Synthesis and biological evaluation of N-heterocyclic carbene-silver(I) acetate complexes derived from 4,5-ditolyl-imidazole

Article abstract of DOI:10.1016/j.ica.2012.10.029

From the reaction of 1,2-bis-(4-methylphenyl)ethane-1,2-dione with formamide, symmetrically substituted 4,5-bis-(4-methylphenyl)-1H-imidazole (1) was synthesised and further reacted with p-benzyl substituted halides to give the symmetrically substituted N-heterocyclic carbene (NHC) precursors 1a-e. The NHC precursors were then reacted with silver(I) acetate to yield NHC-silver(I) acetate complexes 1,3-bis-(benzyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2a), 1,3-bis-(4-methylbenzyl)-4,5-bis-(4-methylphenyl)- imidazole-2-ylidene silver(I) acetate (2b), 1,3-bis-(4-methoxylbenzyl)-4,5-bis- (4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2c), 1,3-bis-(4-methoxycarbonylbenzyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2d) and 1,3-bis-(4-cyanobenzyl)-4,5-bis-(4-methylphenyl)- imidazole-2-ylidene silver(I) acetate (2e). Two NHC-silver acetate complexes 2a and 2e were characterised by single crystal X-ray diffraction. The preliminary in vitro antibacterial activity of the NHC-silver complexes 2a-e was investigated against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli using the qualitative Kirby-Bauer disk-diffusion method. The areas of clearance determined for the maximum dose (4.3 μM) range between 1 mm and 7 mm for MSSA and 0 mm and 7 mm for E. coli. All of the newly synthesised silver(I) acetate complexes were tested for their cytotoxicity by MTT based in vitro tests on the human renal cancer cell line Caki-1 and human breast cancer cell line MCF-7 in order to determine their IC₅₀ values. The NHC-silver complexes 2a-e were found to have IC ₅₀ values of 3.0 (±0.6), 0.51 (±0.07), 4.2 (±1.2), 9.0 (±0.6), 26 (±2) μM, against the renal cancer cell-line Caki-1 and IC₅₀ values of 2.3 (±0.4), 1.4 (±0.2), 3.0 (±0.5), 3.4 (±1.2) and 14 (±2) μM against the breast cancer cell line MCF-7, respectively. Compared to our lead compound SBC3 (1,3-bisbenzyl-4,5-bisphenyl-imidazole-2-ylium silver(I) acetate) (IC₅₀ value = 14 (±1) μM against Caki-1 and 5.8 (±0.6) μM against MCF-7) these values represent improved cytotoxicity against both cell lines, especially for the silver complexes 2a and 2b. These two compounds are not only more active than SBC3 but also exhibit in the case of 2b a 7 times higher biological activity than cisplatin (IC₅₀ value = 3.3 μM) against Caki-1.

Full text of DOI:10.1016/j.ica.2012.10.029

Inorganica Chimica Acta 395 (2013) 135–144
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis and biological evaluation of N-heterocyclic carbene–silver(I) acetate
complexes derived from 4,5-ditolyl-imidazole
Frauke Hackenberg, Grainne Lally, Helge Müller-Bunz, Francesca Paradisi, Daniela Quaglia,
⇑
Wojciech Streciwilk, Matthias Tacke
Centre for Synthesis and Chemical Biology (CSCB), UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2012
Received in revised form 26 October 2012
Accepted 31 October 2012
Available online 14 November 2012
From the reaction of 1,2-bis-(4-methylphenyl)ethane-1,2-dione with formamide, symmetrically substi-
tuted 4,5-bis-(4-methylphenyl)-1H-imidazole (1) was synthesised and further reacted with p-benzyl
substituted halides to give the symmetrically substituted N-heterocyclic carbene (NHC) precursors 1a–e.
The NHC precursors were then reacted with silver(I) acetate to yield NHC–silver(I) acetate complexes 1,3-
bis-(benzyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2a), 1,3-bis-(4-methylben-
zyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2b), 1,3-bis-(4-methoxylbenzyl)-4,
5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2c), 1,3-bis-(4-methoxycarbonylbenzyl)-4,
5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I) acetate (2d) and 1,3-bis-(4-cyanobenzyl)-4,5-bis-(4-
methylphenyl)-imidazole-2-ylidene silver(I) acetate (2e).
Keywords:
NHC–silver complexes
Caki-1
MCF-7
Two NHC–silver acetate complexes 2a and 2e were characterised by single crystal X-ray diffraction. The
preliminary in vitro antibacterial activity of the NHC–silver complexes 2a–e was investigated against
Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli using the qualita-
tive Kirby–Bauer disk-diffusion method. The areas of clearance determined for the maximum dose
Escherichia coli
Staphylococcus aureus
(4.3 lM) range between 1 mm and 7 mm for MSSA and 0 mm and 7 mm for E. coli. All of the newly synthes-
ised silver(I) acetate complexes were tested for their cytotoxicity by MTT based in vitro tests on the human
renal cancer cell line Caki-1 and human breast cancer cell line MCF-7 in order to determine their IC50 values.
The NHC–silver complexes 2a–e were found to have IC50 values of 3.0 ( 0.6), 0.51 ( 0.07), 4.2 ( 1.2), 9.0
( 0.6), 26 ( 2)
( 0.5), 3.4 ( 1.2) and 14 ( 2)
compound SBC3 (1,3-bisbenzyl-4,5-bisphenyl-imidazole-2-ylium silver(I) acetate) (IC50 value = 14 ( 1)
against Caki-1 and 5.8 ( 0.6) M against MCF-7) these values represent improved cytotoxicity against both
cell lines, especially for the silver complexes 2a and 2b. These two compounds are not only more active than
lM, against the renal cancer cell-line Caki-1 and IC50 values of 2.3 ( 0.4), 1.4 ( 0.2), 3.0
lM against the breast cancer cell line MCF-7, respectively. Compared to our lead
lM
l
SBC3 but also exhibit in the case of 2b a 7 times higher biological activity than cisplatin (IC50 value = 3.3 lM)
against Caki-1.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
are nowadays not only used in organometallic chemistry and catal-
ysis, but more and more also in the design and synthesis of cyto-
toxic and antibacterial transition metal complexes. Most
commonly used transition metals in the development of new anti-
cancer and antibacterial drugs are platinum, palladium, rhodium,
ruthenium, copper, silver and gold [2–15].
Silver salts such as silver nitrate are used for many years as anti-
microbial agents and have exhibited low toxicity for humans, but
recently an increased number of research groups focuses on the
activity of new silver therapeutics as anticancer agents [4,11].
Youngs and co-workers have reported anticancer activity of
NHC–silver complexes derived from 4,5-dichloro-1H-imidazole
against the human cancer cell lines OVCAR-3 (ovarian), MB157
(breast), and HeLa (cervical). Activity was even demonstrated
in vivo for [4,5-dichloro-1,3-dimethylimidazol-2-ylidene] silver(I)
In the treatment of cancer, platinum-based drugs such as cis-
platin, oxaliplatin and carboplatin are widely used, but due to sev-
eral factors resistance against these drugs may develop during
treatment. To overcome resistance many groups focus on the de-
sign of novel platinum based complexes [1] by using specific carri-
ers, or a change from platinum to other transition metals. Novel
carrier ligands must initially meet certain criteria, such as being
easily accessible in few steps, substituents can be widely varied
and their reactivity in biological medium can be easily fine-tuned.
All these criteria are met by N-heterocyclic carbenes (NHCs), which
⇑
Corresponding author.
E-mail address: matthias.tacke@ucd.ie (M. Tacke).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.10.029

136
F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
acetate 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
cytotoxicity of the resulting silver complex was 40-fold (MCF-7
and HL60) to 7-fold (MCF-7R) higher than that of cisplatin [6]. Also
Gust et al. have recently reported that symmetrically and unsym-
metrically substituted NHC–silver(I) halides show promising
activity against different cancer cell lines as well as bacteria strains
[16], which correlates well with the findings in our group [17–26].
Within this paper we present a new series of symmetrically
p-benzyl-substituted N-heterocyclic carbene silver acetate com-
plexes derived from 4,5-ditolyl-imidazole, their synthesis, cytotox-
icity, and antibacterial studies.
alents of K2CO3 (207 mg, 1.50 mmol) were dissolved in acetonitrile
and stirred for 2 d at room temperature. After filtering off the pre-
cipitate and removing the solvent under reduced pressure the ob-
tained sticky white solid was washed with pentane (2 ꢀ 20 mL)
and diethyl ether (2 ꢀ 20 mL) to yield a white powder (280 mg,
0.55 mmol, 55% yield).
1H NMR (d ppm, DMSO, 400 MHz): 9.57 (s, 1H, NCHN), 7.28–
7.26 (m, 4H), 7.16–7.12 (m, 8H), 6.99–6.96 (m, 6H), 5.37 (s, 4H,
CH2), 2.26 (s, 6H, CH3).
13C NMR (d ppm, DMSO, 101 MHz): 140.28 (NCHN), 136.82,
134.63, 132.25, 131.10, 129.83, 129.27, 128.93, 128.17, 122.47
(Cbenzyl + Ctolyl + Cimidazole), 50.82 (CH2), 21.30 (CH3).
IR absorptions (KBr, cmꢁ1): 3421 (m), 3031 (s), 2942 (s), 1619
(s), 1558 (s), 1502 (s), 1452 (s), 817 (s).
MS (m/z, QMS-MS/MS): 509.00 [M+ꢁH].
2. Experimental part
Micro Anal. Calc. for C31H29BrN2 (509.48): C, 73.08; H, 5.74; N,
5.50. Found: C, 72.37, H 5.52, N 5.41%.
2.1. General conditions
Melting point: 203–206 °C
All silver(I) acetate reactions were carried out under exclusion
of light. All solvents used were of analytical grade and were used
without further purification. 4,4-Dimethylbenzoin, formamide,
benzyl bromide, 4-methylbenzyl bromide, 4-methoxybenzyl
chloride, methyl 4-(bromomethyl)benzoate, 4-(bromomethyl)
benzonitrile, silver(I) acetate and K2CO3 were purchased from
Sigma–Aldrich Chemical Company and were used without further
purification.
2.2.3. Synthesis of 1,3-bis-(4-methylbenzyl)-4,5-bis-(4-
methylphenyl)-imidazolium bromide (1b)
4,5-Bis-(4-methylphenyl)-1H-imidazole (248 mg, 1.00 mmol), 2
equivalents of 4-methylbenzyl bromide (371 mg, 2.00 mmol) and
1.5 equivalents of K2CO3 (207 mg, 1.50 mmol) were dissolved in
acetonitrile and stirred for 2 d at room temperature. After filtering
off the precipitate and removing the solvent under reduced pres-
sure the obtained sticky white solid was washed with pentane
(2 ꢀ 20 mL) and diethyl ether (2 ꢀ 20 mL) to yield a white powder
(200 mg, 0.37 mmol, 37% yield).
1H NMR spectra were measured on a Varian 300 MHz spectrom-
eter while 13C spectra were measured on a Varian 400 MHz spec-
trometer. All chemical shifts are reported in ppm and referenced
to TMS. IR spectra were recorded on a Perkin-Elmer Paragon
1000 FT-IR spectrometer employing a KBr disc. ESI MS was per-
formed on a quadrupole tandem mass spectrometer (Quattro
Micro, Micromass/Water’s Corp., USA), using solutions in 100%
methanol. MS spectra were obtained in the ES+ (electron spray
positive ionisation) mode for all compounds. CHN Analysis was
carried out in an Exeter Analytical CE-440 elemental analyzer.
Crystal Data was collected using an Agilent Technologies (former
Oxford Diffraction) SuperNova diffractometer fitted with an Atlas
detector. A suitable crystal of 2a was grown from a saturated solu-
tion of diethyl ether and slow evaporation of the solvent while
crystals of 2e were grown in a saturated dichloromethane solution
with slow infusion of pentane. 2a and 2e were measured with Mo
1H NMR (d ppm, CDCl3, 300 MHz): 11.07 (s, 1H, NCHN), 7.14 (d,
J = 7.7 Hz, 4H), 7.10–7.00 (m, 8H), 6.97 (d, J = 7.8 Hz, 4H), 5.41 (s,
4H, CH2), 2.35 (s, 6H, CH3tolyl), 2.30 (s, 6H, CH3benzyl).
13C NMR (d ppm, CDCl3, 101 MHz): 140.60 (NCHN), 138.86,
136.96, 132.01, 130.64, 130.54, 129.73, 129.70, 128.48, 121.86
(Cmethylbenzyl + Ctolyl + Cimidazole), 50.96 (CH2), 21.39 (CH3tolyl), 21.16
(CH3benzyl).
IR absorptions (KBr, cmꢁ1): 3423 (m), 3010 (s), 2865 (s), 1558
(s), 1515 (s), 1450 (s), 817 (s)
MS (m/z, QMS-MS/MS): 457.64 [M+ꢁBr].
Micro Anal. Calc. for C33H33BrN2 (537.53): C, 73.74; H, 6.19; N,
5.21. Found: C, 73.44; H, 6.12; N, 5.01%.
Melting point: 212–214 °C.
Ka (0.71073 A°) at 100 K. A five-time redundant (2a) or a complete
2.2.4. Synthesis of 1,3-bis-(4-methoxybenzyl)-4,5-bis-(4-
methylphenyl)-imidazolium chloride (1c)
(2e) dataset was collected, assuming that the Friedel pairs are not
equivalent. An analytical absorption correction based on the shape
of the crystal was performed [27]. The structure was solved by
direct methods using SHELXS-97 [28] and refined by full matrix
least-squares on F2 for all data using SHELXL-97 [28]. Hydrogen
atoms were added at calculated positions and refined using a
riding model. Their isotropic thermal displacement parameters
were fixed to 1.2 (1.5 for methyl groups) times the equivalent ones
of the parent atom. Anisotropic thermal displacement parameters
were used for all non hydrogen atoms.
4,5-Bis-(4-methylphenyl)-1H-imidazole (248 mg, 1.00 mmol), 2
equivalents of 4-methoxybenzyl chloride (0.24 mL, 2.00 mmol)
and 1.5 equivalents of K2CO3 (207 mg, 1.50 mmol) were dissolved
in acetonitrile and refluxed for 3 d. After filtering off the precipitate
and removing the solvent under reduced pressure the obtained
sticky white solid was washed with pentane (2 ꢀ 20 mL) and
diethyl ether (2 ꢀ 20 mL) to yield a white powder (200 mg,
0.38 mmol, 38% yield).
1H NMR (d ppm, CDCl3, 300 MHz): 11.57 (s, 1H, NCHN), 7.12 (dd,
J = 15.7, 8.2 Hz, 8H), 6.95 (d, J = 8.1 Hz, 4H), 6.77 (d, J = 8.7 Hz, 4H),
5.39 (s, 4H, CH2), 3.76 (s, 6H, OCH3), 2.36 (s, 6H, CH3).
13C NMR (d ppm, CDCl3, 101 MHz): 159.90 (NCHN), 140.56,
131.69, 130.62, 130.27, 129.74, 128.45, 125.75, 122.00, 114.29,
(Cmethoxybenzyl + Ctolyl + Cimidazole) 55.25 (CH2), 50.67 (OCH3), 21.39
(CH3).
2.2. Synthesis
2.2.1. Synthesis of 4,5-bis(4-methylphenyl)-1H-imidazole (1)
4,5-Bis(4-methylphenyl)-1H-imidazole
was
synthesised
according to literature [29] to give a yield of 66%. The formation
of the product was confirmed by NMR.
IR absorptions (KBr, cmꢁ1): 3421 (m), 3088–2956 (m), 1613 (s),
1514 (s), 1452 (s), 1250 (s), 1178 (s), 821 (s).
2.2.2. Synthesis of 1,3-bis-benzyl-4,5-bis-(4-methylphenyl)-
imidazolium bromide (1a)
4,5-Bis-(4-methylphenyl)-1H-imidazole (248 mg, 1.00 mmol), 2
equivalents of benzyl bromide (0.24 mL, 2.00 mmol) and 1.5 equiv-
MS (m/z, QMS-MS/MS): 489.49 [M+ꢁHCl].
Micro Anal. Calc. for C33H33ClN2O2 (525.08): C, 75.48; H, 6.33; N,
5.34. Found: C, 75.28; H, 6.22; N, 5.13%.
Melting point: 196–198 °C.

F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
137
2.2.5. Synthesis of 1,3-bis-(4-methoxycarbonylbenzyl)-4,5-bis-(4-
methylphenyl)-imidazolium bromide (1d)
IR absorptions (KBr, cmꢁ1): 3480–3416 (m), 2360 (s), 1598 (s),
1440 (s), 1375 (s), 1020 (s), 817 (s).
4,5-Bis-(4-methylphenyl)-1H-imidazole (248 mg, 1.00 mmol), 2
equivalents of 4-methoxycarbonylbenzyl bromide (458 mg,
2.00 mmol) and 1.5 equivalents of K2CO3 (207 mg, 1.50 mmol)
were dissolved in acetonitrile and stirred for 3 d at room tempera-
ture. After filtering off the precipitate and removing the solvent un-
der reduced pressure the obtained sticky white solid was washed
with pentane (2 ꢀ 20 mL) and diethyl ether (2 ꢀ 20 mL) to yield
a white powder (100 mg, 0.16 mmol, 16% yield).
MS (m/z, QMS-MS/MS): 429.61 [M+ꢁAgOAc].
Micro Anal. Calc. for C33H32AgN2O2 (596.49): C, 66.45; H, 5.41;
N, 4.70. Found: C, 66.44; H, 5.39; N, 4.54%.
Melting point: 143–147 °C.
2.2.8. Synthesis of 1,3-bis-(4-methylbenzyl)-4,5-bis-(4-
methylphenyl)-imidazole-2-ylidene silver(I) acetate (2b)
1,3-Bis-(4-methylbenzyl)-4,5-bis-(4-methylphenyl)-imidazolium
bromide (1b) (100 mg, 0.19 mmol) and 2 equivalents of silver(I)
acetate (65 mg, 0.39 mmol) were dissolved in 30 mL of dichloro-
methane and stirred in darkness at room temperature for 2 d. After
filtering off the AgBr by-product, the solvent was reduced to 3 mL
and by adding dropwise 10 mL of pentane a white powder precip-
itated. The solvent was decanted and the white precipitate was
washed with pentane (2 ꢀ 10 mL) and diethyl ether (3 ꢀ 10 mL)
to yield a white powder (90 mg, 0.14 mmol, 78% yield).
1H NMR (d ppm, CDCl3, 300 MHz): 7.91 (d, J = 8.0 Hz, 4H,
CHbenzyl + CHtolyl), 7.04 (t, J = 8.0 Hz, 8H, CHbenzyl + CHtolyl), 6.85 (d,
J = 8.0 Hz, 4H, CHbenzyl + CHtolyl), 5.37 (s, 4H, CH2), 3.90 (s, 6H,
CH3benzyl), 2.29 (s, 6H,CH3tolyl), 2.06 (s, 1H, CH3actetate).
13C NMR (d ppm, CDCl3, 101 MHz): 177.16 (NCN), 138.97 (C@O),
137.60, 133.47, 132.38, 130.52, 129.53, 129.27, 129.22, 128.26,
127.37, 124.95 (CHtolyl + CHbenzyl + CHimidazole), 53.19 (CH2), 21.27
(CH3), 21.09 (CH3).
1H NMR (d ppm, DMSO, 400 MHz): 9.57 (s, 1H, NCHN), 7.89 (d,
J = 8.4 Hz, 4H), 7.25 (d, J = 8.1 Hz, 4H), 7.14 (s, 8H), 5.49 (s, 4H, CH2),
3.83 (s, 6H, CH3), 2.24 (s, 6H, CH3tolyl).
13C NMR (d ppm, DMSO, 101 MHz): 166.19 (C@O), 140.37
(NCHN), 139.83, 132.39, 131.04, 129.96, 129.82, 128.92, 128.38,
124.98, 122.26 (Cmethoxycarbonylbenzyl + Cphenyl + Cimidazole), 52.74
(CH2), 50.55 (OCH3), 21.28 (CH3tolyl).
IR absorptions (KBr, cmꢁ1): 3423 (m), 2950 (s), 1722 (s), 1614
(s), 1434 (s), 1280 (s), 821 (s).
MS (m/z, QMS-MS/MS): 545.54 [M+ꢁBr].
Micro Anal. Calc. for C35H33BrN2O4 (625.55): C, 67.20; H, 5.32; N,
4.48. Found: C, 67.10; H, 5.20; N, 4.80%.
Melting point: 207–211 °C.
2.2.6. Synthesis of 1,3-bis-(4-cyanobenzyl)-4,5-bis-(4-methylphenyl)-
imidazolium bromide (1e)
4,5-Bis-(4-methylphenyl)-1H-imidazole (248 mg, 1.00 mmol), 2
equivalents of 4-cyanobenzyl bromide (391 mg, 2.00 mmol) and
1.5 equivalents of K2CO3 (207 mg, 1.50 mmol) were dissolved in
acetonitrile and stirred for 2 d at room temperature. After filtering
off the precipitate and removing the solvent under reduced pres-
sure the obtained sticky white solid was washed with pentane
(2 ꢀ 20 mL) and diethyl ether (2 ꢀ 20 mL) to yield a white powder
(327 mg, 0.58 mmol, 58% yield).
IR absorptions (KBr, cmꢁ1): 3437 (m), 3028 (s), 2921 (s), 1708
(s), 1517 (s), 819 (s)
MS (m/z, QMS-MS/MS): 451.53 [M+ꢁAgOAc].
Micro Anal. Calc. for C35H36AgN2O2 (624.54): C, 67.31; H, 5.81;
N, 4.49. Found: C, 66.98; H, 5.53; N, 4.09%.
Melting point: 126–128 °C.
2.2.9. Synthesis of 1,3-bis-(4-methoxybenzyl)-4,5-bis-(4-
methylphenyl)-imidazole-2-ylidene silver(I) acetate (2c)
1,3-Bis-(4-methoxybenzyl)-4,5-bis-(4-methylphenyl)-imidazo-
lium chloride (1c) (100 mg, 0.18 mmol) and 2.1 equivalents of
silver(I) acetate (62 mg, 0.38 mmol) were dissolved in 30 mL of
dichloromethane and stirred in darkness at room temperature for
2 d. After filtering off the AgBr by-product, the solvent was reduced
to 3 mL and by adding dropwise 10 mL of pentane a white powder
precipitated. The solvent was decanted and the white precipitate
was washed with pentane (2 ꢀ 10 mL) and diethyl ether
(3 ꢀ 10 mL) to yield a white powder (70 mg, 0.10 mmol, 56% yield).
1H NMR (d ppm, CDCl3, 300 MHz): 7.05 (d, J = 8.0 Hz, 4H), 6.91
(m, 8H), 6.74 (d, J = 8.0 Hz, 4H), 5.22 (s, 4H, CH2), 3.76 (s, 6H,
CH3methoxybenzyl), 2.31 (s, 6H, CH3tolyl), 2.09 (s, 3H, CH3acetate).
13C NMR (d ppm, CDCl3, 101 MHz): 178.62 (NCN), 159.21 (C@O),
138.99, 132.31, 130.54, 129.23, 128.95, 128.51, 124.98, 123.56,
113.95 (Cmethoxybenzyl + Ctolyl + Cimidazole), 55.23 (CH2), 52.98
(OCH3), 22.81 (CH3tolyl), 21.28 (CH3acetate).
1H NMR (d ppm, DMSO, 400 MHz): 9.57 (s, 1H, NCHN), 7.81 (d,
J = 8.3 Hz, 4H), 7.32 (d, J = 8.4 Hz, 4H), 7.13 (s, 8H), 5.50 (s, 4H, CH2),
2.24 (s, 6H, CH3).
13C NMR (d ppm, DMSO, 101 MHz): 140.39 (NCHN), 140.02
(CN), 137.51, 133.06, 132.32, 131.02, 129.82, 129.06, 122.21,
118.86, 111.57 (Ccyanobenzyl + Ctolyl + Cimidazole), 50.47 (CH2), 21.29
(CH3).
IR absorptions (KBr, cmꢁ1): 3461 (m), 2966 (m), 2227 (s), 1614
(s), 1560 (s), 1448 (s), 821 (s).
MS (m/z, QMS-MS/MS): 479.57 [M+ꢁHBr].
Micro Anal. Calc. for C33H27BrN4 (559.50): C, 70.84; H, 4.86; N,
10.01. Found: C, 70.56; H, 4.62; N, 9.85%.
Melting point: 279–281 °C.
2.2.7. Synthesis of 1,3-bis-benzyl-4,5-bis-(4-methylphenyl)-imidazole-
2-ylidene silver(I) acetate (2a)
1,3-Bis-benzyl-4,5-bis-(4-methylphenyl)-imidazolium bromide
(1a) (100 mg, 0.19 mmol) and 2.1 equivalents of silver(I) acetate
(69 mg, 0.41 mmol) were dissolved in 30 mL of dichloromethane
and stirred in darkness at room temperature for 2 d. After filtering
off the AgBr by-product, the solvent was reduced to 3 mL and by
adding dropwise 10 mL of pentane a white powder precipitated.
The solvent was decanted and the white precipitate was washed
with pentane (2 ꢀ 10 mL) and diethyl ether (3 ꢀ 10 mL) to yield
a white powder (90 mg, 0.15 mmol, 77% yield).
IR absorptions (KBr, cmꢁ1): 3398 (m), 3003 (s), 2920 (s), 1576
(s), 1513 (s), 1384 (s), 1252 (s), 820 (s).
MS (m/z, QMS-MS/MS): 489.52 [M+ꢁAgOAc].
Micro Anal. Calc. for C35H36AgN2O4 (656.54): C, 64.03; H, 5.53;
N, 4.27. Found: C, 63.41; H, 5.28; N, 4.03%.
Melting point: 166–168 °C.
2.2.10. Synthesis of 1,3-bis-(4-methoxycarbonylbenzyl)-4,5-bis-(4-
methylphenyl)-imidazole-2-ylidene silver(I) acetate (2d)
1H NMR (d ppm, CDCl3, 300 MHz): 7.25–7.20 (m, 6H), 7.02 (d,
J = 7.9 Hz, 8H), 6.87 (d, J = 7.9 Hz, 4H), 5.30 (s, 4H, CH2), 2.29 (s,
6H, CH3tolyl), 2.07 (s, 3H, CH3acetate).
1,3-Bis-(4-methoxycarbonylbenzyl)-4,5-bis-(4-methylphenyl)-
imidazolium bromide (1d) (100 mg, 0.16 mmol) and 2 equivalents
of silver(I) acetate (56 mg, 0.33 mmol) were dissolved in 30 mL of
dichloromethane and stirred in darkness at room temperature for
2 d. After filtering off the AgBr by-product, the solvent was reduced
to 3 mL and by adding dropwise 10 mL of pentane a white powder
13C NMR (d ppm, CDCl3, 101 MHz): 198.98 (NCN), 164.32 (C@O),
138.07, 135.40, 129.48, 128.93, 128.23, 127.62, 126.91, 126.34,
124.76, 123.81 (Ctolyl + Cimidazole + Cbenzyl), 52.51 (CH2), 20.27
(CH3tolyl
)

138
F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
precipitated. The solvent was decanted and the white precipitate
was washed with pentane (2 ꢀ 10 mL) and diethyl ether
(3 ꢀ 10 mL) to yield a white powder (60 mg, 0.08 mmol, 53% yield).
1H NMR (d ppm, CDCl3, 300 MHz): 7.91 (d, J = 8.0 Hz, 4H), 7.04
(t, J = 8.3 Hz, 8H), 6.85 (d, J = 8.0 Hz, 4H), 5.37 (s, 4H, CH2), 3.91
(s, 6H, CH3methoxycarbonylbenzyl), 2.29 (s, 6H, CH3tolyl), 2.06 (s, 3H,
CH3acetate).
2.4. Cytotoxicity studies
Preliminary in vitro cell tests were performed on the human
cancerous renal cell line Caki-1 in order to compare the cytotoxic-
ity of the compounds presented in this paper. This cell line was
chosen based on its regular and long-lasting growth behaviour,
which is similar to the one shown in kidney carcinoma cells. The
cells were obtained from the ATCC (American Tissue Cell Culture
Collection) and maintained in Dulbecco’s Modified Eagle Medium
containing 10% (v/v) FCS (fetal calf serum), 1% (v/v) penicillin
streptomycin and 1% (v/v) L-glutamine. Cells were seeded in 96-
13C NMR (d ppm, CDCl3, 101 MHz): 179.13 (NCN), 169.67 (C@O),
166.56 (C@O), 141.15, 139.45, 132.75, 130.34, 129.99, 129.87,
129.40, 127.20, 124.31, 53.29 (CH2), 52.16 (OCH3), 22.79
(CH3acetate), 21.27 (CH3tolyl).
IR absorptions (KBr, cmꢁ1): 3434 (m), 2950 (w), 1724 (s), 1579
(m), 1434 (m), 819 (s).
well plates containing 200
lL microtitre wells at a density of
5000-cells/200 L of medium and were incubated at 37 °C for
l
MS (m/z, QMS-MS/MS): 547.45 [M+ꢁAgOAc].
24 h to allow for exponential growth. Then the compounds used
for the testing were dissolved in the minimal amount of DMSO
(dimethylsulfoxide) possible and diluted with medium to obtain
stock solutions of 5 ꢀ 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 37 °C. Then, the solu-
tions were removed from the wells and the cells were washed with
PBS (phosphate buffer solution) and fresh medium was added to
the wells. Following a recovery period of 24 h incubation at
Micro Anal. Calc. for C37H36AgN2O6 (712.56): C, 62.37; H, 5.09;
N, 3.93. Found: C, 61.66; H, 4.78; N, 3.72%.
Melting point: 196–198 °C.
2.2.11. Synthesis of 1,3-bis-(4-cyanobenzyl)-4,5-bis-(4-
methylphenyl)-imidazole-2-ylidene silver(I) acetate (2e)
1,3-Bis-(4-cyanobenzyl)-4,5-bis-(4-methylphenyl)-imidazolium
bromide (1e) (100 mg, 0.18 mmol) and 2.1 equivalents of silver(I)
acetate (63 mg, 0.37 mmol) were dissolved in 30 mL of dichloro-
methane and stirred in darkness at room temperature for 2 d. After
filtering off the AgBr by-product, the solvent was reduced to 3 mL
and by adding dropwise 10 mL of pentane a white powder precip-
itated. The solvent was decanted and the white precipitate was
washed with pentane (2 ꢀ 10 mL) and diethyl ether (3 ꢀ 10 mL)
to yield a white powder (104 mg, 0.16 mmol, 90% yield).
1H NMR (d ppm, CDCl3, 300 MHz): 7.53 (d, J = 7.9 Hz, 4H), 7.08
(dd, J = 11.0, 8.0 Hz, 8H), 6.86 (d, J = 7.9 Hz, 4H), 5.37 (s, 4H, CH2),
2.31 (s, 6H, CH3tolyl), 2.06 (s, 3H, CH3acetate).
37 °C, individual wells were treated with 200 lL of a solution of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) in medium. The solution consisted of 22 mg of MTT in
40 mL of medium. The cells were incubated for 3 h at 37 °C. The
medium was then removed and the purple formazan crystals were
dissolved in 200 lL DMSO per well. For all tests cells with low pas-
sage numbers were used. A Wallac Victor (Multilabel HTS Counter)
Plate Reader was used to measure absorbance at 540 nm. Cell via-
bility was expressed as a percentage of the absorbance recorded for
control wells. The values used for the dose response curves repre-
sent the values obtained from four consistent MTT-based assays for
each compound tested.
13C NMR (d ppm, CDCl3, 101 MHz): 179.13 (NCN), 165.98 (C@O),
141.04 (CN), 139.84, 132.80, 132.53, 130.24, 129.59, 128.12,
123.96, 118.24, 112.20 (Ccyanobenzyl + Ctolyl + Cimidazole), 53.15
(CH2), 22.70 (CH3tolyl), 21.29 (CH3acetate).
IR absorptions (KBr, cmꢁ1): 3430 (s), 2925 (w), 2227 (m), 1581
(s), 1506 (m), 1405 (s), 819 (m).
3. Results and discussion
MS (m/z, QMS-MS/MS): 479.44 [M+ꢁAgOAc].
3.1. Synthesis
Micro Anal. Calc. for C35H30AgN4O2 (646.51): C, 65.02; H, 4.68;
N, 8.67. Found: C, 64.82; H, 4.37; N, 8.28%.
The synthetic route for symmetrically substituted imidazole
starting materials, N-heterocyclic carbenes as ligand precursors
and their corresponding silver complexes described in here is given
in Scheme 1. The initial symmetrically 4,5-diaryl-substituted imid-
azole starting material was prepared by reacting 2-bis(4-methyl-
phenyl)ethane-1,2-dione with formamide to form 4,5-bis(4-
methylphenyl)-1H-imidazole (1) according to literature [29], with
60% yield.
Melting point: 161–163 °C.
2.3. Antibacterial studies
The silver(I) acetate complexes were screened in preliminary
in vitro antibacterial tests 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-nega-
tive bacteria.
To assess the biological activity of compounds 2a–e the qualita-
tive Kirby–Bauer disk-diffusion method was applied [30]. All bac-
teria were individually cultured from a single colony in sterile LB
medium [31] overnight at 37 °C in an orbital shaker incubator.
All the work carried out was performed under sterile conditions.
The symmetrically substituted NHC precursors 1,3-bis-benzyl-
4,5-bis-(4-methylphenyl)-imidazolium bromide (1a), 1,3-bis-(4-
methylbenzyl)-4,5-bis-(4-methylphenyl)-imidazolium bromide (1b),
1,3-bis-(4-methoxycarbonylbenzyl)-4,5-bis-(4-methylphenyl)-imi-
dazolium bromide (1d) and 1,3-bis-(4-cyanobenzyl)-4,5-bis-(4-
methylphenyl)-imidazolium bromide (1e) were prepared by
stirring 4,5-bis(4-methoxylphenyl)-1H-imidazole (1) with 2 equiv-
alents of appropriately p-substituted benzyl bromide in the pres-
ence of K2CO3 in acetonitrile at room temperature for 1–5 d with
35, 99, 38 and 50% yields, respectively. The symmetrically substi-
tuted NHC precursor 1,3-bis-(4-methoxybenzyl)-4,5-bis-(4-meth-
ylphenyl)-imidazolium chloride (1c) was synthesised by stirring
4,5-bis(4-methoxylphenyl)-1H-imidazole (1) with 2 equivalents
of 4-methoxybenzyl chloride in the presence of K2CO3 in acetoni-
trile at 80 °C temperature for 9 d with 38% yield (ii).
For each strain, 70 lL 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 solutions (9:1 DMSO:H2-
O) of every compound were prepared at 2.2 and 4.4
to test the effect of different concentrations. Each plate was then
tested with 5 and 7 L of 2.2 M solution and 5 and 10 L for
the 4.4 M solution. The plates were covered and placed in an
lM to be able
l
l
l
l
incubator at 37 °C for 24 h. The plates were then removed and
the area of clearance, which is defined as the distance between
the edge of the filter paper disc and the beginning of the bacterial
growth, was measured for each sample in mm.
The NHC precursors were characterised by spectral (1H, 13C
NMR, IR and MS) and elemental analysis studies. The 1H NMR spec-
tra of all NHC precursors 1a–e show a characteristic downfield

F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
139
O
N
R
R
R
R
X-
O
(ii)
(i)
(iii)
Ag
H
C+
HN
N
N
N
N
O
O
CH3NO, CH2O2
CH3CN
K2CO3
CH2Cl2
AgOAc
NaHSO4, H2O, NaOH
1
1a-e
2a-e
R =
H
(2a)
(2b)
R/X = H / Br
CH3 / Br
(1a)
(1b)
CH3
OCH3
COOCH3
CN
(2c)
(2d)
(2e)
OCH3 / Cl
COOCH3 / Br
CN / Br
(1c)
(1d)
(1e)
Scheme 1. General reaction scheme for the synthesis of (i) 4,5-bistolyl-imidazole (1), (ii) 1,3-bis-(p-substituted benzyl)-4,5-bistolyl imidazolium halides (1a–e) and (iii) 1,3-
bis-(p-substituted benzyl)-4,5-bistolyl imidazole-2-ylium silver acetates (2a–e).
shift in the range d = 9.27–11.05 ppm for the NCHN proton attrib-
utable to the positive charge of the molecule [32,33] .In addition,
their identities have also been confirmed by a base peak for the
[M+ꢁBr] or [M+ꢁCl] fragments in their positive mode ESI mass
spectra.
The NHC–silver complexes 1,3-bis-benzyl-4,5-bis-(4-methyl-
phenyl)-imidazole-2-ylidene silver(I) acetate (2a), 1,3-bis-(4-meth-
ylbenzyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I)
acetate (2b), 1,3-bis-(4-methoxybenzyl)-4,5-bis-(4-methylphenyl)-
imidazole-2-ylidene silver(I) acetate (2c), 1,3-bis-(4-methoxycar-
bonylbenzyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I)
acetate (2d) and 1,3-bis-(4-cyanobenzyl)-4,5-bis-(4-methyl-
phenyl)-imidazole-2-ylidene silver(I) acetate (2e) were syn-
thesised by the reaction of 1a–e with 2.1 equivalents of silver
acetate in dichloromethane. The reaction mixture was stirred for
1–3 d at room temperature to afford the NHC–silver acetate com-
plexes as off white solid in 77, 78, 56, 53 and 90% yields,
respectively.
The complexes were characterised by spectral (1H, 13C NMR, IR,
UV–Vis and MS) and elemental analysis studies. Furthermore, the
C29
C30
C31
C28
C27
C26
C24
C20
C19
C32
C33
C21
O2
C25
N1
C18
N2
C22
O1
C8
C23
C10
Ag
C16
C9
C11
C7
C15
C12
C1
C6
C5
C14
C13
C2
C3
C17
C4
Fig. 1. X-ray diffraction structure of 2a; thermal ellipsoids are drawn on the 50% probability level.

140
F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
C66
C67
C68
N8
C62
C65
C64
C58
C70
C60
O3
C57
C61
N7
C63
Ag2A
C59
C69
C44
C56
C53
C54
C55
O4
N6
C43
C41
C45
C36
C47
C48
C46
C37
C51
C50
C40
C49
C38
C42
N5
C39
C52
Fig. 2. X-ray diffraction structure of 2e showing the major occupied Ag position; thermal ellipsoids are drawn on the 50% probability level.
solid state structures of 2a and 2e were analysed by single crystal
X-ray diffraction. Absence of a downfield NCHN signal in the range
between 11.57–9.57 ppm and presence of new signals at 2.09–
2.06 ppm for the acetate protons in all 1H NMR spectra for 2a–e
however, indicates a successful complex formation. The 13C NMR
resonances of the carbene carbon atoms in complexes 2a–e occur
in the range d 198.98–178.16 ppm respectively. These signals are
shifted downfield compared to the corresponding precursors of
1a–e carbene carbons resonance in the range of 159.90–
140.28 ppm respectively, which further demonstrates the
formation of the expected NHC–silver acetate complexes. Finally,
positive mode ESI mass spectra of all five NHC–silver complexes
(2a–e) are dominated by [M+ꢁO2CCH3] fragment peaks arising
from the loss of the acetate ligand.
C66
C67
C62
N8
C68
C65
C64
C58
C70
O3
C60
C57
C56
C61
C63
Ag2A
C59
C69
N7
C44
C53
C54
C55
O4
Ag2B
N6
C45
C36
C43
C41
C47
C48
C46
C37
C51
C50
C40
C49
C38
C42
N5
C39
C52
Fig. 3. X-ray diffraction structure of 2e showing both Ag positions; thermal ellipsoids are drawn on the 50% probability level, Ag2B with fixed radius.

F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
141
Table 1
Crystal data and structure refinement for complexes 2a and 2e.
Table 2
Selected bond lengths (Å) and bond angles (°) for complexes 2a and 2e.
2a
2e
2a
Bond length (Å)
2e
Bond length (Å)
Empirical formula
Formula weight (g/
mol)
Crystal system
Space group
Unit cell dimensions
a (Å)
C
33H32N2O2Ag
C35H29N4O2Ag
645.49
Ag–C(8)
Ag–O(1)
2.0548(17)
2.1100(13)
1.354(2)
1.397(2)
1.355(2)
1.392(2)
1.363(2)
Ag(1)–C(9)
Ag(1)–O(1)
N(2)–C(9)
N(2)–C(10)
C(9)–N(3)
C(10)–C(18)
C(18)–N(3)
C(9)–Ag(1)–O(1)
N(3)–C(9)–N(2)
N(2)–C(9)–Ag(1)
2.057(2)
2.1101(16)
1.356(3)
1.396(3)
1.348(3)
1.365(3)
1.391(3)
176.66(9)
104.6(2)
127.33(17)
595.47
N(1)–C(8)
N(1)–C(9)
C(8)-N(2)
C(10)–N(2)
C(9)–C(10)
monoclinic
P21/c (#14)
triclinic
ꢀ
P1 (#2)
12.1787(1)
8.77455(7)
25.4346
7.8609(1)
16.5810(3)
23.7243(4)
89.668(2)
89.978(1)
89.995(1)
3092.21(9)
4
b (Å)
c (Å)
a
(°)
b (°)
98.4644(8)
2a
Bond angle (°)
2e
Bond angle (°)
c
(°)
V (Å3)
2688.40(4)
4
1.471
C(8)–Ag–O(1)
N(1)–C(8)–N(2)
N(1)–C(8)–Ag
178.18(6)
C(9)–Ag(1)–O(1)
N(3)–C(9)–N(2)
N(2)–C(9)–Ag(1)
N(3)–C(9)–Ag(1)
C(18)–C(10)–N(2)
C(10)–C(18)–N(3)
C(9)–N(2)–C(10)
176.66(9)
104.6(2)
127.33(17)
128.00(18)
105.8(2)
104.76(14)
128.30(12)
126.93(12)
106.01(14)
106.59(14)
111.24(14)
107.35(11)
124.14(17)
120.71(17)
115.12(16)
Z
Density (mg/m3)
(calc.)
1.387
N(2)–C(8)–Ag
C(10)–C(9)–N(1)
C(9)–C(10)–N(2)
C(8)–N(2)–C(10)
C(32)–O(1)–Ag
O(2)–C(32)–O(1)
O(2)–C(32)–C(33)
O(1)–C(32)–C(33)
Absorption
coefficient
(mmꢁ1)
0.783
0.689
106.5(2)
111.50(19)
F(000)
1224
1320
Crystal size (mm3)
h (°)
0.2205 ꢀ 0.1377 ꢀ 0.0790 0.2042 ꢀ 0.1250 ꢀ 0.0356
C(34)–O(1)–Ag(1)
O(2)–C(34)–O(1)
O(2)–C(34)–C(35)
O(1)–C(34)–C(35)
107.87(15)
124.8(2)
119.0(2)
116.2(2)
2.83–29.36
ꢁ16 6 h 6 16
ꢁ11 6 k 6 12
ꢁ34 6 l 6 34
58286
2.99–26.41
ꢁ9 6 h 6 9
ꢁ20 6 k 6 20
ꢁ29 6 l 6 29
52284
Index ranges
Reflections
collected
Independent
reflections Rint
Completeness to
hmax (%)
7058 (0.0314)
95.6
12634 (0.0427)
99.6
Table 3
Selected bond lengths (Å) and bond angles (°) for both types of species of complex 2e.
2e (95% species)
Bond length (Å) 2e (5% species)
Bond length (Å)
Maximum and
minimum
transmission
Data/restraints/
parameters
Goodness-of-fit
(GOF) on F2
Final R indices
0.949 and 0.891
0.976 and 0.905
Ag(2A)–C(44)
Ag(2A)–O(3)
2.049(2)
2.1103(17)
Ag(2B)–C(44)
Ag(2B)–O(4)
Ag(2B)–O(3)
N(6)–C(44)
N(6)–C(45)
C(44)–N(7)
C(45)–C(53)
C(53)–N(7)
2.202(6)
2.274(6)
2.479(6)
1.361(3)
1.402(3)
1.346(3)
1.357(4)
1.397(3)
7058/0/346
1.094
12634/0/769
1.063
N(6)–C(44)
N(6)–C(45)
C(44)–N(7)
C(45)–C(53)
C(53)–N(7)
1.361(3)
1.402(3)
1.346(3)
1.357(4)
1.397(3)
R1 = 0.0264
R1 = 0.0254
[I > 2r(I)]
wR2 = 0.0645
R1 = 0.0318
wR2 = 0.0542
R1 = 0.0294
2e (95% species)
Bond angle (°)
2e (5% species)
Bond angle (°)
R Indices (all data)
wR2 = 0.0676
Largest difference in 0.516 and ꢁ0.472
wR2 = 0.0561
0.596 and ꢁ0.501
C(44)–Ag(2A)–
O(3)
170.37(9)
C(44)–Ag(2B)–
O(4)
C(44)–Ag(2B)–
O(3)
177.3(3)
peak and hole
124.5(3)
(e Åꢁ3
)
O(4)–Ag(2B)–O(3)
N(7)–C(44)–N(6)
N(6)–C(44)–
Ag(2B)
N(7)–C(44)–
Ag(2B)
C(44)–N(6)–C(45)
C(53)–C(45)–N(6)
C(44)–N(7)–C(53)
C(45)–C(53)–N(7)
C(69)–O(3)–
Ag(2B)
55.66(13)
104.5(2)
110.5(2)
N(7)–C(44)–N(6)
N(6)–C(44)–
Ag(2A)
N(7)–C(44)–
Ag(2A)
C(44)–N(6)–C(45)
C(53)–C(45)–N(6)
C(44)–N(7)–C(53)
C(45)–C(53)–N(7)
C(69)–O(3)–
Ag(2A)
104.5(2)
131.33(19)
3.2. Structural discussion
124.14(18)
141.3(2)
Suitable crystals for X-ray crystallography to determine the
molecular structure of 2a were formed from a saturated solution
of diethyl ether and slow evaporation of the solvent. 2e was grown
from a saturated solution of chloroform with slow infusion of
pentane.
111.3(2)
106.0(2)
111.7(2)
106.6(2)
107.00(15)
111.3(2)
106.0(2)
111.7(2)
106.6(2)
84.9(2)
Compound 2a crystallises in the monoclinic space group P21/c
(#14) while 2e crystallised in the triclinic space group P1 (#2).
C(69)–O(4)–
Ag(2B)
94.8(2)
ꢀ
Both compounds 2a and 2e crystallise with 4 molecules in the unit
cell. The molecular structures of the compounds 2a and 2e are
shown in Figs. 1–3. The crystal data and refinement details for
the compounds mentioned above are tabulated in Table 1, while
selected bond lengths and bond angles are compiled in Tables 2
(2a) and 3 (2e).
The NHC–silver complexes 2a and 2e are mononuclear com-
plexes. In the NHC–silver complex 2a (Fig. 1) the bond lengths
and bond angles within the ligand agree with literature data for
complexes of related ligands [34–38]. Complex 2e shows two inde-
pendent molecules in the unit cell, which are shown in Figs. 2 and
3. Selected bond lengths (Å) and bond angles (°) for both types of
species for complex 2e are shown in Table 3.
O(4)–C(69)–O(3)
O(4)–C(69)–C(70)
O(3)–C(69)–C(70)
124.5(2)
120.0(2)
115.5(2)
O(4)–C(69)–O(3)
O(4)–C(69)–C(70)
O(3)–C(69)–C(70)
124.5(2)
120.0(2)
115.5(2)
In 2e one molecule appears as expected [4,17,18,26], and one is
split into a major (95%) and a minor (5%) species, therefore suffer-
ing from a disorder in the solid state. In the major species the Ag–C
bond length is 2.049(2) Å, the Ag–O one is 2.110(2) Å and the
C–Ag–O angle is 170.37(9)° as seen in Table 3. This agrees very well
with the first molecule, the geometry in 2a and previously reported
examples of this family of compounds reported by our group [17].
The rather short Ag–O distance together with the monodentate

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F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
1.2
1.0
0.8
0.6
0.4
0.2
0.0
coordination of the acetate to the silver, resulting in a twofold lin-
ear coordination of the Ag atom, suggests a predominantly cova-
lent character of the Ag(2A)–O(3) bond.
In the minor species of the second molecule (5%, see Fig. 3) the
silver atom binds to both oxygen atoms of the acetate group. The
bond length to the carbene carbon atom increases (Ag(2B)–C(44):
2.202(6) Å; Table 3). The bond lengths to the oxygen atoms are
elongated as well (Ag(2B)–O(4): 2.274(6) Å, and Ag(2B)–O(3):
2.479(6) Å), increasing the coordination number of silver to 3.This
bonding pattern is characteristic for a predominantly ionic coordi-
nation of the acetate to the silver. Both of these modes have been
observed earlier [17,26] suggesting a rather small energy differ-
ence between the major and minor form.
2a, IC50 = 3.6 (+/- 1.0) 10-6
2b, IC50 = 0.51 (+/- 0.07) 10-6
M
M
2c, IC50 = 4.1 (+/- 1.0) 10-6
2d, IC50 = 9.5 (+/- 0.5) 10-6
M
M
2e, IC50 = 26 (+/- 2) 10-6
SBC3, IC50 = 14 (+/- 1) 10-6
M
3.3. Biological evaluation
M
In previous studies, we already synthesized and biologically
evaluated symmetrically substituted 1,3-bis-(p-substituted-ben-
zyl)-4,5-diarylimidazole silver acetates ([17,21]). From these stud-
ies, the earliest reported complex SBC3 (1,3-bisbenzyl-4,5-
bisphenyl-imidazole-2-ylium silver acetate) [21] worked as a lead
compound for further substituent optimisation to enhance anti-
bacterial and cytotoxic activity.
1E-9
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
Log10 drug concentration (M)
Fig. 4. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 2a–e on the viability of Caki-1 cells.
1.2
1.0
0.8
0.6
3.4. Cytotoxicity studies
The log dose response curves for complexes 2a–e against the
human renal cancer cell line Caki-1 and the human breast cancer
cell line MCF-7 are shown in Figs. 4 and 5 respectively. All NHC–sil-
ver acetate complexes exhibit unique IC50 values against both cell
lines which indicates that the cytotoxic activity of the compounds
is directly influenced by the different p-substituents (–H, –CH3,
–OCH3, COOCH3, –CN) on the benzyl rings.
2a, IC50 = 2.3 (+/- 0.4) 10-6
2b, IC50 = 1.4 (+/- 0.1) 10-6
2c, IC50 = 2.1 (+/- 0.7) 10-6
2d, IC50 = 5.6 (+/- 1.0) 10-6
2e, IC50 = 9.1 (+/- 0.5) 10-6
M
M
M
M
M
0.4
0.2
0.0
The highest activity is observed for compound 2b with an IC50
value of 0.51 ( 0.07)
( 0.1) M against MCF-7. Compared to our previously reported
NHC–silver(I) acetate complexes [17] and especially compared to
our lead compound SBC3 (IC50 = 14 ( 1) M against Caki-1 and
IC50 = 5.8 ( 0.6) M against MCF-7), complex 2b exhibits a remark-
lM against Caki-1 and an IC50 = 1.4
l
l
SBC3, IC50 = 5.8 (+/- 0.6) 10-6
M
l
1E-9 1E-8 1E-7 1E-6
1E-5
1E-4
1E-3
able increase in cytotoxic activity. By removing the CH3 groups in
Log10 drug concentration (M)
the para position on the benzyl rings, the cytotoxic activity drops
by a factor of 6 (2a, IC50 = 3.0 ( 0.6)
groups leads to a decrease in activity of up to 50-fold (2e, IC50 = 26
( 2) M). Additionally the IC50 value of the NHC precursor 1b
lM) and replacing it with other
Fig. 5. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 2a–e on the viability of MCF-7 cells.
l
Fig. 6. Area of clearance on Escherichia coli (Gram ꢁve) by 2a–e.

F. Hackenberg et al. / Inorganica Chimica Acta 395 (2013) 135–144
143
Fig. 7. Area of clearance on Staphylococcus aureus (Gram +ve) by 2a–e.
against Caki-1 was determined to evaluate the effect of silver ace-
tate on the activity of the compound. With an IC50 = 4.8 ( 0.3)
1b shows a high cytotoxic potential by itself, but in combination
with silver acetate, forming the NHC silver(I) complex 2b the syn-
ergistic effect of both parts leads to a superior activity against this
particular cell line.
complexes 2a–e yielded IC50 values of 3.0 ( 0.6), 0.51 ( 0.07), 4.2
( 1.2), 9.0 ( 0.6), 26 ( 2) M against the Caki-1 renal cancer cell-
line (SBC3 IC50 = 14 ( 1) M). At the same time these NHC–silver
complexes were found to have IC50 values against the human
breast cancer cell line MCF-7 of 2.3 ( 0.4), 1.4 ( 0.2), 3.0 ( 0.5),
lM
l
l
3.4 ( 1.2), 14 ( 2) lM, respectively (SBC3 IC50 = 5.8 ( 0.6) lM).
One of the compounds presented in this paper, 1,3-bis-(4-meth-
ylbenzyl)-4,5-bis-(4-methylphenyl)-imidazole-2-ylidene silver(I)
acetate (2b) shows the highest cytotoxic activity [IC50 = 0.51
3.5. Antibacterial testing
( 0.07) lM] compared to all N-heterocyclic carbene–silver com-
Using the Kirby–Bauer disk diffusion method, the antibacterial
activity of the NHC–silver acetate complexes was tested and sum-
marised in Figs. 6 and 7.
The metal salt (silver(I) acetate) used to prepare the complexes
and the solvent (DMSO) used to prepare the stock solutions played
no role in growth inhibition on the same bacteria as previously re-
ported [23,26]. An area of clearance of 0 mm was considered as no
activity, areas of 1–3 mm as low, 4–7 mm as medium, and areas of
clearance P8 mm as high activity.
plexes synthesised by our research group (see [17–22,24–26]).
Here the activity of an already cytotoxic imidazolium is strongly
enhanced by deprotonation and coordination to silver acetate
resulting in a water-soluble and neutral drug molecule. This indi-
cates its high potential as an anticancer drug and gives a good
insight into possible further substitution patterns.
Acknowledgement
The primary aim was to synthesise a derivative of SBC3, that
would exhibit better antimicrobial properties than this leading
compound against Gram-positive S. aureus (SBC3, Area of clear-
ance = 7 mm) and Gram-negative E. coli (SBC3, Area of clear-
ance = 10 mm) bacteria strains. Almost no antibacterial activity
was observed for compounds 2b, 2d and 2e against both bacteria
strains. Medium antibacterial activity was observed for compound
2c against Gram-positive S. aureus (Area of clearance = 5 mm) but
no antibacterial activity was observed against Gram-negative bac-
teria E. coli. The best antibacterial activity was observed for the
compound 2a against both bacteria strains with an area of clear-
ance of 7 mm (Figs. 6 and 7). Therefore no improvement in antibac-
terial activity for the herein presented compounds 2a–e over SBC3
was observed.
This project was funded by the UCD School of Chemistry and
Chemical Biology.
Appendix A. Supplementary material
CCDC 897777 and 897778 contains the supplementary crystal-
lographic data for 2a and 2e. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.ica.2012.10.029.
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