Non-symmetrically substituted N-heterocyclic carbene-Ag(I) complexes of benzimidazol-2-ylidenes: Synthesis, crystal structures, anticancer activity and transmetallation studies

Article abstract of DOI:10.1016/j.ica.2012.09.013

Non-symmetrically substituted benzimidazolium salts (1 and 2) having bromide counterion undergo metallation with Ag₂O at stiochiometric ratio, giving mononuclear bis-carbene Ag(I) complexes (3 and 4), active as anticancer agents against Human C

Full text of DOI:10.1016/j.ica.2012.09.013

Inorganica Chimica Acta 394 (2013) 519–525
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Non-symmetrically substituted N-heterocyclic carbene–Ag(I) complexes
of benzimidazol-2-ylidenes: Synthesis, crystal structures, anticancer activity
and transmetallation studies
a
a
a
Rosenani A. Haque a, , Mohammed Z. Ghdhayeb , Srinivasa Budagumpi , Abbas Washeel Salman ,
⇑
Mohamed B. Khadeer Ahamed b, Amin Malik Shah Abdul Majid b
a The School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
b EMAN Research and Testing Laboratory, The School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2012
Received in revised form 6 September 2012
Accepted 19 September 2012
Available online 29 September 2012
Non-symmetrically substituted benzimidazolium salts (1 and 2) having bromide counterion undergo
metallation with Ag2O at stiochiometric ratio, giving mononuclear bis-carbene Ag(I) complexes (3 and
4), active as anticancer agents against Human Colorectal (HCT 116) cell lines. Both the complexes provide
good activity (IC50 = 13.9
l
M for 3 and 14.6
l
M for 4) in the anticancer studies and so distinctly better
M for 2). The molecular structure of Ag com-
than the ligand precursors (IC50 = 119.3
l
M for 1, and 70.0
l
plexes 3 and 4 is elucidated by X-ray diffraction studies. The Pd(II)– and Au(I)–NHC complexes (5 and 6)
were synthesized from 4 via the technique of transmetallation. However, attempts to produce transmet-
allated complexes using 3 were unsuccessful.
Keywords:
Anticancer agent
Ag(I)–carbene complex
N-Heterocyclic carbene
Transmetallation
X-ray diffraction
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
N-Allyl substituted versions of these popular ligand precursors
for the preparation of transition metal–carbene complexes are rap-
Benzimidazole derivatives are important constituents in most
pharmacologically, catalytically, and biologically active com-
pounds, and therefore correspond to significant synthetic targets
[1,2]. Transition metal derivatives of N-heterocyclic carbene
(NHC) ligands encompassing benzimidazole hub demonstrate
great utility across a wide variety of metal based drugs [3]. Imidaz-
ole and benzimidazole derived NHC precursors are known in the
field of organometallic chemistry for more than five decades, and
imidazole-based derivatives may have been the first NHC precur-
sors ever used by the organometallic chemists. However, transition
metal derivatives of benzimidazole-derived NHCs have a rich his-
tory in catalysis and pharmacology, starting with pioneering con-
tributions by Hahn and co-workers at the end of twentieth
century [4,5]. Among the wide range of biological applications,
N-substituted benzimidazolium salts are known to inhibit a vast
number of cytochrome P450 enzymes employed in hepatic drug
metabolism and steroid biosynthesis. In particular, N-substituted
benzimidazolium salts have been reported to poses antithrom-
botic, herbicidal, anthelmintic and catalytic properties since long
back [6].
idly emerging, however, only a few unique mono- and bis-NHC
architectures exist [7]. Interestingly, N-allyl-substituted dib-
enzotetraazafulvalenes react through rearrangement and produce
dealkylated products [8]. With the successful applications of sym-
metric and asymmetric allyl/alkyl substituted ligands in prepara-
tion of biologically active compounds, the rational design and
such ligands has been an important strategy for the development
of silver based drugs, which can significantly reduce the toxicity
and other side effects. In the recent years researchers have ex-
plored silver, gold and platinum-based NHCs having benzimidazole
core as excellent antitumor and anticancer agents in the form of
complexes. Their various biological applications with fewer side ef-
fects are now attracting the world’s attention [9]. Group-XI ele-
ments are so medicinal that simple amine derivatives of which
are known as ‘‘Drugs’’ in cancer treatment (cisplatin, carboplatin,
etc.) [10]. Recently, palladium complexes are also used for the
treatment of cancer with fewer side effects [11]. Complexes de-
signed for this particular work are on the basis of active sites of cis-
platin and its analogs used for cancer. Following cisplatin
administration, one of the chlorides is displaced by water, and
forms aqua-coordinated platinum, which is responsible to bind to
guanine bases. Either in a similar way or by the slow displacement
of one of the NHCs by water to form aqua-Ag-complex, which can
⇑
Corresponding author. Tel.: +60 19 4118 262, +60 4 653 3578.
E-mail address: rosenani@usm.my (R.A. Haque).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.09.013

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R.A. Haque et al. / Inorganica Chimica Acta 394 (2013) 519–525
further target the DNA of an infected cell. Most notable among the
similarities of reported complexes with cisplatin are the linear C–
Ag–C fragment (N–Pt–Cl in cisplatin) and the basicity of the Ag
ion to bind DNA of the infected cell to arrest cell growth. A litera-
ture survey revealed that the Ag(I)–NHC complexes of imidazole
and benzimidazole show an appreciable level of anticancer activity
against different cancer cell lines (Chart 1). Associated activity of
the complexes is mainly due to the presence of number of
(benz)imidazolium units and the nature of coordinated anions.
Herein we report synthesis, crystal structure, anticancer activity
and transmetallation studies of two new Ag(I)–NHC complexes,
in which the ligand scaffolds have been modified by installation
of allyl/butyl substituents.
tions, structure refinement, molecular graphics and the material
for publication were performed using the SHELXTL and PLATON (Spek,
2009) software packages.
2.2. Syntheses
2.2.1. Synthesis of Ag–carbene complex 3
To a suspension of Ag2O (0.46 g, 0.2 mmol) and 1 (0.249 g,
0.1 mmol) in methanol (20 mL) was stirred at room temperature
for 10 h. The flask was wrapped with aluminum foil to avoid light.
The reaction mixture was filtered through a pad of Celite, and the
resulted colorless filtrate was concentrated to 5 mL under vacuum.
The white solid was afforded by addition of 75 mL diethyl ether. So
obtained solid was redissolved in acetonitrile and 150 mL of
diethyl ether was added to reprecipitate the solid, isolation by fil-
tration and vacuum dried to yield 3. Yield: 0.149 g, 71.8%, M.P.:
212°. 1H NMR (500 MHz, d6-DMSO, 298 K): d 5.05 (2H, d, N–CH2,
J = 7.5 Hz), 5.16 (1H, m, CH2–CH@CH2), 5.59 (2H, s, N–CH2 benzyl),
5.96 (2H, m, CH@CH2), and 7.16–7.37 (5H, m, Ar–H). 13C{1H}NMR
(125 MHz, d6-DMSO, 298 K): d 50.01 (N–CH2CH), 51.4 (N–CH2–
CH@CH2), 114.8 (N–CH2Ar), 121.5 (CH@CH2), 127.6, 129.8 (ArC),
131.8, 134.3 (benzimidazolium ArC), and 189.07 (Ccarbene–Ag). FTIR
2. Experimental
2.1. Reagents and instruments
All chemicals and solvents were obtained from commercial
sources and used without further purifications. The benzimidazole,
Ag2O, benzyl bromide, allyl bromide, butyl bromide, 5-fluorouracil
(standard), and 3-[4,5-yl]-2,5-diphenyltetrazolium bromide for the
MTT assay were purchased from Sigma–Aldrich. 1-Benzylimida-
zole and 1-benzylbenzimidazole were prepared according to the
literature method with slight modifications. The carbene ligand
precursors, 1-allyl-3-benzylbenzimidazole bromide (1) and 1-ben-
zyl-3-butylbenzimdazole bromide (2) were prepared according to
an established procedure [12,13] with slight modifications. The
FTIR spectra of the compounds were recorded in potassium bro-
mide disks using a Perkin Elmer 2000 system spectrometer in
the range 4000 to 400 cmꢀ1. The NMR spectra were recorded in
d6-DMSO using Bruker 500 MHz Ascend spectrometer at ambient
temperature with TMS as an internal reference. All the compounds
were analyzed for C, H, and N using a PerkinElmer 2400 Series II
microanalyzer. The X-ray diffraction data were collected using a
Bruker SMART APEX2 CCD area-detector diffractometer. Calcula-
(KBr disc) cmꢀ1: 2868, 2923
m(C–H), 1476, 1192 m(benzimidazole
ring vibration). Anal. Calc. for C34H34N4AgBr: C, 59.5; H, 5.0; N,
8.2. Found: C, 59.8; H, 5.2; N, 7.9%.
2.2.2. Synthesis of Ag–carbene complex 4
This compound was prepared in a manner analogous to that for
3, only with 2 (0.347 g, 1 mmol) instead of 1. Yield: 0.16 g, 71.1%,
M.P.: 236 °C. 1H NMR (500 MHz, d6-DMSO, 298 K): d 0.96 (3H, t,
CH3, J = 7.25 Hz), 1.35 (6H, m, CH2–CH2–CH3), 1.93 (5H, m, CH2–
CH2–CH3), 4.53 (2H, t, N–CH2, J = 7.75 Hz), 5.79 (2H, s, N–CH2–
Ar), 7.38–7.66 (5H, m, Ar–H), and 7.96 (4H, t, benzimidazole ArH,
J = 7.5 Hz). 13C{1H}NMR (125 MHz, d6-DMSO, 298 K): d 13.3 (CH3),
19.2 (CH3–CH2–CH2), 30.5 (CH3–CH2–CH2), 46.4 (N–CH2), 50.2
(N–CH2, benzyl), 113.3, 126.5.7, 128.6 (Ar–C), 131.2–133.6
Chart 1. Anticancer potential of (benz)imidazole based silver complexes (I–V against Caki-1, and VI and VII against HCT cells from MTT assays).

R.A. Haque et al. / Inorganica Chimica Acta 394 (2013) 519–525
521
(benzimidazolium ArC), and 188.4, 189.0 (Ccarbene–Ag). FTIR (KBr
1493, 1181 m(benzimidazole ring vibration). Anal. Calc. for C18H21-
N2AuCl: C, 43.4; H, 4.3; N, 5.6. Found: C, 43.9; H, 5.9; N, 5.8%.
disc) cmꢀ1: ꢁ2860, 2925
m(C–H), 1488, 1187
m(benzimidazole ring
vibration). Anal. Calc. for C39H48N4AgBrO: C, 60.3; H, 6.2; N, 7.2.
Found: C, 60.6; H, 6.0; N, 7.8%.
2.3. Anticancer activity
2.2.3. Synthesis of Pd–carbene complex 5
2.3.1. Preparation of cell culture
A mixture of Ag–carbene complex 4 (0.194 g, 0.25 mmol) and
Pd(COD)Cl2 (0.071 g, 0.25 mmol) in dichloromethane (25 mL) was
stirred for 10 h at room temperature, and a pale yellow solution
was formed. The resulting yellow solution was filtered through a
pad of Cilite, and concentrated to 5 mL, and petroleum ether
(50 mL) was added to precipitate the pale yellow colored powder.
So obtained solid was redissolved in acetonitrile (10 mL) and
150 mL of diethyl ether was added to reprecipitate the solid, isola-
tion by filtration and vacuum dried to yield 5. Yield: 0.114 g, 64.9%.
M.P.: 244 °C. 1H NMR (500 MHz, d3-CD3CN, 298 K): d 0.82 (3H, t,
CH3, J = 6.75 Hz), 1.14 (3H, t, CH3, J = 6.5 Hz), 1.28 (6H, m, CH2–
CH2–CH3), 1.65 (6H, m, CH2–CH2–CH3), 2.15 (5H, m, CH2–CH2–
CH3), 2.31 (5H, m, CH2–CH2–CH3), 4.77 (2H, t, N–CH2, J = 7.0 Hz),
4.95 (2H, t, N–CH2, J = 6.5 Hz), 6.10 (2H, s, N–CH2–Ar), 6.24 (2H,
s, N–CH2–Ar), 7.13–7.68 (5H, m, Ar–H), and 7.90–8.19 (4H, t, benz-
imidazole ArH, J = 6.25 Hz). 13C{1H}NMR (125 MHz, d6-DMSO,
298 K): d 13.5 (CH3), 20.7 (CH3–CH2–CH2), 32.6 (CH3–CH2–CH2),
48.5 (N–CH2), 52.9 (N–CH2, benzyl), 110.6, 123.2, 127.9 (Ar–C),
134.2, 135.1 (benzimidazolium ArC), and 182.1 (Ccarbene–Pd). FTIR
Initially, cells (HCT 116) were allowed to grow under optimal
incubator conditions. Cells that have reached a confluency of 70–
80% were chosen for cell plating purposes. Old medium was aspi-
rated out of the plate. Next, cells were washed using sterile phos-
phate buffered saline (PBS) (pH 7.4), 2–3 times. PBS was
completely discarded after washing. Following this, trypsin was
added and distributed evenly onto cell surfaces. Cells were incu-
bated at 37 °C in 5% CO2 for 1 min. Then, the flasks containing
the cells were gently tapped to aid cells segregation and observed
under inverted microscope (if cells segregation is not satisfying,
the cells will be incubated for another minute). Trypsin activity
was inhibited by adding 5 ml of fresh complete media (10% FBS).
Cells were counted and diluted to get a final concentration of
2.5 ꢂ 105 cells/mL, and inoculated into wells (100
lL cells/well). Fi-
nally, plates containing the cells were incubated at 37 °C with an
internal atmosphere of 5% CO2.
2.3.2. MTT assay
(KBr disc) cmꢀ1: ꢁ2860, ꢁ2920
m(C–H), 1496, 1182 m(benzimid-
azole ring vibration). Anal. Calc. for C36H42N4PdCl2: C, 61.1; H,
6.0; N, 7.9. Found: C, 61.6; H, 6.3; N, 8.1%.
Cancer cells (100
l
L cells/well, 1.5 ꢂ 105 cells/mL) were inocu-
lated in wells of microtitre plate. Then the plate is incubated in
CO2 incubator for overnight in order to allow the cell for attach-
ment. One hundred microliters of test substance were added into
each well containing the cells. Test substance was diluted with
media into the desired concentrations from the stock. The plates
were incubated at 37 °C with an internal atmosphere of 5% CO2
2.2.4. Synthesis of Au–carbene complex 6
This compound was prepared in a manner analogous to that for
5, only with [AuCl(PPh3)2] (0.189 g, 0.25 mmol) instead of
Pd(COD)Cl2. Yield: 0.151 g, 79.0%, M.P.: 210 °C. 1H NMR
(500 MHz, d6-DMSO, 298 K): d 0.91 (3H, t, CH3, J = 6.25 Hz), 1.35
(6H, m, CH2–CH2–CH3), 1.96 (5H, m, CH2–CH2–CH3), 4.58 (2H, t,
N–CH2, J = 7.0 Hz), 5.76 (2H, s, N–CH2–Ar), 7.28–7.65 (5H, m, Ar–
H), and 7. 89 (4H, t, benzimidazole ArH, J = 6.25 Hz). 13C{1H}NMR
(125 MHz, d6-DMSO, 298 K): d 13.0 (CH3), 19.5 (CH3–CH2–CH2),
31.8 (CH3–CH2–CH2), 48.4 (N–CH2), 51.5 (N–CH2, benzyl), 112.0,
125.2, 127.0 (Ar–C), 133.2, 135.6 (benzimidazolium ArC), and
for 72 h. After this treatment period, 20
added into each well and incubated again for 4 h. After this incuba-
tion period, 50 L of MTT lysis solution (DMSO) was added into the
lL of MTT reagent was
l
wells. The plates were further incubated for 5 min in CO2 incuba-
tor. Finally, plates were read at 570 and 620 nm wavelengths using
a standard ELISA microplate reader. Data were recorded and ana-
lyzed for the assessment of the effects of test substance on cell via-
bility and growth inhibition. The percentage of growth inhibition
was calculated from the optical density (OD) that was obtained
190.3 (Ccarbene–Au). FTIR (KBr disc) cmꢀ1: 2862, 2932
m(C–H),
Scheme 1. Synthesis of allyl/alkyl substituted benzimidazol-2-ylidenes and their Ag–carbene complexes.
Scheme 2. Synthetic pathway to Pd– and Au–carbene complexes via transmetallation reactions.

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R.A. Haque et al. / Inorganica Chimica Acta 394 (2013) 519–525
Table 1
lic chemistry and carbene transfer reactions which can be used to
get access to a number of other late transition/inner transition me-
tal–carbene complexes [3]. In the present study, we describe the
preparations of carbene complexes and also investigated antican-
cer activity of benzimidazolium salts and their Ag complexes.
Reaction of the precursor 1/2 with Ag2O in a 2:1 molar ratio in
methanol gave the complex 3/4 in good yield, after recrystalliza-
tion from methanol–diethyl ether. Synthetic routes to the com-
plexes 3 and 4 are shown in Scheme 1.
Further, Pd(II)– and Au(I)-carbene complexes (5 and 6) were syn-
thesized in quantitative yields at a milder condition by transmetalla-
tion using Ag(I) complex 4 as a carbene transfer reagent (Scheme 2).
Similar transmetallation reactions of Ag(I)–carbene complexes hav-
ing cyclic and acyclic carbene ligands with [AuCl(SMe2)] in acetoni-
trile have been reported [14–16]. However, all our attempts to get
transmetallated products using Ag–carbene complex 3 were unsuc-
cessful, even variable amounts of starting materials were used. Diffu-
sion of diethyl ether into an acetonitrile solution of complex 4
afforded colorless block crystals suitable for single crystal X-ray dif-
fraction studies. The benzimidazolium salts, Ag(I)–, Pd(II)–, and
Au(I)–carbene complexes are thermally stable up to their melting
points and are nonhygroscopic in nature. The complexes are insolu-
ble in water, but are readily soluble in DMSO, DMF, (m)ethanol and
acetonitrile. Both the ligands and their Ag–carbene complexes were
evaluated for their in vitro cytotoxicity towards Human Colorectal
(HCT116) cell lines. Cytotoxic activitywas evaluated by colorimetric
MTT assay to calculate cell viability and growth inhibition.
Crystal data and structure refinement details for Ag–carbene complex 4.
3
4
Formula
C
37H34AgBrN5O
C39H48N4AgBrO
701.66
triclinic
Formula weight
Crystal system
Space group
Unit cell dimensions
a (Å)
697.19
triclinic
P1
ꢀ
ꢀ
P1
10.5279(2)
10.7966(3)
14.6437(2)
108.4830(10)
91.926(2)
100.9250(10)
1542.15(6)
2
12.3083(4)
13.0893(5)
13.5459(4)
115.974(2)
102.260(2)
103.788(2)
1775.58(10)
2
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dcalc (g/cm3)
1.501
0.698
1.312
0.604
Absorption coefficient
(mmꢀ1
)
F(000)
Crystal size (mm)
T (K)
Radiation (Å)
h Minimum, maximum (°)
Dataset
717
732
0.39 ꢂ 0.25 ꢂ 0.13
0.56 ꢂ 0.5 ꢂ 0.17
293
293
Mo K
a
0.71073
Mo Ka 0.71073
1.98, 30.12
ꢀ14:14, ꢀ15:15,
ꢀ20:20
1.79, 34.18
ꢀ19:18, ꢀ20:20,
ꢀ21:21
Total; unique data
Rint
Nref, Npar
32999
0.0253
51931
0.0679
8981, 725
0.0894, 0.2182, 1.318
14382, 772
0.0679, 0.1771, 1.085
R, wR2, S
from MTT assay, i.e., hundredth multiple of subtracted OD value of
control and survived cells over OD of control cells.
Structures of both, ligand precursors and carbene complexes, in
solution were deduced from the 1H, 13C and HMQC NMR spectra.
The benzimidazolium C(2)–H showed a typical absorption in the
1H NMR spectra of 1 and 2 at d 10.05, which is consistent with those
of other reported benzimidazolium salts [17]. Signals due to allyl
and butyl protons in the spectra of 1 and 2 resonated at d 5.42,
5.44, 5.82 and d 0.96, 1.35, 1.93, 4.53, respectively. Singlets at d
5.82 and 5.79 and multiplates in the range d 7.38–7.72 are attrib-
uted to the benzylic methylene and aromatic protons, respectively.
As a simple indicator for the success of the formation of carbene
3. Results and discussion
3.1. Syntheses and structural characterization
Ag–carbene complexes are known to adopt different architec-
tures, depending on the reaction conditions employed, but above
all they are the most popular compounds used in bioorganometal-
Fig. 1. A perspective view of the complex 3 with the displacement ellipsoids drawn at 50% probability. Bromide and solvent molecules were omitted due to clarity. Selected
bond lengths (Å): C1–Ag1 1.73(3); C18–Ag1 2.051(16); C1–N1 1.27(2); C1–N2 1.366(17); N3–C18 1.37(2); N4–C18 1.381(15); N1–C8 1.34(5); N2–C15 1.47(3); N3–C25
1.57(2); N4–C32 1.41(2). Selected bond angles (°): C1–Ag1–C18 172.2(14); N1–C1–N2 124(2); N3–C18–N4 108.7(14); N1–C8–C9 107.3(18); N2–C15–C16 113.4(17); N3–
C25–C26 113(3); N4–C32–C33 114.5(13).

R.A. Haque et al. / Inorganica Chimica Acta 394 (2013) 519–525
523
Fig. 2. Perspective views of two crystallographically independent structural units of 4 in a single crystal, displacement ellipsoids drawn at 50% probability. Selected bond
lengths (Å): Ag(1)–C(8A) 2.096(18); Ag(1)–C(26A) 2.111(11); C(8A)–N(1A) 1.325(14); C(8A)–N(2A) 1.38(2); C(26A)–N(3A) 1.267(15); C(26A)–N(4A) 1.415(14); Ag(1)–O
2.608; O–Br 2.657. Selected bond angles (°): C(26A)–Ag(1)–C(8A) 157.1(6); C(8A)–Ag(1)–O 101.33; C(26A)–Ag(1)–O 99.69; N(1A)–C(8A)–Ag(1) 127.1(14); N(2A)–C(8A)–
Ag(1) 126.3(9); N(3A)–C(26A)–Ag(1) 130.9(9); N(4A)–C(26A)–Ag(1) 121.9(9). Almost similar bond lengths and angles are found for other crystallographically different unit.
120.0
100.0
100.0
80.0
80.0
60.0
60.0
40.0
40.0
20.0
20.0
0.0
1.5625
3.125
6.25
12.5
25
50
100
0.0
-20.0
-40.0
-60.0
-80.0
-
-
-
-20.0
-40.0
-60.0
1
2
3
4
0
20
40
60
80
100
1
2
3
4
Fig. 4. Effects of increasing amounts of 1–4 on the percentage inhibition of cell
proliferation.
Concentrations (µM)
peaks are appeared in the complex spectra with negligible change
in peak position. HMQC spectra of the complexes showed correla-
tion peaks corresponding to the proposed structures. FTIR spectra
of the ligands showed a band of medium intensity in the range
Fig. 3. MTT assay results of NHC precursors 1 and 2, and Ag–carbene complexes 3
and 4 vs. the HCT 116 cell lines.
complexes 3–6, the disappearance of benzimidazoliumC(2)–H in
the corresponding 1H NMR spectrum could be used, proving that
during the complex formation the acidic proton of the benzimid-
azole was deprotonated followed by subsequent coordination to
the Ag(I), Au(I), and Pd(II) [18–20]. In the 13C NMR spectra, for the
same solutions, a signal in the range d 182–190.4 is attributed to
the carbene carbon bound to transition metal ion. Most likely due
to the unsymmetrical arrangement and steric bulk of the NHCs,
complex 5 exist in solution as a mixture of two rotamers.
1560–1590 cmꢀ1
, which is assigned to benzimidazole ring
m
(C@N) vibrations. In the complex spectra this band showed a shift
of ꢁ120 cmꢀ1, indicating the formation of free C–N module in the
benzimidazole ring. Finally, the IR spectra of all compounds con-
tain prominent bands at around 2900, 1220 and 1190 cmꢀ1, and
are assigned to m(C–H) and benzimidazole ring vibrations.
Crystal data and structure refinement details for carbene com-
plexes 3 and 4 are tabulated in Table 1. Single crystals of 3/4 suit-
able for X-ray diffraction analysis were obtained from slow
diffusion of diethyl ether into a solution of 3/4 in acetonitrile at
ambient temperature. A perspective view of complexes 3 and 4
are shown in Figs. 1 and 2, respectively. Complex 3 crystallizes in
In 13C NMR spectrum of 4, the chelating carbene carbon is ob-
served as two doublets centered at d 188.3 with a C–Ag coupling
constant (1JC–Ag = 197.12 Hz), which is consistent with the litera-
ture [21]. These doublets are attributed to carbene carbons bound
to 107Ag and 109Ag centers. Apart from this major change all other
ꢀ
the triclinic space group P1 containing one bis-carbene complex

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R.A. Haque et al. / Inorganica Chimica Acta 394 (2013) 519–525
Fig. 5. Images of the control HCT116 cells, cells treated with 5-FU and compounds 1–4 after 72 h of incubation.
cation, one bromide anion and one acetonitrile molecule in an
asymmetric unit. Both, the carbon and hydrogen atoms, of the gen-
eral-position benzyl and allyl substitutions are disordered over two
sets of sites. In the complex cation, the central Ag(I) is coordinated
by two carbene carbon atoms, making an almost linear C1–Ag1–
C18 bond with the bond angle 172.2(14)°. The carbene ligands
adopt a syn-arrangement around the metal center, probably due
to the sterically demanding nature of the benzyl substituents. Both
the benzimidazole rings are twisted out of the C–Ag–C axis, form-
ing a dihedral angle of 7.8(6)°. The internal ring angle at the car-
bene center (N–C–N) are 108(2) and 108.7(14) for N1–C1–N2
and N3–C18–N4, respectively, which agree well with the reported
complexes [22,23]. In the extended crystal structure, the bromide
anions link the Ag-complex cations into three-dimensional net-
works by C–Hꢃ ꢃ ꢃBr hydrogen bonds with bond distances ranging
two carbene carbon coordination (Ag–C(8A) = 2.096(18) Å, Ag–
C(26A) = 2.111(11) Å) as well as an acetone oxygen interaction.
The benzimidazole rings are twisted out of the plane of benzyl ben-
zene ring, and showing a dihedral angle of 22.8(4)°, which is quite
large to the reported complexes. The C(8A)–Ag–C(26A) angle in 4 is
reduced to 157.1(6)° compared to the somewhat larger angle in the
previously reported Ag complexes having similar ligand architec-
tures [22,23]. The interaction bond length between Ag atom and
oxygen is being 2.608 Å, which is consistent with the reported sim-
ilar isoelectronic complexes [24,25]. However, the interaction dis-
tance is adequately shorter than the sum of van der Waals radii
(3.24 Å) of the respective elements for an Ag–O interaction. Proba-
bly, this insight may be useful in determining the mode of action of
4 as anticancer agent, as it may form strong interaction with DNA of
an infected cell. In the extended structure, the 4 units are connected
through C–H(benzimidazole)ꢃ ꢃ ꢃBr and O(acetone)ꢃ ꢃ ꢃBr hydrogen
bonds with bond distances ranging from 2.405 to 2.657 Å.
from 2.86 to 3.170 Å, and are further consolidated by
interactions with the bond distances 3.619 Å.
p–p stacking
Complex 4 has been defined as a well ordered bromide salt. One
unit of the complex formula comprises the asymmetric unit, i.e.,
one complex cation, one bromide anion and an acetone molecule,
the former disposed about a crystallographic center of symmetry.
While, the carbene ligands adopt a syn-arrangement, probably
due to the sterically demanding nature of the benzyl substituents.
An interesting result was obtained in complex 4, viz, oxygen atom
of acetone has a strong interaction with the Ag center. Therefore,
the metal is not in an essentially linear fashion. The Ag atom is in
a three-coordinate distorted trigonal-planar environment, with
3.2. Anticancer activity
Ag–carbene complexes have been sought for antimicrobial and
anticancer treatments [26]. Numerous Ag complexes have dis-
played interesting anticancer potentials; however, their medicinal
values have always been hampered by their poor solubility and
higher cost of preparation. To increase the anticancer potential
and solubility of Ag complexes, we looked for NHC ligand systems
that could form the target complex in ionic form. Also, strong

R.A. Haque et al. / Inorganica Chimica Acta 394 (2013) 519–525
525
r
-donor effect of NHCs can stabilize the carbene-complexes,
Acknowledgments
which, in turn helps to avoid demetallation. Almost linear C–Ag–
C module assists to target DNA directly according to the cisplatin
paradigm, which ultimately trigger cell death.
The ability of the NHC precursors 1 and 2 and their respective
Ag–carbene complexes 3 and 4 to kill human derived cancer cell
was investigated using HCT 116 cell line and a standard bioassay,
R.A.H. thanks Universiti Sains Malaysia (USM) for the Short term
Grant (203/PKIMIA/6311123) and the Research University (RU).
Grant 1001/PKIMIA/811217. M.Z.G. thanks USM for the RU grant
1001/PKIMIA/844137 and Kufa University, Najaf, Iraq for a post-
graduate research scholarship. S.B. thanks USM for a post-doctoral
research fellowship.
MTT method. HCT116 cells were treated with 5 lM of 5-fluoroura-
cil (5FU) alone for 72 h, however, this concentration represents IC50
(72 h) value for 5FU in this cell line. Similarly, HCT 116 cell lines
were continuously exposed to test compounds 1–4 for 72 h, and
their effects on cellular viability were calculated. The profiles of
percentage inhibition of cell proliferation against the concentration
of the test compound were established to calculate IC50 values of
each derivative. Percentage inhibition of cell proliferation was
determined at different concentrations of the test compounds
Appendix A. Supplementary material
CCDC 881402 and 853548 contain the supplementary crystallo-
graphic data for complexes 3 and 4, respectively. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/data_request/cif. Supple-
mentary data associated with this article can be found, in the on-
line version, at http://dx.doi.org/10.1016/j.ica.2012.09.013.
ranging from 1.5625 to 100 lM. Figs. 3 and 4 depict the effect of
concentration of test compounds on HCT 116 cell lines after 72 h
incubation time. The results, expressed as concentrations of the
benzimidazolium salts 1 and 2, required to inhibit the infected cell
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