference compound. b pIC50 error are found in the ESI.†
c n.a. = not available.
Table 4 IC50 values (mM) of [(L)2Au]+ against human leukaemia (K562),
human colon carcinoma (Hct116) and rat hepatoma cell lines (H4IIE)a
Compound
K 562
Hct116
H4IIE
Cisplatin
[(3)2Au]PF6
[(5)2Au]Cl
18.0
>10
0.93
37.5
21.4
11.0
34.8
38.3
4.94
a pIC50 error are found in the ESI.†
Fig. 2 Molecular structure of [(1)(AuCl)2]. Projection along the crystal-
lographic b-axis. Displacement ellipsoids are drawn at the 50% level and
˚
H-atoms are omitted for clarity. Selected bond lengths [A] and angles
[◦]: Au1–P1 2.2284(12), Au1–Cl1 2.2848(12), P1–Au1–Cl1 177.04(4),
Table 5 Relative activities of [(L)2Au]+ in different cell lines referenced to
cisplatin (IC50 Cisplatin/IC50 compound
)
P1–C1–C1a–P1a 180.00.
Compound
A2780 sens.
A2780 cis.
K 562
Hct116 H4IIE
[(3)2Au]PF6
[(5)2Au]Cl
3.32
2.96
16.5
6.15
< 1.8
19.2
1.75
3.41
1.20
6.02
a wide range with distribution coefficients (logD7.4) between -1.73
and +0.79 (Table 2). The distribution coefficients of the ligands
as well as the values for the corresponding gold(I) complexes
are summarized in Table 4. Due to the poor solubility of the
benzannulated ligands 4 and 6 in n-octanol and water distribution
coefficients of these ligands as well as the corresponding complexes
were not determined.
The gold(I) compounds were screened for their cytotoxicity
against cisplatin sensitive and resistant A2780 human ovarian
cancer cell lines (Table 3). The gold complexes show IC50 values
ranging from 0.4 to 40 mM. Especially, complexes bearing the
thiazolyl ligand 5 and the asymmetric imidazolyl ligand 3, respec-
tively show considerably higher cytotoxic activity than cisplatin.
Complex [(3)2Au]PF6 shows highest cytotoxicity in both cisplatin
sensitive and resistant A2780 cell line (Tables 3 and 5). Complex
[(5)2Au]Cl also shows a higher cytotoxic activity than cisplatin
in both A2780 cell lines, but here partial resistance is observed.
Interestingly complex [(3)(AuCl)2] shows a significant difference
in the cytotoxicity towards the cisplatin sensitive and resistant
A2780 cell lines, with a selectivity in the order found for cisplatin,
whereas the other complexes of the type [(L)(AuCl)2] show much
lower selectivity.
Fig. 3 Molecular structure of [(3)(AuCl)2]·0.5EtOH·0.4CH2Cl2. The
configurations of the stereocenters at the phosphorus atoms are of opposed
sign. Displacement ellipsoids are drawn at the 50% level. Solvent molecules
˚
and H-atoms are omitted for clarity. Selected bond lengths [A] and angles
[◦]: Au1–P1 2.2345(17), Au1–Cl1 2.2970(15), Au2–P2 2.2388(16), Au2–Cl2
2.2984(16), Au1–Au2 3.1218(3), P1–Au1–Cl1 179.25(7), P2–Au2–Cl2
176.54(7), P1–C– C–P2 64.4(7).
lead to a gauche conformation. Complex [(3)(AuCl)2], with the
asymmetric ligand 3, crystallised as an ethanol/dichloromethane
solvate in the space group P21 and racemic twinning was found.
The synthesized tetrahedral gold(I) complexes [(diphos)2Au]+
are more hydrophilic compared to the highly lipophilic and toxic
parent compound [Au(dppe)2]+ (logP = 1.41).29 The nature of the
substituents, the length of the spacer between the phosphorous
atoms and the structure of the resulting complexes finely tune
solvent interactions and their hydrophilicity/lipophilicity covers
Within the series of gold(I) complexes [(3)2Au]PF6 and
[(5)2Au]Cl as well as [(3)(AuCl)2] exhibit highest antitumour
activity against the human ovarian cancer cell line A2780sens.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 9212–9220 | 9215
©

View Article Online
Table 6 Inhibition of thioredoxin reductase (TrxR) and glutathione
The high activity correlates with the intermediate lipophilicity
of these compounds. These complexes are significantly more
lipophilic (logD7.4 = +0.25, +0.21and +0.79)than the water-soluble
complexes [(1)2Au]Cl and [(2)2Au]Cl (logD7.4 = -1.73 and -1.38)
and more hydrophilic than the highly toxic parent compound
[Au(dppe)2]+ (logP = +1.41). Consequently, an optimal balance
of the lipophilic and hydrophilic nature of those complexes is
one of the most important factors for antitumor activity as
was previously shown for analogous pyridinyl-based complexes
by Berners-Price.35 Only the complexes with an intermediate
lipophilicity (between 0.21 and 0.79) show promising cytotoxicity,
while the more hydrophilic complexes are inactive against these
cell lines.
The most active compounds [(3)2Au]PF6 and [(5)2Au]Cl were
further screened for their cytotoxicity against human leukaemia
(K562), human colon carcinoma (Hct116) and rat hepatoma
(H4IIE) cell lines (Table 4). Here, the two complexes show
some significant differences in their antitumor activities, especially
against the leukaemia cell line K562. While the thiazole-based
complex [(5)2Au]Cl shows cytotoxcity against all tested cell lines,
complex [(3)2Au]PF6 only exhibits high antitumor activity against
the ovarian cancer cell lines, considerable less against the human
colon carcinoma and rat hepatoma cell line and almost no
cytotoxicity against the leukaemia cell line K562. This clearly
demonstrates, that this compound is not toxic in general but shows
selectivity towards different cancer cell lines.
reductase (GR)a
Compound IC50 (TrxR)/mM IC50 (GR)/mM IC50(GR)/IC50(TrxR)
[(1)2Au]Cl
[(2)2Au]Cl
0.14
0.12
0.60
1.13
4.23
0.46
4
10
3
[(3)2Au]PF6 1.22
[(5)2Au]Cl 0.14
3
a pIC50 error are found in the ESI.†
bioactive gold(I) species.12,55 The activity of GR was also inhibited
significantly but with lower efficiency. In complex [(3)2Au]PF6 two
heteroaryl ligands were replaced by phenyl resulting in a significant
decrease of activity against both enzymes.
The most potent compounds [(3)2Au]PF6 and [(5)2Au]Cl are of
immediate lipophilicity (logD7.4 = 0.25 and 0.21) and inhibit both
GR and TrxR. Although complexes [(1)2Au]Cl and [(2)2Au]Cl are
as potent TrxR inhibitors as [(5)2Au]Cl, their cytotoxic activity is
much lower. For [(5)2Au]Cl this is consistent with the proposed
mechanism of action for gold(I) compounds of the type [(L)2Au]+,
as these lipophilic cations have to cross membranes before they can
interact with cytosolic or mitochondrial TrxR as cellular targets.
[(3)2Au]PF6 shows in some cell lines the highest cytotoxicity
(A2780sens. and cis.), although the inhibition of TrxR is poor.
This could indicate a different mode of action for this compound
in these cell lines.
Cell death can be induced by apoptosis and necrosis. Apoptosis
is the process of programmed cell death whereas necrosis is the
premature death of cells and living tissue. The mode of cell death
induction of compounds [(3)2Au]PF6 and [(5)2Au]Cl in H4IIE
cells was determined using a DNA fragmentation assay.52–54 Fig. 4
shows gel electrophoresis chromatograms of the DNA fragments
after incubation of H4IIE cells at 37 ◦C for 24 h with different
concentrations of [(3)2Au]PF6 and [(5)2Au]Cl, respectively. Both
compounds show the characteristic ladder pattern starting at
2.5 mM, which indicates apoptosis.
Conclusion
We prepared a series of gold(I) complexes [(diphos)(AuCl)2] and
[(diphos)2Au]X with azol-based diphos type ligands. Therefore
two novel imidazole-based ligands bis(di-1-methylimidazol-
2-ylphosphino)propane
(2)
and
bis(1-methylimidazol-2-
ylphenylphosphino)ethane (3), have been prepared. The rac
and meso isomers of [(3)(AuCl)2] could be separated due to their
different solubility in chloroform. In the solid state structure
of [(3)(AuCl)2] intramolecular aurophilic contacts are found.
The complexes [(diphos)(AuCl)2] and [(diphos)2Au]X have
distribution coefficients (logD7,4) between -1.73 and 0.79 and
are more hydrophilic than the highly toxic parent compound
[Au(dppe)2]Cl. Of the number complexes screened, only the
bis-chelated gold(I)-complexes [(3)2Au]PF6 and [(5)2Au]Cl with
intermediate lipophilicity (logD7.4 = 0.21 und 0.25) showed high
cytotoxcities. But there are differences in the selectivity of these
two complexes. While complex [(5)2Au]Cl showed a similar
cytotoxicity towards all tested cell lines, complex [(3)2Au]PF6
is inactive against the leukaemia cell line K562. Therefore the
complexes [(3)2Au]PF6 and [(5)2Au]Cl do not share a common
toxic moiety. This observation could be confirmed by proving the
occurrence of apoptosis.
As reported recently by Rackham et al. bischelated gold(I)
phosphine complexes can be effective inhibitors of TrxR and act
as antimitochondrial agents.56 Here we showed that complexes
of this type are also able to inhibit the activity of GR but with
lower potency. The main difference between the two proteins is the
presence of a selenocysteine–cysteine bridge in the active site of
TrxR, which is replaced by a cysteine–cysteine bridge in GR. The
preferential inhibition of TrxR over GR might be a consequence
Fig. 4 Gel electrophoresis of DNA treated with various concentrations
of a) [(3)2Au]PF6 and b) [(5)2Au]Cl after 24 h incubation on H4IIE cells at
37 ◦C (M = marker, NC = negative control, PC = positive control).
The potential of the complexes [(L)2Au]Cl (L = 1, 2, 5) and
[(3)2Au]PF6 to inhibit the activity of the disulfide reductases TrxR
and GR was studied on the isolated enzymes using an estab-
lished procedure (DTNB reduction assay, Table 6).55 Generally
[(1)2Au]Cl, [(2)2Au]Cl and [(5)2Au]Cl displayed strong inhibitory
activities with EC50 values for TrxR comparable to that of other
9216 | Dalton Trans., 2011, 40, 9212–9220
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
of this difference and has also been observed with other gold
species.10,36
Preparation of complexes [(L)(AuCl)2]
[(tht)AuCl] (0.31 mmol) and the corresponding ligand (0.16 mmol)
were stirred in dichloromethane at least for 2 h. The resulting
precipitate was filtered, washed twice with dichloromethane or
chloroform and diethyl ether and dried in vacuo.
Experimental section
The compounds [(tht)AuCl], 1-methyl-2-trimethylsilylimidazole,
ligands 1, 4, 5 and 6 were prepared according to literature
procedures.46 The preparations were carried out in Schlenk tubes
under an atmosphere of dry nitrogen using anhydrous solvents
purified according to standard procedures. The metal complexes
were prepared using wet solvents. All chemicals were used as
[(1)(AuCl)2]
Yield: 0.11 g (78%). 1H NMR (dmso-d6): d = 3.28 (vt, 4H, (CH2)2),
3.79 (s, 12H, NCH3), 7.20 (s, 4H, H4im), 7.60 (s, 4H, H5im). 31P{ H}
1
NMR (dmso-d6): d = -13 (s). ESI+ (MeOH): m/z (%) = 333 [L-
imNMe]+, 612 [LAu]+, 808 [LAu2]+, 843 [LAu2Cl]+. MALDI MS
(CHCl3): m/z (%) = 843 [LAu2Cl]+. C18H24N8P2Au2Cl2·2CH2Cl2
(1049.08): calc. C 22.90, H 2.69, N 10.68; found C 22.8, H 2.7, N
10.7. Distribution coefficient: logD7.4 = -1.05 0.03.
1
purchased. 1H and 31P{ H}NMR spectra were recorded on a
1
Bruker DRX 200 and 13C{ H} NMR spectra on a Bruker
1
DRX 500 spectrometer. The 1H and 13C{ H} NMR spectra were
calibrated against the residual proton signals and the carbon
signals of the solvents as internal references (CDCl3: dH = 7.30 ppm
and dC = 77.0 ppm; MeOD-d4: dH = 3.31 ppm and dC = 49.1 ppm,
dmso-d6: dH = 2.50 ppm and dC = 39.5 ppm; D2O: dH = 4.79) while
[(2)(AuCl)2]
1
the 31P{ H} NMR spectra were referenced to external 85% H3PO4.
Yield: 97 mg (68%). 1H NMR (dmso-d6): d = 2.07 (s (br), 2H, CH2-
(CH2)-CH2), 3.08 (br, 4H, CH2-(CH2)-CH2), 3.73 (s, 12H, NCH3),
The signals are referred to as singlet (s), doublet (d), virtual triplets
(vt), multiplets (m) and broad signals (br). The MALDI mass
spectra were recorded on a Bruker Ultraflex MALDI-TOF mass
spectrometer and ESI mass spectra with a Finnigan LCQ Deca
Ion-Trap-API mass spectrometer, The elemental composition of
the compounds was determined with a Perkin Elmer Analysator
2400 at the Institut fu¨r Pharmazeutische und Medizinische
Chemie, Heinrich-Heine-Universita¨t Du¨sseldorf.
7.19 (s, 4H, H4im), 7.57 (s, 4H, H5im). 31P{ H}NMR (dmso-d6):
1
d = -10 (s). ESI+ (H2O): m/z (%) = 347 [L-im]+, 626 [LAu]+.
MALDI MS (MeOH): m/z (%) = 626 [LAu]+, 857 [LAu2Cl]+.
C19H26N8P2Au2Cl2·0.5CH2Cl2 (935.72): calc. C 25.03, H 2.91, N
11.98; found C 25.3, H 2.6, N 11.6. Distribution coefficient:
logD7.4 = -1.51 0.09.
[(3)(AuCl)2]
Bis(di-1-methylimidazol-2-ylphosphino)propane (2)
Yield: 0.12 g (86%).1H NMR (CDCl3): d = 2.77 (vt, 4H, (CH2)2),
3.81 (s, 6H, NCH3), 7.10 (s, 2H, H4im), 7.22 (s, 2H, H5im), 7.41–
1,3-Bis(dichlorophosphino)propane (1.0 g, 4.1 mmol) was added
to 1-methyl-2-trimethylsilanylimidazole (2.8 g, 0.02 mol) at 0 C
◦
1
7.76 (m, 10H, Ph). 31P{ H} NMR (dmso-d6): d = 5.5 (s), 6.7 (s).
drop-wise. The suspension was stirred for 1 h at 0 ◦C and for 17 h
at ambient temperature. The resulting (CH3)3SiCl was removed in
vacuo and the residue washed with n-hexane. The solid was filtered
off and dried in vacuo. Yield: 1.4 g (82%). 1H NMR (MeOD-d4):
d = 1.63 (m, 2H, CH2(CH2)2CH2),2.59 (vt, 4H, (CH2)2), 3.57 (s,
12H, NCH3), 7.10 (d, 3JHH = 1.2 Hz 4H, H4im), 7.23 (s, 4H, H5im).
MALDI MS (CDCl3): m/z (%) = 835 [LAu2Cl]+, 1705 [LAu2Cl2]2 .
+
C22H24N4P2Au2Cl2·3 CHCl3 (1229.38): calc. C 24.43, H 2.21, N
4.56; found C 24.3, H 2.4, N 4.7. Distribution coefficient: logD7.4
=
0.79 0.08.
[(4)(AuCl)2]
1
1
31P{ H}NMR (MeOD-d4): d = -59 (s). 13C{ H} NMR (MeOD-
d4): d = 22.6 (CH2 (b)), 26.1 (CH2 (a)), 33.9 (NCH3), 124.4 (C5im),
130.5 (C4im), 144.0 (C2im). MALDI MS (CHCl3): m/z (%) = 429
[M+H]+. C19H26N8P2·0.5CH2Cl2(470.88): calc. C 49.74, H 5.87, N
Yield: 92 mg (53%). 1H NMR (CDCl3): d = 3.66 (d, J = 5.44 Hz,
4H, (CH2)2), 4.14 (s, 12H, NCH3), 7.29–7.46 (m, 12H, Hph),
3
1
7.70 (d, JHH = 7.72 Hz, 4H, Hph). 31P{ H} NMR (CDCl3):
d = -6 (s). MALDI MS (CHCl3): m/z = 1043 [LAu2Cl]+.
C34H32N8P2Au2Cl2·2CH2Cl2 (1249.32): calc. 34.61, H 2.90, N 8.97;
found: C 34.4, H 2.9, N 9.0. Distribution coefficient: logD7.4 = n.d.
23.8, found C 49.9, H 6.4, N 23.6. Distribution coefficient: logD7.4
-0.74 0.09.
=
Bis(1-methylimidazol-2-ylphenylphosphino)ethane (3)
[(5)(AuCl)2]
1,2-Bis(chloro-2-ylphenylphosphino)-ethane (0.71 g, 46 mmol)
was added to 1-methyl-2-trimethylsilanylimidazole (0.73 g, 0.023
m◦ol) at 0 ◦C drop-wise. The suspension was stirred for 1 h at
0 C and 17 h at ambient temperature. The resulting (CH3)3SiCl
was removed in vacuo. The residue was dissolved in chloroform
and the product was precipitated upon addition of diethyl ether.
Yield: 0.11 g (77%). 1H NMR (dmso-d6): d = 3.32 (d, 4H,
(CH2)2), 8.32 (s, 4H, Hth). 31P{ H} NMR (dmso-d6): d = 10 (s).
1
FAB MS: m/z = 343 [L-C3H3NS]+, 623 [LAu]+, 855 [LAu2Cl]+.
C14H12N4S4P2Au2Cl2 (891.32): calc. C 18.87, H 1.36, N 6.29; found
C 19.0, H 1.4, N 6.0. Distribution coefficient: logD7.4 = n.d.
1
Yield: 0.31 g (33%). H NMR (dmso-d6): d = 2.08–2.35 (m, 4H,
(CH2)2), 3.57 (s, 6 H, NCH3), 7.10 (s, 2H, H4im), 7.30 (s, 2H, H5im),
[(6)(AuCl)2]
1
7.33–7.39 (m, 10H, Ph). 31P{ H} NMR (dmso-d6): d = -38.3 (s),
-38.5 (s). MALDI MS (CHCl3): m/z (%) = 407 [L]+, 423 [LO]+,
439 [LO2]+. C22H24N4P2·0.5CHCl3(466.09):calc. C 57.98, H 5.30,
N 12.02, found C 57.7, H 5.5, N 12.3. Distribution coefficient:
logD7.4 = 1.65 0.03.
Yield: 94 mg (54%). H NMR (CDCl3): d = 3.76 (d, J = 5.1 Hz,
1
4H, (CH2)2), 7.57–7.68 (m, 8H, Hph), 8.13–8.27 (m, 8H, Hph).
1
31P{ H} NMR (CDCl3): d = 16 (s). MALDI MS (CHCl3): m/z =
823 [LAu]+, 1055 [LAu2Cl]+. C30H20N4S4P2Au2Cl2·2.5CH2Cl2
Dalton Trans., 2011, 40, 9212–9220 | 9217
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
1
12H, NCH3), 6.96–7.46 (m, 28H, Him/Ph). 31P{ H} NMR (dmso-
d6): d = 4.2 (s), -143 (q, PF6). ESI+ (MeOH): m/z (%) = 603 [LAu]+,
1009 [L2Au]+. MALDI MS (MeOH): m/z (%) = 407 [L+H]+,
603 [LAu]+, 1009 [L2Au]+. C44H48N8P5AuF6·0.5CH2Cl2·1.5 H2O
(1224.20): calc. C 43.66, H 4.28, N 9.15; found C 43.4, H 4.69, N
9.9. Distribution coefficient: logD7.4 = 0.25 0.03.
(1303.88): calc. C 29.94, H 1.93, N 4.30; found C 29.8, H 1.7,
N 4.3. Distribution coefficient: logD7.4 = n.d.
Preparation of complexes [(L)2Au]Cl
[(tht)AuCl] (320.59 g mol-1, 0.16 mmol) and the corresponding
ligand (0.31 mmol) were stirred in dichloromethane for 17 h. The
volume of the solution was reduced to 1/3 and layered with diethyl
ether. The resulting precipitate was filtered, washed with diethyl
ether and dried in vacuo.
X-ray crystallography
Crystallographic data were collected at 183(2) K on an Oxford
Diffraction Xcalibur system with a Ruby detector using Mo-
˚
Ka radiation (l = 0.7107 A) that was graphite-monochromated.
[(1)2Au]Cl
Suitable crystals were covered with oil (Infineum V8512, formerly
known as Paratone N), mounted on top of a glass fibre and
immediately transferred to the diffractometer. The program suite
CrysAlisPro was used for data collection, semi-empirical absorption
correction and data reduction.57 Structures were solved with direct
methods using SIR97 and were refined by full-matrix least-squares
methods on F2 with SHELXL-97.58,59 [(3)(AuCl)2] crystallized in
the space group P21. A racemic twinning was found and refined
at a ratio of 56 : 44. The asymmetric unit additionally contained
0.5 equiv. ethanol and 0.4 equiv. dichloromethane. The structure
of [(4)(AuCl)2] contained undefined residual electronic density
caused by disordered solvent molecules. This was treated with
the program utility SQUEEZE of the Platon program suite.60
The structure of [(3)2Au]PF6 contained a disorder between one
phenyl and one N-methylimidazole group bound to the same
phosphorous atom. The occupancies were refined as a ratio of
6 : 4. In one case, two atoms of the imidazole and the phenyl
overlaid almost perfectly. All the structures were checked for
higher symmetry with help of the program Platon.60
Yield: 68 mg (40%). 1H NMR (MeOD-d4): d = 3.02 (s, 8H, (CH2)2),
1
3.44 (s, 24H, NCH3), 7.32 (s, 8H, H4im), 7.43 (s, 8H, H5im). 31P{ H}
NMR (D2O): d = -16 (s). ESI+ (MeOH): m/z (%) = 333 [L-
imNMe]+, 612 [LAu]+, 1026 [L2Au]+, 1062 [L2AuCl]+. MALD IMS
(CHCl3): m/z (%) = 333 [L-imNMe]+, 415 [L]+, 612 [LAu]+, 1026
[L2Au]+. C36H48N16P4AuCl·3CH2Cl2 (1315.98): calc. C 35.60, H
4.14, N 17.03, found C 35.4, H 4.4, N 16.8. Distribution coefficient:
logD7.4 = -1.73 0.03.
[(2)2Au]Cl
1
Yield: 0.1 g (57%). H NMR (dmso-d6): d = 1.78 (s (br), 4H,
CH2-(CH2)-CH2), 2.77 (vt, 8H, CH2-(CH2)-CH2), 3.57 (s, 24H,
NCH3), 7.19 (d, 3JHH = 1.1 Hz,8H, H4im), 7.38 (d, 3JHH = 1.1 Hz,
1
8H, H5im). 31P{ H} NMR (D2O): d = -37 (s). ESI+ (H2O): m/z
(%) = 347 [L-imNMe]+, 626 [LAu]+. MALDI MS (MeOH): m/z
(%) = 626 [LAu]+, 1053 [L2Au]+. C38H52N16P4AuCl·3.5 CH2Cl2
(1386.50): calc. C 35.95, H 4.29, N 16,16; found C 35.5, H 4.2, N
16.4. Distribution coefficient: logD7.4 = -1.38 0.01.
Distribution coefficients (logD)
[(3)2Au]Cl
The n-octanol–water distribution coefficients of the compounds
were determined using a shake-flask method. PBS buffered
bidistilled water (100 mL, phosphate buffer, c(PO43-) = 10 mM,
c(NaCl) = 0.15 M, pH adjusted to 7.4 with HCl) and n-octanol
(100 mL) were shaken together using a laboratory shaker (Perkin
Elmer), for 72 h to allow saturation of both phases. 1 mg of each
compound was mixed in 1 mL of aqueous and organic phase,
respectively for 10 min using a laboratory vortexer. The resultant
emulsion was centrifuged (3000 ¥ g, 5 min) to separate the phases.
The concentrations of the compounds in the organic and aqueous
phases were then determined using UV absorbance spectroscopy
(230 nm). LogDpH was defined as the logarithm of the ratio of the
concentrations of the complex in the organic and aqueous phases
logD = {[compound(org)]/[compound(aq)], the value reported is the
mean of three separate determinations.
1
Yield: 0.12 g (72%). H NMR (D2O): d = 2.58 (br, 8H, (CH2)2),
3.05 (s, 12H, NCH3), 7.08-7.49 (m, 28H, Him/Ph). 31P{ H} NMR
1
(dmso-d6): d = 4.2 (s). ESI+ (MeOH): m/z (%) = 603 [LAu]+.
MALDI MS (MeOH): m/z (%) = 407 [L+H]+, 603 [LAu]+, 1009
[L2Au]. C44H48N8P4AuCl·0.5CH2Cl2 (1087.69): calc. C 49.14, H
4.54, N 10.30; found C 49.1, H 4.9, N 9.8.
[(5)2Au]Cl
Yield: 96 mg (55%). 1H NMR (CDCl3): d = 3.09 (br, 8H, (CH2)2),
7.67 (d, JHH = 2.85 Hz, 8H, H4th), 7.89 (br, 8H, H5th). 31P{ H}
NMR (CDCl3): d = 3 (s). ESI+ (CH3OH): m/z = 342 [L-th]+, 623
[LAu]+. MALDI MS (CHCl3): m/z = 623 [LAu]+, 1049 [L2Au]+.
C28H24N8S8P4AuCl·1.5CH2Cl2 (1212.78): calc. C 29.22, H 2.24, N
3
1
9.24; found C 29.2, H 2.3, N 9.3. Distribution coefficient: logD7.4
=
0.21 0.02.
Materials, cell lines and cell culture
Preparation of [(3)2Au]PF6
The human ovarian carcinoma cell line A2780 was obtained from
European Collection of Cell Cultures (ECACC, Salisbury, UK).
The human chronic myelogenous leukemia cell line K562 was
obtained from the German Collection of Microorganisms and
Cell Cultures (DSMZ, Germany). All other reagents were supplied
by Sigma Chemicals unless otherwise stated. All cell lines were
grown at 37 ◦C under humidified air supplemented with 5% CO2
in RPMI 1640 (Invitrogen, Germany) containing 10% fetal calf
[(tht)AuCl] (39 mg, 0.12 mmol) and ligand 3 (100 mg, 0.25 mmol)
were stirred in dichloromethane for 15 min. To the solution
NH4PF6 (21 mg, 0.12 mmol) in methanol was added and the
mixture stirred for 15 h. The volume of the solution was reduced
to 1/3 and layered with diethyl ether. The resulting precipitate was
filtered, washed with diethyl ether and dried in vacuo. Yield: 0.15 g
(81%). 1H NMR (dmso-d6): d = 2.16–2.31 (br, 8H, (CH2)2), 2.92 (s,
9218 | Dalton Trans., 2011, 40, 9212–9220
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
serum (PAN Biotech, Germany), 100 IU/mL penicillin, 100 mg
mL-1 streptomycin and 2 mM glutamine. The cells were grown to
80% confluency before using them for the MTT cell viability assay.
225 mL of reaction mixture (1000 mL reaction mixture consisted of
500 mL potassium phosphate buffer pH 7.0, 80 mL 100 mM EDTA
solution pH 7.5, 20 mL BSA solution 0.05%, 100 mL of 20 mM
NADPH solution and 300mL of distilled water) were added and
the reaction started by addition of 25 mL of an 20 mM ethanolic
DTNB solution. After proper mixing, the formation of 5-TNB was
monitored with a microplate reader (Perkin Elmer VictortmX4)
at 405 nm in 10 s intervals for 6 min. The increase in 5-TNB
concentration over time followed a linear trend (r2 ≥ 0.99) and
the enzymatic activities were calculated as the slopes (increase in
absorbance per second) thereof. For each tested compound the
non interference with the assay components was confirmed by a
negative control experiment using an enzyme free solution. The
IC50 values were calculated as the concentration of compound
decreasing the enzymatic activity of the untreated control by 50%
and are given as the means and error of repeated experiments.
MTT cell viability assays
The rate of cell-survival under the action of test substances was
evaluated by an improved MTT assay as previously described.61
The assay is based on the ability of viable cells to metabolize
yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT, Applichem, Germany) to violet formazane crystals
that can be detected spectrophotometrically. In brief, A2780 cells
were seeded at a density of 8,000 cells/well and K562 at a density
of 30,000 cells/well in 96well plates (Sarstedt, Germany). After
24 h, cells were exposed to the test compounds at concentrations
of 10-5 M and 10-4 M. Incubation was ended after 72 h and cell
survival was determined by addition of MTT solution (5 mg
mL-1 in phosphate buffered saline). The formazan precipitate
was dissolved in DMSO. Absorbance was measured at 544 nm
and 620 nm in a FLUOstarmicroplate-reader (BMG LabTech,
Offenburg, Germany). The absorbance of untreated control cells
was taken as 100% viability. All tests were performed in triplicate.
Notes and references
1 O. Rixe, W. Ortuzar, M. Alvarez, R. Parker, E. Reed, K. Paull and T.
Fojo, Biochem. Pharmacol., 1996, 52, 1855–1865.
2 A. V. Klein and T. W. Hambley, Chem. Rev., 2009, 109, 4911–4920.
3 I. Ott and R. Gust, Pharm. Unserer Zeit, 2006, 35, 124–133.
4 B. Spingler, D. A. Whittington and S. J. Lippard, Inorg. Chem., 2001,
40, 5596–5602.
5 P. Abada and S. B. Howell, Metal-Based Drugs, 2010, Article ID
317581.
6 G. G. M. D’Souza, M. A. Wagle, V. Saxena and A. Shah, Biochim.
Biophys. Acta, Bioenerg., 2011, 1807, 689–696.
7 P. J. Barnard and S. J. Berners-Price, Coord. Chem. Rev., 2007, 251,
1889–1902.
8 F. Caruso, R. Villa, M. Rossi, C. Pettinari, F. Paduano, M. Penatti,
M. G. Daidone and N. Zaffaroni, Biochem. Pharmacol., 2007, 73, 773–
781.
9 E. A. Pacheco, E. R. T. Tiekink and M. W. Whitehouse, in Gold
Chemistry – Applications and Future Directions in the Life Sciences,
(Ed. F. Mohr), John Wiley & Sons Ltd., 2009.
10 I. Ott, Coord. Chem. Rev., 2009, 253, 1670–1681.
11 S. Y. Ho and E. R. T. Tiekink, in Metallotherapeutic Drugs and Metal-
based Agents - the Use of Metals in Medicine, (Ed. E. R. T. Tiekink and
M. Gielen), John Wiley & Sons Ltd., 2005.
12 V. Gandin, A. P. Fernandes, M. P. Rigobello, B. Dani, F. Sorrentino, F.
Tisato, M. Bjo¨rnstedt, A. Bindoli, A. Sturaro, R. Rella and C. Marzano,
Biochem. Pharmacol., 2010, 79, 90–101.
13 L. K. Webster, S. Rainone, E. Horn and E. R. T. Tiekink, Met.-Based
Drugs, 1996, 3, 63–66.
14 E. R. T. Tiekink, P. D. Cookson, B. M. Linahan and L. K. Webster,
Met.-Based Drugs, 1994, 1, 299–304.
15 C. K. Mirabelli, R. K. Johnson, D. T. Hill, L. F. Faucette, G. R. Girad,
G. Y. Kuo, C.-M. Sung and S. T. Crooke, J. Med. Chem., 1986, 29,
218–223.
16 C. K. Mirabelli, R. K. Johnson, C.-M. Sung, L. Faucette, K. Muirhead
and S. T. Crooke, Cancer Res., 1985, 45, 32–39.
DNA fragmentation analysis52
H4IIE cells (0.5 ¥ 106) were plated in 30 mm cell culture dishes
and allowed to attach for 48 h, then cells were incubated 24 h
with the compounds. Then attached and floating cells were
collected and lysed with 500 mL lysis buffer (10 mM Tris–HCl,
0.6% SDS, 10 mM EDTA) and 5 mL RNase A (10 mg mL-1,
DNase-free) for 40 min at 37 ◦C. Proteins were precipitated by
addition of 125 mL NaCl (5 M) for 1 h at 4 ◦C, followed by
centrifugation (10,000 ¥ g, 15 min). The supernatant was first
extracted with chloroform/phenol (250 mL/500 mL) followed by
a second extraction with chloroform (650 mL). The DNA in the
supernatant was precipitated with ice-cold isopropanol (1 mL)
and stored overnight at -20 ◦C. Afterwards the samples were
centrifuged (15 min, 10,000 ¥ g, 4 ◦C) and the DNA pellet was
resuspended in 30 mL TE-buffer (10 mM Tris–HCl, 1 mM EDTA
in H2O). DNA yield was measured photometrically at 260 nm. The
oligonucleosomal DNA fragmentation as a characteristic feature
of the apoptotic cell death was analysed via electrophoresis (1.75%
agarose, 3.5 h at 60 V, 4 mg DNA/lane). After this the gel was
stained with ethidium bromide and analysed under UV-light using
the Quantity One system from Biorad (Mu¨nchen, Germany).
Inhibition of thioredoxin-reductase
17 S. J. Berners-Price, C. K. Mirabelli, R. K. Johnson, M. R. Mattern,
F. L. McCabe, L. F. Faucette, C.-M. Sung, S. Mong, P. J. Sadler and
S. T. Crooke, Cancer Res., 1986, 46, 5486–5493.
18 S. Tian, F.-M. Siu, S. C. F. Kiu, C.-N. Lok and C.-M. Che, Chem.
Commun., 2011, DOI: 10.1039/c1cc11820j.
19 X. Zhang, Y. Zheng, L. E. Fried, Y. Du, S. J. Montano, A. Sohn, B.
Lefkove, L. Holmgren, J. L. Arbiser, A. Holgren and J. Lu, Free Radical
Biol. Med., 2011, 50, 811–820.
20 J. Armstrong, Br. J. Pharmacol., 2009, 151, 1154–1165.
21 Y. Wang, Q.-Y. He, R. W.-Y. Sun, C.-M. Che and J.-F. Chiu, Cancer
Res., 2005, 65, 11553–11564.
To determine the inhibition of TrxR and GR an established
microplate reader based assay was performed with minor
modifications.55 For this purpose commercially available rat liver
TrxR and baker yeast GR (both from Sigma-Aldrich) were
used and diluted with distilled water to achieve a concentration
of 2.0 U/mL. The compounds were freshly dissolved as stock
solutions in DMF. To each 25 mL aliquots of the enzyme solution
each 25 mL of potassium phosphate buffer pH 7.0 containing the
compounds in graded concentrations or vehicle (DMF) without
compounds (control probe) were added and the resulting solutions
(final concentration of DMF: 0.5% v/v) were incubated with
moderate shaking for 75 min at 37 ◦C in a 96 well plate. To each well
22 S. J. Berners-Price and A. Filipovska, Aust. J. Chem., 2008, 61, 661–
668.
23 S. Davis, M. J. Weiss, J. R. Wong, T. J. Lampidis and L. B. Chen, J. Biol.
Chem., 1985, 260, 13844–13850.
24 J. S. Modica-Napolitano and J. R Aprille, Adv. Drug Delivery Rev.,
2001, 49, 63–70.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 9212–9220 | 9219
©

View Article Online
25 G. D. Hoke, R. A. Macia, P. C. Meunier, P. J. Bugelski, C. K. Mirabelli,
G. F. Rush and W. D. Matthews, Toxicol. Appl. Pharmacol., 1989, 100,
293–306.
26 G. D. Hoke, G. F. Rush, G. E. Bossard, J. V. McArdle, B. D. Jensen and
C. K. Mirabelli, J. Biol. Chem., 1988, 23, 11203–11210.
27 C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G. G. Lobbia,
E. Del Giudice, M. Porchia, F. Tisato and C. Santini, J. Med. Chem.,
2008, 51, 798–808.
42 S. E. Jackson-Rosario and W. T. Self, Metallomics, 2010, 2, 112–116.
43 J. L. Hickey, R. A. Ruhayel, P. J. Barnard, M. V. Baker, S. J.
Berners-Price and A. Filipovska, J. Am. Chem. Soc., 2008, 130, 12570–
12571.
44 S. Urig, K. Fritz-Wolf, R. Re´au, C. Herold-Mende, K. To´th, E.
Davioud-Charvet and K. Becker, Angew. Chem., 2006, 118, 1915–1920.
45 P. C. Kunz, M. U. Kassack, A. Hamacher and B. Spingler, Dalton
Trans., 2009, 7741–7747.
28 R. J. Bowen, J. Caddy, M. A. Fernandes, M. Layh, M. A. Mamo and
R. Meijboom, J. Organomet. Chem., 2006, 691, 717–725.
29 A. S. Humphreys, A. Filipovska, S. J. Berners-Price, G. A.
Koutsantonis, B. W. Skelton and A. H. White, Dalton Trans., 2007,
4943–4950.
30 R. J. Bowen, A. C. Garner, S. J. Berners-Price, I. D. Jenkins and R. E.
Sue, J. Organomet. Chem., 1998, 554, 181–184.
46 P. C. Kunz, C. Wetzel, M. Bongartz, A. L. Noffke and B. Spingler,
J. Organomet. Chem., 2010, 695, 1891–1897.
47 B. M. Berven and G. A. Koutsantonis, Synthesis, 2008, 16, 2626–2630.
48 R. J. Long, D. J. Jones, V. C. Gibson and A. J. P. White, Organometallics,
2008, 27, 5960–5967.
49 S. J. Berners-Price, M. A. Mazid and P. J. Sadler, J. Chem. Soc., Dalton
Trans., 1984, 969–974.
31 S. J. Berners-Price, R. J. Bowen, P. J. Harvey, P. C. Healy and G. A.
Koutsantonis, J. Chem. Soc., Dalton Trans., 1998, 1743–1750.
32 S. J. Berners-Price, R. J. Bowen, P. Galettis, P. C. Healy and M. J.
McKeage, Coord. Chem. Rev., 1999, 185–186, 823–836.
33 S. J. Berners-Price, R. J. Bowen, T. W. Hambley and P. C. Healy, J. Chem.
Soc., Dalton Trans., 1999, 1337–1346.
34 D. Screnci, M. J. McKeage, P. Galettis, T. W. Hambley, B. D. Palmer
and B. C. Baguley, Br. J. Cancer, 2000, 82, 966–972.
35 M. J. McKeage, S. J. Berners-Price, P. Galettis, R. J. Bowen, W. Brouwer,
L. Ding, L. Zhuang and B. C. Baguley, Cancer Chemother. Pharmacol.,
2000, 46, 343–350.
50 S. J. Berners-Price, P. S. Jarrett and P. J. Sadler, Inorg. Chem., 1987, 26,
3074–3077.
51 D. S. Eggleston, D. F. Chodosh, G. R. Girand and D. T. Hill, Inorg.
Chim. Acta, 1985, 108, 221–226.
52 S. Nagata, Exp. Cell Res., 2000, 256, 12–12.
53 P. R. Walker and M. Sikorska, Biochem. Cell Biol., 1997, 75, 287–299.
54 M. M. Compton, Cancer Metastasis Rev., 1992, 11, 105–119.
55 R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can, A. Kitanovic, L. A.
Onambele, M. Stefanopoulou, Y. Geldmacher, W. S. Sheldrick, G.
Wolber, A. Prokop, S. Wo¨lfl and I. Ott, J. Med. Chem., 2010, 53, 8608–
8618.
36 A. Bindoli, M. P. Rigobello, G. Scutari, C. Gabbiani, A. Casini and L.
Messori, Coord. Chem. Rev., 2009, 253, 1692–1707.
56 O. Rackham, S. J. Nichols, P. J. Leedman, S. J. Berners-Price and A.
Filipovska, Biochem. Pharmacol., 2007, 74, 992–1002.
57 CrysAlisPro Software system, Oxford Diffraction Ltd., vers. 171.32
Oxford, UK, 2008.
58 A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C.
Giacovazzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori and R.
Spagna, J. Appl. Crystallogr., 1999, 32, 115–119.
37 S. Jackson-Rosario, D. Cowart, A. Myers, R. Tarrien, R. L. Levine,
R. A. Scott and W. T. Self, J. Biol. Inorg. Chem., 2009, 14, 507–519.
38 E. Vergara, A. Casini, F. Sorrentino, O. Zava, E. Cerrada, M. P.
Rigobello, A. Bindoli, M. Laguna and P. J. Dyson, ChemMedChem,
2010, 5, 96–102.
39 K. P. Bhabak, B. J. Bhuyan and G. Mugesh, Dalton Trans., 2011, 40,
59 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2007,
64, 112–122.
2099–2111.
40 D. Mustacich and G. Powis, Biochem. J., 2000, 346, 1–8.
41 K. Yan, C.-N. Lok, K. Bierla and C.-M. Che, Chem. Commun., 2010,
46, 7691–7693.
60 A. L. Spek, J. Appl. Crystallogr., 2003, 36, 7–13.
61 H. Mu¨ller, M. U. Kassack and M. Wiese, J. Biomol. Screening, 2004,
9, 506–515.
9220 | Dalton Trans., 2011, 40, 9212–9220
This journal is
The Royal Society of Chemistry 2011
©