Synthesis, antitumor, and antibacterial activity of Bis[4,5-diarylimidazol- 2-ylidene]methane derivatives

Article abstract of DOI:10.1002/ardp.201100474

Cationic [bis(1,3-diethyl-4,5-diarylimidazol-2-ylidene)]Au(I) bromide complexes have demonstrated considerable potential as new antitumor agents. In order to investigate whether the gold is crucial for the antitumor activity, the imidazole ligands were connected by a methylene bridge. Biological evaluation revealed that bis[1,3-diethyl-4,5-diarylimidazol-2-ylidene]methane compounds exhibited growth inhibition effects against mammary (MCF-7 and MDA-MB 231) and colon (HT-29) carcinoma cell lines. In comparison with gold complexes, the methylene derivatives showed drastically reduced cell growth inhibitory properties. However, the growth of bacteria was significantly inhibited by bis[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]methane dibromide (4) and opens a new application of this compound type. To investigate whether the gold in cationic [bis(1,3-diethyl-4,5-diarylimidazol-2-ylidene)]Au(I) bromide complexes is crucial for their antitumor activity, the imidazole ligands were connected by a methylene bridge. In comparison with the gold complexes, the methylene derivatives showed drastically reduced cell growth inhibitory properties. However, the growth of bacteria was significantly inhibited by bis[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]methane dibromide (4), opening a new application of this compound type. Copyright

Full text of DOI:10.1002/ardp.201100474

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Synthesis, Antitumor, and Antibacterial Activity of
Bis[4,5-diarylimidazol-2-ylidene]methane Derivatives
Wukun Liu1, Xiaohua Chen2, and Ronald Gust1,3
1
¨
Institute of Pharmacy, Freie Universitat Berlin, Berlin, Germany
2 Bacterial Genetics, Institute of Biology, Humboldt University of Berlin, Berlin, Germany
3 Institute of Pharmacy, University of Innsbruck, Innsbruck, Austria
Cationic [bis(1,3-diethyl-4,5-diarylimidazol-2-ylidene)]Au(I) bromide complexes have demonstrated
considerable potential as new antitumor agents. In order to investigate whether the gold is
crucial for the antitumor activity, the imidazole ligands were connected by a methylene bridge.
Biological evaluation revealed that bis[1,3-diethyl-4,5-diarylimidazol-2-ylidene]methane compounds
exhibited growth inhibition effects against mammary (MCF-7 and MDA-MB 231) and colon (HT-29)
carcinoma cell lines. In comparison with gold complexes, the methylene derivatives showed
drastically reduced cell growth inhibitory properties. However, the growth of bacteria was
significantly inhibited by bis[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]methane
dibromide (4) and opens a new application of this compound type.
Keywords: Antibacterial activity / Antitumor activity / Gold–bisNHC complexes / Methylene bridged imidazoles
Received: December 30, 2011; Revised: February 7, 2012; Accepted: February 16, 2012
DOI 10.1002/ardp.201100474
Introduction
of their strong antiproliferative effects [7–13]. Both phos-
phine complexes exhibit their antitumor activities mainly
due to the inhibition of the enzyme thioredoxin reductase
(TrxR) [13]. The use of NHCs as alternatives to phosphines as
ligands for the soft Au(I) ion resulted in complexes with
different steric and lipophilic properties [2–5, 7–13].
In our group, we intensively studied the significance of
ligands derived from pharmacologically active 4,5-diarylimi-
dazoles on the cytotoxicity of cationic Au–NHC complexes.
Among them the dimeric bis[1,3-diethyl-4,5-diarylimidazol-2-
ylidene]gold(I) complexes (Scheme 1) showed good growth
inhibition properties. They were at least 10-fold more active
than cisplatin against mammary (MCF-7 and MDA-MB 231)
and colon (HT-29) carcinoma cells [7, 8].
In order to overcome the disadvantages (side effects, e.g.,
nausea, alopecia, and development of resistance during
the therapy) of the established platinum-based drugs cispla-
tin, carboplatin, and oxaliplatin which are widely used in the
treatment of cancer, current strategies in the development of
novel metallodrugs more and more focused on the use of
transition metal complexes containing improved organic
ligands [1, 2].
During the last years N-heterocyclic carbenes (NHCs) could
be established as carrier ligands for metal complexes display-
ing high biological effects [3–6]. Especially gold–NHCs which
can be seen as derivatives of auranofin and its chloro analog
Et3PAuCl (Scheme 1) recently gained much attention because
Encouraged by these promising results and in continu-
ation of our structure–activity relationship (SAR) studies,
we extended our research to bis[1,3-diethyl-4,5-diarylimida-
zol-2-ylidene]methane analogs. Due to this structural modi-
fication the relevance of the gold for growth inhibitory
effects can be estimated. Formally, in the cationic Au(I)–
bisNHC complexes, the gold atom was exchanged by a meth-
ylene linker. All synthesized compounds were characterized
by MS, NMR spectra and tested for antitumor as well as
antibacterial potency in vitro.
Correspondence: Prof. Ronald Gust, Institute of Pharmacy, University of
Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria.
E-mail: ronald.gust@uibk.ac.at
Fax: þ43 512 507 58299
Abbreviations: 5-fluorouracil (5-FU); Luria broth (LB); N-heterocyclic
carbenes (NHCs); phosphate buffered saline (PBS); structure–activity
relationship (SAR); thioredoxin reductase (TrxR).
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W. Liu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Scheme
Et3PAuCl, and cationic Au(I)–bisNHC com-
1. Structures
of
auranofin,
plexes (Au1 and Au2).
Results and discussion
phenyl)-1H-imidazole (9) was isolated and reacted with NaH
and ethyl bromide in absolute THF to afford N-alkylation (9!1).
Deprotonation of 1 and reaction with diethyl carbonate at
low temperature using a modified procedure of Ru¨ether and
co-workers [14] yielded the bis[1-ethyl-4,5-bis(4-methoxy-
phenyl)-1H-imidazol-2-yl]methanone 2 (Scheme 2). Due to
the relatively low acidity of the imidazole, BuLi was used
as base.
Chemistry
1-Ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazole (1), the educt for
the preparation of the dimeric compounds 3–6 (see Scheme 2),
was synthesized according to our previously published
methods [6, 7]. As outlined in Scheme 3, reaction of 2 equiv.
of the commercially available 4-methoxybenzaldehyde (7)
under catalysis of thiamine hydrochloride gave 2-hydroxy-
1,2-bis(4-methoxyphenyl)ethanone (8) which was heated in for-
mamide to reflux for 3 h. The resulting 4,5-bis(4-methoxy-
For reduction to the bis[1-ethyl-4,5-bis(4-methoxyphenyl)-
1H-imidazol-2-yl]methane (3), the keton 2 was heated with
hydrazine hydrate and potassium hydroxide [14]. The careful
Scheme 2. Synthetic routes of bis[4,5-
diarylimidazol-2-ylidene]methane derivatives.
Reagents and conditions: (a) n-BuLi, THF,
S808C to rt, 54.0%; (b) KOH, NH2NH2
H2O, 120–1508C, 73.3%; (c) ethyl bromide,
CH3CN, reflux, 48–72 h, 77.6%; (d) BBr3,
CH2Cl2, S608C, then warm to rt, 48 h,
65.5%; (e) ethyl bromide, CH3CN, reflux,
48–72 h, 68.2%.
Scheme 3. Synthetic routes of 1-ethyl-4,5-
bis(4-methoxyphenyl)-1H-imidazole (1).
Reagents and conditions: (a) thiamine
hydrochloride, H2O/ethanol 1:2, rt, 96 h,
58.8%; (b) formamide, reflux, 3 h, 75.3%;
(c) 95% NaH, ethyl bromide, absolute THF,
reflux, 2 h, 87.9%.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Biological Activity of Methylene Bridged Imidazoles
3
Figure 1. (a) 1H NMR spectra of 2 (above) and 3 (below) (400 MHz, CDCl3); (b) 13C NMR spectra of 2 (above) and 3 (below) (100 MHz,
CDCl3).
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Table 1. Growth inhibitory effects against MCF-7, MDA-MB 231,
and HT-29 cells.
control of the temperature is crucial to the relative success of
the reaction. A yellow solid was formed at 608C and the
reaction mixture became clear and colorless when the
temperature reached 1168C. The reaction was kept at
1208C for 1 h followed by heating to 1508C for another
3 h. In experiments in which the temperature was instantly
raised to 1508C, the solution became dark brown and the
yields were low. These observations indicate that the initial
hydrazone formation has to be completed prior to the final
reduction step at elevated temperature.
Compound
Cytotoxicity, IC50a) (mM)
MCF-7
MDA-MB 231
HT-29
2
3
4
5
>20
10.1 ꢀ 1.2
2.6 ꢀ 0.2
9.7 ꢀ 0.4
5.5 ꢀ 0.4
4.7 ꢀ 0.4
0.17
>20
>20
>20
>20
14.1 ꢀ 4.1
7.1 ꢀ 0.4
8.1 ꢀ 0.6
15.5 ꢀ 0.1
7.3 ꢀ 1.0
0.43
11.2 ꢀ 2.2
10.8 ꢀ 1.0
9.6 ꢀ 0.3
0.54
6
5-FU
Au1b)
Au2b)
Following this procedure, 4 could be obtained in high
yield and analytically pure after recrystallization from
THF. Its structure was confirmed by NMR spectroscopy.
Characteristically, the methylene protons were observed at
d ¼ 7.58 in the 1H NMR spectrum of 3 (Fig. 1a). In its 13C NMR
0.30
1.55
0.47
a)
The IC50 value was determined as the concentration causing
50% decrease in cell growth after 72 h of incubation and calcu-
lated as the mean of at least two or three independent
experiments.
–
spectrum the signal of the C O group peak at d ¼ 174.7 as
–
b)
determined for 2 is displaced by a CH2 signal upfield shifted
to d ¼ 49.8 (Fig. 1b).
Data from Ref. [8].
The bis[1-ethyl-4,5-bis(4-hydroxyphenyl)-1H-imidazol-2-yl]-
methane (5) was generated from the corresponding com-
pound 3 by ether cleavage with BBr3. Finally, compounds 3
and 5 were further reacted with ethyl bromide in CH3CN to
yield the bis[1,3-diethyl-4,5-diarylimidazol-2-ylidene]methane
derivatives 4 and 6, respectively.
period of time (72 h). From the concentration–activity (%T/C)
relation the IC50 values were calculated (OriginPro 8) and are
presented in Table 1. Additionally, the time-dependent activi-
ties of 4 on the MCF-7 cell line and of 6 on MDA-MB 231 and
HT-29 cell lines are presented in Fig. 2 as examples.
The methanone
2 was inactive in all cell lines
Biological activity
(IC50 >20 mM), while its methylene analogon 3 reduced
the growth of MCF-7 and HT-29 cells with IC50 ¼ 10.1 and
14.1 mM, respectively. Interestingly, 3 was inactive against
MDA-MB 231 cells (IC50 >20 mM). An additional N-ethyl group
The growth inhibitory potency of compounds 2–6 as well as
the antitumor drug 5-fluorouracil (5-FU) was evaluated in an
established in vitro cytotoxicity assay using MCF-7 and MDA-
MB 231 breast cancer as well as HT-29 colon carcinoma cells
[15, 16]. In this test, a known number of cells were exposed to
increasing concentrations (0.63–20 mM) of the compounds
on 96-well tissue culture plates and incubated for a given
increased the growth inhibitory potency. Although
4
represents a permanent cation it was more active than 5-
FU at the MCF-7 cell line (IC50 ¼ 2.6 mM; 5-FU: IC50 ¼ 4.7 mM)
and comparably active at HT-29 cells (IC50 ¼ 7.1 mM; 5-FU:
120
120
120
100
80
60
40
20
0
100
100
80
80
60
60
40
40
20
20
0
0
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
Incubation time (h)
Incubation time (h)
Incubation time (h)
1.25 µM
2.5 µM
5 µM
10 µM
20 µM
1.25 µM
2.5 µM
5 µM
10 µM
20 µM
0.63 µM
1.25 µM
2.5 µM
5 µM
10 µM
Figure 2. Time–activity curves of 4 on MCF-7 (left) cell line and 6 on MDA-MB 231 (middle) and HT-29 (right) cell lines. Error bars are hidden
behind the symbols in some cases.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Biological Activity of Methylene Bridged Imidazoles
5
Table 2. Sensitivity test of compounds in inhibiting bacterial growth
Test compounds Concentration (mM)
Diameter of the zone of inhibition (mm)
Ervinia amylovora
Ea 273
Escherichia coli
DH5a
Bacillus subtilis
168
Bacillus
megaterium
4
5
10
5
15
17
5
7
8
5
18
30
5
20
23
5
6
10
5
5
5
5
5
5
8
5
9
Au1
10
10
5
12
5
5
11
5
10
9
5
10
12
5
AgNO3
H2O
IC50 ¼ 7.3 mM). Again, no influence was observed on MDA-MB
231 cells (IC50 >20 mM).
378C (Escherichia coli DH5a, Bacillus subtilis 168, and Bacillus
megaterium). When the cell concentration of bacteria was up
to 4 ꢁ 107 CFU/mL, 0.5 mL of bacteria suspensions were mixed
with 20 mL of melting LB agar and cooled below 608C to
prepare the plates. AgNO3 was used as positive control. Fifty
microliters of aqueous solution with increasing compound
concentrations or 50 mL of water (containing 0.1% DMSO) was
loaded into the wells (diameter: 5 mm) and punched in
indicator bacteria plates which were then incubated at 308C
overnight to observe the growth inhibition effects. The anti-
microbial activity of the compounds was determined by
measuring the diameter of the inhibiting zone around wells.
Our testing confirmed for 4 antimicrobial properties at a
level comparable to silver nitrate against E. amylovora Ea 273,
E. coli DH5a, B. subtilis 168, and B. megaterium (see Table 2). The
maximum of antimicrobial activity was observed against the
B. subtilis 168 and B. megaterium. The diameter of the inhi-
bition zone of 4 against the B. subtilis 168 at 10 mM was
almost threefold than that of AgNO3.
Effects on MDA-MB 231 cells can be observed after ether
cleavage resulting in compounds 5 and 6 (IC50 ¼ 10.2 and
10.8 mM, respectively). At the other cell lines 6 was half as
active as its methoxy derivative 4 (IC50 ¼ 5.5 and 2.6 mM at
MCF-7 cells; IC50 ¼ 15.5 and 7.1 mM at HT-29 cells) while 5 (4-
OH) and 3 (4-OCH3) were equipotent at MCF-7 cells (IC50 ¼ 9.7
and 10.1 mM). At HT-29 cells, 5 (IC50 ¼ 8.1 mM) was more
active than 3 (IC50 ¼ 14.1 mM).
These data clearly demonstrate that the exchange of the
gold center by a methylene group decreases the growth
inhibitory effects. The cationic bis[1,3-diethyl-4,5-diarylimi-
dazol-2-ylidene]Au(I) complexes Au1 (IC50 ¼ 0.17 mM, MCF-7;
IC50 ¼ 0.54 mM, MDA-MB 231; IC50 ¼ 0.43 mM, HT-29) and
Au2 (IC50 ¼ 0.30 mM, MCF-7; IC50 ¼ 1.55 mM, MDA-MB 231;
IC50 ¼ 0.47 mM, HT-29) were more than 10–20-fold higher
cytotoxic than the methylene derivatives 4 and 6.
In order to exclude that the cells become resistant during
the treatment, which is indicated by an increased cell
growth after an initial inhibition, we investigated the
time-dependent cell growth. In Fig. 2 the time–activity curves
of 4 and 6 are depicted as representatives.
Unlike compound 4, compound 6 was inactive in our
bacterial growth inhibiting assay, indicating that methoxy
substituents might mediate higher potency than hydroxyl
substituents against bacteria. Moreover, unlike its high anti-
tumor activity, the gold complex Au1 was less active than
compound 4. Au1 reached nearly the activity of AgNO3
against B. subtilis 168 and B. megaterium, but it was even
inactive against E. amylovora Ea 273 and E. coli DH5a.
In both cases, the tumor cells showed marginal recuper-
ation after a prolonged exposition to the drugs. Because the
exponential cell growth is guaranteed for at least 140 h of
incubation, the rise of the growth curve can be an indication
of development of drug resistance. The maximal antiproli-
ferative effects were achieved between 48 and 72 h, so we Conclusion
evaluated the IC50 value of all complexes after an incubation
time of 72 h [17, 18].
In conclusion, bis[1,3-diethyl-4,5-diarylimidazol-2-ylidene]-
Finally, because imidazolium salts and gold complexes were
long term investigated as antimicrobial agents [2, 4], we eval-
uated the in vitro antimicrobial activity of target compounds 4
and 6 compared to the gold complex Au1. A modified agar
diffusion test [6, 19] was used to evaluate the preliminary
antibacterial activities of compounds. Indicator bacteria were
grown in Luria broth (LB) at 308C (Ervinia amylovora Ea273) or
methane compounds were synthesized and tested for bio-
logical activities. They possessed growth inhibitory effects
dependent on the substituents at the aromatic rings. The
lower influence compared with the related gold complexes
documented that the gold core seems to be responsible for
the high cytotoxic activity of cationic bis[1,3-diethyl-4,5-diaryl-
imidazol-2-ylidene]Au(I) complexes. Antibacterial tests showed
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W. Liu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
360 mmol) was added and the flask immersed into an oil bath.
On heating a yellow solid began to form at 608C. The reaction
mixture became clear and almost colorless between 110 and
1208C. Stirring at 1208C was continued for 1 h. The temperature
was then raised to 1508C and stirring was continued for 3 h at
this temperature. Some white solid precipitated at the end of this
period. The reaction mixture was allowed to cool to rt during
which a white waxy solid formed. At this point all following
operations were carried out in air. CH2Cl2 (40 mL) was added and
the solution transferred to a separatory funnel. The CH2Cl2 layer
was separated and the remaining light brown liquid extracted
with CH2Cl2 (2 ꢁ 40 mL). The pooled CH2Cl2 layers were washed
twice with H2O (2 ꢁ 15 mL) to remove excess hydrazine hydrate.
The combined H2O extracts were then repeatedly extracted with
CH2Cl2 (6 ꢁ 20 mL). The combined CH2Cl2 solutions were dried
(Na2SO4), filtered, and rotary evaporated to give an off-white
solid. The product was recrystallized from THF, and colorless
rhombic crystals separated on cooling to rt. Yield: 4.84 g (73.3%)
of a white solid (mp 130–1318C); MS m/z: 628 [M]þ; 1H NMR
(CDCl3): d 1.26 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 3.76 (s, 6H, OCH3),
3.80 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 3.87 (s, 6H, OCH3), 6.75 (d, 4H,
ArH, J ¼ 8.8 Hz), 6.78 (d, 4H, ArH, J ¼ 8.8 Hz), 7.24 (d, 4H, ArH,
J ¼ 8.8 Hz), 7.40 (d, 4H, ArH, J ¼ 8.8 Hz), 7.58 (s, 2H, CH2);
13C NMR (CDCl3): d 16.3 (s, CH3); 40.0 (s, CH2); 49.9 (s, CH2);
55.0, 55.2 (s, OCH3); 113.5, 114.5, 122.8, 127.1, 127.5, 127.7,
132.0, (s, CAr); 135.6, 137.7 (s, NCN); 156.1, 159.7 (s, CAr). Anal.
calcd for C39H40N4O4: C, 74.50; H, 6.41; N, 8.91. Found C, 74.55; H,
6.52; N, 8.73.
that compound 4 exhibited higher activity than AgNO3 and
the gold complex Au1 and open a new application of this type
of compounds. In the next step of this SAR study we will
exchange the methylen/gold center by other metals.
Furthermore, investigations are in progress to get deeper
insight into the mechanism of action of bisNHC–metal
complexes.
Experimental
Chemistry
General
The following instrumentation was used: 1H or 13C NMR 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,
USA; Elemental C, H, N analysis: PerkinElmer 240 B and C
analyzer; Melting point: capillary tube; uncorrected.
Chemicals were obtained from Sigma–Aldrich (Germany) and
used without further purification. Reactions were all monitored
by TLC on silica gel plates 60 F254 (Merck, Darmstadt, Germany),
visualized by UV light. Column chromatography was performed
with Merck silica gel 60H, grain size <0.063 mm, 230 mesh
ASTM (Darmstadt, Germany).
1-Ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazole (1) was synthes-
ized according to Refs. [6, 7].
Bis[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-
ylidene]methane dibromide 4
Bis[1-ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazol-2-yl]-
methanone 2
Ethyl bromide (2.7 g, 25.0 mmol) and 3 (3.14 g, 5.0 mmol) were
combined and refluxed in 50 mL of acetonitrile for 48–72 h.
Removal of the solvent under reduced pressure gave the crude
product which was washed with Et2O (3 ꢁ 30 mL) and dried
in vacuo. Yield: 3.29 g (77.6%) of a white solid (mp 149–1518C);
A solution of 1-ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazole (1)
(20.0 g, 65 mmol) in dry THF (200 mL) was cooled to ꢂ458C
and n-BuLi (2.5 M in hexane) (26 mL, 65 mmol) was slowly added.
The yellow solution was stirred at ꢂ458C for 30 min and then
cooled to ꢂ808C. Diethylcarbonate (3.8 g, 32.5 mmol) was added
dropwise. The color of the solution changed from pale yellow to
purple. After the reaction mixture was allowed to warm to ꢂ208C
during 5 h, the reaction was quenched by addition
of H2O (10 mL). After warming to rt, THF was removed in vacuo
and the residue extracted with CH2Cl2 (3 ꢁ 50 mL). The CH2Cl2
layer was extracted, dried (Na2SO4), filtered, and evaporated to
give a brown orange oil. The pure product was obtained as
colorless crystals after recrystallization from acetone, and from
the resulting mother liquor after slow evaporation in air. Yield:
11.2 g (54.0%) of a white solid (mp 203–2058C); MS m/z: 642 [M]þ;
1H NMR (CDCl3): d 1.35 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 3.77 (s, 6H,
OCH3), 3.89 (s, 6H, OCH3), 4.23 (q, 4H, CH2CH3, J ¼ 7.2 Hz), 6.77
(d, 4H, ArH, J ¼ 8.8 Hz), 7.02 (d, 4H, ArH, J ¼ 8.8 Hz), 7.32 (d, 4H,
ArH, J ¼ 8.8 Hz), 7.49 (d, 4H, ArH, J ¼ 8.8 Hz); 13C NMR (CDCl3): d
16.8 (s, CH3); 40.9 (s, CH2); 55.1, 55.2 (s, OCH3); 113.5, 114.5, 122.8,
126.9, 128.3, 128.6, 132.1 (s, CAr); 140.0, 142.4 (s, NCN); 158.5,
160.2 (s, CAr); 174.6 (s, CO). Anal. calcd for C39H38N4O5: C, 72.88; H,
5.96; N, 8.72. Found C, 72.64; H, 5.71; N, 8.81.
ESI-MS m/z: 686 [M–2Br]2þ 1H NMR (DMSO-d6): d 1.30 (t, 12H,
;
CH2CH3, J ¼ 7.2 Hz), 3.77 (s, 12H, OCH3), 4.07 (q, 8H, CH2CH3,
J ¼ 7.2 Hz), 7.01 (d, 8H, ArH, J ¼ 8.4 Hz), 7.33 (d, 8H, ArH,
J ¼ 8.8 Hz), 9.40 (s, 2H, CH2); 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 C43H50Br2N4O4: C,
61.00; H, 5.95; N, 6.62. Found C, 61.00; H, 5.82; N, 6.77.
Bis[1-ethyl-4,5-bis(4-hydroxyphenyl)-1H-imidazol-2-yl]-
methane 5
A solution of 3 (1.00 mmol, 0.63 g) in 20 mL of dry CH2Cl2 was
cooled to ꢂ608C. At this temperature BBr3 (4.5 mmol, 1.12 g) in
5 mL of dry CH2Cl2 was added under N2 atmosphere. Then the
reaction mixture was allowed to warm to rt 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 to give the product. Yield:
0.374 g (65.5%) of a red solid (mp 304–3068C); MS m/z: 572 [M]þ;
1H NMR (DMSO-d6): d 1.24 (t, 6H, CH2CH3, J ¼ 7.2 Hz), 4.19 (q, 4H,
CH2CH3, J ¼ 7.2 Hz), 6.69 (d, 4H, ArH, J ¼ 8.4 Hz), 6.96 (d, 4H,
ArH, J ¼ 8.4 Hz), 7.25 (d, 4H, ArH, J ¼ 8.4 Hz), 7.30 (d, 4H, ArH,
J ¼ 8.4 Hz), 9.62 (s, 2H, CH2), 10.03 (s, 4H, OH, exchangeable
Bis[1-ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazol-2-yl]-
methane 3
Compound 2 (6.75 g, 10.5 mmol) and KOH (2 g, 35.6 mmol) were
placed into
a Schlenk flask. Hydrazine hydrate (17.5 mL,
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Biological Activity of Methylene Bridged Imidazoles
7
by D2O). Anal. calcd for C35H32N4O4: C, 73.41; H, 5.63; N, 9.78.
Found C, 73.54; H, 5.32; N, 9.55.
mean of at least two or three independent experiments
(OriginPro 8).
Bis[1,3-diethyl-4,5-bis(4-hydroxyphenyl)-imidazol-2-
ylidene]methane dibromide 6
Antimicrobial test
The experiments were performed according to agar diffusion
test with some modifications [6, 19]. Indicator bacteria were
grown in LB at 308C (E. amylovora Ea 273) or 378C (E. coli DH5a,
B. subtilis 168, and B. megaterium). When cell concentration of
bacteria was up to 4 ꢁ 107 CFU/mL, 0.5 mL of bacteria sus-
pensions were mixed with 20 mL of melting LB agar and
cooled below 608C to prepare the plates. Fifty microliters
of a sterile solution (H2O/DMSO 999:1 v/v) in various concen-
trations was loaded into the wells (diameter: 5 mm) and
punched in indicator bacteria plates which were then incu-
bated at 308C overnight to observe the growth inhibition
effects. Equal amount of sterilized water (containing 0.1%
DMSO) was also loaded into indicator bacteria plates as a
negative control. The inhibition zone diameter was then
measured and recorded.
Ethyl bromide (0.27 g, 2.5 mmol) and 5 (0.286 g, 0.50 mmol)
were combined and refluxed in 20 mL of acetonitrile for 48–
72 h. Removal of the solvent under reduced pressure gave the
crude product which was washed with Et2O (3 ꢁ 10 mL)
and dried in vacuo. Yield: 0.269 g (68.2%) of a red solid (mp
295–2978C); ESI-MS m/z: 630 [M–2Br]2þ 1H NMR (DMSO-d6):
;
d 1.21 (t, 12H, CH2CH3, J ¼ 7.2 Hz), 4.15 (q, 8H, CH2CH3,
J ¼ 7.2 Hz), 6.84 (d, 4H, ArH, J ¼ 8.0 Hz), 6.95 (d, 4H, ArH,
J ¼ 8.0 Hz), 7.22 (d, 4H, ArH, J ¼ 8.4 Hz), 7.27 (d, 4H, ArH,
J ¼ 8.4 Hz), 9.46 (s, 2H, CH2), 9.93 (s, 4H, OH, exchangeable
by D2O). Anal. calcd for C39H42Br2N4O4: C, 59.25; H, 5.35; N,
7.09. Found C, 59.15; H, 5.53; N, 6.94.
Biological activity
Cell culture
The human MCF-7 and 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 mono-
layer 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 378C.
This work was supported by the China Scholarship Council (File No.
2008613021) and the Deutsche Forschungsemeinschaft (DFG) within FOR
630 ‘‘Biological function of organometallic compounds’’.
The authors have declared no conflict of interest.
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