Synthesis and biological studies of silver N-heterocyclic carbene complexes derived from 4,5-diarylimidazole

Article abstract of DOI:10.1016/j.ejmech.2011.10.002

A novel class of silver N-heterocyclic carbene complexes (5a-f) were synthesized in high yield by reacting silver(I) oxide with 4,5-diarylimidazolium halides (4a-f). The complexes were characterized using NMR and IR spectroscopy. The structure was confirmed on the example of bromo[1,3-diethyl-4,5-bis(4- fluorophenyl)imidazol-2-ylidene]silver(I) (5c) by crystal structure analysis. The X-ray structure indicated a three-dimensional coordination polymer with a repeating unit consisting of a C_carben-Ag₂-Br ₂-C_carben cluster. Pharmacological investigations revealed that all silver complexes possessed growth inhibitory effects against breast cancer (MCF-7 and MDA-MB-231) as well as colon carcinoma (HT-29) cells. The most active compound 5c was slightly less active against MCF-7 cells, more active against MDA-MB-231 cells and comparable active as cisplatin against HT-29 cells. Further pharmacological investigations were performed with selected compounds on estrogen receptor (ER) binding, DNA intercalation, cyclooxygenase (COX) inhibition and antibacterial activity. The complexes were only marginally active at the DNA, ER and the COX enzymes, so these targets can be excluded to be involved in the mode of action. However, the growth of bacteria was significantly inhibited by 5c and 5f and opens a new application of this complex type.

Full text of DOI:10.1016/j.ejmech.2011.10.002

European Journal of Medicinal Chemistry 46 (2011) 5927e5934
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological studies of silver N-heterocyclic carbene complexes
derived from 4,5-diarylimidazole
Wukun Liu a, Kerstin Bensdorf a, Adelheid Hagenbach b, Ulrich Abram b, Ben Niu c, Aruljothi Mariappan c,
,d,
Ronald Gust a
*
a Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2þ4, 14195 Berlin, Germany
b Institute of Chemistry and Biochemistry, Freie Universität Berlin, Fabeckstrasse 34-36, 14195 Berlin, Germany
c Bacterial Genetics, Institute of Biology, Humboldt Universität Berlin, Chausseestrasse 117, D-10115 Berlin, Germany
d Institute of Pharmacy, University of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of silver N-heterocyclic carbene complexes (5aef) were synthesized in high yield by
reacting silver(I) oxide with 4,5-diarylimidazolium halides (4aef). The complexes were characterized
using NMR and IR spectroscopy. The structure was confirmed on the example of bromo[1,3-diethyl-4,5-
bis(4-fluorophenyl)imidazol-2-ylidene]silver(I) (5c) by crystal structure analysis. The X-ray structure
indicated a three-dimensional coordination polymer with a repeating unit consisting of a Ccarben-Ag2-
Br2-Ccarben cluster. Pharmacological investigations revealed that all silver complexes possessed growth
inhibitory effects against breast cancer (MCF-7 and MDA-MB-231) as well as colon carcinoma (HT-29)
cells. The most active compound 5c was slightly less active against MCF-7 cells, more active against MDA-
MB-231 cells and comparable active as cisplatin against HT-29 cells. Further pharmacological investi-
gations were performed with selected compounds on estrogen receptor (ER) binding, DNA intercalation,
cyclooxygenase (COX) inhibition and antibacterial activity. The complexes were only marginally active at
the DNA, ER and the COX enzymes, so these targets can be excluded to be involved in the mode of action.
However, the growth of bacteria was significantly inhibited by 5c and 5f and opens a new application of
this complex type.
Received 21 April 2011
Received in revised form
26 September 2011
Accepted 2 October 2011
Available online 7 October 2011
Keywords:
Silver N-heterocyclic carbene complexes
Tumor cell growth inhibition
Estrogen receptor binding
COX inhibition
Antibacterial properties
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
physicochemical properties and the reactivity in biological medium
of the final NHC metal complexes. Despite extensive studies on NHC
Platinum-based drugs such as cisplatin or oxaliplatin are widely
used in the treatment of cancer. A great disadvantage is the possible
development of resistance during the therapy. Therefore, many
groups focus their attention on the design of platinum complexes
for the treatment of tumors with intrinsic (e.g the mammary
carcinoma) or acquired resistance [1]. A very promising strategy to
overcome resistance phenomena is the use of specific carriers and
the change from platinum to other transition metals [2e4].
N-Heterocyclic carbenes (NHCs) came into the focus as carrier
ligands for cytotoxic metal complexes because they perfectly fit
prerequisites for an efficient drug design and fast optimization
[2e4]. NHCs are readily accessible in few steps and their substitu-
ents can be widely varied allowing an easy fine-tuning of both the
complexes in organometallic chemistry and catalysis, only
a restricted array of biomedical applications have been reported so
far for gold, palladium, copper, ruthenium, platinum, silver and
rhodium derivatives [2e14].
Silver salts have enjoyed a long history as antimicrobial agents
and have proved to exhibit low toxicity for humans [2e6,9e14].
Recently, an increasing number of reports deal with silver complexes
showing properties relevant for the design of novel anticancer
therapeutics [3,4,9e13]. For instance, Youngs et al. described silver
NHC complexes with 4,5-dichloro-1H-imidazole ligands as selective
cytostatics in ovarian (OVCAR-3) and breast (MB-157) cancer cell
lines. Activity was even demonstrated in vivo for acetato
[4,5-dichloro-1,3-dimethylimidazol-2-ylidene] silver(I) (Scheme 1)
against an ovarian cancer xenograft model [10]. The groups of
Gautier and Morel reported an N,N0-diaryl-substituted carbene of
high lipophilicity as suitable ligand for metal complexes. The cyto-
toxicity of the resulting silver complex (Scheme 1) was 40-fold (MCF-
7 and HL60) to 7-fold (MCF-7R) higher than that of cisplatin [4].
* Corresponding author. Institute of Pharmacy, University of Innsbruck, Innrain
52, A-6020 Innsbruck, Austria. Tel.: þ43 512 507 5245; fax: þ43 512 507 2940.
E-mail address: ronald.gust@uibk.ac.at (R. Gust).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.002

5928
W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
cleavage with BBr3 [16]. Subsequent reaction of 3aee with ethyl
bromide in CH3CN yielded the imidazolium salts 4aee. The same
reaction of 3c with benzyl chloride resulted in 3-benzyl-1-ethyl-
4,5-diarylimidazolium chloride 4f. Finally, the imidazolium salts
4aef were treated with silver oxide in a mixture of CH2Cl2 and
CH3OH at room temperature to form the corresponding silver NHC
complexes 5aef in analogy to the reported procedure by Wang and
Lin [5,6,18].
Scheme 1. Selected silver NHC complexes and 2,4,5-triarylimidazoles.
Precursors and the silver complex were characterized by IR,
NMR and MS spectra. The IR spectra of ligands and complexes are
very similar and not suitable to confirm the binding of NHC to silver.
In silver NHC complexes, the methine proton at C2 is exchanged by
Encouraged of these and other previously reported results of
metal NHC complexes with high antitumor activity [2e10], we
started the examination of the antitumor activity of silver NHC
complexes with 4,5-diarylimidazole ligands (Scheme 1). To achieve
a high accumulation grade in tumor cells, the aromatic rings were
2-F, 3-F, 4-F, 4eOH, or 4eOCH3 substituted. Such substitution
patterns were already very successfully used in the case of [1,2-
diarylethylenediamine]platinum(II) complexes [1,15]. The related
NHC complexes can be considered as plain derivatives of these
compounds.
the metal. Consequently, the NCHN signal at
d
¼ 9e11 disappeared
in the 1H NMR spectra. In accordance with the literature [5,6], the
metal bound carbine (NCNeAg) is not observable in the 13C NMR
spectrum. Furthermore, it should be noted that upon metal binding
the 1H NMR and 13C NMR signals of ethyl, benzyl and phenyl are
nearly unaffected.
All novel silver complexes are sufficiently stable in solid state
and solution. If the complexes were dissolved in CDCl3, methanol-
d4 or DMSO-d6 no change of the spectra was observed during the
storage at room temperature.
2. Chemistry
The formation of the complexes could be supported by positive
mode ESI mass spectrometry. The spectra indicated a base peak
corresponding to the [2M e AgX2]þ fragment for 5aec, 5f and the
[M e AgX]þ fragment for 5aef.
To evaluate the structure of the silver complexes, 5c was crys-
tallized from a MeOH/CH2Cl2 solution and characterized by X-ray
diffraction. Its molecular structure is depicted in Fig. 1. Selected
bond distances and bond angles are given in the figure caption. The
crystallographic experimental data of 5c are presented in Table 1.
The complexes are arranged in the crystal in dimeric units. Each
metal binds one NHC and two bridging bromides (see Fig. 1). The
AgeBr distances are 2.525(2) and 2.809(2) Å, respectively for
Ag(1)eBr(2) and Ag(1)eBr(1). The bond angle of C(1)eAg(1)eBr(2)
In previous studies, we already synthesized substituted 4,5-
diarylimidazoles by oxidation of respective substituted 4,5-diaryl-
2-imidazolines, which were obtained from substituted benzalde-
hydes in a 6 step sequence, unfortunately, in low yields [16].
Therefore, a new synthesis (Scheme 2) based on a procedure re-
ported by Sharpe et al. was chosen [17]. Compounds 1aed were
synthesized from commercially available benzaldehydes via the
catalysis of thiamine. For ring closure, yielding the respective
imidazoles 2aed, the benzoines 1aed were heated in formamide to
reflux for 3 h. Reaction of 2aed with NaH and ethyl bromide in
absolute THF afforded the corresponding N-alkylation (3aed). The
hydroxyl-substituted imidazole 3e was generated from 3d by ether
Compound
1a, 2a, 3a, 4a, 5a
1b, 2b, 3b, 4b, 5b
1c, 2c, 3c, 4c, 5c
1d, 2d, 3d, 4d, 5d
3e, 4e, 5e
R
2-F
R'
Et
X
Br
Br
Br
Br
Br
Cl
3-F
Et
4-F
Et
4-MeO
4-OH
4-F
Et
Et
4f, 5f
PhCH2
Scheme 2. Synthesis routes and structures of intermediates and silver NHC complexes 5aef. Reagents and conditions: (I) thiamine hydrochloride, water/ethanol 1:2, room
temperture (rt), 2e7 days, 52e59%; (II) formamide, reflux, 3 h, 61e80%; (III) 95% NaH, ethyl bromide, absolute THF, reflux, 2 h, 82e89%; (IV) BBr3, CH2Cl2, 48h, 75%; (V) ethyl
bromide or benzyl chloride, CH3CN, reflux, 48e72 h, 76e85% (VI) Ag2O, CH3OH/CH2Cl2 1:1.2, rt, 12 h, 51e82%.

W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
5929
231 breast cancer cells and HT-29 colon cancer cells. The experi-
ments were performed according to established procedures
[19,20]. IC50 values of the silver complexes 5aef and cisplatin are
listed in Table 2.
Cisplatin characteristically reduced the cell growth of MCF-7
(IC50 ¼ 1.6
m
M), MDA-MB-231 (IC50 ¼ 7.8
mM) and HT-29
(IC50 ¼ 4.1
mM) cells. The substituents at the 4,5-standing phenyl
rings of the silver complexes determined the cytotoxicity. The 4-
hydroxyl-substituted compound 5e showed relatively low anti-
proliferative activities with IC50 values of 9.2e16.2 mM, while the
fluoro- and methoxy-substituted compounds 5aed, 5f showed
strong activities in the three cancer cell lines with IC50 values below
10 mM. These results are in accordance with previous results that
the methoxy substituents induced higher cytotoxicity than
hydroxyl substituents [19].
In addition, 5aec, 5f showed comparable activities against
Fig. 1. X-ray molecular structure of 5c. Selected bond lengths [Å] and angles [ꢀ] Ag(1)e
C(1) 2.12(1), Ag(1)eBr(2) 2.525(2), Ag(1)eBr(1) 2.809(2), Ag(1)eAg(4) 3.060(1), C(1)e
Ag(1)eBr(2) 153.6(3), Br(2)eAg(1)eBr(1) 88.90(5), C(1)eAg(1)eBr(1) 111.0(3), C(1)e
Ag(1)eAg(4) 66.0(3), Br(2)eAg(1)eAg(4) 119.35(5), Br(1)eAg(1)eAg(4) 120.79(6),
N(2)eC(1)eAg(1) 126.9(7), N(5)eC(1)eAg(1) 127.7(8), Ag(2)eBr(1)eAg(1) 86.62(6),
Ag(1)eBr(2)eAg(2) 94.01(6), C(91)eAg(4)eAg(1) 67.2(2), Br(4)eAg(4)eAg(1)
117.62(9), Br(3)eAg(4)eAg(1) 127.89(6).
mammary carcinoma cells (IC50 ¼ 3e4
mM). Compared to cisplatin
they were half as active at the MCF-7 cell line and two fold more
active at the MDA-MB-231 cancer cell line. At HT-29 cells only 5c
achieved an activity comparable to cisplatin. All other complexes
(5aeb, 5f) were less active (IC50 ¼ 7e10
demonstrated a preference for MCF-7 cells, with an IC50 ¼ 3.7
compared to 8.5 and 9.9 M determined for the other cell lines.
Time-activity curves of 5c for concentrations ranged from 0.5 to
M are presented in Fig. 2. Compound 5c caused cytocidal effects
(T/Ccorr < 0%) at 10 M and cytostatic effects (T/Ccorr z 0%) at 5 M,
mM). Compound 5d
mM
m
amounts to 153.6(3)ꢀ that of C(1)eAg(1)eBr(1) to 111.0(3)ꢀ. The
C(1)eAg(1) distance is 2.12(1) Å, which is in accordance with
published NHCesilver complexes [5,6,18]. Ag(1) and Ag(4) are
arranged in a distance of 3.060(1) Å which indicates weak inter-
action comparable with metalemetal distance in metallic silver(0)
(2.88 Å) [6].
10
m
m
m
while cisplatin did not reach cytocidal activity. The curves of 5c at
higher concentrations are characterized by a fast onset of activity
with maximal effects already after an incubation time of 48 h.
Cisplatin achieved its maximal effect at the end of the experiment.
Furthermore, Fig. 2 shows in the case of 5c a recuperation of the
tumor cells after a prolonged exposition time. Because exponential
cell growth is guaranteed for at least 140 h of incubation, the rise of
the growth curve can be explained by development of drug resis-
tance [19]. This effect of 5c is more pronounced at MDA-MB-231
and HT-29 cells compared to MCF-7 cells.
It is well accepted that the DNA represents the major target of
antitumor metal complexes [1e4], so we studied the interaction of
5c with calf thymus (CT) DNA by using circular dichroism (CD)
spectroscopy [21]. However, the missing changes of absorbance
(data not showed) exclude a binding of the complex.
During the past years cyclooxygenases (COXs), especially COX-2
became very interesting targets in cancer chemotherapy. We could
already demonstrate that the derivation of the nonsteroidal anti-
inflammatory drug (NSAID) aspirin (e.g. to (prop-2-ynyl)-2-
acetoxybenzoate) and coordination to metals (e.g. [(prop-2-yn)-2-
acetoxy]dicobalthexacarbonyl (Co-ASS)) increased the COX inhibi-
tory properties as well as the cytotoxic potency [22e25]. Because
the NHC ligands designed in this paper are derivatives of
2,4,5-triarylimidazoles (see Scheme 1), which inactivate the COX
enzymes dependent on the substituents in the aromatic rings [26],
it makes sense to take this target into consideration as well.
3. Results and discussion
All complexes as well as the established anticancer drug
cisplatin were screened for cytotoxicity against MCF-7, MDA-MB-
Table 1
Crystallographic experimental data of 5c.
Empirical formula
Wavelength
C77 H74 Ag4 Br4 Cl2 F8 N8
0.71073 Å
Radiation
Mo Ka
Crystal system
Space group
Unit cell dimensions
Monoclinic
P21/n
a ¼ 17.325(5) Å
b ¼ 17.344(5) Å
c ¼ 25.972(5) Å
7767(4) Å3
4
a
b
g
¼ 90ꢀ.
¼ 95.62(1)ꢀ.
¼ 90ꢀ.
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1.784 Mg/m3
3.188 mmꢁ1
4104
Crystal description
Crystal color
Crystal size
No. of reflexions (lattice)
Theta range (lattice)
Theta range for data collection
Index ranges
Needle
Colorless
0.3 ꢂ 0.05 ꢂ 0.05 mm3
98
2.29 to 59.53
1.79 to 25.00
Table 2
ꢁ20< ¼ h < ¼ 20,ꢁ20 < ¼ k < ¼ 20,
Antiproliferative effects against MCF-7, MDA-MB-231 and HT-29 cells after 72 h
incubation.
ꢁ28<¼l<¼30
Reflections collected
Independent reflections
Completeness to theta ¼ 25.00ꢀ
Absorption correction
Decay
35,447
13,042 [R(int) ¼ 0.0995]
Compound
Cytotoxicity IC50, [
m
M]
MDA-MB-231
95.4%
MCF-7
HT-29
None
0.5%
5a
5b
5c
5d
5e
5f
Cisplatin
3.4 ꢃ 0.7
3.5 ꢃ 0.1
3.9 ꢃ 0.2
3.7 ꢃ 0.3
9.2 ꢃ 0.2
3.6 ꢃ 0.1
1.6 ꢃ 0.5
3.6 ꢃ 0.2
4.1 ꢃ 0.7
3.5 ꢃ 0.3
8.5 ꢃ 0.8
12.8 ꢃ 0.2
3.4 ꢃ 0.2
7.8 ꢃ 0.8
7.5 ꢃ 0.5
7.4 ꢃ 0.8
4.4 ꢃ 0.1
9.9 ꢃ 0.1
16.2 ꢃ 0.2
6.8 ꢃ 1.2
4.1 ꢃ 0.3
Refinement method
Full-matrix least-squares on F2
13,042/0/928
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
0.782
R1 ¼ 0.0694, wR2 ¼ 0.1699
R1 ¼ 0.1488, wR2 ¼ 0.2261
0.859 and ꢁ1.609 e.Åꢁ3
Largest diff. peak and hole

5930
W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
Fig. 2. Time activity curves of 5c (above) and cisplatin (below) on MCF-7 (left), MDA-MB-231 (middle) and HT-29 (right) cell line lines. Error bars are hidden behind the symbols in
some cases.
MCF-7 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 [27,28]. HT-29 human colon cancer cells constitutively
express COX-2 [29]. Therefore, the most cytotoxic complex 5c was
selected for the investigation on isolated COX-1 and COX-2
enzymes by ELISA. Co-ASS and aspirin were used for compar-
complexes. Therefore, ER
stably transfected with the plasmid EREwtcluc (MCF-7-2a cells) and
U2eOS cells transiently transfected with the plasmid pSG5-ER
(U2eOS/ ) or pSG5-ER FL (U2eOS/ ) and the reporter plasmid
a-positive MCF-7 breast cancer cells,
a
a
b
b
p(ERE)2-lucþ were used to evaluate ER subtype selectivity on
molecular level [16,30,31].
Among the complexes only 5f induced a low luciferase expres-
ison. At 10
m
M Co-ASS inhibited both COX enzymes to about 50%
sion (conc. 10
mM: activation 90% ER
a and 66% ERb; conc. 1 mM:
(68% (COX-1) and 63% (COX-2)) so we used this concentration and
performed the ELISA without calculation of the IC50 values.
Aspirin was only marginally active at COX-1 (29% inhibition) and
activation 36% ER
slight receptor binding. Therefore, an ER-mediated cytotoxicity can
be excluded.
a
and 20% ER ) which is an indication of only
b
inactive at COX-2. Unfortunately, at a concentration of 10
m
M, 5c
Finally, a number of silver NHC complexes possessing good
antibacterial activities [3,4,10e14] which induced us to evaluate the
effectiveness of 5c and 5f as antibacterial agents in vitro through
a modified agar diffusion test [32].
caused no COX inhibition (COX-1 (4.6%) and COX-2 (1.4%)), so it is
very unlikely that COX enzymes are involved in the inhibition of the
tumor cell growth.
Because the above mentioned 2,4,5-triarylimidazoles were not
only COX inhibitors but also ligands of the estrogen receptor (ER)
[16,26,30], we investigated if the ER might act as carrier for the
Indicator bacteria were grown in Luria broth (LB) at 30 ꢀC
(Erwinia amylovora Ea 273 and Erwinia carotovora) or 37 ꢀC
(Escherichia coli DH5a, Bacillus subtilis 168, Bacillus megaterium,
Table 3
Sensitivity test of the silver compounds in inhibiting bacterial growth.
Test
Concentration (mM)
Diameter of the zone of inhibition (mm)
Compounds
Ervinia amylovora
E. coli
Bacillus
Bacillus
Ea 273
DH5
a
subtilis 168
megaterium
5c
5
10
5
10
5
7
9
8
7
8
6
9
12
15
28
9
7
8
5
18
12
12
5
5f
8
7
AgNO3
H2O
12
12
5
11
11
5
10
9
5

W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
5931
Table 4
5.1.2. General procedure for the synthesis of 1aed
Antimicrobial activity of 5c at 4.2 mM.
2.00 g (5.94 mmol) of thiamine hydrochloride were dissolved in
a mixture of 10 mL of distilled water and 20 mL of ethanol followed
by stirring the solution until it is homogeneous. Then 2.0 M NaOH
was added dropwise to the stirring solution until the pH reached
9e10. After addition of 74.6 mmol of the substituted benzaldehyde,
the reaction was allowed to stand at room temperature. After 2e7
days, the reaction mixture was filtered, the ethanol was evaporated
and the remaining water phase was extracted with dichloro-
methane. After drying the collected organic layers over MgSO4,
removal of the solvent under reduced pressure gave the crude
product, which was then purified by flash column chromatography
(EtOAc/petroleum ether).
Indicator strains
Inhibiting effect (diameter of the
zone of inhibition)
a
Erwinia carotovora
Pseudomonas aeruginosa
Bacillus cereus ATCC 14579
þ
(5c 9.5 mm; H2O 5 mm)
þ (5c 8 mm; H2O 5 mm)
þ (5c 6 mm; H2O 5 mm)
a
"þ" indicates positive results of 5c.
Pseudomonas aeruginosa and Bacillus cereus ATCC 14579). When cell
concentration of bacteria was up to 4 ꢂ 107 CFU/mL 0.5 mL bacteria
suspensions were mixed with 20 mL melting LB agar and cooled
below 60 ꢀC to prepare the plates. AgNO3 was used as positive
control. 50
concentrations or 50
m
L of aqueous solution with increasing complex
5.1.2.1. 1,2-Bis(2-fluorophenyl)-2-hydroxyethanone
(1a). Yield
4.53
mL of water (containing 0.1% DMSO) was
56.4%; pale yellow solid. MS m/z: 248 [M]þ. 1H NMR (CDCl3):
d
loaded into the wells (diameter: 5 mm) and punched in indicator
bacteria plates which were then incubated at 30 ꢀC overnight to
observe the growth inhibition effects. The antimicrobial activity of
the compounds was determined by measuring the diameter of the
inhibiting zone around the wells.
Our assay confirmed that 5c and 5f possessed antimicrobial
properties at a level comparable to silver nitrate against E. amylo-
vora Ea 273, E. coli DH5a, B. subtilis 168 and B. megaterium (see
Table 3). The maximum of antimicrobial activity was observed for
complexes 5c and 5f against the B. subtilis 168 and B. megaterium.
The diameter of the inhibition zone of 5f against the B. subtilis 168
at 10 mM was almost 3-fold than that of AgNO3.
Futhermore, extention testing of 5c against other bacteria
(E. carotovora, P. aeruginosa and B. cereus ATCC 14579) also indicated
positive results in comparison with the negative control (Table 4).
(s,1H, OH, exchangeable by D2O), 6.08 (s,1H, CH), 6.96e7.08 (m, 4H,
ArH), 7.18e7.26 (m, 2H, ArH), 7.45e7.51 (m, 1H, ArH), 7.85e7.89 (m,
1H, ArH).
5.1.2.2. 1,2-Bis(3-fluorophenyl)-2-hydroxyethanone
(1b). Yield
4.21
52.3%; pale yellow solid. MS m/z: 248 [M]þ. 1H NMR (CDCl3):
d
(s,1H, OH, exchangeable by D2O), 5.90 (s,1H, CH), 6.98e7.27 (m, 4H,
ArH), 7.28e7.42 (m, 2H, ArH), 7.58e7.67 (m, 2H, ArH).
5.1.2.3. 1,2-Bis(4-fluorophenyl)-2-hydroxyethanone
(1c). Yield
4.50
58.1%; pale yellow solid. MS m/z: 248 [M]þ. 1H NMR (CDCl3):
d
(s, 1H, OH, exchangeable by D2O), 5.91 (s, 1H, CH), 7.01e7.12 (m, 4H,
ArH), 7.30e7.35 (m, 2H, ArH), 7.90e7.95 (m, 2H, ArH).
5.1.2.4. 2-Hydroxy-1,2-bis(4-methoxyphenyl)ethanone (1d). Yield
58.8%; pale yellow solid. MS m/z: 272 [M]þ. 1H NMR (CDCl3):
d
3.76 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.57 (d, 1H, OH, J ¼ 5.6 Hz,
4. Conclusions
exchangeable by D2O), 5.85 (d, 1H, CH, J ¼ 4.8 Hz), 6.83e6.87
(m, 4H, ArH), 7.23e7.26 (m, 2H, ArH), 7.88e7.91 (d, 2H, ArH,
J ¼ 5.6 Hz).
4,5-Diarylimidazole derivatives showed low antiproliferative
effects in cancer cells. After coordination to Ag-halides, the anti-
proliferative effects in cultured tumor cells greatly increased.
Preliminary in vitro studies showed that 5c and 5f exhibited good
antibacterial activities. Despite the mode of action of silver NHC
complexes remains unclear, these findings indicate that this type of
silver NHC complexes may be useful in anticancer and antibacterial
chemotherapy. Further studies on the pharmacodynamics and
pharmacokinetics as well as antibacterial testing of the complexes
are in progress.
5.1.3. General procedure for the synthesis of 2aed
A solution of the substituted benzoin 1aed (23 mmol) in 30 mL
of formamide was heated to reflux for 3 h. The reaction mixture was
poured into 100 mL of water and stirred vigorously to dissolve the
gummy product. The resulting powder was filtered, washed with
water, and suspended in 5% HCl (200 mL). The solution was heated
to 80e90 ꢀC and filtered hot. The acidic filtrate was treated with an
excess of NH4OH to form a white precipitate, which was filtered and
washed with water and cold acetonitrile.
5. Experimental section
5.1.3.1. 4,5-Bis(2-fluorophenyl)-1H-imidazole (2a). Yield 79.5%; white
1
solid. MS m/z: 256 [M]þ. H NMR (DMSO-d6):
d 7.08e7.36 (m, 8H,
5.1. Chemistry
ArH), 7.59 (s, 1H, N]CHeN), 12.65 (s, 1H, NH, exchangeable by D2O).
5.1.1. General
5.1.3.2. 4,5-Bis(3-fluorophenyl)-1H-imidazole (2b). Yield 80.2%;
The following instrumentation was used: IR spectra (KBr
pellets): Perkin Elmer Model 580 A (Shelton, USA); 1H or 13C NMR
spectra: Bruker ADX 400 spectrometer operated at 400 MHz 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, USA; Elemental C, H, N analysis: Perkin Elmer 240 B and C
analyzer. Chemicals were obtained from SigmaeAldrich (Germany)
and used without further purification. Reactions were all moni-
tored by TLC, performed on silica gel plates 60 F254 (Merck,
Darmstadt/Germany). Visualization on TLC was achieved by UV
light. Column chromatography was performed with Merck silica gel
60H, grain size <0.063 mm, 230 mesh ASTM (Darmstadt/Ger).
white solid. MS m/z: 256 [M]þ. 1H NMR (DMSO-d6):
d 7.07e7.45 (m,
8H, ArH), 7.84 (s, 1H, N]CHeN), 12.66 (s, 1H, NH, exchangeable by
D2O).
5.1.3.3. 4,5-Bis(4-fluorophenyl)-1H-imidazole (2c). Yield 61.5%;
white solid. MS m/z: 256 [M]þ. 1H NMR (DMSO-d6):
d 7.10e7.14 (t,
2H, ArH, J ¼ 8.0 Hz), 7.24e7.28 (t, 2H, ArH, J ¼ 8.0 Hz), 7.41e749 (m,
4H, ArH), 7.77 (s, 1H, N]CHeN), 12.50 (s, 1H, NH, exchangeable by
D2O). Anal. calcd. for C15H10F2N2: C, 70.31; H, 3.93; N, 10.93%. Found
C, 70.32; H, 3.95; N, 10.93%.
5.1.3.4. 4,5-Bis(4-methoxyphenyl)-1H-imidazole (2d). Yield 75.3%;
white solid. MS m/z: 280 [M]þ. 1H NMR (CDCl3):
d 3.82 (s, 6H,

5932
W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
OCH3), 6.82e6.84 (d, 4H, ArH, J ¼ 8.0 Hz), 7.39e7.41 (d, 4H, ArH,
J ¼ 8.0 Hz), 7.59 (s, 1H, N]CHeN), 9.67 (1H, NH, exchangeable by
D2O). Anal. calcd. for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99%. Found
C, 72.95; H, 5.75; N, 10.08%.
for 48e72 h. Then removal of the solvent under reduced pressure
gave the crude product which was washed with diethyl ether
(3 ꢂ 30 mL) and dried in vacuo.
5.1.5.1. 1,3-Diethyl-4,5-bis(2-fluorophenyl)-1H-imidazolium bromide
(4a). Yield 78.6%; white solid. ESI-MS m/z: 313 [M ꢁ Br]þ. IR (KBr,
cmꢁ1): 3119, 2990, 1646, 1618, 1560, 1490, 1451, 1228, 1208, 821,
5.1.4. General procedure for the synthesis of 3aed
To a solution of 15.30 mmol of imidazole 2aed in 50 mL of dry
THF, 16.50 mmol (417 mg, 95% powder) of sodium hydride and
16.50 mmol (1.23 mL, 1782 mg) of ethyl bromide were added and
heated to reflux for 2 h. Subsequently, the reaction mixture was
hydrolyzed with 50 mL of water and the product was extracted
with CHCl3. The organic layer was dried over Na2SO4 and the
solvent was distilled off to give the crude product, which was then
purified by flash column chromatography (diethyl ether/CH2Cl2).
769. 1H NMR (CDCl3):
d
1.53 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 4.30 (q, 4H,
CH2CH3, J ¼ 7.2 Hz), 7.16e7.22 (m, 6H, ArH), 7.48e7.53 (m, 2H, ArH),
11.24 (s, 1H, N]CHeN). 13C NMR (CDCl3):
15.5 (s, CH3); 43.8 (s,
d
CH2); 112.3, 112.4, 116.3, 116.5, 125.3, 127.5, 132.4 (s, CAr); 133.6 (d,
CAr); 137.6 (s, NCHN); 159.0, 161.5 (s, CAr).
5.1.5.2. 1,3-Diethyl-4,5-bis(3-fluorophenyl)-1H-imidazolium bromide
(4b). Yield 80.7%; white solid. ESI-MS m/z: 313 [M ꢁ Br]þ. IR (KBr,
cmꢁ1): 3114, 3057, 2975, 2910, 1580, 1559, 1465, 1437, 1206, 1150,
5.1.4.1. 1-Ethyl-4,5-bis(2-fluorophenyl)-1H-imidazole
(3a). Yield
1.32 (t,
82.5%; pale yellow oil. MS m/z: 284 [M]þ. 1H NMR (CDCl3):
d
885, 804. 1H NMR (CDCl3):
d
1.52 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 4.35 (q,
3H, CH2CH3, J ¼ 7.2 Hz), 3.89 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 6.89e6.94
(t, 1H, ArH, J ¼ 9.6 Hz), 7.07e7.19 (m, 5H, ArH), 7.36e7.42 (m, 1H,
ArH), 7.50e7.54 (m, 1H, ArH), 7.73 (s, 1H, N]CHeN).
4H, CH2CH3, J ¼ 7.2 Hz), 6.95e6.97 (m, 2H, ArH), 7.06 (d, 2H, ArH,
J ¼ 7.6 Hz), 7.18e7.23 (m, 2H, ArH), 7.42e7.47 (m, 2H, ArH), 11.09 (s,
1H, N]CHeN). 13C NMR (CDCl3):
d 15.3 (s, CH3); 43.3 (s, CH2); 117.0,
117.3, 117.4, 117.6, 126.1, 126.2, 126.3 (s, CAr); 130.3 (d, CAr); 130.9,
131.0 (s, CAr); 136.6 (s, NCHN); 160.9, 163.4 (s, CAr).
5.1.4.2. 1-Ethyl-4,5-bis(3-fluorophenyl)-1H-imidazole
(3b). Yield
1.28 (t,
88.8%; pale yellow oil. MS m/z: 284 [M]þ. 1H NMR (CDCl3):
d
3H, CH2CH3, J ¼ 7.2 Hz), 3.84 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 6.81e6.86
(m, 1H, ArH), 7.08e7.22 (m, 6H, ArH), 7.43e7.48 (m, 1H, ArH), 7.63
(s, 1H, N]CHeN).
5.1.5.3. 1,3-Diethyl-4,5-bis(4-fluorophenyl)-1H-imidazolium bromide
(4c). Yield 84.5%; white solid. ESI-MS m/z: 313 [M ꢁ Br]þ. IR (KBr,
cmꢁ1): 3127, 3038, 2984, 2941, 1631, 1597, 1559, 1504, 1226, 1196,
1160, 851, 819. 1H NMR (CDCl3):
d
1.31 (t, 6H, CH2CH3, J ¼ 7.2 Hz),
5.1.4.3. 1-Ethyl-4,5-bis(4-fluorophenyl)-1H-imidazole
(3c). Yield
1.27 (t,
4.10 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 7.33 (t, 4H, ArH, J ¼ 8.8 Hz),
87.6%; pale yellow oil. MS m/z: 284 [M]þ. 1H NMR (CDCl3):
d
7.50e7.53 (m, 4H, ArH), 9.69 (s, 1H, N]CHeN). 13C NMR (CDCl3):
3H, CH2CH3, J ¼ 7.2 Hz), 3.81 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 6.89 (t, 2H,
ArH, J ¼ 8.8 Hz), 7.17 (t, 2H, ArH, J ¼ 8.8 Hz), 7.29e7.32 (m, 2H, ArH,
J ¼ 8.4 Hz), 7.38e7.41 (m, 2H, ArH), 7.61 (s, 1H, N]CHeN). Anal.
calcd. for C17H14F2N2: C, 71.85; H, 4.96; N, 9.85%. Found C, 71.81; H,
4.98; N, 9.88%.
d 15.7 (s, CH3); 43.5 (s, CH2); 116.6,116.8 (s, CAr); 120.9 (d, CAr); 131.1
(s, CAr); 132.7 (d, CAr,); 136.6 (s, NCHN); 162.4, 164.9 (s, CAr). Anal.
calcd. for C19H19BrF2N2: C, 58.03; H, 4.87; N, 7.12%. Found C, 58.04;
H, 4.93; N, 7.13%.
5.1.5.4. 1,3-Diethyl-4,5-bis(4-methoxyphenyl)-1H-imidazolium
bromide (4d). Yield 80.4%; grey solid. ESI-MS m/z: 337 [M ꢁ Br]þ. IR
(KBr, cmꢁ1): 3136, 3065, 2982, 2937, 2837, 1626, 1611, 1561, 1506,
5.1.4.4. 1-Ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazole (3d). Yield
87.9%; pale yellow oil. MS m/z: 308 [M]þ. 1H NMR (CDCl3):
d 1.27 (t,
3H, CH2CH3, J ¼ 7.2 Hz), 3.76 (s, 3H, OCH3), 3.81 (q, 2H, CH2CH3,
J ¼ 7.2 Hz), 3.87 (s, 3H, OCH3), 6.76 (d, 2H, ArH, J ¼ 8.7 Hz), 6.99 (d,
2H, ArH, J ¼ 8.4 Hz), 7.25 (d, 2H, ArH, J ¼ 8.4 Hz), 7.41 (d, 2H, ArH,
J ¼ 8.8 Hz), 7.59 (s, 1H, N]CHeN). Anal. calcd. for C19H20N2O2. 1/6
CH2Cl2: C, 71.37; H, 6.35; N, 8.69%. Found C, 71.13; H, 6.39; N, 8.45%.
1251, 1181, 1021, 847, 807. 1H NMR (CDCl3):
d 1.44 (t, 6H, CH2CH3,
J ¼ 7.2 Hz), 3.77 (s, 6H, OCH3), 4.26 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 6.87
(d, 4H, ArH, J ¼ 8.8 Hz), 7.10 (d, 4H, ArH, J ¼ 8.4 Hz), 10.77 (s, 1H, N]
CHeN). 13C NMR (CDCl3):
d 15.9 (s, CH3); 43.2 (s, CH2); 55.4 (s,
OCH3); 114.8, 116.9, 131.5, 131.8 (s, CAr); 136.2 (s, NCHN); 160.9 (s,
CAr). Anal. calcd. for C21H25BrN2O2: C, 60.44; H, 6.04; N, 6.71%.
Found C, 60.56; H, 6.09; N, 6.75%.
5.1.4.5. 1-Ethyl-4,5-bis(4-hydroxyphenyl)-1H-imidazole
(3e). A
solution of 3d (10.0 mmol, 3.08 g) in 20 mL of dry CH2Cl2 was
cooled to ꢁ60 ꢀC. At this temperature BBr3 (45 mmol,11.2 g) in 5 mL
of dry CH2Cl2 was added under N2 atmosphere. Then the reaction
mixture was allowed to warm to room temperature and was stirred
for further 48 h. After cooling the reaction mixture with an ice bath,
the surplus of BBr3 was hydrolyzed three times with methanol and
the phenolic product was dissolved in 0.1 N NaOH. The alkaline
water phase was filtered and the pH was adjusted to 8 with 2 N HCl.
The precipitate was collected by suction filtration and washed with
CH2Cl2 and acetone. Yield 75.4%; grey solid. MS m/z: 280 [M]þ. 1H
5.1.5.5. 1,3-Diethyl-4,5-bis(4-hydroxyphenyl)-1H-imidazolium
bromide (4e). Yield 76.5%; grey solid. ESIeMS m/z: 309 [M ꢁ Br]þ.
IR (KBr, cmꢁ1): 3600e2300 (OH), 3124, 2997, 1611, 1560, 1508, 1429,
1345, 1270, 1219, 1173, 841. 1H NMR (DMSO-d6):
d 1.30 (t, 6H,
CH2CH3, J ¼ 7.2 Hz), 4.06 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 6.82 (d, 4H,
ArH, J ¼ 8.4 Hz), 7.19 (m, 4H, ArH), 9.44 (s, 1H, N]CHeN), 9.94 (s,
2H, ArOH, exchangeable by D2O). Anal. calcd. for C19H21BrN2O2. 2
CH2Cl2: C, 45.11; H, 4.51; N, 5.01%. Found C, 44.97; H, 4.52; N, 5.24%.
NMR (DMSO-d6):
d
1.12 (t, 3 H, CH2CH3, J ¼ 7.2 Hz), 3.74 (q, 2 H,
5.1.5.6. 3-Benzyl-1-ethyl-4,5-bis(4-fluorophenyl)-1H-imidazolium
chloride (4f). Yield 78.7%; white solid. ESI-MS m/z: 375 [M ꢁ Cl]þ.
IR (KBr, cmꢁ1): 3119, 3032, 3002, 2974, 2941, 1607, 1555, 1506,
CH2CH3, J ¼ 7.2 Hz), 6.56 (d, 2H, ArH, J ¼ 8.4 Hz), 6.85 (d, 2H, ArH,
J ¼ 8.0 Hz), 7.11 (d, 2H, ArH, J ¼ 8.0 Hz), 7.19 (d, 2H, ArH, J ¼ 8.4 Hz),
7.70 (s, 1H, N]CHeN), 9.21 (s, 1H, ArOH, exchangeable by D2O),
9.66 (s, 1H, ArOH, exchangeable by D2O). Anal. calcd. for
C17H16N2O2. 5/12 CH2Cl2: C, 66.26; H, 5.37; N, 8.87%. Found C, 66.09;
H, 5.68; N, 8.81%.
1455, 1294, 1253, 1179, 1022, 843. 1H NMR (CDCl3):
d 1.49 (t, 3H,
CH2CH3, J ¼ 7.2 Hz), 4.32 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 5.55 (s, 2H,
CH2Ar), 7.01e7.14 (m, 8H, ArH), 7.23e7.42 (m, 5H, ArH), 10.97 (s,
1H, N]CHeN). 13C NMR (CDCl3):
d 15.6 (s, CH3); 43.6 (s, CH2);
51.6 (s, ArCH2); 116.3 (s, CAr); 116.5 (d, CAr); 116.7 (s, CAr); 120.9 (d,
CAr); 121.0 (d, CAr); 128.5, 128.8, 129.0 (s, CAr); 131.3 (d, CAr);
132.6, 132.7, 133.0, 133.1, 133.7(s, CAr); 137.5 (s, NCHN); 162.4,
164.9 (s, CAr).
5.1.5. General procedure for the synthesis of 4aef
Ethyl bromide or benzyl chloride (25 mmol) and imidazole 3aee
(5.00 mmol) were combined and refluxed in 50 mL of acetonitrile

W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
5933
5.1.6. General procedure for the synthesis of 5aef
[2M ꢁ AgCl2]þ, 375 [M ꢁ AgCl]þ. IR (KBr, cmꢁ1): 3057, 2983, 2935,
To a stirred solution of 1.6 mmol imidazolium salt in CH2Cl2/
methanol (10:12 v/v) was added silver(I) oxide (203 mg,
0.88 mmol) under N2 and the reaction mixture was allowed to
stir overnight in the dark. The resultant grey precipitate was
filtered over a bed of celite, the colorless filtrate was isolated,
and the volume of the solution was reduced to 5 mL under
vacuum. 30 mL of n-hexane was then added to produce a white
solid which was filtered and recrystallized from CH2Cl2/n-
hexane.
1630, 1502, 1451, 1228, 1159, 840. 1H NMR (CDCl3):
d 1.29 (t, 3H,
CH2CH3, J ¼ 7.2 Hz), 4.15 (q, 2H, CH2CH3, J ¼ 7.2 Hz), 5.24 (s, 2H,
CH2Ar), 6.94 (d, 6H, ArH, J ¼ 6.4 Hz), 7.05 (t, 2H, ArH, J ¼ 8.4 Hz),
7.17e7.26 (m, 5H, ArH). Anal. calcd. for C24H20AgClF2N2: C, 55.68; H,
3.89; N, 5.41%. Found C, 55.58; H, 3.99; N, 5.40%.
5.2. X-ray crystallography
The intensities for the X-ray determinations were collected on
an STOE IPDS 2T instrument with Mo K
a
radiation (
l
¼ 0.71073 Å)
5.1.6.1. Bromo[1,3-diethyl-4,5-bis(2-fluorophenyl)imidazol-2-
ylidene]silver(I) (5a). Yield 79.6%; white solid. ESI-MS m/z: 731
[2M ꢁ AgBr2]þ, 313 [M ꢁ AgBr]þ. IR (KBr, cmꢁ1): 3053, 2978, 2932,
at 200 K. Standard procedures were applied for data reduction and
absorption correction. Structure solution and refinement were
performed with SHELXS97 and SHELXL97 [33]. Hydrogen atom
positions were calculated for idealized positions and treated with
the “riding model” option of SHELXL. More details on data collec-
tions and structure calculations are contained in Table 1 and sup-
porting information.
2739, 1616, 1584, 1445, 1235, 1203, 882. 1H NMR (CDCl3):
d 1.27 (t,
6H, CH2CH3, J ¼ 7.2 Hz), 4.07 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 7.10e7.19
(m, 6H, ArH), 7.38e7.44 (m, 2H, ArH). 13C NMR (CDCl3):
d
17.1 (s,
CH3); 45.4 (s, CH2); 115.4, 115.5, 116.0, 116.2, 124.6, 127.0, 132.1,
132.2, 132.4, 159.2, 161.7 (s, CAr); signal for AgeC was not observed.
Anal. calcd. for C19H18AgBrF2N2: C, 45.63; H, 3.63; N, 5.60%. Found C,
45.87; H, 3.84; N, 5.21%.
5.3. Biological activity
5.3.1. Cytotoxicity
5.1.6.2. Bromo[1,3-diethyl-4,5-bis(3-fluorophenyl)imidazol-2-
ylidene]silver(I) (5b). Yield 81.2%; white solid. ESI-MS m/z: 731
[2M ꢁ AgBr2]þ, 313 [M ꢁ AgBr]þ. IR (KBr, cmꢁ1): 3057, 2981, 2934,
The human MCF-7, MDA MB-231 breast cancer 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), supple-
mented with 5% fetal bovine serum (FBS; Biochrom, Germany) in
a humidified atmosphere (5% CO2) at 37 ꢀC.
The experiments were performed according to established
procedures with some modifications [19,20]. In 96 well plates
100 mL of a cell suspension in culture medium at 7500 cells/mL
1630, 1557, 1452, 1345, 1228, 764. 1H NMR (CDCl3):
d 1.27 (t, 6H,
CH2CH3, J ¼ 7.2 Hz), 4.13 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 6.89e6.93 (m,
2H, ArH), 6.96e7.00 (m, 2H, ArH), 7.09e7.13 (m, 2H, ArH), 7.34e7.39
(m, 2H, ArH). 13C NMR (CDCl3):
d 17.4 (s, CH3); 45.1 (s, CH2); 116.6,
116.8, 117.2, 117.4 (s, CAr); 126.1 (d, CAr); 129.5, 129.6 (s, CAr);
130.7e130.8 (dd, CAr); 161.3, 163.8 (s, CAr); signal for AgeC was not
observed. Anal. calcd. for C19H18AgBrF2N2: C, 45.63; H, 3.63; N,
5.60%. Found C, 45.85; H, 3.98; N, 5.28%.
(MCF-7 and MDA-MB-231) or 3000 cells/mL (HT-29) were plated
into each well and were incubated for three days under culture
conditions. After the addition of various concentrations of the test
compounds, cells were incubated for up to appropriate incubation
time. Then the medium was removed, 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 photometric measurement at 590 nm. Mean values were
calculated and the effects of the compounds were expressed as %
Treated/Controlcorr values according to the following equations:
5.1.6.3. Bromo[1,3-diethyl-4,5-bis(4-fluorophenyl)imidazol-2-
ylidene]silver(I) (5c). Yield 81.3%; white solid. ESI-MS m/z: 731
[2M ꢁ AgBr2]þ, 313 [M ꢁ AgBr]þ. IR (KBr, cmꢁ1): 3053, 2981, 2934,
2874, 1630, 1598, 1505, 1456, 1345, 1227, 847, 820. 1H NMR (CDCl3):
d
1.27 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 4.10 (q, 4H, CH2CH3, J ¼ 7.2 Hz),
7.07 (t, 4H, ArH, J ¼ 8.4 Hz), 7.16e7.19 (m, 4H, ArH). 13C NMR
(CDCl3): d 17.4 (s, CH3); 45.0 (s, CH2); 116.1, 116.3, 123.7, 123.8, 130.9,
132.2, 132.3, 161.8, 164.3 (s, CAr); signal for AgeC was not observed.
Anal. calcd. for C19H18AgBrF2N2. 0.15 n-hexan: C, 46.85; H, 4.05; N,
5.41%. Found C, 47.05; H, 4.15; N, 5.73%.
T ꢁ C0
T=Ccorr½%ꢄ ¼
$100
5.1.6.4. Bromo[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-
ylidene]silver(I) (5d). Yield 58.7%; white solid. ESI-MS m/z: 337
[M ꢁ AgBr]þ. IR (KBr, cmꢁ1): 3036, 2972, 2936, 2838, 1630, 1559,
1519, 1506, 1457, 1345, 1293, 1253, 1180, 1022, 843. 1H NMR (CDCl3):
C ꢁ C0
(C0 control cells at the time of compound addition; C control cells at
the time of test end; T probes/samples at the time of test end).
The IC50 value was determined as the concentration causing 50%
inhibition of cell proliferation and calculated as mean of at least two
or three independent experiments (OriginPro 8).
d
1.26 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 3.80 (s, 6H, OCH3), 4.09 (q, 4H,
CH2CH3, J ¼ 7.2 Hz), 6.86 (d, 4H, ArH, J ¼ 8.4 Hz), 7.11 (d, 4H, ArH,
J ¼ 8.4 Hz). Anal. calcd. for C21H24AgBrN2O2. 0.3 n-hexane: C, 49.79;
H, 5.17; N, 5.09%. Found C, 49.75; H, 4.78; N, 5.42%.
5.3.2. Intercalation with calf thymus DNA
5.1.6.5. Bromo[1,3-diethyl-4,5-bis(4-hydroxyphenyl) imidazol-2-ylidene]
silver(I) (5e). Yield 51.3%; white solid. ESI-MS m/z: 309 [M ꢁ AgBr]þ.
IR (KBr, cmꢁ1): 3600e2300 (OH), 3062, 2983, 2937, 2806, 1630,
Intercalation with calf thymus DNA was performed according to
previously described procedures without modifications [21].
1594, 1498, 1277, 1169, 843. 1H NMR (DMSO-d6):
d
1.23 (t, 6H,
5.3.3. Estrogen receptor interaction studies
CH2CH3, J ¼ 7.2 Hz), 4.06 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 6.75 (d, 4H,
ArH, J ¼ 8.4 Hz), 7.08 (d, 4H, ArH, J ¼ 8.4 Hz), 9.22 (s, 2H, ArOH,
exchangeable by D2O). Anal. calcd. for C19H20AgBrN2O2. 0.5 n-
hexan: C, 49.00; H, 5.05; N, 5.20%. Found C, 48.91; H, 4.77; N, 5.51%.
Estrogen receptor interaction studies were performed according
to previously described procedures without modifications [16,30,31].
5.3.4. Inhibition of COX enzymes
The inhibition of isolated ovine COX-1 and human recombinant
5.1.6.6. Chloro[3-benzyl-1-ethyl-4,5-bis(4-fluorophenyl)imidazol-2-
ylidene]silver(I) (5f). Yield 76.9%; white solid. ESI-MS m/z: 857
COX-2 was determined with 10
ELISA (“COX inhibitor screening assay”, Cayman Chemicals).
mM of the respective compounds by

5934
W. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 5927e5934
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5.3.5. Antibacterial test
The experiments were performed according to agar diffusion
test with some modifications [32]. Indicator bacteria were grown in
LB at 30 ꢀC (E. amylovora Ea 273 and E. carotovora) or 37 ꢀC (E. coli
DH5a, B. subtilis 168, B. megaterium, P. aeruginosa and B. cereus ATCC
14579). When cell concentration of bacteria was up to 4 ꢂ 107 CFU/
mL, 0.5 mL bacteria suspensions were mixed with 20 mL melting LB
agar and cooled below 60 ꢀC to prepare the plates. 50
mL sterile
solution (H2O:DMSO 999:1 v/v) in various concentrations was
loaded into the wells (diameter: 5 mm) and punched in indicator
bacteria plates which were then incubated at 30 ꢀC overnight to
observe the growth inhibition effects. Equal amount of sterilized
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plates as a negative control. The inhibition zone diameter was then
measured and recorded.
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Acknowledgements
This work was supported by the China Scholarship Council
(CSC) and the Deutsche Forschungsgemeinschaft (DFG) within
FOR 630 “Biological function of organometallic compounds”. The
authors are also grateful for technical support by Gerhard Rubner,
Silke Bergemann, Anja Wellner (Institute of Pharmacy, Freie
Universität Berlin); Dr. Xiaohua Chen (Bacterial Genetics, Institute
of Biology, Humboldt University of Berlin) and Prof. Jinpei Zhou
(Department of Medicinal Chemistry, China Pharmaceutical
University).
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53 (2010) 6889e6898.
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Appendix. Supplementary data
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907e913.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.10.002.
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