Synthesis, cytotoxicity and antibacterial studies of novel symmetrically and non-symmetrically p-nitrobenzyl-substituted N-heterocyclic carbene-silver(i) acetate complexes

Article abstract of DOI:10.1002/zaac.201000395

From the reaction of 1H-imidazole (1a), 4,5-dichloro-1H-imidazole (1b), 1H-benzimidazole (1c), 1-methylimidazole (1d), 4,5-dichloro-1-methylimidazole (1e) and 1-methylbenzimidazole (1f) with p-nitrobenzyl bromide (2), symmetrically and non-symmetrically p

Full text of DOI:10.1002/zaac.201000395

ARTICLE
DOI: 10.1002/zaac.201000395
Synthesis, Cytotoxicity and Antibacterial Studies of Novel Symmetrically
and Non-Symmetrically p-Nitrobenzyl-Substituted N-Heterocyclic Carbene–
Silver(I) Acetate Complexes
Siddappa Patil,[a] Anthony Deally,[a] Brendan Gleeson,[a] Frauke Hackenberg,[a]
Helge Müller-Bunz,[a] Francesca Paradisi,[a] and Matthias Tacke*[a]
Keywords: Antitumour agents; Antibacterial drugs; Silver; N-heterocyclic carbenes; Caki-1
Abstract. From the reaction of 1H-imidazole (1a), 4,5-dichloro-1H- silver(I) acetate (4f), respectively. The two NHC–silver(I) acetate com-
imidazole (1b), 1H-benzimidazole (1c), 1-methylimidazole (1d), 4,5- plexes 4a and 4e were characterised by single-crystal X-ray diffraction.
dichloro-1-methylimidazole (1e) and 1-methylbenzimidazole (1f) with All compounds studied in this work were preliminary screened for
p-nitrobenzyl bromide (2), symmetrically and non-symmetrically p- their antimicrobial activities in vitro against Gram-positive bacteria
nitrobenzyl-substituted N-heterocyclic carbene (NHC) [(3a–f)] precur- Staphylococcus aureus, and Gram-negative bacteria Escherichia coli
sors were synthesised. These NHC-precursors were then reacted with using the qualitative Kirby–Bauer disk-diffusion method. All NHC–
silver(I) acetate to yield the NHC-silver acetate complexes [1,3-bis(4- silver(I) acetate complexes exhibited medium to high antibacterial ac-
nitrobenzyl)imidazol-2-ylidene]silver(I) acetate (4a), [4,5-dichloro- tivity with areas of clearance ranging from 3 to 7 mm at the highest
1,3-bis(4-nitrobenzyl)imidazol-2-ylidene]silver(I) acetate (4b), [1,3- amount used, whereas the NHC-precursors showed significantly lower
bis(4-nitrobenzyl)benzimidazol-2-ylidene]silver(I) acetate (4c), [1- activity. In addition, NHC–silver(I) acetate complexes 4a–f had their
methyl-3-(4-nitrobenzyl)imidazole-2-ylidene] silver(I) acetate (4d), preliminary cytotoxicity tests on the human renal-cancer cell line Caki-
[4,5-dichloro-1-methyl-3-(4-nitrobenzyl)imidazole-2-ylidene] silver(I) 1 and showed medium to high cytotoxicity with IC50 values ranging
acetate (4e) and [1-methyl-3-(4-nitrobenzyl)benzimidazole-2-ylidene] from 15 (+/–1) to 27 (+/–2) μM.
cillin. Only when first resistances were reported, silver com-
Introduction
pounds regained importance. Antibacterial activity of silver
compounds is attributed to soluble silver ions, Ag+. Elementary
The biomedical applications of metal complexes based on N-
heterocyclic carbene (NHC)[1–5] are just beginning to unfold,
despite such complexes being phenomenally successful in ho-
mogeneous catalysis.[6–8] N-Heterocyclic carbene complexes
of silver are commonplace in the organometallic literature. The
interest in NHC–silver complexes is largely due to their ease
of synthesis and their ability to serve as useful to other NHC–
metal complexes by NHC transfer reactions.[9] In addition,
their diverse properties in bonding and structure and potential
applications in medicine,[10–15] nanomaterials,[16] liquid
crystals[17] and organic catalysis[18] also contribute to the at-
traction of NHC–silver complexes.
Silver compounds have been used for medicinal applications
for more than hundred years. It was targeted for water purifica-
tion, wound care antiseptics and infections. Silver nitrate was
commonly used as antimicrobial compound since the 17th and
18th century before it lost relevance with the discovery of peni-
silver as well as insoluble silver salt precipitates, such as silver
chloride, do not reveal antibacterial activity. The only side ef-
fect of long term silver treatment is the more or less unpleasant
colour change to grey or blue skin of patients. The effect called
Argyria occurs due to irreversible formation and deposition of
silver sulfide in dermis and eyes.
The introduction of silver sulfadiazine (silvadine) as an ef-
fective antimicrobial agent was reported by Fox in 1968.[19]
High antimicrobial activity and minimal side effects of silver
sulfadiazine have made it a very convenient therapy for the
treatment of infections in burns over the past four
decades.[20–23] Silver–carbene complexes, in particular those of
N-heterocyclic carbenes, have gained a significant amount of
interest in the past few years.[9,18] The first use of silver NHCs
as antimicrobial agents was reported by Youngs et al. in
2004.[24] In this report, two silver(I) complexes [silver(I) 2,6-
bis(ethanolimidazolemethyl)pyridine hydroxide and silver(I)
2,6-bis(propanolimidazolemethyl)pyridine hydroxide] showed
better antimicrobial activity than AgNO3 against the microor-
ganisms, Escherichia coli, Staphylococcus aureus and Pseudo-
monas aeruginosa. Another important contribution by the
Ghosh research group led to the synthesis and antimicrobial
evaluation of NHC–silver complexes derived from 1-benzyl-3-
* Prof. Dr. M. Tacke
E-Mail: matthias.tacke@ucd.ie
[a] 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
386
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2011, 637, 386–396

Bioorganometallic Silver Drugs
tert-butylimidazole.[25] Recently, Gürbüz and colleagues have
shown that the new imidazolidin-2-ylidene silver complexes
displayed effective antimicrobial activity against a series of
bacteria and fungi.[26]
Results and Discussion
Synthesis
The synthetic route for symmetrically and non-symmetrically
p-nitrobenzyl-substituted N-heterocyclic carbenes as ligand
precursors and their corresponding silver(I) acetate complexes
described in this work is given in Scheme 1, Scheme 2 and
Scheme 3, respectively. The symmetrically substituted NHC
precursors 1,3-bis(4-nitrobenzyl)imidazolium bromide (3a)
and 1,3-bis(4-nitrobenzyl)benzimidazolium bromide (3c) were
prepared by stirring 1H-imidazole (1a) and 1H-benzimidazole
(1c) with 2 equivalents of p-nitrobenzyl bromide (2) in the
presence of K2CO3 as a base in CH3CN at room temperature
for 3 d with 93 % and 81 % yields respectively. 4,5-Dichloro-
1,3-bis(4-nitrobenzyl)imidazolium bromide (3b) was prepared
by heating 4,5-dichloro-1H-imidazole (1b) with 2 equivalents
of p-nitrobenzyl bromide (2) in the presence of K2CO3 as a
base in CH3CN for 6 d with a yield of 79 %. The non-symmet-
rically substituted NHC precursors 1-methyl-3-(4-nitroben-
zyl)imidazolium bromide (3d) and 1-methyl-3-(4-nitroben-
zyl)benzimidazolium bromide (3f) were prepared by stirring 1-
methylimidazole (1d) and 1-methylbenzimidazole (1f) with p-
nitrobenzyl bromide (2) in toluene at room temperature for
2 d with 67 % and 80 % yields respectively. 4,5-Dichloro-1-
methylimidazole (1e) is formed in 97 % yield from the depro-
tonation of 4,5-dichloroimidazole (1b) with potassium hydrox-
ide and subsequent methylation with iodomethane in acetoni-
Cancer is a widely spread disease that remains an important
public health problem throughout Europe. According to Inter-
national Agency for Research on Cancer, nearly 2.9 million
new cases of cancer and more than 1.7 million cancer deaths
were reported in the Europe in 2004.[27] The number of new
cases diagnosed each year even increased by 300,000 in
2006.[28] For some sorts of cancer still no treatment exists or
the treatment implies harsh conditions for the patients. Namely
the treatment for the four most mortal cancer forms – lung,
colorectal, breast and stomach cancer-need to make quick and
significant progress.[27] This situation calls for the development
of milder and more specific anticancer drugs.
The introduction of cisplatin into clinics in 1978 started a
broad search for other cytotoxic metal complexes. Despite the
resounding success of cisplatin and closely related platinum
antitumor agents, the movement of other transition metal anti-
cancer drugs towards the clinic has been exceptionally
slow.[29–31] In general, metallocene dichlorides (Cp2MCl2) with
M = Ti, V, Nb and Mo show remarkable antitumor
activity,[32,33] and titanocene dichloride, Cp2MCl2, has espe-
cially become a very promising candidate as an anticancer
drug. Cp2MCl2 shows medium-high cytotoxicity in vitro and a
high efficacy in the animal models. Unfortunately, the efficacy
in Phase II clinical trials in patients with metastatic renal-cell
carcinoma[34] or metastatic breast cancer[35] was too low to be
pursued. Further substitution of the cyclopentadienyl ligand led
to bis[(p-methoxybenzyl) cyclopentadienyl] titanium(IV) di-
chloride (Titanocene Y), that showed to be more effective in
trile.
4,5-Dichloro-1-methyl-3-(4-nitrobenzyl)imidazolium
bromide (3e) is formed in 82 % yield by the reaction of 4,5-
dichloro-1-methylimidazole (5b) with p-nitrobenzyl bromide
(2) in toluene. The NHC precursors were fully characterised
by 1H, 13C NMR, IR, UV/Vis spectroscopy, mass spectrometry
1
and elemental analysis. The H NMR spectra of all precursors
the treatment against xenografted CAKI-1 tumours in mice 3a–f show a characteristic downfield shift in the range 9.46–
than that of cisplatin.[36]
10.87 ppm for the NCHN proton attributable to the positive
charge of the molecule.[37,38] Additionally, their identities have
also been confirmed by a base peak for the [M+ – Br] frag-
ments in their positive mode ESI mass spectra.
Recently, carbene–silver complexes have been reported to
have anticancer activity in vitro. Young 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).[14] These silver complexes were shown to be very
stable and can be synthesized efficiently. We have recently re-
ported the anticancer and antibacterial activity of symmetri-
cally and non-symmetrically p-methoxybenzyl-, p-cyanoben-
zyl- or benzyl-substituted N-heterocyclic carbene–silver
complexes. All the reported NHC–silver complexes have
shown medium to high anticancer and antibacterial
activity.[37,38] This encourages further research on N-heterocy-
clic carbene–silver complexes as cytotoxic drug candidates.
Within this paper, we present the synthesis, preliminary cyto-
toxicity and antimicrobial activity of a series of six novel sym-
metrically and non-symmetrically p-nitrobenzyl-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.
The NHC-silver complexes [1,3-bis(4-nitrobenzyl)imidazol-
2-ylidene]silver(I) acetate (4a), [4,5-dichloro-1,3-bis(4-nitrob-
enzyl)imidazol-2-ylidene]silver(I) acetate (4b), [1,3-bis(4-ni-
trobenzyl)benzimidazol-2-ylidene]silver(I) acetate (4c), 1-
methyl-3-(4-nitrobenzyl)imidazole-2-ylidene) silver(I) acetate
(4d), (4,5-dichloro-1-methyl-3-(4-nitrobenzyl)imidazole-2-yl-
idene) silver(I) acetate (4e) and (1-methyl-3-(4-nitroben-
zyl)benzimidazole-2-ylidene)silver(I) acetate (4f) were synthe-
sized by the reaction of 3a–f with 2 equivalents of silver(I)
acetate in dichloromethane/methanol. The reaction mixture
was stirred for 2–3 d at room temperature to afford the NHC–
silver(I) acetate complexes as off white solids in 53 % to 79 %
yield. The complexes were fully characterised by 1H, 13C
NMR, IR, UV/Vis spectroscopy, mass spectrometry and ele-
mental analysis. Furthermore, the solid-state structures of 4a
and 4e were analysed by single-crystal X-ray diffraction. The
absence of a downfield NCHN signal and presence of new
signals at δ = 2.09–1.76 ppm for the acetate protons in all
1
the H NMR spectra for 4a–f, however, indicates a successful
Z. Anorg. Allg. Chem. 2011, 386–396
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
387

M. Tacke et al.
ARTICLE
Scheme 1. General reaction scheme for the synthesis of symmetrically p-nitrobenzyl-substituted N-heterocyclic carbenes (3a–c) and their corre-
sponding N-heterocyclic carbene–silver(I) acetate complexes (4a–c).
Scheme 2. General reaction scheme for the synthesis of non-symmetrically p-nitrobenzyl-substituted N-heterocyclic carbenes (3d and 3f) and
their corresponding N-heterocyclic carbene–silver(I) acetate complexes (4d and 4f).
Scheme 3. General reaction scheme for the synthesis of non-symmetrically p-nitrobenzyl-substituted N-heterocyclic carbene (3e) and its N-
heterocyclic carbene–silver(I) acetate complex (4e).
complex formation. The 13C NMR resonances of the carbene the formation of the NHC–silver(I) acetate complexes.[1,12,37,38]
carbon atoms in complexes 4a–f occur in the range 183.3– Furthermore, positive mode ESI mass spectra of all six NHC-
179.0 ppm respectively. These signals are shifted downfield silver(I) acetate complexes (4a–f) are dominated by [M+–
compared to the corresponding precursors of 3a–f carbene car- O2CCH3] fragment peaks arising from the loss of one acetate
bons resonance at the range 130.0–141.8 ppm, respectively, ligand.
which further demonstrates the formation of expected NHC–
silver(I) acetate complexes. Also the appearance of the 13C
Structural Discussion
NMR spectroscopic resonances for the carbonyl and methyl
carbon atoms of the acetate group of complexes 4a–f in the
Suitable crystals for X-ray crystallography to determine the
range 163.9–177.4 and 22.0–24.2 ppm respectively showed molecular structure of 4a and 4e were grown from saturated
388 www.zaac.wiley-vch.de
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2011, 386–396

Bioorganometallic Silver Drugs
dichloromethane solutions with slow infusion of pentane. 4a acetate group might be disordered across the twofold axis,
crystallised with four molecules in the unit cell, whilst 4e crys- making the two Ag–O distances slightly different. An attempt
tallised with two molecules in the unit cell. Compounds 4a and to refine this disorder was unsuccessful as the split positions
4e crystallised in the monoclinic space group C2/c (#15) and were too close to each other to be refined independently.
¯
triclinic space group P1 (#2). In compounds 4a and 4e there
is an absence of any lattice held water molecules or organic
solvent molecules in the unit cells of the determined structures.
The molecular structures of the compounds 4a and 4e are
shown in Figure 1 and Figure 2. The crystal data and refine-
ment details for two compounds are tabulated in Table 1,
whereas selected bond lengths and bond angles are compiled
in Table 2.
Biological Evaluation
The in vitro antibacterial activities of symmetrically and non-
symmetrically p-nitrobenzyl-substituted N-heterocyclic car-
benes and their corresponding silver complexes were tested
using the qualitative Kirby–Bauer disk diffusion method
against the chosen Gram-positive bacteria Staphylococcus au-
reus (NCTC 7447), and Gram-negative bacteria Escherichia
coli. The metal salt (silver acetate) used to prepare the com-
plexes and the solvent (DMSO) induced very little growth in-
hibition on the same bacteria as previously reported.[37,40] The
antibacterial activities of the NHC-precursors and their corre-
sponding NHC–silver complexes are summarized in Figure 3,
Figure 4, Figure 5, and Figure 6. The results are tabulated in
Table 3 and Table 4, respectively.
The results indicate that all the NHC-precursors 3a,b and
3d–f (except 3c) show a weak antibacterial activity against the
both Gram-positive bacteria Staphylococcus aureus and Gram-
negative bacteria Escherichia coli, whereas NHC-precursor 3c
has exhibited minimal antibacterial activity with an area of
clearance of 3 mm at 0.43 μmol. Medium antibacterial activity
was observed for all NHC-silver(I) acetate complexes 4a–f
against Escherichia coli but high antibacterial activity was ob-
served towards Staphylococcus aureus with an area of clear-
ance of 7 mm at 0.43 μmol.
Figure 1. X-ray diffraction structure of 4a; molecule; thermal ellip-
I
soids are drawn on the 50 % probability level; symmetry operations:
1 –x, y, 1.5–z.
The previously synthesized NHC–silver(I) acetate complexes
showed an activity of up to 12 mm area of clearance at a con-
centration of 0.46 μmol for (1-benzyl-3-methylbenzimidazole-
2-ylidene) silver(I) acetate.[38] Thus, the presented NHC–sil-
ver(I) acetate complexes 4a–f do not belong to the most prom-
ising drug candidates of the NHC–silver series. Possibly, the
nitro group on the benzyl ligand negatively affected the lipo-
philicity of these compounds. The compounds exhibit antimic-
robial activity when they are capable of penetrating into and
through the lipid membrane of microorganisms, once inside
they are able to interfere with cell metabolism by enzymatic
inhibition, for example. Consequently, higher lipophilicity en-
hances antibacterial activity. For example, complexes 4a–f re-
vealed increased antibacterial activity compared to their pre-
cursors 3a–f because NHC-silver acetate complexes are more
lipophilic than polar NHC-precursors.
Figure 2. X-ray diffraction structure of 4e; molecule; thermal ellip-
soids are drawn on the 50 % probability level.
The NHC–silver(I) acetate complexes 4a and 4e are mono-
nuclear complexes. In the NHC–silver(I) acetate complexes 4b
and 4e reported here, the bond lengths and angles in and di-
rectly around the NHC core agree very well among each other
Cytotoxicity Studies
We are interested in the different types of NHC-silver(I) ace-
and with literature data.[37–39] In 4a (Figure 1), the second oxy- tate complexes as possible anti-cancer drugs. Therefore, the in
gen atom of the acetate is 2.69(1) Å away from the silver vitro cytotoxicity of symmetrically and non-symmetrically p-
which is in our opinion still too far to call it a bond. 4e (Fig- nitrobenzyl-substituted NHC-silver(I) acetate complexes 4a–f
ure 2), however, lies on a twofold axis which makes both Ag– was evaluated by MTT-based assays[41] on the human cancer-
O distances equal. The thermal ellipsoids of the oxygen atoms, ous renal-cell line Caki-1. This test involves a 48 h drug expo-
the silver atom and C(10) suggest that the silver atom and sure period, followed by a 24 h recovery time. The log dose
Z. Anorg. Allg. Chem. 2011, 386–396
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
389

M. Tacke et al.
ARTICLE
Table 1. Crystal data and structure refinement for 4a and 4e.
Identification code
4a
4e
Empirical formula
Molecular formula
Formula weight
Crystal system
C19H17N4O6Ag
C19H17N4O6Ag
505.24
C13H12N3O4Cl2Ag
C13H12N3O4Cl2Ag
453.03
monoclinic
triclinic
¯
Space group
C2/c (#15)
P1 (#2)
Unit cell dimensions
a = 19.8284(5) Å
b = 12.8367(3) Å
c = 7.5132(2) Å
α = 90°
a = 7.8488(7) Å
b = 10.476(1) Å
c = 10.7467(8) Å
α = 65.757(8)°
β = 76.373(7)°
γ = 79.596(8)°
779.52(12) Å3
2
β = 93.235(2)°
γ = 90°
Volume
Z
1909.30(8) Å3
4
Density (calculated)
Absorption coefficient
F(000)
1.758 Mg·m–3
1.930 Mg·m–3
8.890 mm–1
13.750 mm–1
1016
448
Crystal size
0.2167 × 0.0269 × 0.0178 mm
4.10 to 76.97°
–24 ≤ h ≤ 24
–16 ≤ k ≤ 16
–9 ≤ l ≤ 9
0.1321 × 0.0323 × 0.0193 mm
4.59 to 62.49°
–9 ≤ h ≤ 9
Theta range for data collection
Index ranges
–12 ≤ k ≤ 11
–12 ≤ l ≤ 12
Reflections collected
Independent reflections
Completeness to θmax
19665
8806
2009 [R(int) = 0.0388]
99.5 %
2446 [R(int) = 0.0411]
98.1 %
Max. and min. Transmission
Data / restraints / parameters
Goodness-of-fit on F2
0.862 and 0.403
2009 / 0 / 139
1.149
0.811 and 0.395
2446 / 0 / 210
1.182
Final R indices [I > 2σ(I)]
R indices (all data)
R1 = 0.0482, wR2 = 0.1062
R1 = 0.0501, wR2 = 0.1077
0.971 and –2.371 e·Å–3
R1 = 0.0718, wR2 = 0.1475
R1 = 0.0758, wR2 = 0.1504
5.012 and –3.241 e·Å–3
Largest diff. peak and hole
Table 2. Selected bond lengths /Å and angles /° for compounds 4a and 4e.
Bond Lengths /Å
4a
4e
Bond Angles /°
4a
4e
N(2)–C(8)
N(2)–C(9)
C(9)–C(9)#1
Ag–C(8)
1.351(5)
1.386(5)
1.340(8)
2.065(6)
2.438(5)
1.258(5)
1.489(9)
1.362(12)
1.388(12)
N(2)#1–C(8)–N(2)
C(8)–N(2)–C(9)
C(9)#1–C(9)–N(2)
N(2)–C(8)–Ag
104.5(5)
111.1(3)
106.7(2)
127.8(2)
153.14(10)
53.7(2)
111.4(8)
166.8(3)
2.040(9)
2.173(7)
Ag–O(3)
C(8)–Ag–O(3)
O(3)–C(10)
C(10)–C(11)
C(8)–N(3)
C(10)–N(3)
C(9)–C(10)
O(3)–Ag–O(3)#1
C(10)–O(3)–Ag
O(3)–C(10)–O(3)#1
O(3)–C(10)–C(11)
N(2)–C(8)–N(3)
C(8)–N(3)–C(10)
C(9)–C(10)–N(3)
C(10)–C(9)–N(2)
C(12)–O(3)–Ag
O(3)–C(12)–O(4)
O(3)–C(12)–C(13)
O(4)–C(12)–C(13)
92.0(4)
1.379(13)
1.389(12)
1.355(13)
122.3(7)
118.9(4)
104.1(8)
110.6(8)
107.0(8)
106.8(8)
103.4(7)
124.2(10)
117.7(10)
118.1(10)
response curve for NHC–silver(I) acetate complexes 4a–f can and 1.5-fold increase in magnitude when compared with com-
be viewed in Figure 7 and Figure 8, respectively. pounds 4a and 4c respectively. In comparison with cisplatin
Symmetrically p-nitrobenzyl-substituted NHC–silver(I) ace- (IC50 value = 3.3 μM), 4b has an approximately fivefold de-
tate complexes 4a–c, which contains 1H-imidazole, 4,5-di- crease in magnitude. Non-symmetrically p-nitrobenzyl-substi-
chloro-1H-imidazole and 1H-benzimidazole groups, has IC50 tuted NHC-silver(I) acetate complexes 4d–f, which also con-
values 27 (+/–2), 15 (+/–1) and 22 (+/–2) μM, respectively. tains 1-methylimidazole, 4,5-dichloro-1-methylimidazole and
Compound 4b is the most promising candidate in this paper 1-methylbenzimidazole groups, has IC50 values 20 (+/–1), 17
because of lowest IC50 value and has an approximately twofold (+/–1) and 16 (+/–9) μM, respectively. Compounds 4d–f show
390
www.zaac.wiley-vch.de
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2011, 386–396

Bioorganometallic Silver Drugs
Figure 3. Area of clearance on Staphylococcus aureus (Gram +ve) by
3a–c and 4a–c.
Figure 6. Area of clearance on Escherichia coli (Gram -ve) by 3d–f
and 4d–f.
Table 3. Area of clearance for compounds 3a–f in mm.
Compounds
Staphylococcus aureus Escherichia coli
(Gram + ve)
(Gram – ve)
3a /mm 0.11 μmol (45.0 μg)
0.15 μmol (63.0 μg)
1
1
1
1
1
1
2
1
1
2
2
1
2
3
3
1
1
1
2
1
1
2
2
1
1
1
2
0.21 μmol (90.0 μg)
0.43 μmol (180.0 μg) 2
3b /mm 0.11 μmol (52.0 μg)
1
1
0.15 μmol (72.8 μg)
0.21 μmol (104.5 μg) 1
0.43 μmol (209.0 μg) 2
3c /mm 0.11 μmol (50.0 μg)
1
2
0.15 μmol (70.0 μg)
0.21 μmol (100.5 μg) 2
0.43 μmol (201.0 μg) 3
3d /mm 0.11 μmol (32.0 μg)
0.15 μmol (44.8 μg)
1
1
1
Figure 4. Area of clearance on Escherichia coli (Gram -ve) by 3a–c
and 4a–c.
0.21 μmol (64.0 μg)
0.43 μmol (128.0 μg) 2
3e /mm 0.11 μmol (39.2 μg)
0.15 μmol (54.9 μg)
1
1
1
0.21 μmol (78.5 μg)
0.43 μmol (157.0 μg) 2
3f /mm 0.11 μmol (37.2 μg)
0.15 μmol (52.1 μg)
1
1
1
0.21 μmol (74.5 μg)
0.43 μmol (149.0 μg) 2
Symmetrically and non-symmetrically p-nitrobenzyl-substi-
tuted NHC-silver(I) acetate complexes show almost similar
IC50 values; both compound classes are easily soluble in
DMSO and all compounds are stable in saline solution with
respect to silver chloride precipitation. It was also observed
that, compared to known reported NHC-silver complexes from
the literature,[37,38] the NHC-silver complexes (4a–f) have al-
most same cytotoxic activity.
Figure 5. Area of clearance on Staphylococcus aureus (Gram +ve) by
3d–f and 4d–f.
Conclusions and Outlook
a very similar IC50 value and in comparison with cisplatin
In summary, a series of six new symmetrically and non-sym-
(IC50 value = 3.3 μM) the IC50 values for the three compounds metrically p-nitrobenzyl-substituted NHC–silver(I) acetate
are not impressive.
complexes 4a–f were synthesised through the reaction of ap-
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.zaac.wiley-vch.de 391
Z. Anorg. Allg. Chem. 2011, 386–396

M. Tacke et al.
ARTICLE
Table 4. Area of clearance for compounds 4a–f in mm.
Compounds
Staphylococcus aureus Escherichia coli
(Gram + ve)
(Gram – ve)
4a /mm 0.11 μmol (54.2 μg)
4
5
3
4
4
5
3
4
5
6
3
4
4
5
3
4
4
5
3
4
4
5
3
4
4
5
0.15 μmol (75.9 μg)
0.21 μmol (108.5 μg) 6
0.43 μmol (217.0 μg) 7
4b /mm 0.11 μmol (62.5 μg)
4
5
0.15 μmol (87.5 μg)
0.21 μmol (125.0 μg) 5
0.43 μmol (250.0 μg) 7
4c /mm 0.11 μmol (59.5 μg)
4
5
0.15 μmol (83.3 μg)
0.21 μmol (119.0 μg) 6
0.43 μmol (238.0 μg) 7
4d /mm 0.11 μmol (41.2 μg)
0.15 μmol (57.7 μg)
4
4
6
0.21 μmol (82.5 μg)
Figure 8. Cytotoxicity curves from typical MTT assays showing the
effect of compounds 4d–f on the viability of Caki-1 cells.
0.43 μmol (165.0 μg) 7
4e /mm 0.11 μmol (48.5 μg)
0.15 μmol (67.9 μg)
4
5
6
0.21 μmol (97.0 μg)
0.43 μmol (194.0 μg) 7
4f /mm 0.11 μmol (46.5 μg)
0.15 μmol (65.1 μg)
4
5
6
to improve these values by performing formulation experi-
ments to improve solubility of these NHC–silver(I) acetate
complexes, which should allow for in vivo testing of 4b in the
nearby future.
0.21 μmol (93.0 μg)
0.43 μmol (186.0 μg) 7
Experimental Section
Materials and Measurements
All reagents were of analytical grade; they were purchased and used
without further purification. 1H-Imidazole, 4,5-dichloro-1H-imidazole,
1H-benzimidazole, 1-methylimidazole, 1-methylbenzimidazole, p-ni-
trobenzyl bromide, silver acetate, methyl iodide and K2CO3 were pro-
cured commercially from Sigma–Aldrich Chemical Company and
were used without further purification. NMR spectra were measured
with a Varian 400 MHz spectrometer. Chemical shifts are reported in
ppm and are referenced to TMS. IR spectra were recorded with a Per-
kin–Elmer Paragon 1000 FT-IR Spectrometer employing a KBr disc.
UV/Vis spectra were recorded with a Unicam UV4 Spectrometer. Elec-
tron spray mass spectrometry (MS) was performed with a quadrupole
tandem 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 posi-
tive ionisation) mode for compounds 1e, 3a–f and 4a–f. CHN analysis
was done with an Exeter Analytical CE-440 Elemental Analyser. Silver
was estimated by spectrophotometric methods (Atomic absorption
spectra 55B Varian), whereas chlorine and bromine were determined
in mercurimetric titrations. Crystal data were collected using an Oxford
Diffraction SuperNova diffractometer fitted with an Atlas detector.
Figure 7. Cytotoxicity curves from typical MTT assays showing the
effect of compounds 4a–c on the viability of Caki-1 cells.
propriately symmetrically and non-symmetrically p-nitroben-
zyl-substituted N-heterocyclic carbenes 3a–f with silver(I) ace-
tate. The preliminary antibacterial activity of the NHC- Compounds 4a was measured with Mo-Kα (0.71073 Å), and 4e with
Cu-Kα (1.54184 Å). 4a and 4e were collected at 100K. An at least
twice redundant dataset was collected, assuming that the Friedel pairs
are not equivalent. An analytical absorption correction based on the
shape of the crystal was performed.[42] The structures were solved by
direct methods using SHELXS-97[43] and refined by full-matrix least-
squares on F2 for all data using SHELXL-97.[43] All hydrogen atoms
were added at calculated positions and refined using a riding model.
Their isotropic temperature factors were fixed to 1.2 times (1.5 times
for methyl groups) the equivalent isotropic displacement parameters of
the carbon atom the hydrogen atom is attached to. Anisotropic thermal
precursors and their corresponding NHC–silver(I) acetate com-
plexes were tested in vitro against two microbial strains. All
the complexes have shown high antibacterial activity com-
pared to the precursors and it is also clear that, as the com-
plexes concentration increases, the antibacterial activity be-
comes higher. NHC–silver(I) acetate complexes 4a–f yielded
antitumor IC50 values of 27 (+/–2), 15 (+/–1), 22 (+/–2), 20
(+/–1) 17 (+/–1) and 16 (+/–9) μM, respectively, on the Caki-
1 cell line. The complex 4b however gave a superior IC50 value
of 15 (+/–1) μM. Further work is currently underway in order displacement parameters were used for all non-hydrogen atoms. Suita-
392 www.zaac.wiley-vch.de
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2011, 386–396

Bioorganometallic Silver Drugs
ble crystals of 4a and 4e were grown in a saturated dichloromethane stirred for 30 min in acetonitrile (30 mL) before 4-nitrobenzylbromide
solution with slow infusion of pentane. Further details about the data (0.864 g, 4.00 mmol) was added in one portion. The mixture was
collection are listed in Table 1, as well as reliability factors.
stirred at room temperature for 8 h. After the solvent was removed
under reduced pressure, water (50 mL) was added. The precipitate was
filtered off, washed with pentane and diethyl ether. The pink crystalline
powder was dried for 4 h under vacuum to yield the product 3c
(0.765 g, 1.63 mmol, 81.5 % yield).
CCDC-798552 (for 4a), and -798553 (for 4e), respectively, contain the
supplementary crystallographic data for this paper. These data can be
obtained free of charge from the Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
1H NMR ([D6]DMSO, 400 MHz): δ = 10.09 (s, 1 H, NCHN), 8.27
(d, J = 8.0 Hz, 4 H, CHNitrobenzyl), 7.91 (dd, J = 6.3, 3.1 Hz, 2 H,
CHBenzimid), 7.77 (d, J = 8.0 Hz, 4 H, CHNitrobenzyl), 7.63 (dd, J = 6.3,
3.1 Hz, 2 H, CHBenzimid), 5.98 (s, 4 H, CH2). 13C NMR ([D6]DMSO,
100 MHz, proton decoupled): δ = 148.0, 144.1, 141.5, 131.5, 129.9,
127.5, 124.4, 114.4 (NCN+CBenzimid+CNitrobenzyl), 49.7 (CH2). IR
(KBr): 3391 (s), 3031 (w), 1607 (m), 1565 (m), 1523 (s), 1435 (w),
1349 (s), 1200 (w), 1109 (m), 1029 (w), 802 (w), 763 (s), 722 (m),
594 (w), 424 (w) cm–1. UV/Vis (CH3OH, nm): λ 264 (ε 11443), λ 366
(ε 2104). MS (m/z, QMS-MS/MS): 389.2 [M+-Br]. Micro Analysis
Calculated for C21H17N4O4Br (469.29): Calcd.: C, 53.75 %; H,
3.65 %; N, 11.94 %; Br, 17.03 %; Found: C, 53.55 %; H, 3.52 %; N,
12.00 %; Br, 17.14 %.
Syntheses
1,3-Bis(4-nitrobenzyl)imidazolium bromide (3a): A mixture of 1H-
imidazole (0.136 g, 2.00 mmol) and K2CO3 (0.414 g, 3.00 mmol) was
stirred for 15 min in acetonitrile (30 mL). 4-Nitrobenzyl bromide
(0.864 g, 4.00 mmol) was added in one portion and stirring was con-
tinued at room temperature for further 2 d. After the solvent was re-
moved under reduced pressure, water (50 mL) was added. The precipi-
tate was filtered off, washed with diethyl ether and dried in suction at
room temperature for 7 h to yield the product 3a (0.780 g, 1.86 mmol,
93.0 % yield) as brown solid.
1H NMR ([D6]DMSO, 400 MHz): δ = 9.46 (s, 1 H, NCHN), 8.28 (d,
J = 8.8 Hz, 4 H, CHNitrobenzyl), 7.89 (d, J = 1.6 Hz, 2 H, CHImid),
7.67 (d, J = 8.8 Hz, 4 H, CHNitrobenzyl), 5.62 (s, 4 H, CH2). 13C NMR
([D6]DMSO, 100 MHz, proton decoupled): δ = 139.6, 138.6, 130.0,
124.5, 123.1, 121.7 (NCN+CImid+ CNitrobenzyl), 52.2 (CH2). IR (KBr):
3427 (m), 3016 (m), 1605 (w), 1518 (s), 1347 (s), 1214 (w), 1151 (m),
1108 (m), 1018 (w), 858 (m), 807 (m), 756 (w), 729 (m), 619 (m)
cm–1. UV/Vis (CH3OH, nm): λ 259 (ε 10475), λ 368 (ε 2022), λ 443
(ε 1566). MS (m/z, QMS-MS/MS): 339.1 [M+-Br]. Micro Analysis
Calculated for C17H15N4O4Br (419.23): Calcd.: C, 48.70 %; H,
3.61 %; N, 13.36 %; Br, 19.06 %; Found: C, 49.00 %; H, 3.39 %; N,
13.05 %; Br, 19.15 %.
[1,3-Bis(4-nitrobenzyl)imidazol-2-ylidene]silver(I) acetate (4a):
1,3-Bis(4-nitrobenzyl)imidazolium bromide (0.419 g, 1.00 mmol) was
dissolved in dichloromethane (40 mL), silver(I) acetate (0.333 g,
2.00 mmol) was added, and the mixture was stirred at room tempera-
ture for 2 d. The yellow precipitate, presumably silver bromide, was
filtered off, and a clear solution was obtained. The volatile components
were removed in vacuo to produce a colourless sticky solid. The solid
was washed with diethyl ether (3 × 10 mL) and dried under reduced
pressure for 2 h to yield 4a (0.400 g, 0.79 mmol, 79.17 % yield) as a
colourless solid.
1H NMR (CDCl3, 400 MHz): δ = 8.27–8.20 (m, 4 H, CHNitrobenzyl),
7.51–7.44 (m, 4 H, CHNitrobenzyl), 7.06 (s, 2 H, CHImid), 5.46 (s, 4 H,
CH2), 2.09 (s, 3 H, COCH3). 13C NMR (CDCl3, 100 MHz, proton
decoupled): δ = 179.0 (NCN), 172.1 (C=O), 148.1, 141.9, 128.6,
124.4, 122.0 (CImid+CNitrobenzyl), 55.1 (CH2), 22.2 (COCH3). IR (KBr):
3436 (s), 2928 (w), 1577 (m), 1518 (s), 1413 (m), 1348 (s), 1246 (w),
1107 (m), 804 (m), 734 (m), 669 (w), 467 (m) cm–1. UV/Vis (CH3OH,
nm): λ 229 (ε 9292), λ 255 (ε 9033), λ 363 (ε 2306). MS (m/z, QMS-
MS/MS): 446.1 [M+-O2CCH3]. Micro Analysis Calculated for
C19H17N4O6Ag (505.23): Calcd.: C, 45.17 %; H, 3.39 %; N, 11.09 %;
Ag, 21.35 %; Found: C, 44.99 %; H, 3.41 %; N, 11.21 %; Ag,
21.43 %.
4,5-Dichloro-1,3-bis(4-nitrobenzyl)imidazolium bromide (3b): 4,5-
Dichloro-1H-imidazole (0.273 g, 2.00 mmol) and K2CO3 (0.414 g,
3.00 mmol) were stirred for 15 min in acetonitrile (30 mL). 4-Nitrob-
enzylbromide (0.432 g, 2.00 mmol) was added in one portion and stir-
ring was continued at room temperature for further 2 d. After the sol-
vent was removed under reduced pressure, water (50 mL) was added.
The aqueous phase was extracted with CH2Cl2 (4 × 20 mL). Organic
phases were combined and dried with magnesium sulfate. The residue
was obtained after solvent removal under reduced pressure. The result-
ing residue was dissolved in acetonitrile and another portion of 4-
nitrobenzylbromide (0.432 g, 2.00 mmol) was added. The reaction
mixture was heated under reflux for 4 d. The light yellow coloured
precipitate formed was filtered off and washed three times with diethyl
ether and dried in vacuoto yield the product 3b (0.750 g, 1.53 mmol,
76.8 % yield).
[4,5-Dichloro-1,3-bis(4-nitrobenzyl)imidazol-2-ylidene]silver(I) ac-
etate (4b): 4,5-Dichloro-1,3-bis(4-nitrobenzyl)imidazolium bromide
(0.488 g, 1.00 mmol) and silver(I) acetate (0.333 g, 2.00 mmol) were
mixed in methanol (50 mL) and stirred at room temperature for 2 d.
The yellow precipitate, presumably silver bromide, was filtered off 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 diethyl ether and dried under reduced pressure for
3 h to yield 4b (0.350 g, 0.60 mmol, 60.96 % yield) as a colourless
solid.
1H NMR ([D6]DMSO, 400 MHz): δ = 9.70 (s, 1 H, NCHN), 8.29 (d,
J = 8.7 Hz, 4 H, CHNitrobenzyl), 7.71 (d, J = 8.5 Hz, 4 H, CHNitrobenzyl),
5.71 (s, 4 H, CH2). 13C NMR ([D6]DMSO, 100 MHz, proton decou-
pled): δ = 148.1, 140.3, 138.0, 129.9, 124.4, 119.9 (NCN+CCl+
CNitrobenzyl), 51.2 (CH2). IR (KBr): 2978 (m), 1609 (m), 1580 (m),
1530 (s), 1353 (s), 1203 (w), 1146 (m), 1020 (w), 884 (w), 853 (m),
798 (w), 738 (m), 697 (w), 614 (w) cm–1. UV/Vis (CH3OH, nm): λ
255 (ε 10753), λ 366 (ε 2272). MS (m/z, QMS-MS/MS): 408.9 [M+-
Br]. Micro Analysis Calculated for C17H13N4O4Cl2Br (488.12):
Calcd.: C, 41.83 %; H, 2.68 %; N, 11.48 %; Cl, 14.53 %; Br, 16.37 %;
Found: C, 41.68 %; H, 2.38 %; N, 11.21 %; Cl, 14.40 %; Br, 16.58 %.
1H NMR ([D6]DMSO, 400 MHz): δ = 8.20 (d, J = 8.6 Hz, 4 H,
CHNitrobenzyl), 7.53 (d, J = 8.3 Hz, 4 H, CHNitrobenzyl), 5.65 (s, 4 H,
CH2), 1.79 (s, 3 H, COCH3). 13C NMR ([D6]DMSO, 100 MHz, proton
decoupled): δ = 183.3 (NCN), 175.2 (C=O), 147.4, 143.1, 128.6,
124.3, 118.1 (CCl+ CNitrobenzyl), 53.3 (CH2), 24.2 (COCH3). IR (KBr):
1,3-Bis(4-nitrobenzyl)benzimidazolium bromide (3c): 1H-Benzi- 3427 (s), 1572 (s), 1409 (s), 1347 (m), 1108 (w), 1016 (m), 797 (w),
midazole (0.236 g, 2.00 mmol) and K2CO3 (0.414 g, 3.00 mmol) were 735 (w), 650 (w) cm–1. UV/Vis (CH3OH, nm): λ 256 (ε 18567), λ
Z. Anorg. Allg. Chem. 2011, 386–396
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
393

M. Tacke et al.
ARTICLE
359 (ε 2520). MS (m/z, QMS-MS/MS): 515.1 [M+-O2CCH3]. Micro 1H NMR (CDCl3, 400 MHz): δ = 7.36 (s, 1 H, NCHN), 3.61 (s, 3 H,
Analysis Calculated for C19H15N4O6Cl2Ag (574.12): Calcd.: C, N-CH3). 13C NMR (CDCl3, 100 MHz, proton decoupled): δ = 134.6
39.75 %; H, 2.63 %; N, 9.76 %; Cl, 12.35 %; Ag, 18.79 %; Found: (NCN), 125.5, 113.7 (CCl), 32.5 (N-CH3). IR (KBr): 3437 (m), 3099
C, 39.54 %; H, 2.71 %; N, 9.57 %; Cl, 12.21 %; Ag, 18.81 %.
(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) cm–1. UV/Vis (CH3OH, nm): λ 225 (ε 4371), λ 275 (ε 933).
MS (m/z, QMS-MS/MS): 150.9 [M+]. Micro Analysis 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 %.
[1,3-Bis(4-nitrobenzyl)benzimidazol-2-ylidene]silver(I)
acetate
(4c): 1,3-Bis(4-nitrobenzyl)benzimidazolium bromide (0.469 g,
1.00 mmol) was dissolved in dichloromethane (40 mL). Silver(I) ace-
tate (0.333 g, 2.00 mmol) was added and the mixture was stirred at
room temperature for 2 d. The yellow precipitate, presumably silver
bromide, was filtered off and the solution was concentrated to 5 mL. 4,5-Dichloro-1-methyl-3-(4-nitrobenzyl)imidazolium bromide (3e):
Pentane (100 mL) was added and the desired product was filtered off To a solution of 4,5-Dichloro-1-methylimidazole (0.754 g, 5.00 mmol)
as a colourless precipitate. The solid was stirred in diethyl ether in acetonitrile (40 mL), 4-nitrobenzylbromide (1.08 g, 5.00 mmol)
(50 mL) for 30 minutes to remove the acetic acid and the product 4c was added; the resulting solution was stirred for 1 h at room tempera-
(0.375 g, 0.67 mmol, 67.53 % yield) was collected by vacuum filtra- ture and heated for 2 d at 85 °C. Afterwards, the solvent was removed
tion.
under reduced pressure. The resulting yellow residue was saturated
with diethyl ether to get a light yellow precipitate, which was filtered
off, washed with diethyl ether and finally dried in vacuoin order to
give compound 3e (1.50 g, 4.08 mmol, 81.73 % yield).
1H NMR ([D6]DMSO, 400 MHz): δ = 8.18 (d, J = 8.0 Hz, 4 H,
CHNitrobenzyl), 7.68 (dd, J = 6.0, 3.0 Hz, 2 H, CHBenzimid), 7.62 (d, J =
8.0 Hz, 4 H, CHNitrobenzyl), 7.37 (dd, J = 6.0, 3.0 Hz, 2 H, CHBenzimid),
5.94 (s, 4 H, CH2), 1.76 (s, 3 H, COCH3). 13C NMR ([D6]DMSO, 1H NMR ([D6]DMSO, 400 MHz): δ = 9.60 (s, 1 H, NCHN), 8.27 (d,
100 MHz, proton decoupled): δ = 190.5 (NCN), 176.0(C=O), 147.6, J = 8.7 Hz, 2 H, CHNitrobenzyl), 7.67 (d, J = 8.6 Hz, 2 H, CHNitrobenzyl),
144.1, 133.8, 129.0, 124.8, 124.3, 112.8 (CBenzimid+ CNitrobenzyl), 51.7 5.71 (s, 2 H, CH2), 3.86 (s, 3 H, N-CH3). 13C NMR ([D6]DMSO,
(CH2), 23.6 (COCH3). IR (KBr): 3441 (m), 2926 (w), 1575 (s), 1521 100 MHz, proton decoupled): δ = 148.1, 140.6, 137.6, 129.7, 124.3
(s), 1438 (w), 1402 (m), 1347 (s), 1185 (w), 1108 (m), 735 (m) cm–1. (NCN+CNitrobenzyl), 120.5, 118.6 (CCl), 50.8 (CH2), 35.6 (N-CH3). IR
UV/Vis (CH3OH, nm): λ 268 (ε 16956), λ 281(ε 14625), λ 361(ε 2549). (KBr): 3363 (s), 3063 (m), 2949 (w), 1519 (s), 1449 (m), 1349 (s),
MS (m/z, QMS-MS/MS): 496.1 [M+-O2CCH3]. Micro Analysis Cal- 1156 (m), 846 (m), 613 (m), 480 (w) cm–1. UV/Vis (CH3OH, nm): λ
culated for C23H19N4O6Ag (555.29): Calcd.: C, 49.75 %; H, 3.45 %; 229 (ε 8669), λ 255 (ε 8562), λ 368 (ε 1685). MS (m/z, QMS-MS/
N, 10.09 %; Ag, 19.43 %; Found: C, 49.14 %; H, 3.23 %; N, 10.00 %; MS): 287.0 [M+-Br]. Micro Analysis Calculated for C11H10N3Cl2
Ag, 19.15 %.
BrO2 (367.03): Calcd.: C, 36.00 %; H, 2.75 %; N, 11.45 %; Cl,
19.32 %; Br, 21.77 %; Found: C, 36.05 %; H, 2.65 %; N, 11.05 %;
Cl, 18.99 %; Br, 21.82 %.
1-Methyl-3-(4-nitrobenzyl)imidazolium bromide (3d): To a solution
of 1-methylimidazole (0.396 g, 5.00 mmol) in toluene (30 mL), 4-ni-
trobenzylbromide (1.08 g, 5.00 mmol) was added; the resulting solu- 1-Methyl-3-(4-nitrobenzyl)benzimidazolium bromide (3f): 4-Ni-
tion was stirred for 2 d at room temperature. Afterwards, the solvent trobenzylbromide (1.08 g, 5.00 mmol) was added to a solution of 1-
was removed under reduced pressure. The resultant residue was first methylbenzimidazole (0.660 g, 5.00 mmol) in toluene (45 mL). The
washed with pentane and afterwards with diethyl ether. Compound 3d reaction mixture was stirred at room temperature for 2 d. Afterwards,
(1.00 g, 3.35 mmol, 67.0 % yield) was obtained as a colourless solid the solvent was removed under reduced pressure. The resulting residue
after drying under reduced pressure.
was first washed with pentane and afterwards with diethyl ether. Com-
pound 3f (1.40 g, 4.02 mmol, 80.41 % yield) was obtained as a colour-
less solid after drying under reduced pressure.
1H NMR (CDCl3, 400 MHz): δ = 10.87 (s, 1 H, NCHN), 8.24 (d, J =
8.8 Hz, 2 H, CHNitrobenzyl), 7.79 (d, J = 8.8 Hz, 2 H, CHNitrobenzyl), 7.31
(s, 1 H, CHImid), 7.23 (s, 1 H, CHImid), 5.89 (s, 2 H, CH2), 4.08 (s, 3 1H NMR ([D6]DMSO, 400 MHz): δ = 9.91 (s, 1 H, NCHN), 8.26 (d,
H, N-CH3). 13C NMR ([D6]DMSO, 100 MHz, proton decoupled): δ = J = 8.7 Hz, 2 H, CHBenzimid), 8.06 (d, J = 7.6 Hz, 1H CHNitrobenzyl),
139.6,
138.6,
130.0,
124.5,
123.1,
121.7,
109.9
7.90 (d, J = 7.6 Hz, 1 H, CHNitrobenzyl), 7.81–7.57 (m, 4 H, CHBenzimid+
(NCN+CImid+CNitrobenzyl), 52.2 (CH2), 36.9 (N-CH3). IR (KBr): 3362 CHNitrobenzyl), 5.98 (s, 2 H, CH2), 4.13 (s, 3 H, N-CH3). 13C NMR
(s), 3011 (s), 1608 (w), 1758 (w), 1581 (s), 1450 (w), 1352 (s), 1168 ([D6]DMSO, 100 MHz, proton decoupled): δ = 148.0, 143.8, 141.8,
(s), 1105 (m), 1016 (w), 858 (m), 805 (m), 771 (w), 723 (s), 657 (w), 132.5, 131.1, 129.8, 127.2, 127.1, 124.3, 114.2, 113.9
624 (m), 478 (w) cm–1. UV/Vis (CH3OH, nm): λ 254 (ε 7702), λ 366 (NCN+CBenzimid+CNitrobenzyl), 49.3 (CH2), 33.9 (N-CH3). IR (KBr):
(ε 1297). MS (m/z, QMS-MS/MS): 218.1 [M+-Br]. Micro Analysis 3410 (m), 3108 (w), 2963 (s), 1607 (w), 1564 (m), 1518 (s), 1487 (m),
Calculated for C11H12N3BrO2 (298.14): Calcd.: C, 44.31 %; H, 1412 (w), 1346 (s), 1280 (w), 1201 (w), 1103 (m), 1016 (m), 856 (m),
4.06 %; N, 14.09 %; Br, 26.80 %; Found: C, 44.40 %; H, 4.00 %; N, 803 (m), 757 (s), 706 (s), 603 (m), 476 (w), 428 (m) cm–1. UV/Vis
13.90 %; Br, 26.25 %.
(CH3OH, nm): λ 240 (ε 11331), λ 265 (ε 13667). MS (m/z, QMS-MS/
MS): 268.1 [M+-Br]. Micro Analysis Calculated for C15H14N3O2Br
(348.19): Calcd.: C, 51.74 %; H, 4.05 %; N, 12.07 %; Br, 22.95 %;
Found: C, 51.01 %; H, 3.92 %; N, 12.15 %; Br, 23.00 %.
4,5-Dichloro-1-methylimidazole (1e): 4,5-Dichloroimidazole (1.23 g,
9.00 mmol) and potassium hydroxide (2.24 g, 40.0 mmol) were stirred
in acetonitrile (50 mL) for 2 h at room temperature. The excess potas-
sium hydroxide was filtered off from the solution and iodomethane (1-Methyl-3-(4-nitrobenzyl)imidazole-2-ylidene) silver(I) acetate
(0.562 mL, 9.00 mmol) was added. The reaction mixture was stirred (4d): 1-Methyl-3-(4-nitrobenzyl)imidazolium bromide (0.596 g,
at room temperature for 24 h. The volatile components were removed, 2.00 mmol) was dissolved in dichloromethane (50 mL). Silver(I) ace-
and the crude product was re-dissolved in dichloromethane. The solid, tate (0.667 g, 4.00 mmol) was added and the mixture was stirred for
presumably KI, was filtered off and discarded, and the volatile compo- 3 d at room temperature. The precipitate, presumably silver bromide,
nents were removed in vacuo to yield a yellow crystalline solid was filtered off and the solution was concentrated to 2 mL. Diethyl
(1.32 g, 8.72 mmol, 97.0 % yield) (1e).
ether was added and the product was crystallised in the freezer over-
394 www.zaac.wiley-vch.de
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2011, 386–396

Bioorganometallic Silver Drugs
night. The resulting colourless solid was filtered off and dried on a H, 3.71 %; N, 9.68 %; Ag, 24.84 %; Found: C, 46.98 %; H, 3.53 %;
vacuum line to yield the clean, dry product 4d (0.410 g, 1.06 mmol, N, 9.76 %; Ag, 24.51 %.
53.36 % yield).
1H NMR (CDCl3, 400 MHz): δ = 8.16 (d, J = 8.7 Hz, 2 H,
CHNitrobenzyl), 7.49 (d, J = 8.7 Hz, 2 H, CHNitrobenzyl), 7.15 (d, J =
Antibacterial Studies
2.0 Hz, 2 H, CHImid), 5.44 (s, 2 H, CH2), 3.87 (s, 3 H, N-CH3), 2.01
(s, 3 H, COCH3). 13C NMR (CDCl3, 100 MHz, proton decoupled):
δ = 180.1 (NCN), 177.4 (C=O), 147.6, 143.0, 128.5, 124.0, 123.1,
121.5 (CImid+CNitrobenzyl), 54.4 (CH2), 38.7 (N-CH3), 22.7 (COCH3).
IR (KBr): 3402 (m), 1571 (s), 1518 (s), 1409 (s), 1348 (s), 1233 (m),
1162 (s), 1108 (m), 1016 (m), 923 (m), 858 (m), 804 (m), 734 (w),
653 (w), 478 (w) cm–1. UV/Vis (CH3OH, nm): λ 260 (ε 11302), λ
Preliminary in vitro antibacterial activity of symmetrically and non-
symmetrically p-nitrobenzyl-substituted N-heterocyclic carbenes as li-
gand precursors and their corresponding silver(I) acetate complexes
were screened against two bacterial strains. The test organisms in-
cluded Staphylococcus aureus (SA) (NCTC 7447)as a Gram-positive
bacteria and Escherichia coli (E. coli) as Gram-negative bacteria.
356 (ε 1879). MS (m/z, QMS-MS/MS): 325.1 [M+-O2CCH3]. Micro To assess the biological activity of compounds 3a–f and 4a–f, the qual-
Analysis Calculated for C13H14N3O4Ag (384.14): Calcd.: C, 40.65 %; itative Kirby–Bauer disk-diffusion method was applied. All bacteria
H, 3.67 %; N, 10.94 %; Ag, 28.08 %; Found: C, 40.47 %; H, 3.82 %; were individually cultured from a single colony in sterile LB medium
N, 10.77 %; Ag, 28.00 %.
overnight at 37 °C (orbital shaker incubator). All the work carried out
was performed under sterile conditions.
(4,5-Dichloro-1-methyl-3-(4-nitrobenzyl)imidazole-2-ylidene) sil-
ver(I) acetate (4e): A mixture of 4,5-dichloro-1-methyl-3-(4-nitroben-
zyl)imidazolium bromide (0.367 g, 1.00 mmol) and silver(I) acetate
(0.333 g, 2.00 mmol) in dichloromethane (50 mL) was stirred for 2 d
at room temperature. The yellow silver bromide suspension was fil-
tered off to give a colourless solution. The volatile components were
removed in vacuo to produce a colourless sticky solid. The solid was
first washed with pentane and afterwards with diethyl ether and dried
under reduced pressure for 2 h to yield 4e (0.350 g, 0.77 mmol,
77.25 % yield) as a colourless solid.
For each strain, 70 μL of culture were spread evenly on agar-LB me-
dium. Four 5 mm diameter Whatman paper discs were placed evenly
separated on each plate. Two stock solutions (90:10 DMSO:H2O) of
every compound were prepared at 2.1 μM and 4.3 μM to be able to
test the effect of different concentrations. Each plate was tested with
5 μL and 7 μL of 2.1 μM solution and 5 μL and 10 μL for the 4.3 μM
solution. The plates were covered and placed in an incubator at 37 °C
for 24 h. The plates were afterwards removed and the area of clearance
(defined as the distance between the edge of the filter paper disc and
the beginning of the bacterial growth) for each sample was measured
in millimetres.
1H NMR (CDCl3, 400 MHz): δ = 8.23 (d, J = 8.7 Hz, 2 H,
CHNitrobenzyl), 7.53 (d, J = 8.7 Hz, 2 H, CHNitrobenzyl), 5.46 (s, 2 H,
CH2), 3.87 (s, 3 H, N-CH3), 2.09 (s, 3 H, COCH3). 13C NMR (CDCl3,
100 MHz, proton decoupled): δ = 180.9 (NCN), 172.4 (C=O), 148.1,
140.9, 128.6, 124.3, 118.8, 117.3 (CImid+CNitrobenzyl), 53.6 (CH2), 38.1
Cytotoxicity Studies
(N-CH3), 22.6 (COCH3). IR (KBr): 3419 (m), 2946 (w), 1581 (s), Preliminary in vitro cell tests were performed on the human cancerous
1528 (s), 1406 (m), 1347 (s), 1270 (w), 1195 (w), 1110 (m), 1015 (w),
renal cell line Caki-1 in order to compare the cytotoxicity of the com-
842 (m), 806 (m), 739 (w), 707 (m), 567 (w), 484 (w) cm–1. UV/Vis pounds presented in this paper. These cell lines were chosen based on
(CH3OH, nm): λ 259 (ε 9170), λ 367 (ε 1975). MS (m/z, QMS-MS/ their regular and long-lasting growth behaviour, which is similar to the
MS): 393.9 [M+-O2CCH3]. Micro Analysis Calculated for one shown in kidney carcinoma cells. They were obtained from the
C13H12N3O4Cl2Ag (453.03): Calcd.: C, 34.47 %; H, 2.67 %; N, ATCC (American Tissue Cell Culture Collection) and maintained in
9.28 %; Cl, 15.65 %; Ag, 23.81 %; Found: C, 34.32 %; H, 2.53 %; Dulbecco’s Modified Eagle Medium containing 10 % (v/v) FCS (fetal
N, 9.01 %; Cl, 15.34 %; Ag, 23.64 %.
calf serum), 1 % (v/v) penicillin streptomycin and 1 % (v/v) l-gluta-
mine. Cells were seeded in 96-well plates containing 200 μL microtitre
(1-Methyl-3-(4-nitrobenzyl)benzimidazole-2-ylidene)silver(I) ace- wells at a density of 5,000 cells/200 μL of medium and were incubated
tate (4f): 1-Methyl-3-(4-nitrobenzyl)benzimidazolium bromide at 37 °C for 24 h to allow for exponential growth. Afterwards, the
(0.348 g, 1.00 mmol) was dissolved in dichloromethane (60 mL), and compounds used for the testing were dissolved in the minimal amount
silver acetate (0.333 g, 2.00 mmol) was added. The mixture was stirred of DMSO (dimethylsulfoxide) possible and diluted with medium to
at room temperature for 2 d. The yellow precipitate, presumably silver obtain stock solutions of 5 × 10–4 m in concentration and less than
bromide, was filtered off and discarded. The volume of the reaction 0.7 % of DMSO. The cells were afterwards treated with varying con-
mixture was reduced under pressure to 5 mL. Pentane (100 mL) was centrations of the compounds and incubated for 48 h at 37 °C. Subse-
added and the fine colourless precipitate 4f (0.325 g, 0.74 mmol, quently, the solutions were removed from the wells and the cells were
74.85 % yield) was filtered off and washed with diethyl ether (75 mL). washed with PBS (phosphate buffer solution) and fresh medium was
added to the wells. Following a recovery period of 24 h incubation at
1H NMR (CDCl3, 400 MHz): δ = 8.19 (d, J = 8.7 Hz, 2 H, CHBenzimid), 37 °C, individual wells were treated with a solution of MTT (3-(4,5-
7.58–7.21 (m, 6 H, CHBenzimid+ CHNitrobenzyl), 5.74 (s, 2 H, CH2), 4.10 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in medium
(s, 3 H, N-CH3), 2.08 (s, 3 H, COCH3). 13C NMR (CDCl3, 100 MHz, (200 μL) (30 mg MTT in 30 mL medium). The cells were incubated
proton decoupled): δ = 179.0 (NCN), 163.9 (C=O), 147.9, 142.0, for 3 h at 37 °C. Afterwards, the medium was removed and the purple
134.6, 133.3, 128.0, 124.6, 124.6, 124.2, 111.5, 111.4 formazan crystals were dissolved in DMSO (200 μL per well). A Wal-
(CBenzimid+CNitrobenzyl), 52.4 (CH2), 36.0 (N-CH3), 22.6 (COCH3). IR lac–Victor (Multilabel HTS Counter) Plate Reader was used to meas-
(KBr): 3432 (s), 1564 (s), 1460 (s), 1397 (m), 1290 (s), 1106 (w), 1019 ure absorbance at 540 nm. Cell viability was expressed as a percentage
(w), 747 (m), 670 (w) cm–1. UV/Vis (CH3OH, nm): λ 269 (ε 9561), λ of the absorbance recorded for control wells. The values used for the
364 (ε 2131). MS (m/z, QMS-MS/MS): 375.9 [M+-O2CCH3]. Micro dose response curves represent the values obtained from four consist-
Analysis Calculated for C17H16N3O4Ag (434.19): Calcd.: C, 47.03 %; ent MTT-based assays for each compound tested.
Z. Anorg. Allg. Chem. 2011, 386–396
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
395

M. Tacke et al.
ARTICLE
[19] C. L. Fox, Arch. Surg. 1968, 96, 184.
Acknowledgement
The authors thank the Irish Research Council for Science Engineering
and Technology (IRCSET) for funding through a postdoctoral fellow-
ship for Dr. Siddappa Patil.
[20] A. R. C. Lee, H. Leem, J. Lee, K. C. Park, Biomaterials 2005,
26, 4670.
[21] H. S. Rosenkranz, H. S. Carr, Antimicrob. Agents Chemother.
1972, 25, 367.
[22] H. S. Carr, T. J. Wlodkowski, H. S. Rosenkranz, Antimicrob.
Agents Chemother. 1973, 4, 585.
[23] C. L. Fox, S. M. Modak, Antimicrob. Agents Chemother. 1974, 5,
582.
References
[24] A. Melaiye, R. S. Simons, A. Milsted, F. Pingitore, C. Wesdemio-
tis, C. A. Tessier, W. J. Youngs, J. Med. Chem. 2004, 47, 973.
[25] S. Ray, R. Mohan, J. K. Singh, M. K. Samantaray, M. M. Shaikh,
D. Panda, P. Ghosh, J. Am. Chem. Soc. 2007, 129, 15042.
[26] I. Özdemir, E. Ö. Özcan, S. Günal, N. Gürbüz, Molecules 2010,
15, 2499.
[1] A. Kascatan-Nebioglu, A. Melaiye, K. M. Hindi, S. Durmus,
M. J. Panzner, L. A. Hogue, R. J. Mallett, C. E. Hovis, M.
Coughenour, S. D. Crosby, A. Milsted, D. L. Ely, C. A. Tessier,
C. L. Cannon, W. J. Youngs, J. Med. Chem. 2006, 49, 6811.
[2] A. Melaiye, Z. Sun, K. M. Hindi, A. Milsted, D. L. Ely, D. H.
Reneker, C. A. Tessier, W. J. Youngs, J. Am. Chem. Soc. 2005,
127, 2285.
[27] P. Boyle, J. Ferlay, Annals Oncol. 2005, 16, 481.
[28] J. Ferlay, P. Autier, M. Boniol, M. Heanue, M. Colombet, P.
Boyle, Annals Oncol. 2007, 18, 581.
[3] J. C. Garrison, C. A. Tessier, W. J. Youngs, J. Organomet. Chem.
2005, 690, 6008.
[29] A. Gelasco, S. J. Lippard, Top. Biol. Inorg. Chem. 1999, 1, 1.
[30] E. R. Jamieson, S. J. Lippard, Chem. Rev. 1999, 99, 2467.
[31] N. Farrell, Y. Qu, J. D. Roberts, Top. Biol. Inorg. Chem. 1999, 1,
99.
[4] P. J. Barnard, L. E. Wedlock, M. V. Baker, S. J. Berners-Price,
D. A. Joyce, B. W. Skelton, J. H. Steer, Angew. Chem. 2006, 118,
6112; Angew. Chem. Int. Ed. 2006, 45, 5966.
[5] I. Özdemir, A. Denizci, H. T. Öztürk, B. Çetinkaya, Appl. Orga-
nomet. Chem. 2004, 18, 318.
[32] P. Köpf-Maier, H. Köpf, Chem. Rev. 1987, 87, 1137.
[33] F. Caruso, M. Rossi, Met. Ions Biol. Syst. 2004, 42, 353.
[34] G. Lummen, H. Sperling, H. Luboldt, T. Otto, H. Rubben, Cancer
Chemother. Pharmacol. 1998, 42, 415.
[6] W. A. Herrmann, Angew. Chem. 2002, 114, 1342; Angew. Chem.
Int. Ed. 2002, 41, 1290.
[7] E. Peris, R. H. Crabtree, Coord. Chem. Rev. 2004, 248, 2239.
[8] S. D. Gonzalez, N. Maria, S. P. Nolan, Chem. Rev. 2009, 109,
3612.
[35] N. Kröger, U. R. Kleeberg, K. B. Mross, L. Edler, G. Saß, D. K.
Hossfeld, Onkologie 2000, 23, 60.
[36] N. J. Sweeney, O. Mendoza, H. Müller-Bunz, C. Pampillo´n,
F. J. K. Rehmann, K. Strohfeldt, M. Tacke, J. Organomet. Chem.
2005, 690, 4537.
[9] I. J. B. Lin, C. S. Vasam, Coord. Chem. Rev. 2007, 251, 642.
[10] K. M. Hindi, A. J. Ditto, M. J. Panzeri, D. A. Medvetz, S. D.
Han, C. E. Hovis, J. K. Hilliard, J. B. Taylor, Y. H. Yun, C. L.
Cannon, W. J. Youngs, Biomaterials 2009, 30, 3771.
[11] A. Kascatan-Nebioglu, M. J. Panzner, C. A. Tessier, C. L. Can-
non, W. J. Youngs, Coord. Chem. Rev. 2007, 251, 884.
[12] K. M. Hindi, T. J. Siciliano, S. Durmus, M. J. Panzner, D. A.
Medvetz, D. V. Reddy, L. A. Hogue, C. E. Hovis, J. K. Hilliard,
R. Mallet, C. A. Tessier, C. L. Cannon, W. J. Youngs, J. Med.
Chem. 2008, 51, 1577.
[37] S. Patil, J. Claffey, A. Deally, M. Hogan, B. Gleeson, L. M. M.
Méndez, H. Müller-Bunz, F. Paradisi, M. Tacke, Eur. J. Inorg.
Chem. 2010, 1020.
[38] S. Patil, J. Claffey, A. Deally, B. Gleeson, H. Müller-Bunz, F.
Paradisi, M. Tacke, Appl. Organomet. Chem. 2010, 24, 781.
[39] P. De Fremont, N. M. Scott, E. D. Stevens, T. Ramnial, O. C.
Lightbody, C. L. B. Macdonald, J. A. C. Clyburne, C. D. Aber-
nethy, S. P. Nolan, Organometallics 2005, 24, 6301.
[40] B. Gleeson, J. Claffey, D. Ertler, M. Hogan, H. Müller-Bunz, F.
Paradisi, D. Wallis, M. Tacke, Polyhedron 2008, 27, 3619.
[41] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
[42] CrysalisPro, Version 1.171.33.55, Oxford Diffraction Limited,
2010.
[13] K. M. Hindi, M. J. Panzner, C. A. Tessier, C. L. Cannon, W. J.
Youngs, Chem. Rev. 2009, 109, 3859.
[14] D. A. Medvetz, K. M. Hindi, M. J. Panzner, A. J. Ditto, Y. H.
Yun, W. J. Youngs, Met. Based Drugs 2008, 384010.
[15] J. C. Y. Lin, R. T. W. Huang, C. S. Lee, A. Bhattacharyya, W. S.
Hwang, I. J. B. Lin, Chem. Rev. 2009, 109, 3561.
[16] E. Peris, Top. Organomet. Chem. 2007, 21, 83.
[17] C. K. Lee, C. S. Vasam, T. W. Huang, H. M. J. Wang, R. Y.
Yang, C. S. Lee, I. J. B. Lin, Organometallics 2006, 25, 3768.
[18] J. C. Garrison, W. J. Youngs, Chem. Rev. 2005, 105, 3978.
[43] G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112.
Received: November 1, 2010
Published Online: January 27, 2011
396
www.zaac.wiley-vch.de
© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2011, 386–396