NHC gold halide complexes derived from 4,5-diarylimidazoles: Synthesis, structural analysis, and pharmacological investigations as potential antitumor agents

Article abstract of DOI:10.1021/jm201156x

A series of novel neutral NHC gold halide complexes derived from 4,5-diarylimidazoles were synthesized, characterized, and analyzed for biological effects. High growth inhibitory effects in MCF-7 and MDA-MB 231 breast cancer as well as HT-29 colon cancer cell lines depended on the presence of the C4,C5-standing aromatic rings. Methoxy groups at these rings did not change the growth inhibitory properties, while F-substituents in the ortho-position (5d) increased the activity in MCF-7 and MDA-MB 231 cells. The substituents at the nitrogen atoms and the oxidation state of the metal play a subordinate role. The most active bromo[1,3-diethyl-4,5-bis(2-fluorophenyl)-1,3- dihydro-2H-imidazol-2-ylidene]gold(I) (5d) was distinctly more active than cisplatin. All complexes caused thioredoxin reductase (TrxR) inhibition (EC ₅₀ = 374-1505 nM) distinctly lower than auranofin (EC₅₀ = 18.6 nM) excluding this enzyme as main target. Because of the low nuclear content, a participation of DNA interaction on the mode of action is very unlikely. The missing ER binding and the missing correlation of growth inhibition and inactivation of COX enzymes exclude these targets, too. (Figure presented)

Full text of DOI:10.1021/jm201156x

Article
pubs.acs.org/jmc
NHC Gold Halide Complexes Derived from 4,5-Diarylimidazoles:
Synthesis, Structural Analysis, and Pharmacological Investigations
as Potential Antitumor Agents
Wukun Liu,† Kerstin Bensdorf,† Maria Proetto,† Ulrich Abram,‡ Adelheid Hagenbach,‡
and Ronald Gust*,†,§
†Institute of Pharmacy, Freie Universitat Berlin, Konigin-Luise-Strasse, 2+4, 14195 Berlin, Germany
̈
̈
‡Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Fabeckstrasse 34-36, 14195 Berlin, Germany
̈
§Institute of Pharmacy, University of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria
S
* Supporting Information
ABSTRACT: A series of novel neutral NHC gold halide complexes
derived from 4,5-diarylimidazoles were synthesized, characterized, and
analyzed for biological effects. High growth inhibitory effects in MCF-7
and MDA-MB 231 breast cancer as well as HT-29 colon cancer cell
lines depended on the presence of the C4,C5-standing aromatic rings.
Methoxy groups at these rings did not change the growth inhibitory
properties, while F-substituents in the ortho-position (5d) increased the
activity in MCF-7 and MDA-MB 231 cells. The substituents at the nitro-
gen atoms and the oxidation state of the metal play a subordinate role.
The most active bromo[1,3-diethyl-4,5-bis(2-fluorophenyl)-1,3-dihydro-
2H-imidazol-2-ylidene]gold(I) (5d) was distinctly more active than
cisplatin. All complexes caused thioredoxin reductase (TrxR) inhibition (EC50 = 374−1505 nM) distinctly lower than auranofin
(EC50 = 18.6 nM) excluding this enzyme as main target. Because of the low nuclear content, a participation of DNA interaction
on the mode of action is very unlikely. The missing ER binding and the missing correlation of growth inhibition and inactivation
of COX enzymes exclude these targets, too.
indicated that the phosphine ligand is more important for the
biological potency than the halide or the thioglucose. Exchange
of the carbohydrate ligand of auranofin (or the chlorine ligand
of Et3PAuCl) does not lead to a loss of antitumor activity.4,11,12
Of late, there has been considerable interest in N-heterocyclic
carbenes (NHCs) as alternatives to phosphines as ligands for
the soft gold(I) ion. The relative ease of systematic modifica-
tion of the NHC substituents and the comparable donor properties
of NHCs to phosphines render NHCs attractive ligands.3,4,8,13−26
Recently, some NHC gold complexes were developed as
antitumor agents. Cationic gold(I) carbenes are lipophilic com-
pounds that are able to induce mitochondrial membrane
permeabilization of rat liver mitochondria, as shown by
Berners-Price et al.1,3,4,6,8,14−20 Raubenheimer et al. reported
a promising ferrocenylbis(carbene)gold(I) complex with an
IC50 value in the range of those of cisplatin against Jurkat, CoLo
320 DM, and MCF-7 cells.21 Also, antiproliferative activity has
been described for a few simple neutral NHC gold(I) halide
complexes16,20,22−25 as well NHC gold(I) halides bound to
biomolecules.25 The structural modifications of these com-
plexes mainly focus on variation of 1,3-N substituents at the
imidazole core.
INTRODUCTION
■
Platinum-based drugs such as cisplatin or oxaliplatin are widely
used in current tumor chemotherapy. However, severe side
effects and frequent development of resistance phenomena com-
plicate and hamper the clinical application.1,2 A very promising
strategy to overcome these obstacles is the use of specific carriers
and the change from platinum to other transition metals.1−4
Among novel non-platinum based antitumor agents, gold
complexes have recently gained attention because of their
strong antiproliferative effects.1,3−9 Two lead structures are
auranofin, which is well-known for its antiarthritic properties,
and its chloro analogue Et3PAuCl (Scheme 1).
Scheme 1. Structures of Auranofin and Et3PAuCl
Both complexes exhibit their antitumor activities due to
inhibition of the enzyme thioredoxin reductase (TrxR).1,3−11 In
addition, previous structure−activity relationship (SAR) studies
Received: August 30, 2011
Published: November 17, 2011
© 2011 American Chemical Society
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a
Scheme 2. Synthesis Routes and Structures of Intermediates and NHC Gold Complexes
a
Reagents and conditions: (I) thiamine hydrochloride, water/ethanol 1:2, room temp, 2−7 days, 49−62%; (II) formamide, reflux, 3 h, 61−80%;
(III) 95% NaH, ethyl bromide, absolute THF, reflux, 2 h, 68−88%; (IV) ethyl bromide or benzyl chloride, CH3CN, reflux, 48−72 h, 72−84%. (V)
Method A: LiHMDS, Me2SAuCl, LiBr, DMF, room temp, 4 h, 45−84%. Method B: Ag2O, CH3OH/CH2Cl2 1:1.2, room temp, 12 h, then
Me2SAuCl, LiBr, room temp, 6 h, 28-48%. (VI) Br2, CH2Cl2, room temp, 3 h, 83−85%.
a
In a previous study, we showed that NHC silver halide com-
plexes possess good growth inhibitory effects against mammary
and colon carcinoma cells.27 Moreover, replacement of the phos-
phine in Et3PAuCl by an NHC ligand with pharmacological
activity (e.g., hormonal activity, cyclooxygenase inhibitory prop-
erties) could lead to new multitarget antitumor agents including
the TxR.3,5,9,23,25−29 Therefore, we focused our attention on the
design of NHC gold complexes derived from pharmacologically
active 4,5-diarylimidazoles.28 To achieve a high accumulation
grade in tumor cells, the aromatic rings were 2-OCH3, 3-OCH3,
4-OCH3, 2-F, 3-F, 4-F, or 4-OH substituted. Such substitution
patterns were already very successfully used in the case of [1,2-
diarylethylenediamine]platinum(II) complexes.29
Scheme 3. Synthesis Route of 5k
a
Reagents and conditions: (I) ethyl bromide, CH3CN, reflux, 48−72 h,
74.2%. (II) Method A: LiHMDS, Me2SAuCl, LiBr, DMF, room temp,
4 h, 64.3%. Method B: Ag2O, CH3OH/CH2Cl2 1:1.2, room temp, 12 h,
then Me2SAuCl, LiBr, room temp, 6 h, 48%.
N-alkylations (3a−g).27,28 The hydroxyl-substituted imidazole
3h was generated from 3c by ether cleavage with BBr3.28 Sub-
sequent reaction of 3a−h and commercially available 3k with
ethyl bromide in CH3CN yielded the imidazolium salts 4a−g
and 4k. The same reaction of 3c and 3f with benzyl chloride
resulted in 1-benzyl-3-ethyl-4,5-diarylimidazolium chlorides 4i
and 4j.
Two synthetic procedures were investigated for the synthesis
of the NHC gold(I) complexes 5a−k. The first procedure (method
A) involves the treatment of a DMF solution of Me2SAuCl with a
solution containing an equimolar amount of 4a−k (treated for
deprotonation with lithium bis(trimethylsilyl)amide (LiHMDS)).
RESULTS AND DISCUSSION
■
Synthesis and Structural Characterization. The synthetic
route to NHC gold derivatives is outlined in Schemes 2 and 3.
Precursors were synthesized according to our previously
published methods.27,28 Thus, 1a−g were obtained from com-
mercially available substituted benzaldehydes via the catalysis of
thiamine hydrochloride. For ring closure, yielding the respec-
tive imidazoles 2a−g, the benzoines 1a−g were heated in
formamide to reflux for 3 h. Reaction of 2a−g with NaH and
ethyl bromide in absolute THF afforded the corresponding
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This procedure gave 5a−k in high yields.30 The second proce-
dure (method B) involves the NHC−Ag(I) transfer method,
whereby 4a−k were treated with silver oxide in CH2Cl2, and
the resulting NHC−Ag(I) complex was allowed to react with
Me2SAuCl.30,31 This procedure gave 5a−k in moderate yields.
Further amounts of lithium bromide have to be present in both
methods to ensure the exclusive formation of NHCAuBr (previous
reports showed that the chloride in the complexes could be
exchanged for bromide in the solution during the reaction30,32).
The oxidations of gold(I) complexes 5c and 5f were per-
formed with bromine in dichloromethane at room temperature,
resulting in the formation of an orange powder of the desired
gold(III) complexes 6c and 6f.25,33
Figure 1. X-ray molecular structure of 5f. Selected bond lengths (Å)
and angles (deg) of 5f are as follows: Au(1)−C(1) 1.988(9), Au(1)−
Br(1) 2.403(1), C(1)−N(5) 1.32(1), C(1)−N(2) 1.362(12), C(1)−
Au(1)−Br(1) 178.3(2), N(5)−C(1)−N(2) 105.8(7), N(5)−C(1)−
Au(1) 126.8(7), N(2)−C(1)−Au(1) 127.4(6).
Precursors and the gold complexes were characterized by IR,
NMR, and MS spectra. The IR spectra of ligands and com-
plexes are very similar and not suitable to confirm the binding
of NHC to gold. Analogously to NHC silver complexes,27 the
formation of the gold(I) complexes proceeded with the
deprotonation of the (NCHN) proton of the imidazolium bromide
Table 1. Selected X-ray Data for 5f and 6f
1
salt. Consequently, the H NMR spectrum of the respective gold
parameter
crystal system
5f
6f
complex lacks the characteristic NCHN resonance in the downfield
region of the spectrum (Figure S1a in Supporting Information).
The 13C NMR spectrum, however, showed the appearance of the
metal bound carbine (NCN−Au) peak at a much downfield shifted
region (Δδ ≈ 37 ppm) which is comparable to those reported for
other NHC gold(I) complexes (Figure S1b).34 Furthermore, upon
metal binding the 1H NMR and 13C NMR signals of ethyl, benzyl,
and phenyl remain nearly unaffected (Figure S1).
triclinic
P1
orthorhombic
Pbca
space group
̅
unit cell dimensions
a = 10.107(1) Å, α =
93.59(1)°
a = 11.536(1) Å
b = 11.423(1) Å, β =
106.27(1)°
c = 17.576(1) Å, γ =
90.33(1)°
1943.5(3) Å3
b = 15.954(1) Å
c = 23.474(2) Å
volume
4320.3(6) Å3
8
The formation of complexes were further supported by
positive mode ESI mass spectrometry, which documented a
base peak corresponding to the [M + Na]+ fragment for 5a−g,
5i−k and a base peak at m/z = 1089 corresponding to the
[2M − Br]+ fragment for 5h.
Z
4
density (calculated)
absorption coefficient
F(000)
2.014 Mg/m3
9.647 mm−1
1112
2.303 Mg/m3
12.383 mm−1
2784
crystal size
0.50 × 0.23 × 0.03 mm3
0.34 × 0.15 × 0.10
mm3
1
The H NMR spectra of the gold(III) complexes 6c and 6f
θ range for data
collection
1.79−26.00°
1.74−29.27°
were similar to the spectra of the corresponding gold(I) com-
plexes 5c and 5f, while the NCN resonance in the 13C NMR
spectra of 6c and 6f are shifted upfield by 34.8 and 33.8 ppm
compared to 5c and 5f, respectively (Figure S1). This strong
upfield shift is surprising when compared to the downfield shift
of the carbon atoms in the aromatic ring. As expected, this shift
arises from the increased acidity of gold(III) due to an increase
in the oxidation state.25,33
index range
−12≤ h ≤ 12, −14≤ k
≤ 14, −20 ≤ l ≤ 21
−15 ≤ h ≤ 12, −21
≤ k ≤ 21, −31 ≤ l
≤ 32
reflections collected
independent reflections
absorption correction
14995
42667
7458 [Rint = 0.1039]
numerical
5847 [Rint = 0.1481]
integration
max and min
transmission
0.4848 and 0.1871
0.3731 and 0.1138
To gain insight into the coordination chemistry and struc-
tural parameters of these complexes, 5f was isolated as single
crystals by slow evaporation of a concentrated MeOH/CH2Cl2
solution and characterized by X-ray diffraction. The molecular
structure of 5f is depicted in Figure 1 and is a linear mono-
carbene gold(I) complex with common Au−Ccarbene and Au−Br
bond lengths of 1.988(9) and 2.403(1) Å, respectively. The
C(1)−Au(1)−Br(1) bond with an angle of 178.3(2)° is nearly
linear.
data/restraints/
parameters
7458/0/452
5847/0/244
goodness-of-fit on F2
0.840
0.830
final R indices [I >
2σ(I)]
R1 = 0.0535, wR2 =
0.1304
R1 = 0.0550, wR2 =
0.1365
R indices (all data)
R1 = 0.0711, wR2 =
0.1417
3.591 and −2.647 e·Å−3
R1 = 0.1034, wR2 =
0.1713
largest diff peak and hole
0.960 and −3.829
e·Å−3
An X-ray structure of 6f could be obtained using the same
method. 6f has a four-coordinate gold atom in a square-planar
environment, as expected for d8 metals. The Br(3)−Au(1)−
Br(4) and C(1)−Au(1)−Br(2) bonds are nearly linear, with
angles of 175.93(5)° and 176.6(3)°. The C(1)−Au(1) distance
is 2.005(8) Å, which is in accordance with published NHC
gold(III) bromido complexes.25,33 The Br−Au distances were
found to be between 2.411(1) and 2.434(1) Å, with the Au−Br
bond in trans position being slightly longer than other Au−Br
bonds (Figure 2).
No aurophilic interactions have been observed in the solid
state structure of both complexes, which is most likely due to
the steric bulk of the carbene ligand.
Finally, all novel gold complexes are sufficiently stable in
solid state and solution. If the complexes were dissolved in
CDCl3, methanol-d4, methanol-d4/D2O (1:1), or DMSO-d6, no
changes of the spectra were observed during the storage at
room temperature. Furthermore, HPLC analysis of aqueous
solutions demonstrated sufficient stability and no degradation
during 24 h.
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auranofin caused effects comparable to those of cisplatin against
MCF-7 cells (IC50 = 1.1 μM) and was slightly more active against
HT-29 cells (IC50 = 2.6 μM).11 Et3PAuCl was less active at MCF-7
and HT-29 cell lines (IC50 of 3.2 and 5.3 μM, respectively).10,11
Exchange of the triethylphophine ligand by 1,3-diethyl-1,3-
dihydro-2H-imidazol-2-ylidene in Et3PAuCl as well as the exchange
of the chloride by bromide did not change the activity against
MCF-7 cells (5k, IC50 = 3.0 μM) but reduced the effects against
HT-29 cells (5k, IC50 = 7.7 μM). At the MDA-MB 231 cell line
5k caused an IC50 = 6.9 μM, similar to cisplatin. Introduction of
phenyl rings at C4 and C5 of the heterocycle (5k → 5g) increased
the growth inhibitory effects (IC50 = 0.87 μM at MCF-7 cells,
IC50 = 3.1 μM at MDA-MB 231 cells, IC50 = 3.3 μM at HT-29
cells). 5g was even more active than cisplatin.
As outlined in Table 2, methoxy substituents at the aromatic
rings (5g → 5a,b,c) did not change the growth inhibitory prop-
erties of 5g, while F substituents in the ortho-position (5d)
increased the activity against MDA-MB 231 cells (IC50 = 1.7 μM)
and reduced the activity at all cell lines in meta-position (5e,
IC50 = 3.1 μM at MCF-7 cells, IC50 = 6.4 μM at MDA-MB 231
cells, IC50 = 4.2 μM at HT-29 cells). The results of 5f bearing a
4-F substituent are comparable to those of 5g (unsubstituted) and
5c (4-OCH3).
Figure 2. X-ray molecular structure of 6f. Selected bond lengths (Å)
and angles deg) of 6f are as follows: Au(1)−C(1) 2.005(8), Au(1)−
Br(2) 2.434(1), Au(1)−Br(3) 2.411(1), Au(1)−Br(4) 2.421(1),
C(1)−Au(1)−Br(3) 87.5(3), C(1)−Au(1)−Br(4) 88.9(3), Br(3)−
Au(1)−Br(4) 175.93(5), C(1)−Au(1)−Br(2) 176.6(3), Br(3)−
Au(1)−Br(2) 91.45(4), Br(4)−Au(1)−Br(2) 92.24(4), N(1)−
C(1)−Au(1) 127.7(7), N(2)−C(1)−Au(1) 124.5(7).
Antiproliferative Effects. In vitro cytotoxicity assays were
performed to get an insight into the antitumor activity of the
neutral gold complexes. All complexes and the established
antitumor drug cisplatin were screened against MCF-7, MDA-
MB 231 breast cancer cells, and HT-29 colon cancer cells. The
experiments were performed according to established proce-
dures.10,11,35 IC50 values of the selected intermediate 4f, the gold
complexes 5a−k, 6c, 6f, and cisplatin determined after 72 h
of incubation are shown in Table 2. The time dependent
Exchange of the 4-OCH3 groups by more hydrophilic 4-OH
groups (5c → 5h) strongly decreased the cytotoxicity (5h,
IC50 = 4.5 μM at MCF-7 cells, IC50 > 20 μM at MDA-MB 231
cells, IC50 = 17.0 μM at HT-29 cells). This substitution effect
was already observed during the study of other metal complexes
such as [1,2-diarylethylenediamine]platinum(II)29 or [diarylsalene]-
iron complexes.36
The substituents at the nitrogen atoms seem to play a sub-
ordinate role because complexes with either an N1-ethyl chain
or a bulky N1-benzyl moiety yielded nearly identical results
(compare 5c and 5i as well as 5f and 5j, Table 2).
Finally the growth inhibitory effects depended on the pres-
ence of a metal center (the imidazoles were inactive; e.g., see 4f in
Table 2). Interestingly, the relevance of the oxidation state for the
growth inhibition of MCF-7 and MDA-MB 231 cells was low. At
the HT-29 cell line only, the Au(III) complexes 6c and 6f were
less active than their Au(I) congeners 5c and 5f. Moreover,
these novel gold complexes were distinctly more active than the
corresponding silver complexes27 and benzimidazol-2-ylidene
gold complexes23 tested in the same assay.
Time-dependent cytotoxicity studies were performed on the
examples of 5c and 5f. The curves presented in Figure 3 indi-
cate for 5f a recuperation of HT-29 cells at concentrations
below 2.5 μM and of MCF-7 cells below 1 μM. Because expo-
nential cell growth is guaranteed for at least 140 h of incubation,
the rise of the growth curve can be explained by the development
of drug resistance.35,36
In contrast to cisplatin (Figure S2), which reached its maxi-
mum effect toward the end of the test, the onset of anti-
proliferative effects of 5c and 5f was observed much earlier.
Both complexes showed their maximum of activity after 48 h.
In the case of 5c, the growth inhibition holds during the whole
time of incubation (no recuperation of the tumor cells after a
prolonged time of exposition).
Table 2. Growth-Inhibitory Effects against Breast Cancer
and Colon Cancer Cells
a
cytotoxicity (IC50, μM)
compd
4f
MCF-7
>50
MDA-MB 231
HT-29
>50
>50
5a
1.2 ± 0.1
1.6 ± 0.6
1.4 ± 0.1
0.80 ± 0.06
3.1 ± 0.1
1.1 ± 0.3
0.87 ± 0.07
4.5 ± 0.3
2.6 ± 0.3
1.8 ± 0.1
3.0 ± 0.4
1.9 ± 0.1
1.9 ± 0.4
1.1
2.4 ± 0.5
2.9 ± 0.3
3.7 ± 0.9
1.7 ± 0.9
6.4 ± 0.1
3.9 ± 0.1
3.1 ± 0.5
>20
3.1 ± 0.4
4.2 ± 1.0
2.9 ± 0.1
3.3 ± 1.0
4.2 ± 0.3
2.3 ± 0.1
3.3 ± 0.7
17.0 ± 2.8
4.3 ± 0.1
3.2 ± 0.3
7.7 ± 0.8
6.2 ± 1.0
7.5 ± 2.9
2.6
5b
5c
5d
5e
5f
5g
5h
5i
3.3 ± 0.7
2.4 ± 0.1
6.9 ± 0.8
4.4 ± 1.1
4.4 ± 0.7
d
5j
5k
6c
6f
b
auranofin
c
Et3AuCl
3.2
d
5.3
cisplatin
1.6 ± 0.5
7.8 ± 0.8
4.1 ± 0.3
a
The IC50 value was determined as the concentration causing 50%
decrease in cell growth after 72 h of incubation and calculated as the
b
mean of at least two or three independent experiments. Data from ref
c
d
11. Data from refs 10 and 11. Not measured.
antiproliferative effects of 5c and 5f at the three cancer cell lines
are presented in Figure 3.
Cisplatin characteristically reduced the cell growth of MCF-7
(IC50 = 1.6 μM), MDA-MB 231 (IC50 = 7.8 μM), and HT-29
(IC50 = 4.1 μM) cells. According to our previous reports,
Inhibition of the Enzyme TrxR. As discussed above
(Introduction), the TrxR must be considered as target because
it is involved in various tumor related biochemical pathways and
inactivation causes reduced tumor cell growth.3−6,9,23,24,26,37−40
Auranofin and related gold complexes demonstrated strong
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Figure 3. Time-dependent antiproliferative effects of complexes 5c (left) and 5f (right) on MCF-7, MDA-MB 231, and HT-29 cells.
affinity for TrxR leading to inactivation due to the formation of
a covalent bond at the Se center of the enzyme.3−6,9,23,24,26,37−39
The inhibitory potentials of all gold complexes, 4f (as
example of the free ligands) as well as Et3PAuCl and auranofin
(as positive controls), were studied by the dithiobisnitrobenzoic
acid (DTNB) reduction assay using isolated rat liver TrxR. This
assay makes use of the fact that TrxR reduces the disulfide
bonds of DTNB with formation of 2-nitro-5-thionitrobenzoic
acid (TNB), which can be detected photometrically.
With the exception of the complex 5h, which showed low
solubility in the used assay buffer that might explain its sig-
nificantly lower activity, all gold complexes displayed activity against
TrxR (IC50 of 0.374−1.505 μM). They are comparably active as
benzimidazol-2-ylidene gold complexes23 but less active than
Et3PAuCl and auranofin (IC50 of 25.8 and 18.6 nM, respec-
tively). As expected, the inhibitory effect depends on the pre-
sence of a gold center; 4f was completely inactive (IC50 > 50 μM).
A clear structure−activity relationship is not given by the data
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listed in Table 3, and a correlation with the growth inhibition is
missing. Therefore, besides the TrxR interaction, further targets
must be considered as part of the mode of action.3,4,9,40−46
and accumulation, the intracellular drug level in MCF-7 and
HT-29 cells was studied in the examples of 5c, 5f, and Et3PAuCl.
In the present study we used a recently described electro-
thermal atomic absorption spectroscopy (ETAAS) method,
which was developed for the bioanalysis of gold complexes and
based on the standard addition method.10,47 Experiments were
performed using an extracellular concentration of 10 μM and a
short exposure period of 6 h to avoid a loss of cell biomass due
to toxic effects. Results were corrected for the respective protein
contents of the samples, and values are accordingly presented as
ng of metal/mg of protein.
MCF-7 cells incubated with either 5c or 5f showed about a
3-fold higher gold content (5c, 134.2 ng/mg; 5f, 67.2 ng/mg)
than HT-29 cells (5c, 41.4 ng/mg; 5f, 21.9 ng/mg). However,
the individual cellular parameters must be taken into account.
From the mean cellular diameter and the mean protein to
volume ratio of HT-2947 and MCF-7 cells,48 it can be estimated
that 1.0 ng of Au per mg of protein corresponds to a cellular
molar concentration of 1.0 μM in HT-29 cells but to only
0.57 μM in MCF-7 cells. Nevertheless, the cellular molar con-
centrations of 5c and 5f were higher in MCF-7 cells (76.5 and
38.3 μM, respectively) than in HT-29 cells (41.4 and 21.9 μM,
respectively).
The uptake in HT-29 cells corresponded with that of the
references cisplatin and Et3PAuCl, while it strongly differed in
MCF-7 cell: 5f ∼ cisplatin < 5c < Et3PAuCl. The highest
intracellular metal content of 52.4 ng/mg was achieved with
Et3PAuCl.
To get information on whether the complexes might reach
the major target of metal complexes, the DNA, the nuclei of
MCF-7 and HT-29 cells treated with 5c, 5f, or Et3PAuCl were
isolated by a short sucrose gradient and investigated for their
gold content using ETAAS. The results were calculated as ng of
Au/mg of nuclear protein (Table 4).
Table 3. Inhibition of TrxR
a
a
compd
TrxR (IC50, nM)
compd
5h
TrxR (IC50, nM)
4f
>50000
4371.3 ± 322.2
802.7 ± 68.1
374.4 ± 9.0
379.8 ± 105.1
621.6 ± 51.3
903.6 ± 1.1
18.6 ± 7.2
5a
5b
5c
5d
5e
5f
597.5 ± 89.6
668.3 ± 38.1
1505.5 ± 27.3
703.9 ± 61.5
1202.0 ± 110.3
815.4 ± 74.1
1036.1 ± 40.4
5i
5j
5k
6c
6f
auranofin
Et3PAuCl
5g
25.8 ± 13.0
a
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 mean and error of three experiments.
Reaction with Glutathione. It is a well-known fact that
cisplatin and other metal containing drugs including auranofin are
very reactive toward thiols such as the tripeptide glutathione.1,3−5,23
The levels of glutathione in tumor cells reach the low millimolar
range and are made responsible for inactivation and drug resistance
phenomena. Therefore, it was of interest to study the interaction
with glutathione under physiological conditions.
In the used assay, an excess of the respective gold complexes
(166.7 μM) with reduced glutathione was incubated at 37 °C
for 20 and 60 min. Oxidized GSH reduces analogously to TrxR
DTNB to TNB. Me2SAuCl (33% 20 min; 56% 60 min) used as
positive control led to reduced TNB formation under the chosen
conditions (Figure 4), while auranofin (194% 20 min; 212%
Similar to the cellular uptake, the nuclear gold content was
higher in MCF-7 than in HT-29 cells. Interestingly, 5c and
Et3PAuCl caused nearly the same values (212.1 and 176.8 ng/
mg, respectively) which are 2-fold higher than that of 5f (81.4
ng/mg). In the nuclei of HT-29 cells only small amounts of
gold (14.8 ng/mg (5c), 9.5 ng/mg (5f), and 56.6 ng/mg
(Et3PAuCl)) were detected. These findings did not correlate
with the growth inhibitory potency. Therefore, we generally
investigated the DNA binding properties using salmon testes
DNA (Table 4). In this test 5c and 5f showed a moderate
binding to DNA (5c, 2.03 pmol/μg; 5f, 1.03 pmol/μg) after
4 h, not as high as cisplatin (14.2 pmol/μg) but higher than
Et3PAuCl (0.11 pmol/μg).
On the basis of these results, it is not very likely that DNA
interaction causes the cytotoxic effects of the gold complexes.
ER Interaction and the Inhibition of COX. The missing
correlation between cellular uptake, DNA binding, TrxR-
inhibition, and cytotoxicity induced us to search for further
targets of the novel gold complexes.
As the NHC ligands described in this paper are derivatives of
2,4,5-triarylimidazoles designed as estrogen receptor (ER)
ligands,28,49−51 we investigated if the ER is involved in the
mode of action in MCF-7 cells. For this purpose, ERα-positive
MCF-7 breast cancer cells, stably transfected with the plasmid
EREwtcluc (MCF-7-2a cells) and U2-OS cells transiently trans-
fected with the plasmid pSG5-ERα (U2-OS/α) or pSG5-ERβ FL
(U2-OS/β) and the reporter plasmid p(ERE)2-luc+ were used
to evaluate ER subtype selectivity on the molecular level.28,49−51
Unfortunately, all complexes did not induce agonistic or antagonistic
Figure 4. Interaction of gold complexes (166.7 μM) with glutathione
and DTNB. Percentage building of TNB after 20 and 60 min of
incubation.
60 min) strongly increased the TNB release during the reaction.23
A DMSO control was set as 100%.
Interestingly, the complexes determined in the examples of
5c (82%, 20 min; 91%, 60 min) and 5f (85%, 20 min; 90%,
60 min) did not significantly influence the reaction and can thus
be considered as sufficiently stable against thiols under biologically
relevant conditions, a property highly desirable in the drug
development of novel metal coordination compounds.23
Cellular and Nuclear Uptake as Well as DNA Interac-
tion. Because the cytotoxic effects of metal complexes in cell
culture experiments were strongly influenced by cellular uptake
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Table 4. Cellular, Nuclear Uptake, and DNA Binding Efficiency of the Complexes
a
cellular uptake
a
nuclear uptake (ng of Au/mg of nuclear
ng of metal/mg of protein
μM
protein)
b
compd
5c
MCF-7
HT-29
MCF-7
HT-29
MCF-7
HT-29
DNA binding (pmol/μg)
134.2 ± 29.1
67.2 ± 9.3
282.1 ± 22.8
40.9 ± 5.7
41.4 ± 9.9
21.9 ± 2.1
52.4 ± 1.6
28.6 ± 3.6
76.5
38.3
160.8
23.3
41.4
21.9
52.4
28.6
212.1 ± 14.2
81.4 ± 36.5
176.8 ± 80.3
c
14.8 ± 0.8
9.5 ± 4.1
56.6 ± 14.3
c
2.03 ± 0.08
1.03 ± 0.11
0.11 ± 0.02
14.2 ± 1.4
5f
Et3PAuCl
cisplatin
a
b
Results were calculated from the data of three independent experiments. Results were calculated as the mean of at least two independent
c
experiments, which were performed with two replicates. Not measured.
properties (data not shown). Therefore, an ER-mediated
cytotoxicity can be excluded.
EXPERIMENTAL SECTION
■
General. The following instrumentation was used: IR spectra (KBr
pellets), Perkin-Elmer model 580 A (Shelton, U.S.); H or 13C NMR
1
A further possible target of triarylimidazole derivatives are
the cyclooxygenase enzymes (COX-1/2), as we demonstrated
in an earlier study.51 Both subtypes are the targets not only for
the design of anti-inflammatory drugs but also in cancer chemo-
therapy.51 For Co-ASS, a metallodrug of aspirin, a mode of action
involving COX inhibition was postulated. The complex possessed
high cytotoxicity and represented by far a more efficient COX
inhibitor than aspirin.52−54 Recently, it was demonstrated that
auranofin inhibited the formation of eicosanoids related to the
activity of COX-1 and 12-lipoxygenase (LOX) in human
platelets.4
On the basis of these facts, the relative COX inhibition was
determined exemplarily for complex 5f and its ligand 4f. The
effects of the compounds on isolated COX-1 and COX-2
enzymes were quantified by ELISA in a concentration of 10 μM.
Aspirin was used for comparison. At the concentration used
aspirin showed only marginal activity at COX-1 (29% inhibition)
and was inactive at COX-2. Interestingly, in contrast to its ligand
4f which was inactive at both isoenzymes, 5f selectively inhibited
COX-1 (100%). Participation on the growth inhibitory effects
against MCF-7 might be possible, since these cells have a basal
level of COX-1 and a barely detectable and transient COX-2
inducible expression, whereas MDA-MB 231 cells show a low
expression of COX-1 but a constitutive level of COX-2.55 There-
fore, their growth is sensitive to NSAID treatment56,57 The data
listed in Table 2 indicate for 5f a comparable activity against both
cell lines. Therefore, COX-inhibition is also very unlikely as the
mode of action.
spectra, Bruker ADX 400 spectrometer operated at 400 or 100 MHz
(internal standard, tetramethylsilane); electron impact (EI) MS
spectra, Varian CH-7A (70 eV) spectrometer; ESI-TOF spectra,
Agilent 6210 ESI-TOF, Agilent Technologies, Santa Clara, CA, U.S.;
elemental C, H, N analysis, PerkinElmer 240 B and C analyzer. All
complexes reported in the manuscript have a purity of >95%.
Chemicals were obtained from Sigma-Aldrich (Germany) and used
without further purification. Reactions were all monitored by TLC,
performed on silica gel plates 60 F254 (Merck, Darmstadt/Germany).
Visualization on TLC was achieved by UV light. Column chro-
matography was performed with Merck silica gel 60H, grain size of
<0.063 mm, 230 mesh ASTM (Darmstadt/Germany).
General Procedure for the Synthesis of 5a−k. Method A. LiHMDS
(104 mg, 0.62 mmol) was added to a solution of the imidazolium salt
(0.55 mmol) in DMF (5 mL). After the resulting mixture was stirred
for 15 min a solution of Me2SAuCl (162 mg, 0.55 mmol) and LiBr
(477 mg, 5.5 mmol) in DMF (1 mL) was added and the suspension
stirred for additional 4 h. Then water (10 mL) was added and a gray
precipitate formed. The precipitate was collected and washed with
water (3 × 10 mL). Column chromatography on silica gel (CH2Cl2/
petroleum ether) followed by recrystallization from CH2Cl2/n-hexane
yielded pure gold complexes.
Method B. An amount of 0.16 mmol of imidazolium salt in a
mixture of CH2Cl2 (1 mL) and methanol (1.2 mL) was combined with
solid silver(I) oxide (21 mg, 0.09 mmol) under N2 atmosphere and
stirred overnight under protection from light. Then Me2SAuCl (47 mg,
0.16 mmol) and LiBr (139 mg 1.6 mmol) were added and the sus-
pension was stirred for an additional 6 h under N2. The gray precipitate
was separated by filtration over a bed of Celite and the filtrate was
evaporated to dryness under reduced pressure to yield a colorless oil.
Column chromatography on silica gel (CH2Cl2/petroleum ether)
followed by recrystallization from CH2Cl2/n-hexane yielded pure gold
complexes.
Nevertheless it is worthy to note that 5f is an effective COX-1
inhibitor, which makes this class of complexes an interesting
subject for further SAR study.
Bromo[1,3-diethyl-4,5-bis(2-methoxyphenyl)-1,3-dihydro-
2H-imidazol-2-ylidene]gold(I) 5a. Method A: yield 56.7% of
a white solid. ESI-MS m/z: 337 [M − AuBr]+, 635 [M + Na]+, 869
[2M − AuBr2]+, 1145 [2M − Br]+. IR (KBr, cm−1): 3061, 2955, 2921,
CONCLUSION
■
4,5-Diarylimidazole derivatives were synthesized as NHC li-
gands for gold complexes. The inactive ligands modulate the
biological activity of the complexes. NHC gold halides caused
growth inhibitory effects dependent on the substituents at the
aromatic rings against MCF-7 and MDA-MB 231 breast as
well as HT-29 colon cancer cell lines. The influence of N-
substituents and the oxidation state of the metal (Au(I) or
Au(III)) is relatively low. All complexes inhibited the TrxR.
The missing SAR and the missing correlation with cytotoxic
properties indicated the involvement of further targets. On the
basis of the investigations of cellular and nuclear uptake, as well
as the binding to the ER, DNA binding and interference in the
hormonal system can be excluded. The selective inhibition of
the COX-1 enzyme by complex 5f opens a new perspective for
use of NHC gold complexes in medicinal chemistry.
1
2851, 1632, 1603, 1461, 1248, 1179, 1023, 756. H NMR (CDCl3): δ
1.27 (t, 6H, CH2CH3, J = 7.2 Hz), 3.81 (s, 6H, OCH3), 4.10 (q, 4H,
CH2CH3, J = 7.2 Hz), 6.81−6.97 (m, 5H, ArH), 7.10−7.12 (m, 1H,
ArH), 7.30−7.34 (m, 2H, ArH). Anal. (C21H24AuBrN2O2) C, H, N.
Bromo[1,3-diethyl-4,5-bis(3-methoxyphenyl)imidazol-2-
ylidene]gold(I) 5b. Method A: yield 78.9% of a pale yellow solid.
ESI-MS m/z: 635 [M + Na]+, 869 [2M − AuBr2]+, 1145 [2M − Br]+,
1861 [3M + Na]+, 1877 [3M + K]+. IR (KBr, cm−1): 3065, 2955,
2920, 2850, 1630, 1601, 1460, 1238, 1176, 1040, 701. 1H NMR
(CDCl3): δ 1.33 (t, 6H, CH2CH3, J = 7.2 Hz), 3.73 (s, 6H, OCH3),
4.20 (q, 4H, CH2CH3, J = 7.2 Hz), 6.71−6.80 (m, 4H, ArH), 6.89−
6.91 (m, 2H, ArH), 7.26−7.29 (m, 2H, ArH). 13C NMR (CDCl3): δ
17.0 (s, CH3); 44.3 (s, CH2); 55.3 (s, OCH3); 114.7, 116.2, 122.7,
128.9, 129.9, 130.8, 159.6 (s, CAr); 172.7 (s, NCN). Anal.
(C21H24AuBrN2O2) C, H, N.
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Bromo[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-
ylidene]gold(I) 5c. Method A: yield 73.4%. Method B: yield 45.1% of
a white solid. ESI-MS m/z: 635 [M + Na]+, 869 [2M − AuBr2]+, 1145
[2M − Br]+. IR (KBr, cm−1): 3065, 2967, 2930, 2841, 1630, 1505, 1251,
1178, 1026, 843. 1H NMR (CDCl3): δ 1.30 (t, 6H, CH2CH3, J = 7.2 Hz),
3.80 (s, 6H, OCH3), 4.16 (q, 4H, CH2CH3, J = 7.2 Hz), 6.86 (d, 4H,
ArH, J = 8.4 Hz), 7.10 (d, 4H, ArH, J = 8.8 Hz). 13C NMR (CDCl3): δ
16.9 (s, CH3); 44.1 (s, CH2); 55.2 (s, OCH3); 114.3, 119.8, 130.6, 131.7,
160.1 (s, CAr); 172.1 (s, NCN). Anal. (C21H24AuBrN2O2) C, H, N.
Bromo[1,3-diethyl-4,5-bis(2-fluorophenyl)imidazol-2-
ylidene]gold(I) 5d. Method A: yield 79.9% of a white solid. ESI-MS
m/z: 611 [M + Na]+, 627 [M + K]+, 821 [2M − AuBr2]+, 859 [2M −
AuBr2+K]+, 1097 [2M − Br]+, 1217 [2M + K]+. IR (KBr, cm−1):
4.23 (q, 2H, CH2CH3, J = 7.2 Hz), 5.37 (s, 2H, CH2Ar), 6.92−7.08
(m, 8H, ArH), 7.16−7.24 (m, 5H, ArH). 13C NMR (CDCl3): δ 16.9
(s, CH3); 44.4 (s, CH2); 53.0 (s, CH2Ar); 115.8, 116.0, 116.1, 116.4,
123.3, 123.4, 127.4, 128.3, 128.6, 130.7, 130.9, 132.3, 132.4, 132.7,
132.8, 135.4 (s, CAr); 161.9 (d, CAr); 164.4 (d, CAr); 174.1 (s, NCN).
Anal. (C24H20AuBrF2N2) C, H, N.
Bromo(1,3-diethylimidazol-2-ylidene)gold(I) 5k. Method A:
yield 64.3% of a white solid. ESI-MS m/z: 422 [M + Na]+, 721 [2M −
Br]+, 824 [2M + Na]+. IR (KBr, cm−1): 3153, 3120, 2955, 2919, 2851,
1
1629, 1463, 1421, 1377, 1204, 1096, 754. H NMR (CDCl3): δ 1.47
(t, 6H, CH2CH3, J = 7.2 Hz), 4.23 (q, 4H, CH2CH3, J = 7.2 Hz), 6.95
(s, 2H, CH). Anal. (C7H12AuBrN2) C, H, N.
General Procedure for the Synthesis of 6c and 6f. To a
solution of 5c or 5f (0.1 mmol) in CH2Cl2 (2 mL) was added bromine
(23.7 mg, 0.15 mmol). The mixture was stirred for 3 h in the dark, and
the solvent was removed under reduced pressure, as well as the excess
of bromine. The residue was recrystallized from CH2Cl2/n-hexane to
yield 6c or 6f as orange powder.
1
3061, 2955, 2921, 2851, 1632, 1484, 1456, 1229, 1107, 762. H NMR
(CDCl3): δ 1.31 (t, 6H, CH2CH3, J = 7.2 Hz), 4.14 (q, 4H, CH2CH3,
J = 7.2 Hz), 7.10−7.19 (m, 6H, ArH), 7.38−7.44 (m, 2H, ArH). 13C
NMR (CDCl3): δ 16.6 (s, CH3); 44.7 (s, CH2); 115.1, 115.2, 116.1,
116.3, 124.7, 126.4 (s, CAr); 132.3 (d, CAr); 132.5, 159.2, 161.7 (s,
CAr); 174.0 (s, NCN). Anal. (C19H18AuBrF2N2) C, H, N.
Tribromo[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-
ylidene]gold(III) 6c. Yield 83.1% of an orange powder. ESI-MS m/z:
795 [M + Na]+. IR (KBr, cm−1): 3060, 3039, 2979, 2934, 2836, 1606,
Bromo[1,3-diethyl-4,5-bis(3-fluorophenyl)imidazol-2-
ylidene]gold(I) 5e. Method A: yield 84.5% of a white solid. ESI-MS
m/z: 611 [M + Na]+, 627 [M + K]+, 821 [2M − AuBr2]+, 1097 [2M −
Br]+, 1217 [2M + K]+. IR (KBr, cm−1): 3055, 2954, 2919, 2851, 1608,
1
1504, 1472, 1436, 1253, 1178, 1023, 843. H NMR (DMSO-d6): δ
1.25 (t, 6H, CH2CH3, J = 7.2 Hz), 3.76 (s, 6H, OCH3), 4.17 (q, 4H,
CH2CH3, J = 7.2 Hz), 6.98 (d, 4H, ArH, J = 8.4 Hz), 7.36 (d, 4H,
ArH, J = 8.8 Hz). 13C NMR (CDCl3): δ 15.3 (s, CH3); 44.1 (s, CH2);
55.3 (s, OCH3); 114.5, 118.2, 132.0, 133.8 (s, CAr); 137.3 (s, NCN);
160.7 (s, CAr). Anal. (C21H24AuBr3N2O2) C, H, N.
1
1580, 1462, 1187, 791. H NMR (CDCl3): δ 1.35 (t, 6H, CH2CH3, J =
7.2 Hz), 4.23 (q, 4H, CH2CH3, J = 7.2 Hz), 6.92−7.02 (m, 4H, ArH),
7.11−7.16 (m, 2H, ArH), 7.33−7.42 (m, 2H, ArH). 13C NMR (CDCl3):
δ 16.9 (s, CH3); 44.5 (s, CH2); 116.7, 117.0, 117.4, 117.6 (s, CAr); 126.3
(d, CAr); 129.3, 129.4 (s, CAr); 130.1 (d, CAr); 130.7, 130.8, 161.4, 164.1
(s, CAr); 173.8 (s, NCN). Anal. (C19H18AuBrF2N2·0.1 n-hexane) C, H, N.
Bromo[1,3-diethyl-4,5-bis(4-fluorophenyl)imidazol-2-
ylidene]gold(I) 5f. Method A: yield 78.2%. Method B: yield 48.7%
of a white solid. ESI-MS m/z: 611 [M + Na]+, 821 [2M − AuBr2]+. IR
(KBr, cm−1): 3061, 2966, 2924, 2851, 1631, 1601, 1503, 1460, 1228,
Tribromo[1,3-diethyl-4,5-bis(4-fluorophenyl)imidazol-2-
ylidene]gold(III) 6f. Yield 85.6% of an orange powder. ESI-MS m/z:
771 [M + Na]+. IR (KBr, cm−1): 3110, 3062, 2984, 2936, 2875, 1629,
1
1600, 1503, 1474, 1343, 1228, 1159, 848. H NMR (CDCl3): δ 1.36
(t, 6H, CH2CH3, J = 7.2 Hz), 4.26 (q, 4H, CH2CH3, J = 7.2 Hz), 7.11
(t, 4H, ArH, J = 8.4 Hz), 7.23−7.26 (m, 4H, ArH). 13C NMR
(CDCl3): δ 15.3 (s, CH3); 44.3 (s, CH2); 116.5, 116.7 (s, CAr); 121.9
(d, CAr); 132.6, 132.7, 133.2 (s, CAr); 139.4 (s, NCN); 162.3, 164.9 (s,
CAr). Anal. (C19H18AuBr3F2N2·0.15CH2Cl2) C, H, N.
X-ray Crystallography. The intensities for the X-ray determi-
nations were collected on a STOE IPDS 2T instrument with Mo Kα
radiation (λ = 0.710 73 Å) at 200 K. Standard procedures were applied
for data reduction and absorption correction. Structure solution and
refinement were performed with SHELXS97 and SHELXL97.58
Hydrogen atom positions were calculated for idealized positions and
treated with the “riding model” option of SHELXL. More details on
data collections and structure calculations are contained in Table 1 and
Supporting Information.
Cytotoxicity. The human MCF-7, MDA-MB 231 breast cancer
cell, and HT-29 colon cancer cell lines were obtained from the
American Type Culture Collection. All cell lines were maintained as a
monolayer culture in L-glutamine containing Dulbecco’s modified
Eagle’s medium (DMEM) with 4.5 g/L glucose (PAA Laboratories,
Austria), supplemented with 5% fetal bovine serum (FBS; Biochrom,
Germany) in a humidified atmosphere (5% CO2) at 37 °C.
The experiments were performed according to established pro-
cedures with some modifications.10,11,35 In 96-well plates, an amount
of 100 μL of a cell suspension in culture medium at 7500 cells/mL
(MCF-7 and MDA-MB 231) or 3000 cells/mL (HT-29) was plated
into each well and incubated for 3 days under culture conditions. After
the addition of various concentrations of the test compounds, cells
were incubated for up to the appropriate incubation time. Then the
medium was removed, and the cells were fixed with glutardialdehyde
solution 1% and stored under phosphate buffered saline (PBS) at
4 °C. Cell biomass was determined by a crystal violet staining,
followed by extracting of the bound dye with ethanol and a photo-
metric measurement at 590 nm. Mean values were calculated, and the
effects of the compounds were expressed as % treated/controlcorr
values according to the following equation:
1
1159, 1094, 843. H NMR (CDCl3): δ 1.31 (t, 6H, CH2CH3, J = 7.2
Hz), 4.18 (q, 4H, CH2CH3, J = 7.2 Hz), 7.07 (t, 4H, ArH, J = 8.4 Hz),
7.16−7.20 (m, 4H, ArH). 13C NMR (CDCl3): δ 16.9 (s, CH3); 44.3
(s, CH2); 116.2, 116.4 (s, CAr); 123.4 (d, CAr); 130.3, 132.3, 132.4, 161.9,
164.4 (s, CAr); 173.2 (s, NCN). Anal. (C19H18AuBrF2N2) C, H, N.
Bromo[1,3-diethyl-4,5-diphenylimidazol-2-ylidene]gold(I)
5g. Method A: yield 82.2% of a white solid. ESI-MS m/z: 575 [M +
Na]+, 591 [M + K]+, 749 [2M − AuBr2]+, 1025 [2M − Br]+. IR (KBr,
1
cm−1): 3056, 2955, 2923, 2852, 1629, 1487, 1461, 1375, 1026, 700. H
NMR (CDCl3): δ 1.31 (t, 6H, CH2CH3, J = 7.2 Hz), 4.20 (q, 4H,
CH2CH3, J = 7.2 Hz), 7.18−7.21 (m, 4H, ArH), 7.32−7.39 (m, 6H, ArH).
13C NMR (CDCl3): δ 16.9 (s, CH3); 44.3 (s, CH2); 127.7, 128.8, 129.3,
130.5, 131.0 (s, CAr); 172.8 (s, NCN). Anal. (C19H20AuBrN2) C, H, N.
Bromo[1,3-diethyl-4,5-bis(4-hydroxyphenyl)imidazol-2-
ylidene]gold(I) 5h. Method A: yield 45.4%. Method B: yield 28.7%
of a white solid. ESI-MS m/z: 813 [2M − 2Br−Au]+, 1089 [2M −
Br]+. IR (KBr, cm−1): 3600−2500 (OH), 2968, 2926, 1607, 1507,
1379, 1272, 1104, 840. 1H NMR (DMSO-d6): δ 1.20 (t, 6H, CH2CH3,
J = 7.2 Hz), 4.04 (q, 4H, CH2CH3, J = 7.2 Hz), 6.75 (d, 4H, ArH, J =
8.4 Hz), 7.14 (d, 4H, ArH, J = 8.4 Hz), 9.78 (s, 2H, ArOH,
exchangeable by D2O). Anal. (C19H20AuBrN2O2) C, H, N.
Bromo[3-benzyl-1-ethyl-4,5-bis(4-methoxyphenyl)imidazol-
2-ylidene]gold(I) 5i. Method A: yield 71.3% of a white solid. ESI-
MS m/z: 697 [M + Na]+, 993 [2M − AuBr2]+, 1269 [2M − Br]+. IR
(KBr, cm−1): 3062, 3034, 2954, 2921, 2851, 1606, 1503, 1459, 1250,
1
1025, 835. H NMR (CDCl3): δ 1.32 (t, 3H, CH2CH3, J = 7.2 Hz),
3.77 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.21 (q, 2H, CH2CH3, J =
7.2 Hz), 5.35 (s, 2H, CH2Ar), 6.75 (d, 2H, ArH, J = 8.8 Hz), 6.87 (q,
4H, ArH, J = 8.8 Hz), 7.02−7.10 (m, 4H, ArH), 7.21−7.23 (m, 3H,
ArH). 13C NMR (CDCl3): δ 16.9 (s, CH3); 44.2 (s, CH2); 52.7 (s,
CH2Ar); 55.2 (s, OCH3); 114.0, 114.3, 119.7, 127.5, 128.0, 128.5,
131.2, 131.3, 131.7, 132.1, 135.9 (s, CAr); 160.1 (d, CAr); 173.3 (s,
NCN). Anal. (C26H26AuBrN2O2) C, H, N.
Bromo[3-benzyl-1-ethyl-4,5-bis(4-fluorophenyl)imidazol-2-
ylidene]gold(I) 5j. Method A: yield 76.5% of a white solid. ESI-MS
m/z: 673 [M + Na]+, 945 [2M − AuBr2]+, 1221 [2M − Br]+. IR (KBr,
cm−1): 3063, 3034, 2955, 2922, 2852, 1632, 1602, 1501, 1457, 1228,
1
1159, 829. H NMR (CDCl3): δ 1.35 (t, 3H, CH2CH3, J = 7.2 Hz),
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Sample Preparation for Nuclear Uptake Studies. The nuclei
of the tumor cells were isolated according to previously described
procedures10 with some minor modifications. Cells were grown in
175 cm2 cell culture flasks until at least 70% confluency. The medium
was removed and replaced with 10 mL of medium containing 10.0 μM
drug. After 24 h of incubation at 37 °C in a humidified atmosphere,
the drug containing medium was removed, cells were trypsinized, re-
suspended in 10 mL of cell culture medium, and isolated by cen-
trifugation (1500 rpm, 5 min), and 0.5−1.0 mL of 0.9% NaCl solution
was added. After centrifugation (1500 rpm, 5 min) pellets were
resuspended in 300 μL of RSB-1 (0.01 M Tris-HCl, 0.01 M NaCl,
1.5 mM MgCl2, pH 7.4) and left for 10 min in an ice bath. Swollen
cells were centrifuged (2000 rpm, 5 min), resuspended in 300 μL of
RSB-2 (RSB-1 containing each 0.3% v/v Nonidet-P40 and sodium
desoxycholate), and homogenized by 10−15 up/down pushes in a
1 mL syringe with needle. Aliquots of 50 μL of the homogenisate were
removed for determination of the total gold content and mixed with
500 μL of water. The homogenisate was centrifuged at 2500 rpm for
5 min, and the resulting crude nuclei were taken up in 150 μL of 0.25 M
sucrose containing 3 mM CaCl2. The suspension was underlayed with
150 μL of 0.88 M sucrose and centrifuged 10 min at 2500 rpm. The
nuclei pellets were stored at −20 °C or immediately dissolved in
500 μL of water and disrupted by use of a sonotrode. The gold content
of the samples was measured by ETAAS and the protein content by the
Bradford method. Results are expressed as the mean of three
independent experiments as ng of gold per mg of nuclear protein.
Electrothermic Atomic Absorption Spectroscopy (ETAAS). ETAAS
measurements were performed according to a previously published
standard addition procedure with some minor modifications.10,47 In
short, to 100 μL aliquots of the diluted lysates increasing amounts of
aqueous gold standard solutions were added. All probes were adjusted
to a final volume of 200 μL using twice distilled water. For each, 20 μL
of Triton X-100 (1%) and ascorbic acid (1%) were added, and the
probes were measured as described below. The gold content of the
lysates was accessed by the linear extrapolation method. A Vario 6
electrothermal atomic absorption spectrometer (AnalytikJena AG) was
used for the gold measurements. Gold was detected at a wavelength
of 242.8 nm with a bandpass of 0.8 nm. A deuterium lamp was used
for background correction. Probes were injected at a volume of 25 μL
into regular graphite wall tubes. Drying, atomization, and tube
cleaning steps were performed as outlined in more detail in the
literature.10,47 The temperature for pyrolysis was set to 1200 °C. The
mean AUC (area under curve) absorptions of duplicate injections were
used throughout the study. The limit of gold detection using biological
samples as described above was 1.7 μg/L.
DNA Binding Studies. Precipitation of drug DNA adducts was
performed according to a described method35 with some modifica-
tions. Salmon testes DNA (Sigma) were dissolved in phosphate-
buffered saline, pH 7.4, and drugs were added as stock solutions in
DMF. The final solutions contained 40.6 mM drug, 250 μg/mL
salmon testes DNA, and 0.1% v/v DMF. After being vortexed, the
solutions were incubated at 37 °C in a water bath for 4 h. Aliquots of
200 μL were mixed with 100 μL of 0.9 M sodium acetate and three
volumes of ice cold ethanol. Samples were stored at −20 °C for
30 min. The pellets were isolated by centrifugation (5000 U/min,
10 min, 4 °C) and resuspended in 300 μL of 0.3 M sodium acetate. An
amount of 900 μL of ice cold ethanol was added, and the precipitate
was collected after centrifugation (5000 U/min, 10 min, 4 °C).
Samples were washed twice with ice cold ethanol and were stored at
−20 °C. The pellets were dissolved in 500 μL of water (twice
distilled), and the DNA content was determined by absorption reading
at 260 nm in a UV microplate reader (Flashscan AnalytikJena AG).
Salmon testes DNA dissolved in water (twice distilled) was used for
calibration purposes. Samples (200 μL) for gold determination
by atomic absorption spectroscopy were stabilized by addition of
20 μL of Triton X-100 (1%). The amount of drugs bound to DNA was
expressed as pmol of drug per μg of DNA. Results were calculated as
the mean of at least two independent experiments, which were
performed with two replicates.
where C0 is the control cell at the time of compound addition, C
is control cell at the time of test end, and T is the probe/sample
at the time of test end.
The IC50 value was determined as the concentration causing 50%
inhibition of cell proliferation and calculated as the mean of at least
two or three independent experiments (OriginPro 8).
TrxR Inhibition. To determine the inhibition of TrxR, an
established microplate reader based assay was performed with minor
modifications.11,23 For this purpose, commercially available rat liver
TrxR (from Sigma-Aldrich) was used and diluted with distilled water
to achieve a concentration of 2.0 U/mL. The compounds were freshly
dissolved as stock solutions in DMSO. To each, 25 μL aliquots of the
enzyme solution, each 25 μL of potassium phosphate buffer, pH 7.0,
containing the compounds in graded concentrations or vehicle
(DMSO) without compounds (control probe), were added. The
resulting solutions (final concentration of DMSO, 0.5% V/V) were
incubated with moderate shaking for 75 min at 37 °C in a 96-well
plate. To each well, an amount of 225 μL of reaction mixture (1000 μL
of reaction mixture consisted of 500 μL of potassium phosphate buffer,
pH 7.0, 80 μL of 100 mM EDTA solution, pH 7.5, 20 μL of BSA
solution 0.05%, 100 μL of 20 mM NADPH solution, and 300 μL of
distilled water) was added, and the reaction was started by addition of
25 μL of a 20 mM ethanolic DTNB solution. After proper mixing, the
formation of TNB was monitored with a microplate reader (Perkin-
Elmer Victor X4) at 405 nm in 10 s intervals for 6 min. The increase in
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
noninterference with the assay components was confirmed by a nega-
tive 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
mean and error of repeated experiments.
Reaction with Glutathione. The gold complexes were prepared
as stock solutions in DMSO and diluted with potassium phosphate
buffer, pH 7.0, to achieve a final concentration of 500 μM (DMSO,
0.2% v/v). To 25 μL of 250 μM aqueous solutions of reduced
glutathione, each 25 μL of the respective potassium phosphate buffer
solution (containing the compounds or only the DMSO vehicle as
control), an amount of 25 μL of 100 mM aqueous EDTA solution, pH
7.5, was added, and the resulting solutions were incubated with
moderate shaking in a 96-well plate at 37 °C for 20 or 60 min. To each
well, an amount of 200 μL of reaction mixture (1000 μL of reaction
mixture consisted of 620 μL of potassium phosphate buffer, pH 7.0,
80 μL of 100 mM EDTA solution, pH 7.5, and 300 μL of distilled
water) was added, and the reaction was started with the addition of
25 μL of a 20 mM ethanolic solution of DTNB. After proper mixing, the
formation of TNB was monitored in a microplate reader (Perkin-Elmer
Victor X4) at 405 nm. For each tested compound, the noninterference
with the assay components was confirmed by a negative control experi-
ment using a glutathione free solution. Results are presented as the mean
of at least two independent experiments.
Sample Preparation for Cellular Uptake Studies. The cellular
uptake was measured according to a previously described proce-
dure.10,47 In short, cells were grown until at least 70% confluency in
175 cm2 cell culture flasks. Stock solutions of the gold complexes in
DMF were freshly prepared and diluted with cell culture medium to
the desired concentration (final DMF concentration, 0.1% v/v; final
gold complex concentration, 10.0 μM). The cell culture medium of the
cell culture flasks was replaced with 10 mL of the cell culture medium
solutions containing the compounds, and the flasks were incubated at
37 °C/5% CO2 for 6 h. The cell pellets were isolated by trypsinization
and centrifugation (room temperature, 2000 rpm, 5 min), resuspended
in twice distilled water, lysed by using a sonotrode, and appropriately
diluted using twice distilled water. An aliquot was removed for the
purpose of protein quantification by the Bradford method. The
determination of the gold content of the samples was performed by
ETAAS. Results were calculated from the data of three independent
experiments and are given as ng of gold per mg of cellular protein.
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Journal of Medicinal Chemistry
Article
Estrogen Receptor Interaction. Estrogen receptor interaction
studies were performed according to previously described procedures
without modifications.28,49−51,59
Inhibition of COX Enzymes. The inhibition of isolated ovine
COX-1 and human recombinant COX-2 was determined with 10 μM
of the respective compounds by ELISA (“COX inhibitor screening
assay”, Cayman Chemicals). Experiments were performed according to
the manufacturer’s instructions. Absorption was measured at 415 nm
(Victor 2, Perkin-Elmer). Results were calculated as the mean of
duplicate determinations.
as potential anticancer drugs. Coord. Chem. Rev. 2009, 253, 1692−
1707.
(10) Scheffler, H.; You, Y.; Ott, I. Comparative studies on the
cytotoxicity, cellular and nuclear uptake of a series of chloro gold(I)
phosphine complexes. Polyhedron 2010, 29, 66−69.
(11) Ott, I.; Qian, X.; Xu, Y.; Vlecken, D. H.; Marques, I. J.; Kubutat, D.;
Will, J.; Sheldrick, W. S.; Jesse, P.; Prokop, A.; Bagowski, C. P. A gold(I)
phosphine complex containing a naphthalimide ligand functions as a TrxR
inhibiting antiproliferative agent and angiogenesis inhibitor. J. Med. Chem.
2009, 52, 763−770.
(12) Mirabelli, C. K.; Johnson, R. K.; Hill, D. T.; Faucette, L. F.;
Girard, G. R.; Kuo, G. Y.; Sung, C. M.; Crooke, S. T. Correlation of
the in vitro cytotoxic and in vivo antitumor activities of gold(I)
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ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR and 13C NMR spectra of the selected complexes (Figure
S1); antiproliferative effects of cisplatin on MCF-7, MDA-MB 231,
and HT-29 cells in different times and concentrations (Figure S2);
detailed synthetic procedures for 1a−g, 2a−g, 3a−g, and 4a−k;
crystallographic information of 5f and 6f; elementary analysis
results of target compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(13) Krishnamurthy, D.; Karver, M. R.; Fiorillo, E.; Orru, V.;
́
Stanford, S. M.; Bottini, N.; Barrios, A. M. Gold(I)-mediated inhibition
of protein tyrosine phosphatases: a detailed in vitro and cellular study.
J. Med. Chem. 2008, 51, 4790−4795.
(14) Hickey, J. L.; Ruhayel, R. A.; Barnard, P. J.; Baker, M. V.; Berners-
Price, S. J.; Filipovska, A. Mitochondria-targeted chemotherapeutics: the
rational design of gold(I) N-heterocyclic carbene complexes that are
selectively toxic to cancer cells and target protein selenols in preference
to thiols. J. Am. Chem. Soc. 2008, 130, 12570−12571.
(15) Barnard, P. J.; Baker, M. V.; Berners-Price, S. J.; Day, D. A.
Mitochondrial permeability transition induced by dinuclear gold(I)
carbene complexes: potential new antimitochondrial antitumour
agents. J. Inorg. Biochem. 2004, 98, 1642−1647.
AUTHOR INFORMATION
Corresponding Author
*Phone: +43 512 507 5245. Fax: +43 512 507 2940. E-mail:
ronald.gust@uibk.ac.at.
■
(16) Jellicoe, M. M.; Nichols, S. J.; Callus, B. A.; Baker, M. V.;
Barnard, P. J.; Berners-Price, S. J.; Whelan, J.; Yeoh, G. C.; Filipovska,
A. Bioenergetic differences selectively sensitize tumorigenic liver
progenitor cells to a new gold(I) compound. Carcinogenesis 2008, 29,
1124−1133.
(17) Bakerm, M. V.; Barnard, P. J.; Berners-Price, S. J.; Brayshaw, S.
K.; Hickey, J. L.; Skelton, B. W.; White, A. H. Cationic, linear Au(I) N-
heterocyclic carbene complexes: synthesis, structure and anti-mitochondrial
activity. Dalton Trans. 2006, 3708−3715.
ACKNOWLEDGMENTS
■
This work was supported by the China Scholarship Council
(CSC) and the Deutsche Forschungsgemeinschaft (DFG)
within FOR 630 “Biological Function of Organometallic
Compounds”. We are also grateful for technical support by
Heike Scheffler, Gerhard Rubner, and Maxi Wenzel (Institute
of Pharmacy, Freie Universitat Berlin, Germany).
̈
(18) Barnard, P. J.; Wedlock, L. E.; Baker, M. V.; Berners-Price, S. J.;
Joyce, D. A.; Skelton, B. W.; Steer, J. H. Luminescence studies of the
intracellular distribution of a dinuclear gold(I) N-heterocyclic carbene
complex. Angew. Chem., Int. Ed. 2006, 45, 5966−5970.
(19) Siciliano, J. T.; Deblock, C. M.; Hindi, M. K.; Durmus, S.;
Panzner, J. M.; Tessier, A. C.; Youngs, J. W. Synthesis and anticancer
properties of gold(I) and silver(I) N-heterocyclic carbene complexes.
J. Organomet. Chem. 2011, 696, 1066−1071.
ABBREVIATIONS USED
■
TrxR, thioredoxin reductase; ER, estrogen receptor; SAR,
structure−activity relationship; NHC, N-heterocyclic carbene;
LiHMDS, lithium bis(trimethylsilyl)azide; DTNB, dithiobisni-
trobenzoic acid; TNB, 2-nitro-5-thionitrobenzoic acid; ETAAS,
electrothermal atomic absorption spectroscopy; COX, cyclo-
oxygenase; LOX, lipoxygenase
(20) Weaver, J.; Gaillard, S.; Toye, C.; Macpherson, S.; Nolan, S. P.;
Riches, A. Cytotoxicity of gold(I) N-heterocyclic carbene complexes
assessed by using human tumor cell lines. Chem.Eur. J. 2011, 17,
6620−6624.
(21) Horvath, U. E. I.; Bentivoglio, G.; Hummel, M.; Schottenberger,
H.; Wurst, K.; Nell, M. J.; van Rensburg, C. E. J.; Cronje, S.;
Raubenheimer, H. G. A cytotoxic bis(carbene) gold(I) complex of
ferrocenyl complexes: synthesis and structural characterization. New
J. Chem. 2008, 32, 533−539.
(22) Ray, S.; Mohan, R.; Singh, J. K.; Samantaray, M. K.; Shaikh, M. M.;
Panda, D.; Ghosh, P. Anticancer and antimicrobial metallopharmaceutical
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(23) Rubbiani, R.; Kitanovic, I.; Alborzinia, H.; Can, S.; Kitanovic, A.;
Onambele, L. A.; Stefanopoulou, M.; Geldmacher, Y.; Sheldrick, W. S.;
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