Synthesis, cytotoxicity and antibacterial studies of symmetrically and non-symmetrically benzyl- or p-cyanobenzyl-substituted N-Heterocyclic carbene - Silver complexes

Article abstract of DOI:10.1002/aoc.1702

From the reaction of 1H-imidazole (1a), 4,5-dichloro-1H-imidazole (1b) and 1H-benzimidazole (1c) with p-cyanobenzyl bromide (2), symmetrically substituted N-heterocyclic carbene (NHC) [(3a-c)] precursors, 1-methylimidazole (5a), 4,5-dichloro-1-methylimida

Full text of DOI:10.1002/aoc.1702

Full Paper
Received: 12 May 2010
Revised: 9 June 2010
Accepted: 10 June 2010
Published online in Wiley Online Library: 23 August 2010
(wileyonlinelibrary.com) DOI 10.1002/aoc.1702
Synthesis, cytotoxicity and antibacterial
studies of symmetrically and
non-symmetrically benzyl- or
p-cyanobenzyl-substituted N-Heterocyclic
carbene–silver complexes
Siddappa Patil, Anthony Deally, Brendan Gleeson, Helge Mu¨ller-Bunz,
Francesca Paradisi and Matthias Tacke∗
From the reaction of 1H-imidazole (1a), 4,5-dichloro-1H-imidazole (1b) and 1H-benzimidazole (1c) with p-cyanobenzyl
bromide (2), symmetrically substituted N-heterocyclic carbene (NHC) [(3a–c)] precursors, 1-methylimidazole (5a), 4,5-
dichloro-1-methylimidazole (5b) and 1-methylbenzimidazole (5c) with benzyl bromide (6), non-symmetrically substituted
N-heterocyclic carbene (NHC) [(7a–c)] precursors were synthesized. These NHC–precursors were then reacted with silver(I)
acetate to yield the NHC-silver complexes [1,3-bis(4-cyanobenzyl)imidazole-2-ylidene] silver(I) acetate (4a), [4,5-dichloro-1,3-
bis(4-cyanobenzyl)imidazole-2-ylidene] silver(I) acetate (4b), [1,3-bis(4-cyanobenzyl)benzimidazole-2-ylidene] silver(I) acetate
(4c), (1-methyl-3-benzylimidazole-2-ylidene) silver(I) acetate (8a), (4,5-dichloro-1-methyl-3-benzylimidazole-2-ylidene) silver(I)
acetate (8b) and (1-methyl-3-benzylbenzimidazole-2-ylidene) silver(I) acetate (8c) respectively. The four NHC-precursors 3a–c,
7c and four NHC–silver complexes 4a–c and 8c were characterized by single crystal X-ray diffraction. The preliminary
antibacterial activity of all the compounds was studied against Gram-negative bacteria Escherichia coli, and Gram-positive
bacteria Staphylococcus aureus using the qualitative Kirby-Bauer disc-diffusion method. All NHC–silver complexes exhibited
medium to high antibacterial activity with areas of clearance ranging from 4 to 12 mm at the highest amount used, while the
NHC-precursors showed significantly lower activity. In addition, all NHC–silver complexes underwent preliminary cytotoxicity
tests on the human renal-cancer cell line Caki-1 and showed medium to high cytotoxicity with IC50 values ranging from 53 ( 8)
c
to 3.2 ( 0.6) µM. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Keywords: anticancer drugs; antibacterial drugs; silver acetate; NHC; Caki-1; Staphylococcus aureus; Escherichia coli
Introduction
benzyl-substituted N-heterocyclic carbene–silver complexes.[19]
All the reported NHC–silver complexes are light- and water-stable
and can be synthesized in high yield and in high purity from cheap
commercially available starting materials.
Keeping this in mind and in continuation of our studies
on the stability and solubility of N-heterocyclic carbene–silver
complexes and the search for new potential anticancer and
antibacterial agents, here we present the synthesis, preliminary
cytotoxicity and antibacterial studies of a series of six novel
symmetrically substituted and non-symmetrically substituted
NHC–silver acetate derivatives. These compounds were tested
on the human cancerous renal-cell line Caki-1 as well as on
the Gram-positive bacteria Staphylococcus aureus and the Gram-
negative bacteria Escherichia coli. In particular, even though still
N-Heterocycliccarbenes(NHCs)arecycliccarbenesthatareusually
derived from the deprotonation of imidazolium salts. Discovered
[1]
by Ofele and Wanzlick[2] in 1968 and isolated in the free state
¨
by Arduengo[3] in 1991, N-heterocyclic carbenes have become
extremelypopularsupportingligandsinorganometallicchemistry
and homogeneous catalysis.[4–6] More recently, NHCs have found
an application in NHC–silver complexes exhibiting antimicrobial
activity, in particular for the possible treatment of cystic fibrosis
andchroniclung infections[7–9] andpossiblyeveninthetreatment
of cancer.[10]
A literature survey revealed that the silver(I) complexes of
phosphines, carboxylates, thio groups and thioamides, tripodal
thioglycosidesandthenaturalproductcoumarinshowahighlevel
of anticancer activity against a variety of different cell lines.[11–18]
Youngs and co-workers have recently reported anticancer activity
of N-heterocyclic carbene–silver complexes derived from 4,5-
dichloro-1H-imidazole against the human cancer cell lines OVCAR-
3 (ovarian), MB157 (breast) and HeLa (cervical).[10] These silver
complexes have been shown to be very stable and can be
synthesized efficiently. Very recently, we reported the anticancer
and antibacterial activity of p-methoxybenzyl-substituted and
∗
Correspondence to: 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
ConwayInstituteofBiomolecularandBiomedicalResearch,CentreforSynthesis
and Chemical Biology (CSCB), UCD School of Chemistry and Chemical Biology,
University College Dublin, Belfield, Dublin 4, Ireland
c
Appl. Organometal. Chem. 2010, 24, 781–793
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

S. Patil et al.
on qualitative screening, the antibacterial activity has significantly
improved with respect to our previously reported compounds.[19]
Synthesis of 1,3-bis(4-cyanobenzyl)imidazolium bromide (3a)
1H-Imidazole (0.39 g, 5.84 mmol) and K2CO3 (1.21 g, 8.76 mmol)
were stirred for 15 min in 30 ml of acetonitrile. 4-
Cyanobenzylbromide (2.28 g, 11.7 mmol) was added in one por-
tion and stirring was continued at room temperature for further
3 days. After the solvent was removed under reduced pressure,
75 ml of water was added. The precipitate was filtered out and
was washed with diethyl ether to yield (1.49 g, 3.93 mmol, 67.4%
yield) 3a as white powder and dried in vacuo.
1H NMR (δ ppm DMSO-d6, 400 MHz): 9.48 (s, 1H, NCHN), 7.91
(d, J = 8.2 Hz, 4H, CHBenzyl), 7.88 (d, J = 1.4 Hz, 2H, CHImid), 7.60
(d, J = 8.2 Hz, 4H, CHBenzyl), 5.57 (s, 4H, CH2). 13C NMR (δ ppm
DMSO-d6, 100 MHz, proton decoupled): 140.4, 137.5, 133.3, 129.6,
123.6, 118.8, 111.9 (NCN + CN + CImid + CBenzyl), 51.9 (CH2). IR
absorptions (KBr, cm−1): 3436 (w), 3133 (w), 3063 (m), 2982 (s),
2851 (w), 2227 (s), 1609 (m), 1568 (s), 1506 (w), 1412 (m), 1356 (w),
1209 (m), 1161 (s), 1019 (w), 858 (m), 824 (w), 783 (s), 635 (m), 557
(m). UV–vis (CH3OH, nm): λ 207 (ε 13 692), λ 225 (ε 9946), λ 268
(ε 4828). MS (m/z, QMS-MS/MS): 299.43 [M+−Br]. Microanalysis
calculated for C19H15N4Br (379.26): calcd–C, 60.17%; H, 3.98%; N,
14.77%; Br, 21.06%; found: C, 59.87%; H, 4.01%; N, 14.60%; Br,
21.19%.
Experimental
General Conditions
All the solvents used were of analytical grade and were used with-
out further purification. 1H-Imidazole, 4,5-dichloro-1H-imidazole,
1H-benzimidazole, 1-methylimidazole, 1-methylbenzimidazole, p-
cyanobenzyl bromide, benzyl bromide, silver acetate, methyl
iodide and K2CO3 were purchased from Sigma-Aldrich Chemi-
cal Company. NMR spectra were measured on a Varian 400 MHz
spectrometer. Chemical shifts are reported in ppm and are refer-
enced to TMS. IR spectra were recorded on a Perkin Elmer Paragon
1000 FT-IR spectrometer employing a KBr disc. UV–vis spectra
were recorded on a Unicam UV4 spectrometer. Electron spray
mass spectrometry (MS) was performed on a quadrupole tan-
dem mass spectrometer (Quattro Micro, Micromass/Water’s Corp.,
USA), using solutions made up in 50% dichloromethane and 50%
methanol. MS spectra were obtained in the ES+ (electron spray
positive ionization) mode for compounds 3a–c, 4a–c, 7a–c and
8a–c. CHN analysis was done with an Exeter Analytical CE-440 Ele-
mental Analyser. Ag was estimated by spectrophotometry (atomic
absorption spectra 55B Varian), while Cl and Br were determined
in mercurimetric titrations. X-ray diffraction data for compounds
3a–c,4a–c,7cand8cwerecollectedusingMo–Kα radiationanda
Bruker Smart APEX CCD area detector diffractometer. A full sphere
of reciprocal space was scanned by phi-omega scans. Pseudo-
empirical absorption correction based on redundant reflections
was performed by the program SADABS.[20] The structures were
solved by direct methods using SHELXS-97[21] and refined by full
matrix least-squares on F2 for all data using SHELXL-97.[21] In 3a
all hydrogen atoms were located in the difference Fourier map
and allowed to refine freely. The same applies to the ‘carbenic’
hydrogen atom in 3c. In 4c the O–H bond distances in the water
molecules were restrained to be 0.84 Å, and the thermal displace-
ment parameters of the water protons were fixed to be 1.5 times
the equivalent thermal displacement parameter of the oxygen
atom. In 7c the hydrogen atoms of the water molecule could not
be detected. All other hydrogen atoms were added at calculated
positions and refined using a riding model. Their isotropic temper-
ature factors were fixed to 1.2 times (1.5 times for methyl groups)
the equivalent isotropic displacement parameters of the parent
carbon atom. Anisotropic thermal displacement parameters were
used for all non-hydrogen atoms. Further details about the data
collection are listed in Table 1, as well as reliability factors. Further
details are available free of charge from the Cambridge Crystal-
lographic Data Centre via www.ccdc.cam.ac.uk/data request/cif
under the CCDC numbers 764 016, 764 017, 764 018, 764 019,
764 020, 764 021, 764 022 and 764 023 for 3a, 3b, 7c, 3c, 4c, 4a, 4b
and 8c respectively. Suitable crystals of 3a–c, 4a–c, 7c and 8c for
X-ray studies were grown from the slow evaporation of a saturated
CH2Cl2 and methanol solutions respectively at room temperature.
Synthesis of 4,5-Dichloro-1,3-bis(4-cyanobenzyl)imidazolium
bromide (3b)
4,5-Dichloro-1H-imidazole (0.79 g, 5.84 mmol) and K2CO3 (1.21 g,
8.76 mmol) were stirred for 15 min in 30 ml of acetonitrile.
4-Cyanobenzylbromide (1.14 g, 5.84 mmol) was added in one
portion and stirring was continued at room temperature for
further 2 days. After the solvent was removed under reduced
pressure 75 ml of water were added. The aqueous phase was
extracted with CH2Cl2 (4 × 20 ml). Organic phases were combined
and dried over magnesium sulfate. The residue was obtained after
solventremovalunderreducedpressure.Theresultingresiduewas
dissolved in CH3CN and another portion of 4-cyanobenzylbromide
(1.14 g, 5.84 mmol) was added. The reaction mixture was heated
under reflux for 6 days. The light yellow coloured precipitate
formed was filtered off and washed several times with diethyl
ether and dried in vacuo to yield (0.75 g, 1.67 mmol, 28.7%
yield) 3b.
1H NMR (δ ppm DMSO-d6, 400 MHz): 9.60 (s, 1H, NCHN), 7.95 (d,
J = 8.2 Hz, 4H, CHBenzyl), 7.62 (d, J = 8.2 Hz, 4H, CHBenzyl), 5.63 (s,
4H, CH2). 13C NMR (δ ppm DMSO-d6, 100 MHz, proton decoupled):
138.5, 137.9, 133.3, 129.4, 119.9, 118.8, 112.0 (NCN + CN + CCl
+ CBenzyl), 51.4 (CH2). IR absorptions (KBr, cm−1): 3442 (w), 2913
(s), 2234 (s), 1577 (m), 1546 (m), 1507 (w), 1449 (w), 1420 (s), 1340
(m), 1198 (w), 1144 (w), 1023 (w), 871 (w), 821 (s), 617 (w), 552
(m). UV–vis (CH3OH, nm): λ 209 (ε 22 871), λ 230 (ε 25 134), λ 279
(ε 9688). MS (m/z, QMS-MS/MS): 368.29 [M+−Br]. Microanalysis
calculated for C19H13N4Cl2Br (448.14): calcd–C, 50.92%; H, 2.92%;
N, 12.50%; Br, 17.82%; Cl, 15.82%; found–C, 50.43%; H, 2.73%; N,
12.35%; Br, 17.20%; Cl, 15.64%.
Synthesis of 1,3-bis(4-cyanobenzyl)benzimidazolium bromide (3c)
Synthesis
1H-Benzimidazole (0.68 g, 5.84 mmol) and K2CO3 (1.21 g,
8.76 mmol) were stirred for 15 min in 30 ml of acetonitrile. 4-
Cyanobenzylbromide (2.28 g, 11.68 mmol) was added in one
portion and stirring was continued at room temperature for fur-
ther3 days. Afterthesolventwasremovedunderreducedpressure
75 ml of water was added. The precipitate was filtered, washed
The synthesis of 4,5-dichloro-1-methylimidazole (5b) was car-
ried out according to the literature procedure[7] where as
1-methyl-3-benzylimidazolium bromide (7a) and 1-methyl-3-
benzylbenzimidazolium bromide (7c) were carried out according
to our new and milder procedures instead of using literature
procedures.[22–24]
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Benzyl- or p-cyanobenzyl-substituted NHC-silver complexes
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Appl. Organometal. Chem. 2010, 24, 781–793
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S. Patil et al.
with diethyl ether and dried in suction at room temperature for
4 h to yield (1.52 g, 3.54 mmol, 61.1% yield) 3c as white solid.
1H NMR (δ ppm DMSO-d6, 400 MHz): 10.06 (s, 1H, NCHN),
7.92–7.89 (m, 6H, CHBenzimid + CHBenzyl), 7.70 (d, J = 8.3 Hz, 4H,
CHBenzyl), 7.64–7.60 (m, 2H, CHBenzyl), 5.90 (s, 4H, CH2). 13C NMR (δ
ppm DMSO-d6, 100 MHz, proton decoupled): 143.9, 139.7, 133.3,
C
22H19N4O3Cl2Ag (566.18): calcd–C, 46.67%; H, 3.38%; N, 9.89%;
Cl, 12.52%; Ag, 19.05%; found–C, 46.52%; H, 2.91%; N, 9.93%; Cl,
12.38%; Ag, 18.92%.
Synthesisof(1,3-bis(4-cyanobenzyl)benzimidazole-2-ylidene)silver(I)
acetate (4c)
131.5, 129.6, 127.4, 118.8, 114.4, 111.9 (NCN + CN + CBenzimid
+
CBenzyl), 49.9 (CH2). IR absorptions (KBr, cm−1): 2966 (w), 2230 (s),
1609 (w), 1562 (s), 1477 (m), 1417 (s), 1374 (m), 1198 (m), 1129
(m), 1025 (m), 841 (w), 822 (m), 785 (m), 754 (s), 684 (m), 613 (w).
UV–vis (CH3OH, nm): λ 224 (ε 32 267), λ 230 (ε 32 882), λ 269 (ε
11 682), λ 280 (ε 8339). MS (m/z, QMS-MS/MS): 349.42 [M+−Br].
MicroanalysiscalculatedforC23H17N4Br(429.32):calcd–C, 64.34%;
H, 3.99%; N, 13.05%; Br, 18.61%; found–C, 63.95%; H, 3.97%; N,
12.92%; Br, 18.40%.
1,3-bis(4-Cyanobenzyl)benzimidazolium
bromide
(0.429 g,
1.00 mmol) was dissolved in methanol (40 ml) and silver acetate
(0.333 g, 2.00 mmol) was added. The mixture was stirred at room
temperature for 1 day. The pale yellow precipitate, presumably
AgBr, was filtered and a clear solution was obtained. The volatile
components were removed in vacuo to produce a white sticky
solid. The solid was washed with pentane and diethyl ether
and dried under reduced pressure for 2 h to yield a white solid
(0.400 g, 0.776 mmol, 77.6% yield) 4c.
1H NMR 1H NMR (δ ppm DMSO-d6, 400 MHz): 7.77 (d, J = 8.0 Hz,
4H, CHBenzyl), 7.67 (dd, J = 6.0, 3.1 Hz, 2H, CHBenzimid), 7.49 (d,
J = 7.7 Hz, 4H, CHBenzyl), 7.37 (dd, J = 6.1, 3.0 Hz, 2H, CHBenzimid),
5.86 (s, 4H, CH2), 1.74 (s, 3H, CH3). 13C NMR (δ ppm DMSO-d6,
100 MHz, proton decoupled): 180.0 (NCN), 175.2 (C O), 142.2,
Synthesis of (1,3-bis(4-cyanobenzyl)imidazole-2-ylidene) silver(I)
acetate (4a)
1,3-bis(4-cyanobenzyl)imidazolium bromide (0.379 g, 1.00 mmol)
was dissolved in methanol (40 ml) and silver acetate (0.333 g,
2.00 mmol) was added. The mixture was stirred at reflux for 1 day.
The pale yellow precipitate, presumably AgBr, was filtered and
a clear solution was obtained. The volatile components were
removed in vacuo to produce an off-white sticky solid. The solid
was washed with diethyl ether and dried under reduced pressure
for 1 h to yield a white solid (0.250 g, 0.537 mmol, 53.7% yield) 4a.
1H NMR (δ ppm DMSO-d6, 400 MHz): 7.82 (d, J = 8.2 Hz, 4H,
CHBenzyl), 7.60 (s, 2H, CHImid), 7.49 (d, J = 8.2 Hz, 4H, CHBenzyl), 5.43
(s, 4H, CH2), 1.80 (s, 3H, CH3). 13C NMR (δ ppm DMSO-d6, 100 MHz,
proton decoupled): 179.8 (NCN), 175.0 (C O), 143.0, 133.1, 128.9,
123.3, 118.9, 111.2 (CN + CImid + CBenzyl), 54.1 (CH2), 23.3 (CH3).
IR absorptions (KBr, cm−1): 3399 (w), 3082 (w), 2977 (w), 2360 (s),
2228 (s), 1576 (s), 1506 (w), 1411 (s), 1352 (w), 1239 (m), 1160
(m), 1103 (w), 1019 (m), 822 (m), 787 (w), 647 (w), 549 (s). UV–vis
(CH3OH, nm): λ 210 (ε 14 792), λ 230 (ε 11 802), λ 268 (ε 5045). MS
(m/z, QMS-MS/MS): 406.32 [M+-CH3OH-O2CCH3]. Microanalysis
calculated for C22H21N4O3Ag (497.30): calcd–C, 53.13%; H, 4.25%;
N, 11.26%; Ag, 21.69%; found–C, 52.90%; H, 4.10%; N, 10.45%; Ag,
21.04%.
133.8, 133.1, 128.5, 124.8, 118.8, 112.8, 111.2 (CN + CBenzimid
+
CBenzyl), 51.83 (CH2), 23.97 (CH3). IR absorptions (KBr, cm−1): 3441
(s), 3060 (w), 2924 (w), 2360 (m), 2230 (s), 1576 (s), 1477 (w), 1400
(s), 1352 (w), 1261 (w), 1182 (w), 1100 (w), 1020 (m), 819 (m), 798
(m), 745 (m), 547 (w).
UV–vis (CH3OH, nm): λ 229 (ε 13 200), λ 275 (ε 6289), λ
284 (ε 5313). MS (m/z, QMS-MS/MS): 456.28 [M+-H2O-O2CCH3].
Microanalysis calculated for C25H21N4O3Ag (533.33): calcd–C,
56.30%; H, 3.96%; N, 10.50%; Ag, 20.22%; found–C, 57.50%; H,
3.85%; N, 10.53%; Ag, 20.78%.
Synthesis of 1-methyl-3-benzylimidazolium bromide (7a)
Benzyl bromide (11.89 ml, 100 mmol) was added in one portion
to a stirred suspension of 1-methylimidazole (3.96 ml, 50.0 mmol)
in 150 ml of toluene. The mixture was stirred for 24 h at room
temperature. The toluene was decanted and the colourless
semisolid mass was washed two times with pentane and three
times with diethyl ether. Compound 7a (11.5 g, 45.4 mmol, 90.9%
yield) was obtained as a colourless wax after drying under reduced
pressure.
1H NMR (δ ppm CDCl3, 400 MHz): 9.52 (s, 1H, NCHN), 7.19 (s, 1H,
CHImid), 7.13 (s, 1H, CHImid), 7.05–6.98 (m, 2H, CHBenzyl), 6.80–6.74
(m, 3H, CHBenzyl), 5.09 (s, 2H, CH2), 3.46 (s, 3H, N-CH3). 13C NMR
(δ ppm CDCl3, 100 MHz, proton decoupled): 136.0, 133.2, 128.7,
128.4, 127.7, 123.5, 121.8 (NCN + CImid + CBenzyl), 52.3 (CH2), 36.2
(N-CH3). IR absorptions (KBr, cm−1): 3422 (w), 3142 (w), 3081 (w),
1627 (m), 1572 (s), 1497 (s), 1455 (s), 1335 (w), 1295 (w), 1161 (s),
1083 (m), 1029 (m), 855 (w), 822 (m), 722 (s), 662 (m), 569 (w),
464 (w). UV–vis (CH3OH, nm): λ 213 (ε 6852), λ 259 (ε 2003). MS
(m/z, QMS-MS/MS): 173.40 [M+-Br]. Microanalysis calculated for
C11H13N2Br (253.14): calcd–C, 52.19%; H, 5.17%; N, 11.06%; Br,
31.56%; found–C, 51.93%; H, 4.98%; N, 10.98%; Br, 31.68%.
Synthesis of (4,5-dichloro-1,3-bis(4-cyanobenzyl)imidazole-2-
ylidene) silver(I) acetate (4b)
4,5-Dichloro-1,3-bis(4-cyanobenzyl)imidazolium
bromide
(0.448 g, 1.00 mmol) was dissolved in methanol (40 ml) and
silver acetate (0.333 g, 2.00 mmol) was added. The mixture was
stirred at room temperature for 1 day. The pale yellow precipitate,
presumably silver bromide, was filtered and discarded. The
volume of the reaction mixture was reduced under reduced
pressure. The residue was washed with pentane and diethyl ether
and dried under reduced pressure to yield a white solid (0.425 g,
0.794 mmol, 79.4% yield) 4b.
1H NMR (δ ppm DMSO-d6, 400 MHz): 7.83 (d, J = 8.2 Hz, 4H,
CHBenzyl), 7.46 (d, J = 8.1 Hz, 4H, CHBenzyl), 5.59 (s, 4H, CH2), 1.71 (s,
3H, CH3). 13C NMR (δ ppm DMSO-d6, 100 MHz, proton decoupled):
185.6 (NCN), 175.3 (C O), 141.2, 133.1, 128.4, 118.8, 118.1, 111.4
(CN + CCl + CBenzyl), 53.5 (CH2), 24.1(CH3). IR absorptions (KBr,
cm−1): 3433 (s), 2360 (s), 2229 (s), 1578 (s), 1507 (w), 1389 (s), 1338
(w), 1210 (m), 1120 (m), 1019 (m), 820 (s), 547 (m). UV–vis (CH3OH,
nm):λ207(ε 20 921), λ228(ε 14 765), λ274(ε 5795). MS(m/z, QMS-
MS/MS): 475.19 [M+ CH3OH-O2CCH3]. Microanalysis calculated for
Synthesis of 4,5-dichloro-1-methylimidazole (5b)
4,5-Dichloroimidazole (1.23 g, 9.00 mmol) and potassium hydrox-
ide (2.24 g, 40.0 mmol) were stirred in acetonitrile (50 ml) for 2 h
at room temperature. The excess potassium hydroxide was fil-
tered from the solution and iodomethane (0.562 ml, 9.00 mmol)
was added. The reaction mixture was stirred at room temperature
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Benzyl- or p-cyanobenzyl-substituted NHC-silver complexes
for 24 h. The volatile components were removed, and the crude
product was re-dissolved in dichloromethane. The solid, presum-
ably KI, was filtered and discarded, and the volatile components
were removed in vacuo to yield a yellow crystalline solid (1.32 g,
8.72 mmol, 97.0% yield) (5b).
1H NMR (δ ppm CDCl3, 400 MHz): 7.36 (s, 1H, NCHN), 3.61 (s, 3H,
N-CH3). 13CNMR(δ ppmCDCl3, 100 MHz, protondecoupled):134.6
(NCN), 125.5, 113.7 (CCl), 32.5 (N-CH3). IR absorptions (KBr, cm−1):
3437 (m), 3099 (s), 2953 (w), 1663 (w), 1521 (s), 1494 (s), 1463 (w),
1362 (m), 1262 (s), 1209 (m), 1126 (s), 988 (s), 835 (m), 721 (m), 666
(s), 623 (m), 540 (m). UV–vis (CH3OH, nm): λ 225 (ε 4371), λ 275 (ε
933). MS (m/z, QMS-MS/MS): 150.92 [M+]. Microanalysis calculated
for C4H4N2Cl2 (150.99): calcd–C, 31.81%; H, 2.67%; N, 18.55%; Cl,
46.95%; found–C, 31.37%; H, 2.59%; N, 18.05%; Cl, 46.40%.
Synthesis of (1-methyl-3-benzylimidazole-2-ylidene) silver(I) acetate
(8a)
1-Methyl-3-benzylimidazolium bromide (0.253 g, 1.00 mmol) was
dissolved in dichloromethane (40 ml), silver acetate (0.333 g,
2.00 mmol) was added, and the mixture was refluxed for 2 d.
The precipitate, presumably AgBr, was filtered and a clear solution
was obtained. The volatile components were removed in vacuo
to produce an off-white sticky solid. The solid was washed with
pentane and dried under reduced pressure to yield (0.080 g,
0.235 mmol, 23.6% yield) 8a.
1H NMR (δ ppm CDCl3, 400 MHz): 7.42–7.24 (m, 5H, CHBenzyl),
6.97 (d, J = 1.7 Hz, 1H, CHImid), 6.92 (d, J = 1.7 Hz, 1H, CHImid),
5.27 (s, 2H, CH2), 3.85 (s, 3H, N-CH3), 2.07 (s, 3H, COCH3). 13C NMR
(δ ppm CDCl3, 125 MHz, proton decoupled): 179.9 (NCN), 177.9
(C O), 135.5, 129.0, 128.5, 127.9, 122.5, 121.0 (CBenzyl + CImid), 55.7
(CH2), 38.8 (N-CH3), 22.5 (COCH3). IR absorption (KBr, cm−1): 3382
(w), 3135 (w), 3096 (w), 1579 (s), 1406 (s), 1158 (s), 1082 (w), 1018
(m), 919 (m), 819 (w), 721 (s), 646 (m), 621 (w), 463 (w). UV–vis
(CH3OH, nm): λ 219 (ε 4075), λ 230 (ε 3008), λ 269 (ε 1878). MS
(m/z, QMS-MS/MS): 280.10 [M+-O2CCH3]. Microanalysis calculated
for C13H15N2O2Ag (339.14): calcd–C, 46.04%; H, 4.45%; N, 8.26%;
Ag, 31.80%; found–C, 45.86%; H, 5.01%; N, 8.31%; Ag, 30.39%.
Synthesis of 4,5-dichloro-1-methyl-3-benzylimidazolium bromide
(7b)
Benzyl bromide (2.37 ml, 20.0 mmol) was added in one portion
to a stirred suspension of 4,5-dichloro-1-methylimidazole (1.50 g,
10.0 mmol) in 40 ml of toluene. The mixture was stirred for 48 h
at room temperature. Afterwards the solvent was removed under
reduced pressure. The resulting yellow residue was saturated with
diethyl ether to get a light yellow precipitate, which was filtered,
washed with diethyl ether and finally dried in vacuo in order to
give compound 7b (2.05 g, 6.36 mmol, 63.7% yield).
Synthesis of (4,5-dichloro-1-methyl-3-benzylimidazole-2-ylidene)
silver(I) acetate (8b)
1H NMR (δ ppm CDCl3, 400 MHz): 11.29 (s, 1H, NCHN), 7.56 (d,
J = 6.2 Hz, 2H, CHBenzyl), 7.47–7.34 (m, 3H, CHBenzyl), 5.64 (s, 2H,
CH2), 4.08 (s, 3H, N-CH3). 13C NMR (δ ppm CDCl3, 100 MHz, proton
decoupled): 138.0 (NCN), 131.6, 129.6, 129.4, 128.9 (CBenzyl), 119.9,
118.9 (CCl), 52.6 (CH2), 35.6 (N-CH3). IR absorptions (KBr, cm−1):
3410 (s), 3363 (s), 3121 (w), 3068 (m), 2918 (m), 2865 (w), 1629 (w),
1583 (m), 1560 (m), 1455 (s), 1346 (w), 1261 (w), 1160 (s), 1096 (w),
1029 (w), 801 (w), 732 (m), 703 (m), 621 (w). UV–vis (CH3OH, nm):
λ 228 (ε 4894), λ 274 (ε 2665). MS (m/z, QMS-MS/MS): 242.13 [M+-
Br]. Microanalysis calculated for C11H11N2Cl2Br (322.03): calcd–C,
41.02%; H, 3.44%; N, 8.69; Br, 24.81%; Cl, 22.01%; found–C, 41.78%;
H, 3.43%; N, 8.57%; Br, 24.37%; Cl, 21.79%.
4,5-Dichloro-1-methyl-3-benzylimidazolium bromide (0.253 g,
1.00 mmol) was dissolved in CH2Cl2 (40 ml) and silver acetate
(0.333 g, 2.00 mmol) was added. The mixture was stirred at room
temperature for 1 day. The yellow precipitate of AgBr was filtered
and a clear solution was obtained. The volatile components were
removed in vacuo to produce an off-white sticky solid. The solid
was washed with diethyl ether and dried under reduced pressure
to yield (0.150 g, 0.367 mmol, 36.8% yield) 8b as a white solid.
1H NMR (δ ppm CDCl3, 400 MHz): 7.47–7.01 (m, 5H, CHBenzyl),
5.25 (s, 2H, CH2), 3.76 (s, 3H, N-CH3), 1.99 (s, 3H, COCH3). 13C NMR
(δ ppm CDCl3, 100 MHz, proton decoupled): 180.1 (NCN), 179.0
(C O), 134.1, 129.0, 128.7, 127.7, 118.2, 117.4 (CCl + CBenzyl), 54.5
(CH2), 38.0 (N-CH3), 22.6 (COCH3). IR absorptions (KBr, cm−1): 3443
(s), 3064 (w), 1575 (s), 1405 (m), 1338 (w), 1261 (w), 1130 (w), 1097
(w), 1022 (w), 800 (w), 746 (w), 704 (m), 468 (w). UV–vis (CH3OH,
nm): λ 216 (ε 13 422), λ 241 (ε 8362), λ 280 (ε 4414). MS (m/z,
QMS-MS/MS): 348.22 [M+-O2CCH3]. Microanalysis calculated for
C13H13N2O2Cl2Ag (408.03): calcd–C, 38.26%; H, 3.21%; N, 6.86%;
Cl, 17.37%; Ag, 26.43%; found–C, 37.85%; H, 3.13%; N, 6.66%; Cl,
17.25%; Ag, 26.21%.
Synthesis of 1-methyl-3-benzylbenzimidazolium bromide (7c)
Benzyl bromide (2.37 ml, 20.0 mmol) was added in one portion to
a stirred suspension of 1-methylbenzimidazole (1.32 g, 10.0 mmol)
in 40 ml of toluene. The mixture was stirred for 4 days at room
temperature. Afterwards the solvent was removed under reduced
pressure. The resultant residue was washed four times with diethyl
ether and dried in vacuo to yield (2.75 g, 9.06 mmol, 90.7% yield) a
colourless solid 7c.
1H NMR (δ ppm CDCl3, 400 MHz): 11.38 (s, 1H, NCHN), 7.76 (d,
J = 8.2 Hz, 1H, CHBenzimid), 7.66–7.53 (m, 5H, CHBenzyl), 7.38–7.30
(m, 3H, CHBenzimid), 5.89 (s, 2H, CH2), 4.29 (s, 3H, N-CH3). 13C NMR (δ
ppmCDCl3,100 MHz,protondecoupled):142.0,131.7,131.1,129.9,
Synthesis of (1-methyl-3-benzylbenzimidazole-2-ylidene) silver(I)
acetate (8c)
1-Methyl-3-benzylbenzimidazolium bromide (0.322 g, 1.00 mmol)
was dissolved in dichloromethane (50 ml) and silver acetate
(0.333 g, 2.00 mmol) was added. The mixture was stirred at room
temperature for 1 day. The yellow precipitate, presumably AgBr,
was filtered and discarded. The volume of the reaction mixture
was reduced under pressure to 5 ml. Pentane (200 ml) was added
and the fine white precipitate was filtered out, washed with diethyl
etheranddriedinvacuotoyieldawhitesolid(0.260 g, 0.668 mmol,
66.8% yield) 8c.
128.3, 128.1, 127.3, 126.2, 124.2, 112.7, 112.0 (NCN + CBenzimid
+
CBenzyl), 50.3 (CH2), 33.0 (CH3). IR absorptions (KBr, cm−1): 3410 (s),
3131 (w), 3065 (m), 2965 (w), 1562 (s), 1490 (w), 1458 (m), 1424
(w), 1384 (m), 1348 (w), 1277 (w), 1195 (m), 1094 (w), 1018 (w),
874 (w), 757 (s), 713 (m), 659 (w). UV–vis (CH3OH, nm): λ 222 (ε
7831), λ 250 (ε 6382), λ 269 (ε 6270). MS (m/z, QMS-MS/MS): 223.41
[M+-Br-H2O]. Microanalysis calculated for C15H17N2BrO (321.21):
calcd–C, 56.09%; H, 5.33%; N, 8.72%; Br, 24.88%; found–C, 56.31%;
H, 5.08%; N, 8.98%; Br, 24.15%.
1H NMR (δ ppm CDCl3, 400 MHz): 7.59–7.09 (m, 9H, CHBenzimid
CHBenzyl), 5.61 (s, 2H, CH2), 4.08 (s, 3H, N-CH3), 2.11 (s, 3H, COCH3).
13C NMR (δ ppm CDCl3, 125 MHz, proton decoupled): 180.0 (NCN),
+
c
Appl. Organometal. Chem. 2010, 24, 781–793
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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S. Patil et al.
179.2 (C O), 134.9, 134.6, 133.5, 129.0, 128.4, 127.2, 124.1, 111.9,
111.2 (CBenzimid + CBenzyl), 53.4 (CH2), 35.9 (N-CH3), 22.7 (COCH3).
IR absorptions (KBr, cm−1): 3433 (w), 1669 (w), 1580 (s), 1445 (w),
1403 (m), 1345 (w), 1262 (w), 1193 (w), 1094 (w), 1024 (w), 806 (w),
747 (s), 703 (m), 649 (w). UV–vis (CH3OH, nm): λ 206 (ε 21 306), λ
274 (ε 3421), λ 285 (ε 4640). MS (m/z, QMS-MS/MS): 330.18 [M+-
O2CCH3]. Microanalysis calculated for C17H17N2O2Ag (389.20):
calcd–C,52.46%;H,4.40%;N,7.19%;Ag,27.71%;found–C,51.92%;
H, 4.36%; N, 7.01%; Ag, 27.40%.
Culture Collection) and maintained in Dulbecco’s modified Eagle
medium containing 10% (v/v) fetal calf serum, 1% (v/v) penicillin
streptomycin and 1% (v/v) L-glutamine. Cells were seeded in
96-well plates containing 200 µl microtitre wells at a density of
5000 cells per 200 µl of medium and were incubated at 37 ◦C
for 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 × 10−4 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 solutions 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 37 ◦C, individual wells were treated with
a 200 µl of a solution of MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] in medium. The solution consisted
of 30 mg of MTT in 30 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 µl 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.
Antibacterial studies
Qualitative antibacterial activity of symmetrically substituted
and non-symmetrically substituted N-heterocyclic carbenes and
their corresponding silver complexes were screened against two
bacterial strains. The test organisms included Staphylococcus
aureus(SA)(NCTC7447)asaGram-positivebacteriaandEscherichia
coli as Gram-negative bacteria.
Toassessthebiologicalactivityofcompounds3a–c,4a–c,7a–c
and 8a–c, the Kirby–Bauer disc-diffusion method was applied.[25]
All bacteria were individually cultured from a single colony in
sterile LB medium[26] overnight at 37 ◦C (orbital shaker incubator).
All the work carried out was performed under sterile conditions.
For each strain, 70 µl of culture were spread evenly on agar-LB
medium. Four 5 mm diameter paper discs were placed evenly
separated on each plate. Two stock solutions (90 : 10 DMSO : H2O)
of every compound were prepared at 2.3 µM and 4.6 µM to be
able to test the effect of different concentrations. Each plate was
then tested with 5 and 7 µl of 2.3 µM solution and 5 and 10 µl
for the 4.6 µM solution. The plates were covered and placed in an
incubator at 37 ◦C for 24 h. The plates were then removed and the
zone 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 millimetres and are summarized in
Tables 3 and 4.
Results and Discussion
Synthesis
The synthetic route for symmetrically substituted and non-
symmetrically substituted N-heterocyclic carbenes as ligand pre-
cursors and their corresponding silver complexes described in this
work is given in Schemes 1–3. We did not follow the synthetic pro-
cedure for the non-symmetrically substituted NHCs precursors,
1-methyl-3-benzylimidazolium bromide (7a), and 1-methyl-3-
benzylbenzimidazoliumbromide(7c),[22–24] butsynthesizedthem
according to new and milder procedures. On the other hand, the
synthesis of 4,5-dichloro-1-methylimidazole (5b) was carried out
according to a literature procedure.[7] The symmetrically substi-
tutedNHCprecursors1,3-bis(4-cyanobenzyl)imidazoliumbromide
(3a) and 1,3-bis(4-cyanobenzyl)benzimidazolium bromide (3c)
werepreparedbystirring1H-imidazole(1a)and1H-benzimidazole
Cytotoxicity Studies
Preliminary in vitro cell tests were performed on the human
cancerousrenalcelllineCaki-1inordertocomparethecytotoxicity
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 that shown in kidney carcinoma
cells. They were obtained from the ATCC (American Tissue Cell
CN
CN
Br
O
R
R
R
N
N
N
N
NH
K2CO3
2AgOAc
CH2Cl2
Ag
H
Br
O
H
+ 2
CH3CN
RI
RI
RI
N
CH3
CN
1a-c
1a, R=RI=H
1b, R=RI=Cl
1c, R/RI=Benzo
2
CN
CN
3a-c
4a-c
3a, R=RI=H
3b, R=RI=Cl
3c, R/RI=Benzo
4a, R=RI=H
4b, R=RI=Cl
4c, R/RI=Benzo
Scheme 1. Generalreaction scheme forthe synthesis of symmetrically substituted N-heterocyclic carbenes(3a–c) and theircorrespondingN-heterocyclic
carbene–silver complexes (4a–c).
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2010, 24, 781–793

Benzyl- or p-cyanobenzyl-substituted NHC-silver complexes
Br
CH3
N
CH3
CH3
N
O
R
R
R
N
2AgOAc
CH2Cl2
Toluene
Br
H
Ag
O
H
+
RI
RI
RI
N
N
N
CH3
6
5a and 5c
5a, R=RI=H
5c, R/RI=Benzo
7a and 7c
8a and 8c
7a, R=RI=H
7c, R/RI=Benzo
8a, R=RI=H
8c, R/RI=Benzo
Scheme 2. General reaction scheme for the synthesis of non-symmetrically substituted N-heterocyclic carbenes (7a and 7c) and their corresponding
N-heterocyclic silver–carbene complexes (8a and 8c).
Br
CH3
N
CH3
N
Cl
Cl
Cl
Cl
Cl
Cl
NH
KOH, CH3I
CH3CN
Toluene
Br
H
H
H
+
N
N
N
1b
6
5b
7b
CH3
N
Cl
Cl
O
2 AgOAc
CH3CN
Ag
O
N
CH3
8b
Scheme 3. General reaction scheme for the synthesis of non-symmetrically substituted N-heterocyclic carbene (7b) and its N-heterocyclic carbene–silver
complex (8b).
(1c) with 2 equivalents of p-cyanobenzyl bromide (2) in the pres-
ence of K2CO3 as a base in CH3CN at room temperature for
3 days with 67 and 61% yields respectively. 4,5-Dichloro-1,3-
bis(4-cyanobenzyl)imidazolium bromide (3b) was prepared by
heating 4,5-dichloro-1H-imidazole (1b) with 2 equivalents of p-
cyanobenzyl bromide (2) in the presence of K2CO3 as a base
in CH3CN for 6 d with a yield of 29%. The non-symmetrically
substituted NHC precursors 1-methyl-3-benzylimidazolium bro-
mide (7a) and 1-methyl-3-benzylbenzimidazolium bromide (7c)
were prepared by stirring 1-methylimidazole (5a) and 1-
methylbenzimidazole (5c) with benzyl bromide (6) in toluene
at room temperature for 2–4 days with 91 and 90% yields respec-
tively. 4,5-Dichloro-1-methylimidazole (5b) is formed in 98% yield
from the deprotonation of 4,5-dichloroimidazole (1b) with potas-
sium hydroxideandsubsequentmethylationwithiodomethanein
acetonitrile. 4,5-Dichloro-1-methyl-3-benzylimidazolium bromide
(7b) is formed in 64% yield by the reaction of 4,5-dichloro-1-
methylimidazole (5b) with benzyl bromide in toluene.
downfield shift in the range δ = 9.48–11.38 ppm for the NCHN
proton attributable to the positive charge of the molecule.[27,28]
Additionally, their identities were also confirmed by a base peak
for the [M+−Br] fragments in their positive mode ESI mass spectra.
The NHC–silver complexes [1,3-bis(4–cyanobenzyl)imidazole-
2-ylidene] silver(I) acetate (4a), [4,5-dichloro-1,3-bis(4-
cyanobenzyl)imidazole-2-ylidene] silver(I) acetate (4b), [1,3-bis(4-
cyanobenzyl)benzimidazole-2-ylidene] silver(I) acetate (4c), (1-
methyl-3-benzylimidazole-2-ylidene) silver(I) acetate (8a), (4,5-
dichloro-1-methyl-3-benzylimidazole-2-ylidene) silver(I) acetate
(8b) and (1-methyl-3-benzylbenzimidazole-2-ylidene) silver(I) ac-
etate (8c) were synthesized by the reaction of 3a–c and 7a–c with
2 equivalents of silver acetate in dichloromethane/methanol. The
reaction mixture was stirred for 1–2 days at room temperature or
refluxed for 2–4 days to afford the NHC–silver acetate complexes
as off-white solid in 23–79% yield. The complexes were fully
characterized by 1H, 13C NMR, IR, UV–visible, mass spectroscopy
and elemental analysis. Furthermore, the solid-state structures of
4a–c and 8c were analysed by single crystal X-ray diffraction. The
absence of a downfield NCHN signal and presence of new signals
at 2.11–1.71 ppm for the acetate protons in all the 1H NMR spectra
for 4a–c and 8a–c; however, indicates a successful complex for-
mation. The 13C NMR resonances of the carbene carbon atoms in
The NHC precursors were fully characterized by 1H, 13C NMR, IR,
UV–visible, massspectroscopyandelementalanalysis. Inaddition,
the solid-state structure of the NHC precursors 3a–c and 7c
1
was determined by single crystal X-ray diffraction. The H NMR
spectra of all precursors 3a–c and 7a–c show a characteristic
c
Appl. Organometal. Chem. 2010, 24, 781–793
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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S. Patil et al.
Br
C17
C3
C16
C18
C12
C4
N4
C1
N1
C2
C19
C5
C8
C9
N2
C15
N3
C13
C7
C14
C6
C11
C10
Figure 1. X-Ray diffraction structure of 3a; molecule; thermal ellipsoids are drawn on the 50% probability level.
complexes 4a–c and 8a–c occur in the range δ 185.6–179.8 ppm,
respectively. These signals are shifted downfield compared with
the corresponding precursors of 3a–c and 7a–c carbene carbons’
Cl1
C10
resonance at the range 143.9–136.0 ppm, respectively, which fur-
C8
Cl2
N2
ther demonstrates the formation of expected NHC–silver acetate
complexes. Also the appearance of the 13C NMR resonances for the
carbonyl and methyl carbons of the acetate group of complexes
4a–c and 8a–c in the range 175.0–179.9 and 22.5–24.1 ppm re-
spectively showed the formation of the NHC–silver complexes.[7,9]
Furthermore, positive mode ESI mass spectra of all six NHC–silver
complexes (4a–c and 8a–c) are dominated by [M+ –O2CCH3]
fragment peaks arising from the loss of one acetate ligand.
C11
C5
N3
C12
C18
C4
C9
H9
C6
C17
C16
C3
C7
C13
C14
C19
C2
N4
C1
N1
C15
Br
Structural Discussion
The crystal structures of the compounds 3a–c, 4a–c, 7c and 8c
were determined. The molecular structures of the compounds
3a–c, 4a–c, 7c and 8c are depicted in Figs 1–9. The crystal data
andrefinementdetailsforalleightcompoundsaregiveninTable 1,
whereas selected bond lengths and bond angles are displayed in
Table 2.
Figure 2. X-Ray diffraction structure of 3b; molecule; thermal ellipsoids are
drawn on the 50% probability level.
Br
The X-ray structures of compounds 3a–c and 7c reveal
that the molecules are nonplanar. In the five-membered ring
(NCNCC) of compounds 3a–c and 7c, the bond distances
and angles are in good agreement with the bond dis-
tances found in the similar compounds 1-(2,4,6-trimethylphenyl)-
3-(N-phenylacetamido)imidazolium chloride, 1,3-dimethyl-4,5-
dichloroimidazolium iodide and 1,3- diisopropylbenzimidazolium
bromide reported in the literature (see Table 2).[7,29,30] In com-
pounds 3a–c there is an absence of any lattice-held water
molecules or organic solvent molecules in the unit cells of the
determined structures. Compound 7c has one lattice held water
molecule in the unit cell of the determined structure.
C2
C21
N1
C23
C20
C3
C1
C7
N4
C22
C16
C9
C19
C4
C6
C17
N3
C5
C8
N2
C18
C10
C11
C15
C14
C12
C13
Also in all the silver complexes reported here, bond lengths
and angles in and directly around the NHC core agree very well
among each other and with literature data.[31–35] Comparing
precursor 3a–c and 7c with the corresponding complexes 4a–c
and 8c (see Table 2), one finds a slight increase of both the C(9)–N
distances. NHC silver complexes 4a–c and 8c are mononuclear
complexes. Compounds 4a and 4b have lattice-held methanol
molecules where as compound 4c has lattice held water molecule.
In the X-ray structure of 4a–c and 8c the acetate moiety acts as a
monodentate ligand.
Figure 3. X-Ray diffraction structure of 3c; molecule; thermal ellipsoids are
drawn on the 50% probability level.
The results are displayed in Tables 3 and 4. With respect to our
previously tested compounds,[19] the concentration of the stock
solutions is reduced 4-fold, hence the amount of compound used
in each test is significantly less, indicating a stronger biological ac-
tivity. Minimal antibacterial activity was observed for compounds
3a–c and 7a–c against both Gram-positive bacteria Staphylo-
coccus aureus and Gram-negative bacteria Escherichia coli using
the Kirby–Bauer disc diffusion method. Improved activity was
observed with compounds 4a, 4b and 8a against Gram-positive
Biological Evaluation
The biological activities of the NHC-precursors and their corre-
sponding NHC–silver complexes are summarized in Figs 10–13.
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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Benzyl- or p-cyanobenzyl-substituted NHC-silver complexes
C21
O1
C20
C3
N1
O2
C4
C7
H1O3
C22
O3
Ag
C5
C2
C1
C6
C9
N2
C12
C13
C14
C8
C15
N3
C11
C10
C19
C18
C16
N4
C17
Figure 4. X-Ray diffraction structure of 4a; molecule; thermal ellipsoids are drawn on the 50% probability level.
C21
C20
O1
H1O3
O3
O2
C22
Ag
C16
C15
N4
C17
C18
C19
C9
C12
N3
C13
C5
C8
C14
C4
C6
N2
C1
C7
C11
N1
C10
Cl1
C3
C2
Cl2
Figure 5. X-Ray diffraction structure of 4b; molecule; thermal ellipsoids are drawn on the 50% probability level.
bacteria Staphylococcus aureus while 4c, 8b and 8c compounds
have shown the best activity so far. In case of Gram-negative
bacteria Escherichia coli 4a, 4b, 8a and 8b compounds showed
moderate activity while the best result was obtained with 4c and
8c. The NHC–silver complexes 4a, 4b and 8a showed medium
antibacterial activity with areas of clearance 4–5 mm against both
Gram-positive bacteria Staphylococcus aureus and Gram negative-
bacteriaEscherichiacoli whereastheNHC–silvercomplexes4cand
8c showed highest antibacterial activity with areas of clearance
9–12 mm against both Gram-positive bacteria Staphylococcus
aureus and Gram negative-bacteria Escherichia coli at the highest
amount(0.46 µmol)used(seeTables 3and 4).ThustheNHC–silver
complexes 4c and 8c are the most promising antibacterial drug
candidates in this paper. The metal salt (silver 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 reported.[19,36]
N1
C7
O2
C3
C25
C2
C24
C1
C4
O1
Ag
C5
C6
C8
N2
C9
C10
C18
N3
C19
C11
C16
C15
C23
N4
C17
C22
C14
C20
C21
C12
Thus, on the basis of the above observation it can be
stated that (i) the NHC–silver complexes 4a–c and 8a–c are
more active against both Gram-positive bacteria Staphylococcus
aureus and Gram-negative bacteria Escherichia coli than the free
NHC-precursors 3a–c and 7a–c. (ii) It was concluded that, as
C13
Figure 6. X-Ray diffraction structure of 4c; molecule; thermal ellipsoids are
drawn on the 50% probability level.
c
Appl. Organometal. Chem. 2010, 24, 781–793
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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S. Patil et al.
b
c
a
Figure 7. Hydrogen bonded chain of 4c running along [1 0 0].
C2
C9
N2
C3
C1
C2
C8
Br1
C10
C1
C6
C3
C4
C11
C12
C6
N1
C7
C5
C14
C7
N1
C15
C4
C13
C5
C9
C13
C14
Ag
O1
C12
Figure 8. X-Ray diffraction structure of 7c; molecule; thermal ellipsoids are
drawn on the 50% probability level.
C8
N2
C10
C17
C16
C11
C15
the NHC-precursors and NHC–silver complexes concentration
increases, the antimicrobial activity becomes higher; and (iii) it
was also observed that, compared with the NHC-precursors the
NHC–silver complexes exhibited an approximately 6-fold increase
in magnitude of antibacterial activity (see Figs 10–13), which is
due to the synergistic effect of the increased lipophilicity of the
complexes.Chelationdecreasesthepolarityofthemetalion,which
further leads to the enhanced lipophilicity of the complex. Since
the microorganism cell wall is surrounded by a lipid membrane
which favours the passage of lipid soluble materials, increased
lipophilicity allows the penetration of complex into and through
the membrane and deactivates the active enzyme sites of the
microorganisms.[37]
O2
Figure 9. X-Ray diffraction structure of 8c; molecule; thermal ellipsoids are
drawn on the 50% probability level.
In comparison with the known reported NHC-precursors and
NHC–silver complexes from the literature,[19] the NHC-precursors
(3a–c and 7a–c) and their corresponding NHC–silver complexes
(4a–c and 8a–c) have shown high antibacterial activity.
Cytotoxicity Studies
The in vitro cytotoxicity of compounds 4a–c and 8a–c were
determined by MTT-based assays[38] involving a 48 h drug
Figure 10. AreaofclearanceonStaphylococcusaureus(Gram+ve)by3a–c
and 4a–c.
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2010, 24, 781–793

Benzyl- or p-cyanobenzyl-substituted NHC-silver complexes
Table 2. Selected bond lengths (Å) and angles (deg) for compounds 3a–c, 4a–c, 7c and 8c
Bond lengths (Å)
3a
3b
3c
4a
4b
4c
Bond lengths (Å)
7c
8c
N(2)–C(9)
1.326(2)
1.380(2)
1.335(5)
1.378(5)
1.342(5)
1.382(5)
1.353(5)
1.336(2)
1.393(2)
1.328(2)
1.357(5)
1.375(5)
1.351(5)
1.373(5)
1.339(6)
1.354(2)
1.379(2)
1.357(2)
1.378(2)
1.348(2)
1.355(7)
1.400(8)
1.349(8)
N(1)–C(8)
C(8)–N(2)
N(2)–C(10)
C(10)–C(15)
N(1)–C(15)
C(9)–C(10)
N(1)–C(9)
Ag–C(8)
1.323(6)
1.329(6)
1.393(6)
1.380(7)
1.394(5)
1.359(5)
1.349(5)
1.399(6)
N(2)–C(10)
C(9)–N(3)
1.3275(19)
1.3813(19)
1.351(2)
N(3)–C(11)
C(10)–C(11)
N(3)–C(15)
1.394(2)
1.398(3)
1.400(8)
1.397(8)
1.378(6)
1.396(5)
2.048(4)
2.097(3)
1.278(5)
1.232(5)
1.525(6)
C(10)–C(15)
C(10)–Cl(1)
1.691(4)
1.688(4)
1.6864(18)
1.6987(18)
2.0625(17)
2.1181(14)
1.269(2)
C(11)–Cl(2)
Ag–O(1)
Ag–C(9)
2.077(4)
2.164(3)
1.269(5)
1.246(5)
1.513(6)
2.073(6)
2.085(5)
O(1)–C(16)
O(2)–C(16)
C(16)–C(17)
Ag–O(1)
O(1)–C(20)
O(2)–C(20)
1.244(2)
C(20)–C(21)
O(1)–C(24)
1.512(3)
1.253(9)
1.214(9)
O(2)–C(24)
C(24)–C(25)
Bond angles (deg)
N(2)–C(9)–N(3)
C(9)–N(2)–C(10)
C(9)–N(3)–C(11)
C(10)–C(11)–N(3)
C(11)–C(10)–N(2)
C(9)–N(3)–C(15)
N(3)–C(15)–C(10)
N(2)–C(10)–C(15)
C(9)–Ag–O(1)
C(20)–O(1)–Ag
C(24)–O(1)–Ag
1.532(11)
Bond angles (deg)
N(1)–C(8)–N(2)
C(8)–N(2)–C(10)
C(15)–C(10)–N(2)
C(10)–C(15)–N(1)
C(8)–N(1)–C(15)
C(9)–C(10)–N(2)
C(10)–C(9)–N(1)
C(8)–N(1)–C(9)
C(8)–Ag–O(1)
108.04(13)
109.16(13)
109.25(13)
106.61(14)
106.93(14)
108.2(3)
108.6(3)
108.9(3)
106.6(3)
107.8(3)
110.28(15)
108.25(15)
103.7(3)
111.2(3)
111.7(3)
106.5(4)
107.0(4)
104.65(14)
111.12(14)
110.58(14)
107.21(15)
106.43(15)
106.1(5)
111.0(5)
110.1(4)
108.2(4)
106.6(4)
106.5(4)
108.5(4)
105.7(3)
111.1(3)
108.64(15)
106.25(15)
106.58(15)
111.3(5)
105.7(5)
105.9(5)
170.1(2)
106.0(4)
106.5(4)
110.7(3)
163.36(13)
105.6(3)
174.90(6)
176.82(14)
110.2(3)
111.65(12)
C(16)–O(1)–Ag
128.3(5)
Figure 11. Area of clearance on Escherichia coli (Gram −ve) by 3a–c and
4a–c.
Figure 12. AreaofclearanceonStaphylococcusaureus(Gram+ve)by7a–c
and 8a–c.
exposure period, followed by 24 h of recovery time. Compounds
were tested for their activity on the human cancerous renal-cell
lineCaki-1andtheresultsareshowninFigs 14and 15,respectively.
Symmetrically substituted NHC–silver acetate complexes 4a–c,
which contain 1H-imidazole, 4,5-dichloro-1H-imidazole and 1H-
benzimidazole groups, have IC50 values of 29 ( 5), 30 ( 7) and
53 ( 8) µM respectively. Compounds 4a and 4b show a very
similar IC50 values and a 2-fold increase in magnitude when
compared with 4c and, in comparison with cisplatin (IC50 value
=
3.3 µM), the IC50 values for the three compounds are not
impressive. Non-symmetrically substituted NHC–silver acetate
complexes 8a–c, which also contain 1-methylimidazole, 4,5-
dichloro-1-methylimidazole and 1-methylbenzimidazole groups,
exhibit IC50 values of 3.2 ( 0.6), 24 ( 7) and 34 ( 3) µM
respectively. Compound8aisthemostpromisingoneinthispaper
because of its lowest IC50 value; 8a exhibits an approximately 8-
c
Appl. Organometal. Chem. 2010, 24, 781–793
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

S. Patil et al.
Table 3. Area of clearance for compounds 3a–c and 4a–c in mm
Table 4. Area of clearance for compounds 7a–c and 8a–c in mm
Staphylococcus
Staphylococcus
aureus
aureus
Escherichia coli
Escherichia coli
Compounds
(Gram + ve)
(Gram −ve)
Compounds
(Gram +ve)
(Gram −ve)
3a (mm) 0.11 µmol (44.3 µg)
0.16 µmol (62.0 µg)
0.23 µmol (88.6 µg)
0.46 µmol (177.2 µg)
3b (mm) 0.11 µmol (51.2 µg)
0.16 µmol (71.7 µg)
0.23 µmol (104.7 µg)
0.46 µmol (209.5 µg)
3c (mm) 0.11 µmol (50.1 µg)
0.16 µmol (70.1 µg)
0.23 µmol (100.2 µg)
0.46 µmol (200.5 µg)
4a (mm) 0.11 µmol (54.3 µg)
0.16 µmol (76.1 µg)
0.23 µmol (108.7 µg)
0.46 µmol (217.5 µg)
4b (mm) 0.11 µmol (62.5 µg)
0.16 µmol (87.5 µg)
0.23 µmol (125.0 µg)
0.46 µmol (250.0 µg)
4c (mm) 0.11 µmol (60.1 µg)
0.16 µmol (84.2 µg)
1
1
1
1
1
1
1
1
1
1
2
2
5
6
6
8
2
4
4
6
5
6
8
10
1
1
1
2
1
1
1
2
1
1
1
3
1
2
3
4
1
2
2
4
3
4
7
10
7a (mm) 0.11 µmol (29.5 µg)
0.16 µmol (41.3 µg)
1
1
1
2
1
1
1
1
1
1
1
1
3
4
4
5
4
5
7
9
4
6
7
9
1
1
0.23 µmol (59.0 µg)
1
0.46 µmol (118.0 µg)
7b (mm) 0.11 µmol (37.5 µg)
0.16 µmol (52.5 µg)
2
1
1
0.23 µmol (75.0 µg)
1
0.46 µmol (150.0 µg)
7c (mm) 0.11 µmol (35.25 µg)
0.16 µmol (49.35 µg)
0.23 µmol (70.5 µg)
0.46 µmol (141.0 µg)
8a (mm) 0.11 µmol (39.5 µg)
0.16 µmol (55.3 µg)
2
1
2
2
3
3
4
0.23 µmol (79.0 µg)
5
0.46 µmol (158.0 µg)
8b (mm) 0.11 µmol (47.5 µg)
0.16 µmol (66.5 µg)
6
3
4
0.23 µmol (95.0 µg)
5
0.46 µmol (190.0 µg)
8c (mm) 0.11 µmol (45.2 µg)
0.16 µmol (63.3 µg)
6
5
8
0.23 µmol (120.3 µg)
0.46 µmol (240.7 µg)
0.23 µmol (90.5 µg)
0.46 µmol (181.0 µg)
10
12
22
20
18
16
14
12
10
8
4a, IC50 = 29 (+/-5).10-6
4b, IC50 = 30 (+/-7).10-6
4c, IC50 = 53 (+/-8).10-6
M
M
M
6
4
2
0
1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3
Log10 Drug Concentration (M)
Figure 13. Area of clearance on Escherichia coli (Gram −ve) by 7a–c and
8a–c.
Figure 14. Cytotoxicity curves from typical MTT assays showing the effect
of compounds 4a–c on the viability of Caki-1 cells (4a and 4b have
overlapping response curves around their IC50 concentrations).
and 11-fold increase in cytotoxicity when compared with 8b and
8c and ranges therefore in the cytotoxicity class of platinum-based
drugs.
Conclusions and Outlook
Symmetrically and non-symmetrically substituted NHC–silver
acetate complexes show very similar IC50 values; both compound
classes are easily soluble in DMSO and all compounds are stable
in saline solution for 24 h with respect to silver chloride or silver
precipitation. It was also observed that, compared with known
reported NHC–silver complexes from the literature,[10,19] the
NHC–silver complexes (4a–c and 8a–c) have almost the same
cytotoxic activity.
A series of six novel symmetrically substituted and non-
symmetrically substituted N-heterocyclic carbene–silver acetate
derivatives (4a–c and 8a–c) were synthesized through the
reaction of appropriately symmetrically substituted and non-
symmetrically substituted N-heterocyclic carbenes (3a–c and
7a–c) with silver acetate. Almost all the complexes have shown
high antibacterial activity compared with the precursors and it is
also clear that, as the precursors and complexes concentration
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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Benzyl- or p-cyanobenzyl-substituted NHC-silver complexes
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1.0
0.8
0.6
0.4
8a, IC50
= M
3.2 (+/-0.6).10-6
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34 ( 3) µM respectively on the Caki-1 cell line. The complex 8a,
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The authors thank the Irish Research Council for Science
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