Synthesis, cytotoxicity and antibacterial studies of novel symmetrically and nonsymmetrically 4-(methoxycarbonyl)benzyl-substituted n-heterocyclic carbene-silver acetate complexes

Article abstract of DOI:10.1002/hlca.201000310

From the reaction of 1H-imidazole (1a), 4,5-dichloro-1H-imidazole (1b), 1H-benzimidazole (1c), 1-methyl-1H-imidazole (1d), and 1-methyl-1H-benzimidazole (1f) with methyl 4-(bromomethyl)benzoate (2), symmetrically and nonsymmetrically 4-(methoxycarbonyl)be

Full text of DOI:10.1002/hlca.201000310

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Synthesis, Cytotoxicity and Antibacterial Studies of Novel Symmetrically and
Nonsymmetrically 4-(Methoxycarbonyl)benzyl-Substituted
N-Heterocyclic Carbene – Silver Acetate Complexes
by Siddappa Patil, Karolin Dietrich, Anthony Deally, Brendan Gleeson, Helge Mꢀller-Bunz,
Francesca Paradisi, and Matthias Tacke*
Conway Institute of Biomolecular and Biomedical Research, Centre for Synthesis and Chemical Biology
(CSCB), UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield,
Dublin 4, Ireland (e-mail: matthias.tacke@ucd.ie)
From the reaction of 1H-imidazole (1a), 4,5-dichloro-1H-imidazole (1b), 1H-benzimidazole (1c), 1-
methyl-1H-imidazole (1d), and 1-methyl-1H-benzimidazole (1f) with methyl 4-(bromomethyl)benzoate
(2), symmetrically and nonsymmetrically 4-(methoxycarbonyl)benzyl-substituted N-heterocyclic car-
bene (NHC) precursors, 3a – 3f, were synthesized. These NHC precursors were then reacted with
silver(I) acetate (AgOAc) to yield the NHC – silver acetate complexes (acetato-kO){1,3-bis[4-(methoxy-
carbonyl)benzyl]imidazol-2-ylidene}silver (4a), (acetato-kO){4,5-dichloro-1,3-bis[4-(methoxycarbonyl)-
benzyl]-2,3-dihydro-1H-imidazol-2-yl}silver (4b), (acetato-kO){1,3-bis[4-(methoxycarbonyl)benzyl]-
2,3-dihydro-1H-benzimidazol-2-yl}silver (4c), (acetato-kO){1-[4-(methoxycarbonyl)benzyl]-3-methyl-
2,3-dihydro-1H-imidazol-2-yl}silver (4d), (acetato-kO){4,5-dichloro-1-[4-(methoxycarbonyl)benzyl]-3-
methyl-2,3-dihydro-1H-imidazol-2-yl}silver (4e), and (acetato-kO){1-[4-(methoxycarbonyl)benzyl]-3-
methyl-2,3-dihydro-1H-benzimidazol-2-yl}silver (4f), respectively. The three NHC – AgOAc complexes
4a, 4c, and 4d were characterized by single-crystal X-ray diffraction. All compounds studied in this work
were preliminarily screened for their antimicrobial activities in vitro against Gram-positive bacteria
Staphylococcus aureus, and Gram-negative bacteria Escherichia coli using the qualitative disk-diffusion
method. All NHC – AgOAc complexes exhibited weak-to-medium antibacterial activity with areas of
clearance ranging from 4 to 7 mm at the highest amount used, while the NHC precursors showed
significantly lower activity. In addition, NHC – AgOAc complexes 4a and 4b, and 4d – 4f exhibited in
preliminary cytotoxicity tests on the human renal-cancer cell line Caki-1 medium-to-high cytotoxicities
with IC50 values ranging from 3.3 ꢀ 0.4 to 68.3 ꢀ 1 mm.
Introduction. – N-Heterocyclic carbenes (NHCs) were introduced to organo-
metallic and inorganic chemistry by the synthesis and isolation of chromium and
mercury NHC complexes by ꢀfele [1], and Wanzlick and Schçnherr [2] in 1968. The
subsequent synthesis and characterization of numerous carbeneꢁmetal complexes by
Lappert and co-workers was a significant contribution to this area of chemistry [3].
NHCs have become an important area of research after the isolation of the first stable
carbene by Arduengo et al. in 1991 [4]. In recent years, researchers have developed a
variety of convenient routes to prepare NHC complexes of main group and transition
metals [5][6]. Metal NHC complexes have been known particularly as catalysts [7 – 11]
for various chemical transformations; later discoveries revealed that silver and gold
derivatives of NHCs can be used in medicinal applications [12 – 15].
The antimicrobial activity of elemental silver and silver salts has been known for
centuries [16][17]. In many ancient societies, drinking H2O was purified and stored
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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using elemental silver [16]. The introduction of 2% AgNO3 solution to prevent the
bacterial eye infection, ophthalmia neonatorum, in newborn babies was reported in
1881 [18]. The use of silver has also been reported for a variety of medicinal
applications such as for treatment of wounds and infections [19][20]. The introduction
of silver sulfadiazine (silvadine) as an effective antimicrobial agent was reported by Fox
in 1968 [21]. High antimicrobial activity and minimal side effects of silver sulfadiazine
have made it a very convenient therapy for the treatment of infections in burns over the
past four decades [19][22 – 24]. Silver– carbene complexes, in particular those of
NHCs, have attracted a significant amount of interest in the past few years [25][26].
The first use of silver NHCs as antimicrobial agents was reported by Youngs and co-
workers in 2004 [12]. In this report, two AgI complexes (silver(I) – 2,6-bis(ethanol-
imidazolemethyl)pyridine hydroxide and silver(I) – 2,6-bis(propanolimidazoleme-
thyl)pyridine hydroxide) showed better antimicrobial activity than AgNO3 against
the microorganisms Escherichia coli, Staphylococcus aureus, and Pseudomonas
aeruginosa. Another important contribution by Ghosh and co-workers led to the
synthesis and antimicrobial evaluation of NHC – silver complexes derived from 1-
benzyl-3-(tert-butyl)-1H-imidazole [27]. Very recently, Gꢁrbꢁz and co-workers have
shown that the new imidazolidin-2-ylidene – silver complexes displayed effective
antimicrobial activity against a series of bacteria and fungi [28].
In the chemotherapeutic treatment of cancer, the discovery of the cytotoxic activity
of cisplatin by Rosenberg et al. induced a new research field of organometallic
complexes as drug candidates [29]. Cisplatin was accepted as drug in 1978 and proved
to be especially successful against testicular and bladder cancer, where the probability
of healing increased by 90% [30]. The drug did not only successfully treat 30,000
patients per year but also satisfied the pharmaceutical industry with the highest
turnover for a cytostatic drug in the US in 1983. Second-generation cytostatic drug
carboplatin with less side effects was introduced to the UK market in 1990 [31]. In a
simplified mechanistic explanation, the Pt complex binds to the DNA strings of the
cancer cell and prohibits further DNA replication and, therefore, further tumor growth.
Soon, other transition metal complexes were considered as possible anticancer
drugs. Especially treatments for cancer types that did not react to cisplatin treatment
were required. Promising candidates were the titanium complexes titanocene
dichloride [30], which showed in vitro activity against breast, lung, and intestinal
carcinoma but failed in clinical trials phase II [32], and also its successor budotitane
(cis-diethoxybis(1-phenylbutane-1,3-dionato)titanium(IV)), which also failed to go
beyond phase I after excellent preclinical results [33]. Further substitution of the
cyclopentadienyl ligand led to bis[(p-methoxybenzyl)cyclopentadienyl]titanium(IV)
dichloride (Titanocene Y), which was more effective in the treatment of xenografted
CAKI-1 tumors in mice than cisplatin [34].
Recently, Ag complexes have been reported to have anticancer activity in vitro.
Egan and co-workers have reported that Ag complexes of coumarin derivatives possess
anticancer activity against certain types of cancer [35]. Zhu et al. has reported that
silver carboxylate dimers possess anticancer activity against human carcinoma cells
[36]. McKeage and co-workers have shown phosphine complexes of silver to be active
anticancer agents, even against cisplatin-resistant cell lines [37]. Youngs and co-
workers have reported anticancer activities of NHC – silver complexes derived from

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4,5-dichloro-1H-imidazole against the human cancer cell lines OVCAR-3 (ovarian),
MB157 (breast), and HeLa (cervical) [38]. These Ag complexes have been shown to be
very stable and can be synthesized efficiently. We have recently reported the anticancer
and antibacterial activities of symmetrically p-methoxybenzyl-substituted and benzyl-
substituted NHC – silver complexes. All the reported NHC – silver complexes have
shown medium-to-high anticancer and antibacterial activities [39]. This encourages
further research on NHC – silver complexes as cytotoxic drug candidates.
Within this article, we present a new series of symmetrically and nonsymmetrically
4-(methoxycarbonyl)benzyl-substituted NHC – silver acetate complexes, their syn-
thesis, cytotoxicity, and antibacterial studies.
Results and Discussion. – Synthesis of 3a – 3f and 4a – 4f. The synthetic route for
symmetrically and nonsymmetrically 4-(methoxycarbonyl)benzyl-substituted NHCs as
ligand precursors and their corresponding Ag complexes described in this work is given
in Schemes 1 – 3. The symmetrically substituted NHC precursors 1,3-bis[4-(methoxy-
carbonyl)benzyl]-1H-imidazol-3-ium bromide (3a), 4,5-dichloro-1,3-bis[4-(methoxy-
carbonyl)benzyl]-1H-imidazol-3-ium bromide (3b), and 1,3-bis[4-(methoxycarbonyl)-
Scheme 1. General Reaction Scheme for the Synthesis of Symmetrically Substituted N-Heterocyclic
Carbenes 3a – 3c and Their Corresponding N-Heterocyclic Carbene – AgOAc Complexes 4a – 4c
Scheme 2. General Reaction Scheme for the Synthesis of Nonsymmetrically Substituted N-Heterocyclic
Carbenes 3d and 3f and Their Corresponding N-Heterocyclic Carbene – AgOAc Complexes 4d and 4f

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Scheme 3. General Reaction Scheme for the Synthesis of Nonsymmetrically Substituted N-Heterocyclic
Carbene 3e and Its N-Heterocyclic Carbene – AgOAc Complex 4e
benzyl]-1H-3,1-benzimidazol-3-ium bromide (3c) were prepared by stirring 1H-
imidazole (1a), 4,5-dichloro-1H-imidazole (1b), and 1H-benzimidazole (1c) with 2
equiv. of methyl 4-(bromomethyl)benzoate (2) in the presence of K2CO3 as a base in
MeCN at room temperature or refluxed for 3 d with 73, 19 and 44 yields, respectively
(Scheme 1). The nonsymmetrically substituted NHC precursors 3-[4-(methoxycarbo-
nyl)benzyl]-1-methyl-1H-imidazol-3-ium bromide (3d) and 3-[4-(methoxycarbonyl)-
benzyl]-1-methyl-1H-3,1-benzimidazol-3-ium bromide (3f) were prepared by stirring
1-methyl-1H-imidazole (1d) and 1-methyl-1H-benzimidazole (1f) with methyl 4-
(bromomethyl)benzoate (2) in toluene at room temperature for 3 – 4 d with 82 and
79% yields, respectively (Scheme 2). Methyl 4-[(4,5-dichloro-1H-imidazol-1-yl)me-
thyl]benzoate (1e) was formed in 83% yield from the deprotonation of 4,5-dichloro-
1H-imidazole (1b) with K2CO3 and subsequent alkylation with methyl 4-(bromome-
thyl)benzoate (2) in MeCN (Scheme 3). 4,5-Dichloro-1-[4-(methoxycarbonyl)benzyl]-
3-methyl-1H-imidazol-3-ium iodide (3e) was prepared by heating 1e with MeI in
MeCN for 1 d with a yield of 65%.
1
The NHC precursors were fully characterized by UV, IR, and H- and 13C-NMR
spectroscopy, mass spectrometry, as well as elemental analysis. The 1H-NMR spectra of
all precursors 3a – 3f show a characteristic downfield shift in the range of d(H) 9.55 –
12.01 for HꢁC(2) attributable to the positive charge of the molecule [40][41].
Additionally, their identities have also been confirmed by a base peak for the [M ꢁ Br]þ
fragments in ESI mass spectra.
The NHC – AgOAc complexes (acetato-kO){1,3-bis[4-(methoxycarbonyl)benzyl]-
2,3-dihydro-1H-imidazol-2-yl}silver (4a), (acetato-kO){4,5-dichloro-1,3-bis[4-(me-
thoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazol-2-yl}silver (4b), (acetato-kO){1,3-
bis[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazol-2-yl}silver (4c), (ace-
tato-kO){1-[4-(methoxycarbonyl)benzyl]-3-methyl-2,3-dihydro-1H-imidazol-2-yl}silver

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(4d),
(acetato-kO){4,5-dichloro-1-[4-(methoxycarbonyl)benzyl]-3-methyl-2,3-dihy-
dro-1H-imidazol-2-yl}silver (4e), and (acetato-kO){1-[4-(methoxycarbonyl)benzyl]-3-
methyl-2,3-dihydro-1H-benzimidazol-2-yl}silver (4f) were synthesized by the reaction
of 3a – 3f with 2 equiv. of AgOAc in CH2Cl2. The mixture was stirred for 2 – 4 d at room
temperature or refluxed for 2 – 4 d to afford the NHC – AgOAc complexes as off white
solids in 58 – 89% yield. The complexes were fully characterized by UV/VIS, IR, and
1H- and 13C-NMR spectroscopy, mass spectrometry, as well as elemental analysis.
Furthermore, the solid-state structures of 4a, 4c, and 4d were analyzed by single crystal
X-ray diffraction. The absence of a downfield shift for HꢁC(2) signal and presence of
1
new signals at 3.89 – 1.99 ppm for the acetate H-atoms in all the H-NMR spectra for
4a – 4f, however, indicates a successful complex formation. The 13C-NMR resonances of
the carbene C-atoms in complexes 4a – 4f appear in the range of d(C) 182.4 – 179.9.
These signals are shifted downfield compared to those of the corresponding precursors
3a – 3f (d(C) 137.8 – 131.2), which further evidences the formation of expected NHC –
AgOAc complexes. Also the appearance of the 13C-NMR resonances for the CO and
Me C-atoms of the AcO group of complexes 4a – 4f in the range of d(C) 179.4 – 177.3
and 22.9 – 15.2, respectively, indicated the formation of the NHC – AgOAc complexes
[42][43]. Furthermore, the ESI mass spectra of 4a – 4f are dominated by [M ꢁ AcO]þ
fragment peaks arising from the loss of one AcO ligand.
Structural Discussion. The crystal structures of the NHC – AgOAc complexes 4a, 4c,
and 4d have been determined. The molecular structures of 4a, 4c, and 4d are shown in
Figs. 1 – 4. The crystal data and refinement details for all three complexes are listed in
Table 1, whereas selected bond lengths and bond angles are compiled in Table 2.
Suitable crystals for X-ray crystallography to determine the molecular structure of 4a
and 4c were grown from the slow evaporation of a saturated MeOH solution, while
crystals of 4d were formed in a saturated CH2Cl2 solution with slow infusion of pentane.
The complexes 4a, 4c, and 4d crystallized in the monoclinic space group C2/c (#15), Cc
(#9), and P21 (#4), respectively. In 4a, a H2O molecule placed on the twofold axis links
two complexes by H-bonding, forming isolated moieties of (4a)2 ꢂ H2O. The crystals of
the complexes 4c and 4d contain no solvent.
Fig. 1. X-Ray diffraction structure of 4a (thermal ellipsoids are drawn at the 50% probability level)

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Fig. 2. H-Bonded moiety of 4a (thermal ellipsoids are drawn at the 50% probability level)
The NHC – AgOAc complexes 4a, 4c, and 4d are mononuclear complexes. In the
complexes 4a, 4c, and 4d reported here, the bond lengths and angles in and directly
around the NHC core agree very well among each other and with literature data
[39][44]. In the X-ray structure of 4a, 4c, and 4d, the AgOAc moiety acts as a
monodentate ligand. In 4a, 4c, and 4d, Ag is two-coordinated in an approximately
linear fashion.
Biological Evaluation. Symmetrically and nonsymmetrically 4-(methoxycarbonyl)-
benzyl-substituted NHCs and their corresponding Ag complexes were preliminarily
screened for their antibacterial activities in vitro against the selected Gram-positive
bacteria Staphylococcus aureus (NCTC 7447) and Gram-negative bacteria Escherichia
coli using the qualitative disk-diffusion method. The results of the antibacterial
activities of NHC precursors and their corresponding NHC – AgOAc complexes are
given in Tables 3 and 4, respectively. The metal salt (AgOAc) used to prepare the
complexes and the solvent (DMSO) played no role in growth inhibition on the same
bacteria as previously reported [39][45].
Almost no antibacterial activity was observed for NHC precursors 3a – 3f against
both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria
Escherichia coli. The NHC – AgOAc complexes 4a – 4c were less soluble in DMSO
(1.0 – 1.2 mg in 100 ml instead of 2.1 – 2.5 mg in 100 ml) and, therefore, showed minimal
antibacterial activity towards both Gram-positive bacteria Staphylococcus aureus and
Gram-negative bacteria Escherichia coli. Weak antibacterial activity was observed for
NHC – AgOAc complexes 4d – 4f against Gram-negative bacteria Escherichia coli, but

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Fig. 3. X-Ray diffraction structure of 4c (with 50% probability ellipsoids)
medium antibacterial activity was observed against Gram-positive bacteria Staph-
ylococcus aureus with an area of clearance of 7 mm at 0.40 mmol.
The previously synthesized NHC – AgOAc complexes in our laboratory showed an
activity of up to 12 mm area of clearance at a concentration of 0.46 mmol for (1-benzyl-
3-methylbenzimidazol-2-ylidene)silver(I) acetate [46]. Thus, the presented NHC – A-
gOAc complexes 4a – 4f do not belong to the most promising drug candidates of the
NHC – Ag series. Possibly, the methyl ester group on the benzyl ligand did not
sufficiently increase the lipophilicity of these compounds. The compounds exhibit

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Fig. 4. X-Ray diffraction structure of 4d (with 50% probability ellipsoids)
antimicrobial activity, when they are capable of penetrating into and through the lipid
membrane of microorganisms. Inside, they can prohibit active enzyme sites of the
microorganisms. Consequently, higher lipophilicity enhances antibacterial activity. For
example, NHC – AgOAc complexes 4a – 4f revealed increased antibacterial activity
compared to NHC precursors 3a – 3f, because 4a – 4f are more lipophilic than polar
NHC precursors.
In some previous complexes, the polarity of the Ag ion was even more reduced by
chelation. The AcO moiety coordinated via two O-atoms to the Ag centre in a
bidentate fashion leading to highly active complexes [39]. Despite the lack of this
coordination, the presented complexes 4a – 4f showed a moderate antibacterial activity.
Cytotoxicity Studies. The different types of NHC – AgOAc complexes are possible
anticancer drug candidates. Therefore, the in vitro cytotoxicities of symmetrically and
nonsymmetrically 4-(methoxycarbonyl)benzyl-substituted complexes 4a and 4b, and
4d – 4f were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT)-based assays [47] on the human cancerous renal-cell line Caki-1. This
test involves a 48-h drug exposure period, followed by a 24-h recovery time. The log
dose-response curves for 4a and 4b, and 4d – 4f are displayed in Fig. 5 and 6,
respectively.
Two symmetrically substituted NHC – AgOAc complexes 4a and 4b, which contain
1H-imidazole and 4,5-dichloro-1H-imidazole groups exhibit IC50 values 13.5 ꢀ 2 and
68.3 ꢀ 1 mm, respectively. The third symmetrically substituted complex 4c, which
contains 1H-benzimidazole group, was not completely soluble in DMSO and thus not
reliable for anticancer-activity studies. Compound 4a has an approximately fivefold
increase in magnitude when compared with 4b, and, in comparison to cisplatin (IC50

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Table 1. Crystal Data and Structure Refinement for 4a, 4c, and 4d
4a
4c
4d
Empirical formula
Molecular formula
Formula weight
Crystal system
Space group
Unit cell dimensions [ꢂ] a
C46H48Ag2N4O13
C27H25AgN2O6
C15H17AgN2O4
C15H17AgN2O4
397.18
Monoclinic
P21 (#4)
4.6273(1)
14.8477(3)
11.1590(2)
908
(C23H23AgN2O6)2 ꢂ H2O C27H25AgN2O6
1080.62
Monoclinic
C2/c (#15)
34.431(2)
7.6357(2)
19.534(1)
908
581.36
Monoclinic
Cc (#9)
23.9033(5)
14.4382(2)
7.3241(2)
908
b
c
a
b
121.446(8)8
908
105.684(2)8
908
96.543(2)8
908
g
Volume [ꢂ3]
Z
4381.3(4)
4
1.638
7.779
2200
2433.58(9)
4
1.587
0.874
1184
761.68(3)
2
1.732
1.342
400
Density [Mg/m3] (calc.)
Absorption coefficient [mmꢁ1
F(000)
]
Crystal size [mm3]
0.3492 ꢂ 0.2741 ꢂ 0.0259 0.1567 ꢂ 0.1053 ꢂ 0.0842 0.2526 ꢂ 0.1212 ꢂ 0.0873
Theta range for data collection 4.53 to 67.018
Index ranges
3.33 to 29.458
ꢁ 32 ꢃ h ꢃ 26
ꢁ 19 ꢃ k ꢃ 19
ꢁ 10 ꢃ l ꢃ 9
11822
3.30 to 33.018
ꢁ 7 ꢃ h ꢃ 6
ꢁ 22 ꢃ k ꢃ 21
ꢁ 16 ꢃ l ꢃ 16
18906
ꢁ 36 ꢃ h ꢃ 40
ꢁ 8 ꢃ h ꢃ 9
ꢁ 22 ꢃ l ꢃ 23
13955
Reflections collected
Independent reflections
Completeness to qmax
3854 (R(int) ¼ 0.0317)
4581 (R(int) ¼ 0.0316)
5185 (R(int) ¼ 0.0284)
98.8%
99.5%
99.4%
Max. and min. transmission
Data/restraints/parameters
Goodness-of-fit on F2
0.897 and 0.452
3854/1/300
1.080
0.937 and 0.911
4581/2/329
1.040
0.898 and 0.771
5185/1/202
1.091
Final R indices (I > 2s(I))
R1 ¼ 0.0224,
wR2 ¼ 0.0605
R1 ¼ 0.0237,
wR2 ¼ 0.0613
R1 ¼ 0.0277,
wR2 ¼ 0.0693
R1 ¼ 0.0309,
wR2 ¼ 0.0705
R1 ¼ 0.0329,
wR2 ¼ 0.0846
R1 ¼ 0.0364,
wR2 ¼ 0.0858
R Indices (all data)
Largest diff. peak and hole
0.423 and ꢁ 0.656 e ꢂꢁ3 0.903 and ꢁ 0.468 e ꢂꢁ3 3.478 and ꢁ 0.724 e ꢂꢁ3
value 3.3 mm), it represents an ca. 20-fold decrease in magnitude. The nonsymmetrically
substituted complexes 4d – 4f, which also contain 1-methyl-1H-imidazole, methyl 4-
[(4,5-dichloro-1H-imidazol-1-yl)methyl]benzoate, and 1-methyl-1H-benzimidazole
groups, have IC50 values 3.3 ꢀ 0.4, 9.4 ꢀ 1, and 13.2 ꢀ 1 mm, respectively. Compound
4d turned out to be the most promising candidate in this study because of the highest
IC50 value and has an approximately three- and fourfold increase in magnitude when
compared with 4e and 4f, respectively. Compared with cisplatin (IC50 3.3 mm), 4d has an
approximately equal activity in magnitude in terms of the IC50 value.
Compared to symmetrically substituted complexes 4a and 4b, the nonsymmetrically
substituted complexes 4d – 4f have shown almost high cytotoxic activities because of
their solubility factor. Symmetrically substituted complexes are less soluble in DMSO
compared to nonsymmetrically substituted complexes. The complexes 4a and 4b, and
4d – 4f are stable in saline solution with respect to AgCl precipitation. It was also

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Helvetica Chimica Acta – Vol. 93 (2010)
Table 2. Selected Bond Lengths [ꢂ] and Angles [8] for Compounds 4a, 4c, and 4d
Bond Lengths [ꢂ] 4a
4c
4d
Bond Angles [8]
4a
4c
4d
N(1)ꢁC(10)
C(10)ꢁN(2)
N(1)ꢁC(11)
C(12)ꢁN(2)
C(11)ꢁC(12)
AgꢁC(10)
1.353(3)
1.340(4)
N(1)ꢁC(10)ꢁN(2) 104.39(17) 105.8(2)
1.354(3)
1.383(2)
1.380(3)
1.350(3)
2.067(2)
2.1557(14) 2.093(2)
1.290(2)
1.236(3)
1.368(4) 1.377(5) C(10)ꢁN(2)ꢁC(12) 111.18(16)
1.388(3)
C(10)ꢁN(1)ꢁC(11) 111.28(16) 111.2(2)
C(12)ꢁC(11)ꢁN(1) 106.40(17)
C(11)ꢁC(12)ꢁN(2) 106.75(17)
2.055(3)
C(10)ꢁAgꢁO(5)
171.80(6) 171.10(10)
AgꢁO(5)
O(6)ꢁC(22)ꢁO(5) 123.32(19)
C(10)ꢁN(2)ꢁC(16)
N(1)ꢁC(11)ꢁC(16)
N(2)ꢁC(16)ꢁC(11)
O(6)ꢁC(26)ꢁO(5)
N(2)ꢁC(8)ꢁN(1)
O(5)ꢁC(22)
O(6)ꢁC(22)
C(22)ꢁC(23)
C(16)ꢁN(2)
C(11)ꢁC(16)
O(5)ꢁC(26)
O(6)ꢁC(26)
C(26)ꢁC(27)
N(1)ꢁC(8)
C(8)ꢁN(2)
N(1)ꢁC(9)
C(9)ꢁC(10)
AgꢁC(8)
110.7(2)
106.1(2)
106.2(2)
122.2(3)
1.509(3)
1.378(4)
1.391(5)
1.273(4)
1.244(4)
104.1(2)
111.5(2)
111.7(2)
107.0(3)
105.6(3)
176.10(10)
125.0(3)
C(8)ꢁN(2)ꢁC(10)
C(8)ꢁN(1)ꢁC(9)
1.503(4)
C(9)ꢁC(10)ꢁN(2)
1.354(3) C(10)ꢁC(9)ꢁN(1)
1.352(4) C(8)ꢁAgꢁO(3)
1.389(4) O(4)ꢁC(14)ꢁO(3)
1.357(4)
2.067(3)
2.137(2)
1.269(4)
1.236(4)
1.519(4)
AgꢁO(3)
O(3)ꢁC(14)
O(4)ꢁC(14)
C(14)ꢁC(15)
observed that, compared to known NHC – AgOAc complexes [39][46], the complexes
4a and 4b, and 4d – 4f have almost the same cytotoxicity.
Conclusions and Outlook. – In summary, we have synthesized a series of six new
symmetrically and nonsymmetrically 4-(methoxycarbonyl)benzyl-substituted N-hetero-
cyclic carbene – silver acetate complexes 4a – 4f through the reaction of appropriately
symmetrically and nonsymmetrically substituted N-heterocyclic carbenes 3a – 3f with
AgOAc. The antibacterial activities of the all compounds were tested in vitro against
both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria
Escherichia coli. Almost all the complexes have shown high antibacterial activity
compared to the precursors, and it is also clear that, as the precursor and complex
concentration increases, the antibacterial activity becomes higher. NHC – AgOAc
complexes 4a and 4b, and 4d – 4f exhibited antitumor IC50 values of 13.5 ꢀ 2, 68.3 ꢀ 1,
3.3 ꢀ 0.4, 9.4 ꢀ 1, and 13.2 ꢀ 1 mm, respectively, against the Caki-1 cell line. The most
promising compound in this study, 4d, is in the cytotoxicity range of cisplatin against
Caki-1, indicating its high potential as an anticancer drug. Further work is currently
underway in order to improve these values by performing formulation experiments to
increase solubility of these complexes, which should allow for in vivo testing of 4d in
the nearby future.

Helvetica Chimica Acta – Vol. 93 (2010)
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Table 3. Area of Clearance [mm] in Antibacterial Activity of Compounds 3a – 3f
Staphylococcus aureus (Gram-pos.)
Escherichia coli (Gram-neg.)
3a
3b
3c
3d
3e
3f
0.10 mmol (45.2 mg)
0.14 mmol (63.8 mg)
0.20 mmol (91.2 mg)
0.41 mmol (182.5 mg)
0
0
0
1
0
0
0
1
0.10 mmol (52.7 mg)
0.14 mmol (73.7 mg)
0.20 mmol (105.4 mg)
0.41 mmol (210.8 mg)
0
1
1
2
1
1
1
2
0.10 mmol (50.7 mg)
0.14 mmol (71.0 mg)
0.20 mmol (101.5 mg)
0.41 mmol (203.1 mg)
0
0
1
3
1
2
2
3
0.10 mmol (31.8 mg)
0.14 mmol (44.6 mg)
0.20 mmol (63.7 mg)
0.41 mmol (127.5 mg)
0
0
0
0
0
0
0
0
0.10 mmol (43.7 mg)
0.14 mmol (61.2 mg)
0.20 mmol (87.5 mg)
0.41 mmol 175.0 mg)
0
1
1
1
0
0
0
0
0.10 mmol (45.8 mg)
0.14 mmol (64.1 mg)
0.20 mmol (91.6 mg)
0.41 mmol (183.3 mg)
1
1
1
1
0
0
0
0
Experimental Part
General. All the solvents used were of anal. grade and were used without further purification. 1H-
Imidazole (1a), 4,5-dichloro-1H-imidazole (1b), 1H-benzimidazole (1c), 1-methyl-1H-imidazole (1d), 1-
methyl-1H-benzimidazole (1f), methyl 4-(bromomethyl)benzoate (2), AgOAc, MeI, and K2CO3 were
purchased from Sigma – Aldrich Chemical Company and were used without further purification. UV/VIS
Spectra: Unicam UV4 spectrometer; l (e) in nm. IR Spectra: Perkin-Elmer Paragon 1000 FT-IR; KBr
disc; n˜ in cmꢁ1. NMR Spectra: Varian 400 MHz spectrometer; chemical shifts d in ppm referenced to
TMS, J in Hz. ESI-MS: quadrupole tandem mass spectrometer (Quattro Micro, Micromass/Waterꢂs
Corp., USA), using solns. made up in 50% CH2Cl2 and 50% MeOH; m/z (rel.). ESI-MS (pos.-ion) for 1e,
3a – 3f, and 4a – 4f. CHN Analysis: Exeter Analytical CE-440 elemental analyser. Ag was estimated by
spectrophotometric method (atomic absorption spectra 55B Varian), while Cl, Br, and I were determined
by mercurimetric titrations. Crystal data were collected using an Oxford Diffraction SuperNova
diffractometer fitted with an Atlas detector. Complex 4a: with CuKa (1.54184 ꢂ); 4c and 4d: with MoKa
(0.71073 ꢂ); 4c was collected at 200 K, 4a and 4c at 100 K. An at least complete data set was collected,
assuming that the pairs are not equivalent. An analytical absorption correction based on the shape of the
crystal was performed [48]. The structures were solved by direct methods using SHELXS-97 [49] and
refined by full-matrix least-squares on F2 for all data using SHELXL-97 [49]. The H-atoms of the H2O
molecules in 4a were located in the difference Fourier map. Their thermal displacement parameters were
fixed to 1.5 times the equivalent one of the parent O-atom. OꢁH Bond lengths were restrained to be
0.84 ꢂ using DFIX. All other H-atoms were added at calculated positions and refined using a riding
model. Their isotropic temp. factors were fixed to 1.2 times (1.5 times for Me groups) the equivalent

2358
Helvetica Chimica Acta – Vol. 93 (2010)
Table 4. Area of Clearance [mm] in Antibacterial Activity of Compounds 4a – 4f
Staphylococcus aureus (Gram-pos.)
Escherichia coli (Gram-neg.)
4a
4b
4c
4d
4e
4f
0.10 mmol (54.4 mg)
0.14 mmol (76.2 mg)
0.20 mmol (108.9 mg)
0.41 mmol (217.8 mg)
3
4
4
5
3
4
4
5
0.10 mmol (62.5 mg)
0.14 mmol (87.5 mg)
0.20 mmol (125.0 mg)
0.41 mmol (250.0 mg)
3
4
4
4
3
4
4
4
0.10 mmol ( 59.5 mg)
0.14 mmol (83.4 mg)
0.20 mmol (119.1 mg)
0.41 mmol (238.3 mg)
3
3
3
4
3
3
4
4
0.10 mmol (40.7 mg)
0.14 mmol (56.9 mg)
0.20 mmol (81.4 mg)
0.41 mmol (162.8 mg)
3
4
5
7
3
4
4
6
0.10 mmol (47.7 mg)
0.14 mmol (66.8 mg)
0.20 mmol (95.5 mg)
0.41 mmol (191.0 mg)
3
4
5
7
3
4
4
6
0.10 mmol (45.8 mg)
0.14 mmol (64.1 mg)
0.20 mmol (91.6 mg)
0.41 mmol (183.3 mg)
3
4
4
7
2
3
3
4
isotropic displacement parameters of the C-atom to which the H-atom is attached. Anisotropic thermal
displacement parameters were used for all non-H-atoms. Suitable crystals of 4a and 4c were formed from
the slow evaporation of a sat. MeOH soln., while crystals of 4d were grown in a sat. CH2Cl2 soln. with
slow infusion of pentane. Further details about the data collection, as well as reliability factors, are listed
in Table 1.
CCDC-788649 (for 4a), -788650 (for 4c), and -788651 (for 4d) contain the supplementary
crystallographic data. These data can be obtained free of charge from the Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
1,3-Bis[4-(methoxycarbonyl)benzyl]-1H-imidazol-3-ium Bromide (3a). Compound 1a (0.136 g,
2.00 mmol) and K2CO3 (0.415 g, 3.00 mmol) were stirred for 15 min in 100 ml of MeCN. Compound 2
(0.916 g, 4.00 mmol) was added in one portion, and stirring was continued at r.t. for further 2 d. After the
solvent was removed under reduced pressure, 150 ml of H2O were added, and the mixture was stirred for
30 min. The white solid was filtered, washed with Et2O (3 ꢂ 20 ml), and dried in suction.
The obtained methyl 4-(1H-imidazol-1-ylmethyl)benzoate (0.623 g, 2.88 mmol) and 2 (0.398 g,
2.88 mmol) were refluxed in 70 ml of MeCN for 1 d. The solvent was removed under reduced pressure.
After drying for 2 h under vacuum, 3a was obtained (0.749 g, 2.11 mmol, 73%). White crystalline powder.
UV/VIS (MeOH): 230 (10495), 276 (1835). IR: 1716s, 1614m, 1550w, 1439m, 1385m, 1318w, 1282s,
1185m, 1150m, 1106s, 1022m, 874s, 734s. 1H-NMR (CDCl3): 9.55 (s, NCHN); 8.32 (d, J ¼ 8.0, 4 arom. H);
7.96 (s, 2 CH¼); 7.79 (d, J ¼ 8.0, 4 arom. H); 5.80 (s, 2 CH2); 4.16 (s, 2 MeO). 13C-NMR (CDCl3): 166.3
(C¼O); 138.7, 136.7, 130.8, 130.0, 128.3, 123.0 (NCN, CH¼, arom. C); 52.3 (MeO); 51.4 (CH2). MS
(QMS-MS/MS): 365.15 ([M ꢁ Br]þ). Anal. calc. for C21H21BrN2O4 (445.31): C 56.64, H 4.75, N 6.29, Br
17.94; found: C 56.03, H 4.78, N 5.79, Br 17.28.

Helvetica Chimica Acta – Vol. 93 (2010)
2359
Fig. 5. Cytotoxicity curves from typical MTT assays showing the effect of compounds 4a and 4b on the
viability of Caki-1 cells
Fig. 6. Cytotoxicity curves from typical MTT assays showing the effect of compounds 4d – 4f on the
viability of Caki-1 cells

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Helvetica Chimica Acta – Vol. 93 (2010)
4,5-Dichloro-1,3-bis[4-(methoxycarbonyl)benzyl]-1H-imidazol-3-ium Bromide (3b). Compound 1b
(598 mg, 4.37 mmol) and K2CO3 (905 mg, 6.55 mmol) were stirred for 15 min in 200 ml of MeCN.
Compound 2 (2.00 g, 8.73 mmol) was added, and the mixture was stirred at r.t. for 4 d. The yellow
suspension was filtered; 2 (520 mg, 2.27 mmol) was added again, and the mixture was refluxed for 4 d.
The solvent was removed under reduced pressure, washed with H2O (100 ml), Et2O (50 ml), and toluene
(2 ꢂ 100 ml): 3b (0.420 g, 0.817 mmol, 19%). White crystalline powder. UV/VIS (MeOH): 275 (6874),
285 (5800). IR: 1724s, 1614s, 1583m, 1551w, 1440m, 1278s, 1109m, 1017s, 759m. 1H-NMR (CDCl3): 12.01
(s, NCHN); 8.05 (d, J ¼ 8.4, 4 arom. H); 7.62 (d, J ¼ 8.4, 4 arom. H); 5.75 (s, 2 CH2); 3.92 (s, 2 MeO).
13C-NMR (CDCl3): 166.2 (C¼O); 138.3, 137.8, 130.4, 130.2, 128.9, 119.9 (NCN, CCl, arom. C); 52.8
(MeO); 51.6 (CH2). MS (QMS-MS/MS): 434.95 ([M ꢁ Br]þ). Anal. calc. for C21H19BrCl2N2O4 (514.20):
C 49.05, H 3.72, N 5.45, Br 15.54, Cl 13.79; found: C 49.16, H 3.42, N 5.31, Br 15.3, Cl 15.98.
1,3-Bis[4-(methoxycarbonyl)benzyl]-1H-benzimidazol-3-ium Bromide (3c). Compound 1c (516 mg,
4.37 mmol) and K2CO3 (905 mg, 6.55 mmol) were stirred for 15 min in 230 ml of MeCN before 2 (2.00 g,
8.73 mmol) was added in one portion. The mixture was stirred at r.t. for 5 d. After the solvent was
removed under reduced pressure, 300 ml of H2O were added. The precipitate was filtered, and washed
with Et2O and MeCN. The white crystalline powder was dried for 4 h under vacuum to yield 3c (0.960 g,
1.94 mmol, 44%). UV/VIS (MeOH): 270 (2852), 280 (2542). IR: 1719s, 1709s, 1553s, 1431m, 1287s,
1189m, 1152m, 1107m, 736m, 612m. 1H-NMR (CDCl3): 11.99 (s, NCHN); 8.04 (d, J ¼ 8.4,
C6H4ꢁCOOMe); 7.58 (d, J ¼ 8.4, C6H4ꢁCOOMe); 7.51 (m, 4 CH¼); 6.00 (s, 2 CH2); 3.90 (s, 2 MeO).
13C-NMR (CDCl3): 166.1 (C¼O); 137.1, 131.2, 131.1, 130.6, 130.0, 128.2, 127.4, 113.6 (NCN, CH¼,
C6H4ꢁCOOMe); 52.3 (MeO); 51.2 (CH2). MS (QMS-MS/MS): 415.08 ([M ꢁ Br]þ). Anal. calc. for
C25H23BrN2O4 (495.37): C 60.62, H 4.68, N 5.66, Br 16.13; found: C 60.42, H 4.78, N, 5.42, Br 16.25.
(Acetato-kO){1,3-bis[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazol-2-yl}silver (4a). Com-
pound 3a (94 mg, 0.212 mmol) and AgOAc (76 mg, 0.450 mmol) were refluxed in CH2Cl2 (70 ml) for 1 d.
The flask was covered with aluminium foil to avoid light exposure. The colourless soln. was filtered, and
the solvent was reduced under vacuum to 4 ml. Et2O (50 ml) was added to the colourless sticky oily
liquid, and the mixture was stirred for 1 h. The solvent was removed with a syringe, and the resulting solid
compound was dried for 4 h under reduced pressure: 4a (0.090 mg, 0.169 mmol, 79%). White crystalline
powder. UV/VIS (MeOH): 230 (10153), 276 (1942). IR: 1719s, 1612m, 1584m, 1433m, 1416m, 1283s,
1
1178m, 1016m, 799s, 676w. H-NMR (CDCl3): 8.04 (d, J ¼ 8.3, 4 arom. H); 7.34 (d, J ¼ 8.3, 4 arom. H);
6.96 (s, 2 CH¼); 5.39 (s, 2 CH2); 3.92 (s, 2 MeO); 2.08 (s, Me). 13C-NMR (CDCl3): 180.2 (NCN); 178.9
(C¼O); 166.4 (C¼O); 140.1, 130.6, 130.4, 127.8, 121.7 (CH¼, arom. C); 55.5 (MeO); 52.3 (CH2); 22.7
(Me). MS (QMS-MS/MS): 472.27 ([M ꢁ AcO]þ). Anal. calc. for C23H23AgN2O6 (531.31): C 51.99, H
4.36, N 5.27, Ag 20.30; found: C 51.90, H 4.32, N 5.04, Ag 19.98.
(Acetato-kO){4,5-dichloro-1,3-bis[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazol-2-yl}silver
(4b). Compound 3b (400 mg, 0.778 mmol) was stirred for 10 min in CH2Cl2 (100 ml). AgOAc (260 mg,
1.56 mmol) was added, and the suspension was stirred at r.t. for 5 d. The flask was covered with
aluminium foil to avoid light exposure. The solvent was removed under reduced pressure, and the white
grayish powder was partly dissolved in CHCl3 (300 ml) to obtain better solubility. The resulting
suspension was filtered over Celite to remove AgBr. The solvent of the colourless soln. was removed
under reduced pressure. The residue was dried for 2 h: 4b (0.371 g, 0.618 mmol, 79%). White crystalline
powder. UV/VIS (MeOH): 230 (16784), 282 (4083). IR: 1720s, 1614m, 1581m, 1512s, 1436s, 1293s,
1183m, 1019m, 753m. 1H-NMR (CDCl3): 8.00 (d, J ¼ 7.7, 4 arom. H); 7.35 (d, J ¼ 7.7, 4 arom. H); 5.45 (s,
2 CH2); 3.92 (s, 2 MeO); 2.03 (s, Me). 13C-NMR (CDCl3): 179.9 (NCN); 177.3 (C¼O); 165.3 (C¼O);
138.0, 129.6, 129.4, 126.5, 117.2 (CCl, arom. C); 53.3 (CH2); 51.3 (MeO); 21.6 (Me). MS (QMS-MS/MS):
541.16 ([M ꢁ AcO]þ). Anal. calc. for C23H21AgCl2N2O6 (600.20): C 46.03, H 3.53, N 4.67, Cl 11.81, Ag
17.97; found: C 46.25, H 3.51, N, 4.58, Cl 11.64, Ag 17.82.
(Acetato-kO){1,3-bis[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazol-2-yl}silver (4c). 3c
(103 mg, 0.208 mmol) and AgOAc (69.2 mg, 0.415 mmol) were refluxed in CH2Cl2 (50 ml) for 2 d. The
flask was covered with aluminium foil to avoid light exposure. The suspension was cooled to r.t., and
charcoal was added to bind AgBr. A colourless soln. was obtained after filtering over Celite. The solvent
was removed under vacuum: 4c (0.070 g, 0.120 mmol, 58%). Beige crystalline powder. UV/VIS
(MeOH): 230 (16787), 275 (7423), 285 (6805). IR: 1719s, 1613m, 1575m, 1481m, 1434m, 1396m, 1016m,

Helvetica Chimica Acta – Vol. 93 (2010)
2361
1
743m, 672w. H-NMR (CDCl3): 7.92 (d, J ¼ 8.1, 4 C6H4ꢁCOOMe); 7.28 (d, J ¼ 8.1, 4 C6H4ꢁCOOMe);
7.20 (s, 4 CH¼); 5.66 (s, 2 CH2); 3.83 (s, 2 MeO); 1.99 (s, Me). 13C-NMR (CDCl3): 180.4 (NCN); 179.4
(C¼O); 166.3 (C¼O); 139.6, 133.8, 130.5, 130.4, 127.1, 124.6, 112.1 (CH¼, C6H4ꢁCOOMe); 53.3
(MeO); 52.2 (CH2); 22.7 (Me). MS (QMS-MS/MS): 522.33 ([M ꢁ AcO]þ). Anal. calc. for C27H25Ag-
N2O6 (581.37): C 55.78, H 4.33, N 4.82, Ag 18.55; found: C 55.25, H 4.44, N 4.43, Ag 18.33.
3-[4-(Methoxycarbonyl)benzyl]-1-methyl-1H-imidazol-3-ium Bromide (3d). Compound 2 (0.458 g,
2 mmol) was added to a soln. of 1d (0.158 g, 2 mmol) in toluene (40 ml). The mixture was stirred at r.t. for
4 d. Afterwards, the solvent was removed under reduced pressure. The resultant residue was first washed
with pentane and then with Et2O. Compound 3d (0.510 g, 1.63 mmol, 82%) was obtained after drying
under reduced pressure. White solid. UV/VIS (MeOH): 230 (98352), 275 (4031), 367 (1341). IR: 3450s,
1
3138w, 3054m, 1712s, 1613m, 1442m, 1291s, 1157m, 856w, 764m, 621m, 477w, 356w. H-NMR (CDCl3):
10.44 (s, NCHN); 8.00 (d, J ¼ 8.3, 2 arom. H); 7.62 (d, J ¼ 8.2, 2 arom. H); 7.55 – 7.50 (m, 2 CH¼); 5.75 (s,
CH2); 4.08 (s, MeN); 3.90 (s, MeO). 13C-NMR (CDCl3): 166.2 (C¼O); 137.9, 137.5, 131.0, 130.4, 128.9,
123.6, 122.1 (NCN, CH¼, arom. C); 52.6 (MeO); 52.3 (CH2); 36.8 (MeN). MS (QMS-MS/MS): 231.2
([M ꢁ Br]þ). Anal. calc. for C13H15BrN2O2 (311.17): C 50.18, H 4.86, N 9.00, Br 25.68; found: C 49.98 H
5.00, N 8.97, Br 25.45.
Methyl 4-[(4,5-Dichloro-1H-imidazol-1-yl)methyl]benzoate (1e). Compound 1b (0.273 g, 2 mmol)
and K2CO3 (0.414 g, 3 mmol) were stirred for 30 min in 40 ml of MeCN. Compound 2 (0.458 g, 2 mmol)
was added in one portion, and stirring was continued at r.t. for further 4 d. After the solvent was removed
under reduced pressure, 70 ml of H2O were added. The aq. phase was extracted with CH2Cl2 (4 ꢂ 25 ml).
Org. phases were combined and dried (MgSO4). Compound 1e (0.475 g, 1.66 mmol, 83%) was obtained
after solvent removal under reduced pressure. White solid. UV/VIS (MeOH): 227 (13078), 275 (4956).
IR: 3431w, 3121w, 2954w, 1714s, 1611w, 1481m, 1428m, 1287s, 1180m, 1112m, 976w, 804w, 734m, 664w,
536w, 484w. 1H-NMR (CDCl3): 8.05 (d, J ¼ 8.3, 2 arom. H); 7.43 (s, NCHN); 7.22 (d, J ¼ 8.5, 2 arom. H);
5.15 (s, CH2); 3.92 (s, MeO). 13C-NMR (CDCl3): 166.3 (C¼O); 139.2, 134.4, 130.5, 130.4, 127.0, 126.7,
113.6 (NCN, CCl, arom. C); 52.2 (MeO); 49.3 (CH2). MS (QMS-MS/MS): 285.0 (Mþ). Anal. calc. for
C12H10Cl2N2O2 (285.13): C 50.55, H 3.54, N 9.82, Cl 24.87; found: C 50.43, H 3.27, N 9.53, Cl 24.98.
4,5-Dichloro-1-[4-(methoxycarbonyl)benzyl]-3-methyl-1H-imidazol-3-ium Iodide (3e). MeI
(0.560 ml, 9.00 mmol) was added in one portion to a stirred suspension of 1e (0.285 g, 1 mmol) in
40 ml of MeCN. The mixture was heated under reflux for 2 d. Afterwards, the solvent was removed under
reduced pressure. The resultant residue was first washed with pentane and then with Et2O. Compound 3e
(0.280 g, 0.655 mmol, 65%) was obtained after drying under reduced pressure. Yellow solid. UV/VIS
(MeOH): 225 (14904), 278 (6014). IR: 3424m, 3031s, 1715s, 1581w, 1436w, 1278s, 1194w, 1105m, 1021w,
964w, 877w, 764m, 710m, 601w, 482w. 1H-NMR (CDCl3): 10.89 (s, NCHN); 8.05 (d, J ¼ 8.3, 2 arom. H);
7.64 (d, J ¼ 8.3, 2 arom. H); 5.72 (s, CH2); 4.06 (s, MeN); 3.91 (s, MeO). 13C-NMR (CDCl3): 166.1
(C¼O); 137.1, 135.8, 131.2, 130.5, 128.9, 120.5, 119.3 (NCN, CCl, arom. C); 52.3 (MeO); 52.2 (CH2); 36.1
(MeN). MS (QMS-MS/MS): 300.1 ([M ꢁ I]þ). Anal. calc. for C13H13Cl2IN2O2 (427.06): C 36.56, H 3.07, N
6.56, Cl 16.60, I 29.72; found: C 36.34, H 3.12, N 6.42, Cl 16.57, I 29.69.
3-[4-(Methoxycarbonyl)benzyl]-1-methyl-1H-3,1-benzimidazol-3-ium Bromide (3f). Compound 2
(0.458 g, 2 mmol) was added to a soln. of 1f (0.264 g, 2 mmol) in toluene (40 ml). The mixture was stirred
at r.t. for 4 d. Afterwards, the solvent was removed under reduced pressure. The resultant residue was
first washed with pentane and then with Et2O. Compound 3f (0.575 g, 1.59 mmol, 79%) was obtained
after drying under reduced pressure. White solid. UV/VIS (MeOH): 230 (9238), 270 (6544), 368 (1588).
IR: 3421w, 3016w, 2951m, 1710s, 1612w, 1565m, 1426m, 1289s, 1191w, 1112m, 1019w, 759s, 687w, 601w,
1
467w. H-NMR (CDCl3): 11.71 (s, NCHN); 8.01 (d, J ¼ 8.3, 2 CH¼); 7.71 (d, J ¼ 8.3, CH¼); 7.67 – 7.51
(m, 1 CH¼, C6H4ꢁCOOMe); 6.03 (s, CH2); 4.30 (s, MeN); 3.89 (s, MeO). 13C-NMR (CDCl3): 166.1
(C¼O); 143.7, 137.3, 132.1, 130.9, 130.9, 130.5, 128.2, 127.3, 127.3, 113.5, 112.8 (NCN, CH¼,
C6H4ꢁCOOMe); 52.2 (MeO); 50.9 (CH2); 33.9 (MeN). MS (QMS-MS/MS): 281.1 ([M ꢁ Br]þ). Anal.
calc. for C17H17BrN2O2 (361.23): C 56.52, H 4.74, N 7.75, Br 22.12; found: C 56.34, H 4.73, N 7.66, Br 22.18.
(Acetato-kO){1-[4-(methoxycarbonyl)benzyl]-3-methyl-2,3-dihydro-1H-imidazol-2-yl}silver (4d).
Compound 3d (0.311 g, 1.00 mmol) was dissolved in CH2Cl2 (35 ml), AgOAc (0.333 g, 2.00 mmol) was
added, and the mixture was stirred at r.t. for 2 d. The yellow precipitate, presumably AgBr, was filtered,
and a clear soln. was obtained. The volatile components were removed in vacuo to produce a white sticky

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Helvetica Chimica Acta – Vol. 93 (2010)
solid. The solid was washed with Et2O (3 ꢂ 10 ml) and dried under reduced pressure for 2 h to yield 4d
(0.300 g, 0.755 mmol, 75%). White solid. UV/VIS (MeOH): 239 (12230), 277 (5445), 362 (1841). IR:
3087w, 2950w, 1711s, 1610m, 1578s, 1405s, 1312w, 1284s, 1185m, 1162m, 1111s, 1018m, 920w, 743m, 732w,
1
647, 620w. H-NMR (CDCl3): 8.02 (d, J ¼ 8.3, 2 arom. H); 7.32 (d, J ¼ 8.2, 2 arom. H); 7.01 – 6.93 (m,
2 CH¼); 5.35 (s, CH2); 3.91 (s, MeN); 3.87 (s, MeO); 2.08 (s, Me). 13C-NMR (CDCl3): 180.5 (NCN);
179.1 (C¼O); 166.3 (C¼O); 140.3, 130.4, 130.3, 127.7, 122.8, 121.0 (CH¼, arom. C); 55.3 (MeO); 52.2
(CH2); 38.8 (MeN); 22.9 (Me). MS (QMS-MS/MS): 338.2 ([M ꢁ AcO]þ). Anal. calc. for C15H17AgN2O4
(397.17): C 45.36, H 4.31, N 7.05, Ag 27.16; found: C 44.99, H 3.95, N 7.01, Ag 26.96.
(Acetato-kO){4,5-dichloro-1-[4-(methoxycarbonyl)benzyl]-3-methyl-2,3-dihydro-1H-imidazol-2-
yl}silver (4e). Compound 3e (0.427 g, 1.00 mmol) was dissolved in CH2Cl2 (40 ml), and AgOAc (0.333 g,
2.00 mmol) was added. The mixture was stirred at r.t. for 2 d. The yellow AgBr suspension was filtered to
give a light yellow coloured soln. The volatile components were removed in vacuo to produce a light
yellow sticky solid. The sticky solid was washed with pentane (3 ꢂ 10 ml) and dried under reduced
pressure for 4 h to yield 4e (0.405 g, 0.868 mmol, 87%). Colourless solid. UV/VIS (MeOH): 230 (14964),
279 (6144). IR: 3436m, 2956w, 1717s, 1615w, 1576s, 1428m, 1384m, 1282s, 1180w, 1109m, 1018w, 801w,
757w, 668w. 1H-NMR (CDCl3): 8.03 (d, J ¼ 8.3, 2 arom. H); 7.39 (d, J ¼ 8.3, 2 arom. H); 5.40 (s, CH2); 3.91
(s, MeN); 3.87 (s, MeO); 2.09 (s, Me). 13C-NMR (CDCl3): 180.5 (NCN); 179.3 (C¼O); 166.3 (C¼O);
138.9, 130.6, 130.3, 127.6, 118.5, 117.5 (CCl, arom. C); 54.2 (MeO); 52.2 (CH2); 38.1(MeN); 15.2 (Me).
MS (QMS-MS/MS): 407.0 ([M ꢁ AcO]þ). Anal. calc. for C15H15AgCl2N2O4 (466.06): C 38.66, H 3.24, N
6.01, Cl 15.21, Ag 23.14; found: C 38.42, H 3.52, N 5.97, Cl 15.13, Ag 23.10.
(Acetato-kO){1-[4-(methoxycarbonyl)benzyl]-3-methyl-2,3-dihydro-1H-benzimidazol-2-yl}silver
(4f). Compound 3f (0.361 g, 1.00 mmol) was dissolved in CH2Cl2 (35 ml), and AgOAc (0.333 g,
1.00 mmol) was added. The mixture was stirred at r.t. for 2 d. The yellow AgBr suspension was filtered to
give a colourless soln. The volatile components were removed in vacuo to produce off white sticky solid.
The solid was first washed with pentane and then with Et2O, and dried under reduced pressure for 2 h to
yield 4f (0.400 g, 0.894 mmol, 89%). Colourless solid. UV/VIS (MeOH): 230 (12177), 275 (8183), 285
(7285). IR: 3447m, 1717s, 1613w, 1576s, 1434w, 1392w, 1347w, 1285s, 1707m, 1019w, 745s, 668m. 1H-NMR
(CDCl3): 7.99 (d, J ¼ 8.3, 2 CH¼); 7.58 – 7.13 (m, CH¼, C6H4ꢁCOOMe); 5.67 (s, CH2); 4.10 (s, MeN);
3.89 (s, MeO); 2.09 (s, Me). 13C-NMR (CDCl3): 182.4 (NCN); 179.1 (C¼O); 166.3 (C¼O); 139.8, 134.6,
133.4, 130.3, 130.3, 127.1, 124.3, 124.3, 111.7, 111.3 (CH¼, C6H4ꢁCOOMe); 53.1 (MeO); 52.2 (CH2); 35.9
(MeN); 22.7 (Me). MS (QMS-MS/MS): 388.1 ([M ꢁ AcO]þ). Anal. calc. for C19H19AgN2O4 (447.23): C
51.03, H 4.28, N 6.26, Ag 24.12; found: C 50.97, H 4.18, N, 6.34, Ag 24.13.
Antibacterial Studies. In vitro antibacterial activity of symmetrically and nonsymmetrically 4-
(methoxycarbonyl)benzyl-substituted N-heterocyclic carbenes and their corresponding Ag complexes
were screened preliminarily against two bacterial strains. The test organisms included Staphylococcus
aureus (SA) (NCTC 7447) as a Gram-positive bacteria and Escherichia coli (E. coli) as Gram-negative
bacteria.
To assess the biological activity of compounds 3a – 3f and 4a – 4f, the qualitative Kirby – Bauer disk-
diffusion method was applied. All bacteria were individually cultured from a single colony in sterile LB
(lysogeny broth) medium overnight at 378 (orbital shaker incubator). All the work carried out was
performed under sterile conditions.
For each strain, 70 ml of culture were spread evenly on agar-LB medium. Four 5-mm diameter
Whatman paper discs were placed evenly separated on each plate. Two stock solns. (DMSO/H2O 90 :10)
of every compound were prepared at 2.0 and 4.1 mm to be able to test the effect of different
concentrations. Each plate was then tested with 5 ml and 7 ml of 2.0 mm soln., and 5 ml and 10 ml for the
4.1 mm soln. The plates were covered and placed in an incubator at 378 for 24 h. The plates were then
removed, and the area of clearance (defined as the distance between the edge of the filter paper disc, and
the beginning of the bacterial growth) for each sample was measured in mm (see Tables 3 and 4).
Cytotoxicity Studies. Preliminary in vitro cell tests were performed on the human cancerous renal cell
line Caki-1 in order to compare the cytotoxicity of the compounds presented in this paper. These cell lines
were chosen based on their regular and long-lasting growth behaviour, which is similar to the one shown
in kidney carcinoma cells. They were obtained from the ATCC (American Tissue Cell Culture Collection)
and maintained in Dulbeccoꢃs Modified Eagle (DME) medium containing 10% (v/v) FCS (fetal calf

Helvetica Chimica Acta – Vol. 93 (2010)
2363
serum), 1% (v/v) penicillin streptomycin, and 1% (v/v) l-glutamine. Cells were seeded in 96-well plates
containing 200-ml microtitre wells at a density of 5000 cells/200 ml of medium and were incubated at 378
for 24 h to allow for exponential growth. Then, the compounds used for the testing were dissolved in the
minimal amount of DMSO possible and diluted with medium to obtain stock solns. of 5 ꢂ 10ꢁ4 m in
concentration and less than 0.7% of DMSO. The cells were then treated with varying concentrations of
the compounds and incubated for 48 h at 378. Then, the solns. were removed from the wells, and the cells
were washed with PBS (phosphate buffer soln.), and fresh medium was added to the wells. Following a
recovery period of 24-h incubation at 378, individual wells were treated with 200 ml of a soln. of MTT
(¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) in medium. The soln. consisted of
30 mg of MTT in 30 ml of medium. The cells were incubated for 3 h at 378. The medium was then
removed, and the purple formazan crystals were dissolved in 200 ml of DMSO per well. A Wallac Victor
(Multilabel HTS Counter) plate reader was used to measure absorbance at 540 nm. Cell viability was
expressed as a percentage of the absorbance recorded for control wells. The values used for the dose-
response curves represent the values obtained from four consistent MTT-based assays for each
compound tested.
The authors thank the Irish Research Council for Science Engineering and Technology (IRCSET) for
funding through a postdoctoral fellowship for S. P.
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Received August 16, 2010