Synthesis, crystal structures, in vitro anticancer, and in vivo acute oral toxicity studies of bis-imidazolium/benzimidazolium salts and respective dinuclear Ag(I)- N -heterocyclic carbene complexes

Article abstract of DOI:10.1080/00958972.2013.831406

The synthesis, spectral (FT-IR and NMR), and structural studies of 1,1′-methylene linked 3,3′-2-cyanobenzyl bis-imidazolium salt (L1) and respective dinuclear Ag(I)-NHC complex (C1) are reported The structures of both compounds were established through single-crystal X-ray diffraction C1 has a short Ag-Ag separation of 3.16 A Both L1 and C1 were tested for potential against leukemia (k562) cell line For comparison, para-xylyl linked bis-benzimidazolium salts (L2-L4) and their dinuclear Ag(I)-NHC complexes (C2-C4) were synthesized and tested against the same cell line (K562) The IC50 values proved that L2-L4 and C2-C4 are many fold more active than L1 and C1 The mechanism of action and structure activity relationship are discussed In vivo oral acute toxicity study (sighting study) was carried out which depicts that 2000 mg/kg dose of selected compounds is an appropriate and safe dose for conducting main study on animals 2013

Full text of DOI:10.1080/00958972.2013.831406

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Synthesis, crystal structures, in vitro
anticancer, and in vivo acute oral
toxicity studies of bis-imidazolium/
benzimidazolium salts and respective
dinuclear Ag(I)-N-heterocyclic carbene
complexes
Rosenani A. Haquea, Noorhafizah Hasanudina, Muhammad Adnan
Iqbala, Ashfaq Ahmadc, Suzana Hashimb, Ams Abdul Majidb &
Mohamed B. Khadeer Ahamedb
a The School of Chemical Sciences, Universiti Sains Malaysia,
Minden, Malaysia
b EMAN Testing and Research Laboratory, The School of
Pharmaceutical Sciences, Universiti Sains Malaysia, Minden,
Malaysia
c The School of Pharmaceutical Sciences, Universiti Sains
Malaysia, Minden, Malaysia
Accepted author version posted online: 02 Aug 2013.Published
online: 02 Sep 2013.
To cite this article: Rosenani A. Haque, Noorhafizah Hasanudin, Muhammad Adnan Iqbal, Ashfaq
Ahmad, Suzana Hashim, Ams Abdul Majid & Mohamed B. Khadeer Ahamed (2013) Synthesis,
crystal structures, in vitro anticancer, and in vivo acute oral toxicity studies of bis-imidazolium/
benzimidazolium salts and respective dinuclear Ag(I)-N-heterocyclic carbene complexes, Journal of
Coordination Chemistry, 66:18, 3211-3228, DOI: 10.1080/00958972.2013.831406
To link to this article: http://dx.doi.org/10.1080/00958972.2013.831406
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Journal of Coordination Chemistry, 2013
Vol. 66, No. 18, 3211–3228, http://dx.doi.org/10.1080/00958972.2013.831406
Synthesis, crystal structures, in vitro anticancer, and in vivo
acute oral toxicity studies of bis-imidazolium/
benzimidazolium salts and respective dinuclear
Ag(I)-N-heterocyclic carbene complexes
ROSENANI A. HAQUE*†, NOORHAFIZAH HASANUDIN†, MUHAMMAD ADNAN
IQBAL*†, ASHFAQ AHMAD§, SUZANA HASHIM‡, AMS ABDUL MAJID‡ and
MOHAMED B. KHADEER AHAMED‡
†The School of Chemical Sciences, Universiti Sains Malaysia, Minden, Malaysia
‡EMAN Testing and Research Laboratory, The School of Pharmaceutical Sciences, Universiti Sains
Malaysia, Minden, Malaysia
§The School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Malaysia
(Received 3 May 2013; accepted 19 July 2013)
The synthesis, spectral (FT-IR and NMR), and structural studies of 1,1′-methylene linked 3,3′-2-cya-
nobenzyl bis-imidazolium salt (L1) and respective dinuclear Ag(I)-NHC complex (C1) are reported.
The structures of both compounds were established through single-crystal X-ray diffraction. C1 has
a short Ag–Ag separation of 3.16 Å. Both L1 and C1 were tested for potential against leukemia
(k562) cell line. For comparison, para-xylyl linked bis-benzimidazolium salts (L2–L4) and their di-
nuclear Ag(I)-NHC complexes (C2–C4) were synthesized and tested against the same cell line
(K562). The IC50 values proved that L2–L4 and C2–C4 are many fold more active than L1 and C1.
The mechanism of action and structure activity relationship are discussed. In vivo oral acute toxicity
study (sighting study) was carried out which depicts that 2000 mg/kg dose of selected compounds is
an appropriate and safe dose for conducting main study on animals.
Keywords: Ag(I)-N-heterocyclic carbene; Anticancer; K562; Crystal structure; Ag–Ag separation;
SAR; Acute oral toxicity
Introduction
N-heterocyclic carbenes (NHCs) are readily accessible in a few steps from azolium
(imidazolium, benzimidazolium, etc.) salts which are stable compounds [1]. In these
carbenes, a divalent carbon is flanked by two π-donor nitrogens. The strong σ-donating and
weak π-accepting properties of NHCs make them excellent ligands for d-block [2] and
f-block elements [3, 4]. NHCs have been bonded to almost all transition metals using
different synthetic routes, and the complexes have been tested for catalytic and biomedical
applications [2, 4–10]. Among various metal NHC complexes, Ag(I)-NHC complexes have
been synthesized most widely [11].
*Corresponding authors. Email: rosenani@usm.my (R.A. Haque); adnan_chem38@yahoo.com (M.A. Iqbal)
© 2013 Taylor & Francis

3212
R.A. Haque et al.
Since the first synthesis of a Ag(I)-N-heterocyclic carbene complex in 1993 [12], this
class of compounds has been extensively used for transmetallation reactions where direct
synthesis using other metal ions was difficult or impossible [9]. These complexes are
valuable due to their ease of synthesis, ease of handling, and air and moisture stability. Ag
(I)-NHC complexes were tested for catalytic potential [13–15] but were less effective com-
pared to other organometallic compounds [6, 16–19]. However, these compounds have vital
antimicrobial activity [20–24]. In 2007, Youngs et al. [25] reviewed these compounds as a
new class of antibiotics. These compounds were tested and corroborated by recent reports
[26–28] and reviews [29–31]. Several mononuclear Ag(I)-NHC complexes with coordina-
tion motifs A-C (chart 1) have been studied [20, 27, 32–35]. Dinuclear Ag(I)-NHC com-
plexes of structure D have recently been introduced as potential anticancer agents [36–46].
Dinuclear Ag(I)-NHC complexes, derived from para-xylyl linked bis-benzimidazolium
salts, exhibited anticancer activity against human colon cancer (HCT 116) and leukemia
(HL-60) [36, 43, 47]. We reported synthesis, crystal structure, and anticancer potential of a
dinuclear Ag(I)-NHC complex, where the dinuclear silver carbene complex was aggregated
in pair with a short Ag–Ag separation [44]. In the present study, we present the structure of
another imidazole-based dinuclear Ag(I)-NHC complex (C1), where the two neighboring sil-
ver ions within the same molecule have a close interaction of 3.16 Å. L1 and C1 were tested
for their antiproliferative potential against chronic myelogenous leukemia (K562) cells. For
comparison, other para-xylyl linked bis-benzimidazolium salts (L2–L4) and dinuclear silver
(I)-NHC complexes (C2–C4) were synthesized and applied on the same cell line (K562).
According to the global cancer statistic report for all types of cancer, 10.9 million new
cancer cases were registered in 2002 and 6.7 million patients died; whereas in 2008, this
figure increased to 12.6 million new cancer cases and 7.5 million deaths [48]. From leuke-
mia (blood cancer), every year 300,000 patients are registered and about 222,000 patients
die [48]. The ratio of deaths/cases (74%) represents the fatality of this specific type of can-
cer. Rapid development in anticancer drug discovery is necessary and the current study is
an effort to contribute.
Experimental
Reagents and instruments
Nuclear magnetic resonance spectra were recorded on a Bruker 500 MHz UltrashieldTM
1
spectrometer at ambient temperature. H and 13C NMR peaks are labeled as singlet (s),
Chart 1. Silver(I)-N-heterocyclic carbene complexes with different coordination motifs studied for their anticancer
potential. Structure D, the un-specified positions represent ortho/meta/para units. R is either alkyl or aryl substitu-
tion.

Dinuclear Ag(I)-N-heterocyclic carbenes
3213
doublet (d), triplet (t), and multiplet (m). Chemical shifts were referenced with respect to
solvent signals. FT-IR spectra were recorded on a Perkin–Elmer-2000. Elemental analysis
was carried out on a Perkin–Elmer series II, 2400 microanalyzer. X-ray diffraction data
were taken with a Bruker SMART APEX2 CCD area-detector diffractometer. The melting
and boiling points were assessed by using a Stuart Scientific SMP-1 (UK) instrument.
Chemicals and solvents were used as received.
RPMI 1640 media were purchased from ScienCell, USA. Heat inactivated fetal bovine
serum (HIFBS) was obtained from GIBCO, UK. Phosphate-buffered saline (PBS),
penicillin/streptomycin (PS) solution, MTS reagent and the reference standard, 5-fluoroura-
cil were purchased from Sigma–Aldrich, Germany. All other chemicals used in this study
were analytical grade.
Cell line and culture conditions
Human immortalized myelogenous leukemia cell line (K562) was purchased from American
Type Culture Collection (Rockville, MD, USA). K562 cell line is the erythroleukemia type
and is derived from a female chronic myelogenous leukemia patient in blast crisis. The cells
were maintained in RPMI 1640 culture medium with 10% HIFBS and 1% PS. Cells were
cultured in 5% CO2-humidified atmosphere at 37 °C.
Synthesis
Synthesis of 1,1′-methylene bis(imidazole). To a stirring solution of imidazole (3.0 g,
44 mM) in 100 mL of dichloromethane, potassium hydroxide (4.94 g, 88 mM) was added.
The mixture was refluxed at 50 °C for 12 h. The solvent was decanted and volatiles were
removed to afford 1,1-methylene bis(imidazole) as a white solid, washed with ether and
dried. General reaction involved in the preparation of the title compound is shown in
1
scheme 1. White powder. Yield: 3.82 g (60%). H NMR (500 MHz, DMSO-d6): 6.21 (2H,
s, 1 × CH2), 6.91 (2H, s, 1 × CH, imidazole), 7.39 (2H, s, 1 × CH, imidazole), 7.9 (2H, s,
2 × NCHN); 13C{1H NMR} (125.1 MHz, DMSO-d6): 54.8 (2 × CH2, methylene), 119.0
(2 × CH, imidazole-C4), 129.1 (2 × CH, imidazole-C5), 137.5 (2 × NCN).
Synthesis of 1,1′-methylenebis{3-(2-cyanobenzyl)-imidazolium} dibromide (L1). 2-(Bro-
momethylene)-benzonitrile (2.65 g, 13.6 mM) was added to a stirring solution of 1,1′-meth-
ylene bis(imidazole) (1.0 g, 6.8 mM) in 1,4-dioxane (100 mL). The mixture was refluxed at
100 °C for 24 h and the solution was evaporated under vacuum to yield a brownish thick
fluid (L1). To purify, the bromide salt was directly converted to its hexafluorophosphate
–
(PF6 ) salt by metathesis using potassium hexafluorophosphate (KPF6) (1.25 g, 6.8 mM) in
50 mL of methanol; the mixture was stirred for 3 h. The salt was obtained as a white crys-
talline solid. Single crystals suitable for X-ray diffraction were obtained by exposing a satu-
rated solution of L1.2PF6 in acetonitrile to the vapors of diethyl ether at room temperature.
For characterization, the PF6 salt of L1 was used whereas for further synthesis halide salt
1
was used. Colorless cubes. Yield 1.8 g (49%), mp: 192–194 °C. H NMR (500 MHz,
DMSO-d6): 5.74 (4H, s, 2 × Nimid–CH2–Ar), 6.67 (4H, s, 2 × Nimid–CH2–Nimid), 7.53 (2H,
d, 2 × CH imidazolium H5′, J = 7.5 Hz), 7.63 (2H, t, Ar-H), 7.66 (2H, t, Ar–H), 7.82 (2H,
m, Ar–H), 7.89 (2H, t, Ar–H), 7.99 (2H, d, 2 × CH imidazolium H4ʹ, J = 7.5 Hz), 8.05

3214
R.A. Haque et al.
Step-I
KOH
N
NH
+
Cl
Cl
N
N
DCM, reflux
12 h
N
N
1,1'-methylene bis(imidazole)
Step-II
N
N
N
N
N
N
N
N
N
N
N
2
+
Br 1,4-dioxane (40 mL)
reflux at 110 oC, 3 days
2Br-
1,1'-methylene bis(imidazole) 2-(bromomethyl)benzonitrile
L1
Step-III
N
N
N
N
N
Ag+
N
N
N
N
N
N
N
Ag2O
N
N
Ag+
MeOH (100 mL)
stirring at RT (25-27 oC)
5 days
N
N
N
N
2Br-
2Br-
KPF6
MeOH (100 mL)
stirring RT
1 day
N
N
N
N
N
N
Ag+
Ag+
N
N
N
N
N
-
2PF6
N
C1
Scheme 1. Synthesis of 1,1′-methylene bis(imidazole), 1,1′-methylenebis{3-(2-cyanobenzyl)-imidazolium}dibro-
mide (L1), and respective dinuclear Ag-NHC complex (C1).
(2H, s, Ar–H), 9.52 (2H, s, 2 × NCHN); 13C{1H NMR} (125.1 MHz, DMSO-d6): 50.7
(2 × Nimid–C–Ar, methylene), 58.5 (2 × Nimid–C–Nimid, methylene), 122.8, 129.6 (imidazo-
lium C5′ and C4′), 111.1 (CN), 116.7, 133.7, 134.0, 136.8 (Ar–C), 138.4 (NCN). FT-IR
(KBr): υ (cm−1); 3422 (Caliph–Nimid); 3159, 3104, 3061 (C–Harom); 2972 (C–Haliph); 2227
(C=N) 1604, 1578, 1555 (Carom–Carom); 1446, 1410, 1361, 1335 (Cimid–Nimid). Anal. Calcd
for C23H22F12N6O1P2: C, 41.21; H, 3.01; N, 12.54%. Found: C, 41.18; H, 3.04; N,
12.40%.

Dinuclear Ag(I)-N-heterocyclic carbenes
3215
3,3′-(1,4-Phenylenebis(methylene))bis(1-alkyl-benzimidazolium) salts (L2–L4). Synthesis
and characterization of para-xylyl linked bis-benzimidazolium salts (L2–L4) were carried
out according to the literature procedure (scheme S1) [39].
Synthesis of imidazole-based dinuclear Ag(I)-NHC complex (C1). L1.2Br (1.5 g, 4 mM)
was dissolved in methanol (100 mL) along with Ag2O (0.64 g, 3 mM) with exclusion of
light by covering the flask with aluminum foil. The reaction mixture was stirred for 5 days
at room temperature. The reaction mixture was filtered by Celite 545 to collect a clear solu-
tion, converted directly to its hexafluorophosphate counterpart by metathesis reaction using
KPF6 (0.56 g, 3 mM) in 40 mL of methanol/water mixture (1 : 1). The white precipitate
was collected and washed with fresh methanol (2 × 3 mL) to give the product as a white
powder that was further recrystallized by acetonitrile/water. Single crystals suitable for
X-ray diffraction were obtained by exposing a saturated solution of C1.2PF6 in acetonitrile
to the vapors of diethyl ether at room temperature. Colorless cubes. Yield 2.7 g (58%), mp:
1
198–200 °C. H NMR (500 MHz, DMSO-d6): 5.44 (8H, s, 4 × Nimid–CH2–Ar), 6.67 (8H,
s, 4 × Nimid–CH2–Nimid), 7.15 (2H, d, 2 × CH imidazolium H5′, J = 8.0 Hz), 7.45 (2H, t,
Ar–H), 7.52 (2H, s, Ar–H), 7.62 (2H, m, Ar–H), 7.75 (2H, d, 2 × CH imidazolium H4′,
J = 7.5 Hz), 7.90 (2H, s, Ar–H), 7.90 (2H, s, Ar-H); 13C{1H NMR} (125.1 MHz,
DMSO-d6): 52.7 (4 × Nimid–C–Ar, methylene), 63.6 (4 × Nimid–C–Nimid, methylene),
122.5, 128.9 (imidazolium C5′ and C4′), 110.1 (CN), 116.9, 133.4, 134.4, 139.2 (Ar-C) and
1
1
181.6 & 183.1 [d, J(Ag109-Ccarbene) =183.75 Hz & d, J(Ag107-Ccarbene) =208.78 Hz]. FT-IR
(KBr): υ (cm−1); 3422 (Caliph–Nimid); 3176, 3140, 3117 (C–Harom); 2979 (C–Haliph); 2231
(C=N); 1601, 1578 (Carom–Carom); 1453, 1423, 1401, 1374, 1351, 1335 (Cimid–Nimid). Anal.
Calcd for C46H36Ag2F12N12P2: C, 43.762; H, 3.18; N, 13.28%. Found: C, 43.62; H, 3.00;
N, 13.30%.
Synthesis of benzimidazole-based dinuclear Ag(I)-NHC complexes (C2–C4). Synthesis
and characterization of benzimidazole-based silver(I) N-heterocyclic carbene complexes
(C2–C4) were carried out according to the literature procedure (scheme S1) [39].
Preparation of cell culture
Initially, K562 cell line was allowed to grow under optimal incubator conditions. Cells that
reached a confluence of 70–80% were chosen for cell plating purposes. Old medium was
replaced centrifugally with fresh medium after washing the cells using sterile PBS (pH 7.4)
2–3 times. Cells were incubated at 37 °C in 5% CO2 for 1 min. Then, the flasks containing
the cells were observed under inverted microscope (to confirm adequate proliferation of
cells). Cells were counted and diluted to get a final concentration of 2.5 × 105 cells/mL and
inoculated into wells (100 μL cells/well). Finally, plates containing the cells were incubated
at 37 °C with an internal atmosphere of 5% CO2.
MTS assay
Cancer cells (100 μL cells/well, 1.5 × 105 cells/mL) were inoculated in a 96-well microtiter
plate. Then, the plate was incubated overnight in a CO2 incubator to allow for acclimatiza-
tion. Various concentrations of 100 μL of test substance were added into each well containing

3216
R.A. Haque et al.
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. After 48 h
treatment period, 50 μL of MTS reagent (2 mg/mL) was added into each well and incubated
again for 4 h. Following the incubation period, the medium containing MTS was aspirated
from each well, and 100 μL of DMSO was added to dissolve formazan and read the absor-
bance of soluble formazan. The absorbance of soluble formazan was read at 570 and 620 nm
wavelengths using a high-end Tecan M200Pro multimode microplate reader. Data were
recorded and analyzed for the assessment of the effects of test substance on cell viability and
growth inhibition. The percentage of growth inhibition was calculated from the optical den-
sity obtained from MTS assay. 5-FU was used as the standard reference drug.
In vivo acute oral toxicity tests
Selection, preparation and grouping of animals. Rodents like rats are used for acute oral
toxicity dose methods with females more sensitive than males. Female rats were obtained
from Animals Research and Service Center (ARASC) USM Main campus under approval
of Grant No. USM/Animals Ethics Approval/2012/(76) (364). Female rats were selected
randomly, marked for identification, and kept in cages for at least 5 days in an animal tran-
sient room. Animals were acclimatized to laboratory conditions prior to the start of dosing
for acute oral toxicity sighting study. All the animals were nulliparous and non-pregnant,
and their weight was 210 g 10. The rats were divided into five groups. Two were marked
with (benz)imidazolium salts L1 and L2 and next two with C1 and C2, respectively. Finally,
the last group was treated for vehicle containing water and minute quantity of Tween 80.
Preparation of doses. Solubility of these substances was enhanced by using Tween-80 in
limited doses to avoid toxicity originating from it. However, care was exercised to avoid
final volume not more than 1 mL/100 g of the body weight of animals as mentioned in
OECD guidelines 420. Doses were prepared shortly before use as their stability of prepara-
tion should be known.
Acute oral toxicity tests. Purpose of this sighting study was to select the safe and appropri-
ate dose for main study. An oral gavage tube was used to insert the single dose of selected
compounds. Animals were fasted for 3–4 h before dose but water supply was not withheld.
Test compounds were given to single dose in sequential manner following the flow chart in
figure shown below as per OECD guidelines. The starting dose for the sighting study is
selected from the fixed dose levels of 5, 50, 300, and 2000 mg/kg and considered as a dose
expected to produce some evident toxicity based, when possible, on data obtained from
in vivo and in vitro studies of structurally related compounds. A period of at least 24 h will
be allowed between the dosing of each animal and they were observed for 14 days.
Results and discussion
Synthesis
Synthesis of C1 was carried out in three steps shown in scheme 1. In step-I, imidazole was
converted to its potassium salt by refluxing two equivalents of potassium hydroxide with

Dinuclear Ag(I)-N-heterocyclic carbenes
3217
one equivalent of imidazole. The potassium salt of imidazole was collected as white powder
after decanting the reaction medium (1,4-dioxane) and directly reacted with dichlorometh-
ane under reflux. Solvent was evaporated; residue was washed with diethyl ether and dried
at ambient temperature. In step-II, two equivalents of 2-(bromomethyl)-benzene were
allowed to react with one equivalent of product obtained in step-I. The mixture was refluxed
for 3 days in 1,4-dioxane. The use of 1,4-dioxane as a reaction medium for the synthesis of
bis-(benz)imidazolium salts has advantages over other solvents (acetonitrile, dimethyl sulf-
oxide, etc.) due to its moderate polarity and low boiling point. Using 1,4-dioxane as a reac-
tion medium, the bis-azolium salts can be either obtained as thick fluids at the bottom of
the flask or powders. The desired compound was obtained either by decanting the reaction
medium (thick fluid) or by filtering (white powder) [39, 43, 44]. Similarly, in the current
synthesis, L1 appeared as a thick yellowish fluid that was further converted to its PF6 salt
by metathesis in methanol to get it a white powder. Metathesis is usually performed to
achieve further purity of such compounds as well as for the ease of handling. However, for
the synthesis of respective dinuclear silver complexes, we prefer halide salts of these
ligands. Hence, in step-III, one equivalent of halide salt of L1.2Br was dissolved in 100 mL
of methanol along with silver oxide (1 equivalent) and the mixture was stirred for 5 days at
room temperature with exclusion of light. Celite 545 was used to filter the mixture; filtration
is usually repeated to get a crystal clear solution which is converted to its hexafluorophos-
phate salt (scheme 1).
Synthesis and characterization of L2–L4 and C2–C4 were carried out according to the
reported procedure with minor modifications (scheme S1) [36].
FT-IR spectra of the compounds
Dinuclear Ag(I)-NHC complexes show some peaks comparable to ligands [39, 40, 43, 44].
For L1, strong to weak but sharp vibrations (3005–3449 cm−1) indicate aromatic Csp2–H
stretches. The pure Csp3–H stretch for L1 appeared at 2972 cm−1 (C–Haliph), weak because of
very few methylene hydrogens. A strong and sharp vibration is at 2227 cm−1 for nitrile
(C≡N) stretch (Supplementary material; figure S1). Weak to strong peaks at
1555–1604 cm−1 represent the symmetric aromatic ring stretches (C=C). The stretch at
1200–1500 cm−1 specific for −HC=N− [39, 43] indicates N-heterocyclic carbene carbon
bonding with silver and a characteristic “four fingers (f.fs)” pattern appears for dinuclear sil-
ver NHC complexes [39, 43]. Previously, for a number of benzimidazole-based dinuclear sil-
ver NHC complexes, such “four fingers (f.fs)” pattern was reported [39, 40, 43, 44]. For C1,
such patterns were not observed; however, some changes compared to L1 were observed.
For example, in L1, four peaks at 1335, 1361, 1410, and 1446 cm−1 changed to six peaks,
1335, 1351, 1374, 1410, 1423, and 1453 cm−1 (Cimid–Nimid). Also, some changes can be
observed from 1555–1604 cm−1 as well as 3061–3159 cm−1 (figure S1). These ranges are
for C=C and C–H stretches of aromatic systems and typical for such complexes [39, 43].
FT-NMR spectra of the compounds
1
FT-NMR characteristics of L1 and C1 were analyzed in DMSO-d6 from 0 to 12 ppm for H
1
NMR and 0–200 ppm for 13C NMR studies. H NMR spectrum of L1 has a sharp singlet at
9.51 δ ppm ascribed to the imidazolium ring (NCHN) acidic proton, in accord with previ-
ous reports [38, 43, 49]. Peaks for benzylic (Ar–CH2–Nimid
) and methylene

3218
R.A. Haque et al.
(Nimid–CH2–Nimid) protons appear at 5.74 and 6.67 δ ppm, respectively (figure S2). Synthe-
sis of C1 was confirmed by disappearance of acidic (NCHN) proton and changes in ben-
zylic signals [50]. Methylene (Nimid–CH2–Nimid) signals in C1 were suppressed (figure S2),
probably because NMR radiations (RF radiations) cannot interact properly with protons
close to the metal ions and hence sharp signals are not generated.
L1 and C1 were further confirmed by 13C NMR spectra. The spectrum of L1 displayed a
peak at 138.4 δ ppm ascribed to the imidazole ring carbon (NCN). This signal for benz-
imidazole ligands is at 142–144 δ ppm [39, 43, 51]. Upon complexation with Ag, two dou-
blets appear at ca. δ 182 for C1 with Ag–C coupling constants ca. 208 and 183 Hz
(figure S3). These doublets appear in dimeric complexes of structure [L2Ag2]2+ due to car-
bene carbon bonding to C–Ag107 and C–Ag109, respectively [39, 50, 52]. For benzimid-
azole-based Ag-NHC complexes, such doublets appear at ca. δ 189 with splitting patterns
180 Hz for Ag107 and 204 Hz for Ag109 [27, 53]. The signals for benzylic (Ar–C–Nimid
)
and methylene (Nimid–C–Nimid) carbons for L1 are at 50.7 and 58.6 ppm, respectively.
These signals moved 2–4 ppm downfield in C1 (52.7 and 63.6 ppm), in accord with previ-
ous reports [38, 39, 43, 52, 54–56].
Crystallography
Single crystals of L1 and C1 suitable for X-ray diffraction were grown by exposing satu-
rated solution of each in acetonitrile (0.5 mL) to diethyl ether at room temperature (vapor
diffusion method). Single crystals appeared as colorless blocks. Crystal refinement data for
L1 and C1 are provided in table 1; selected bond lengths and angles for C1 are tabulated in
table 2. Distances and angles for L1 are provided in the supplementary data file (table S1).
C1 crystallizes in the monoclinic space group with P-21/c (No. 14) symmetry whereas L1
grew in monoclinic space group with P-1 (No. 2) symmetry. A perspective view of L1 and
C1 has been shown in figure 1. Figure 1(a) shows that the ligand is comprised of a bis-imi-
dazolium cation and two bromides with a water bonded through P–F - - H–O hydrogen
bonding.
The presence of water in bis-imidazolium hexafluorophosphate salts is rarely observed
whereas in similar bis-imidazolium/benz-imidazolium bromide salts it is very frequent
[57–62]. This is consistent with their polarity compared to the salts with hexafluorophos-
phate as counter anions (less polar). Figure 1(a) shows that both imidazolium units are on
either side of methylene, facing opposite directions at 110.0 (14)°. The internal ring angles
at N2–C9–N3 and N4–C15–N5 are 107.78 (15) and 108.55 (16), respectively. The crystal
packing (figure S4) shows that the cationic and anionic components are connected through
C–H–F hydrogen bonding in the 3-D network.
The Ag(I)-NHC complex (figure 1(b)) is comprised of two silver cations sandwiched by
two L1 through NHC carbon and two hexafluorophosphates. Two neighboring silver ions
have a close interaction of 3.16 Å. Similar interactions have been previously observed by us
[44] and others [63] for various structures. The internal ring angles of imidazolium units
N1–C1–N2 and N4–C12–N5 are at 104.0 (2)° and 104.02 (19)°, respectively. These internal
ring angles are 3–4° smaller than compared to the angles in L1 whereas angles at each
nitrogen increase 2–3°. The NHC carbon, after bonding with Ag(I), shifts its charge density
to the metal due to which the shape of imidazole ring deviates, as previously reported
[39, 40]. Bond lengths between the Ag ion and carbene are Ag1–C1/C12A = 2.101(2)/
2.097(2) Å, in accord with those of other Ag-NHC complexes [26, 27, 43, 53, 56, 64].

Dinuclear Ag(I)-N-heterocyclic carbenes
3219
Table 1. Crystal data and structure refinement details of L1 and C1.
L1
C1
Formula
Formula weight
C23H20 N6F12 P2.H2O
688.41
C46H36Ag2N12 F12P2
1262.55
Crystal system
Triclinic
Monoclinic
Space group
P-1(No. 2)
P-21/c (No. 14)
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α (°)
8.4636(1)
11.6581(2)
15.5520(2)
69.641(1)
84.252(1)
70.027(1)
1351.82(4)
2
12.1787(15)
16.182(2)
14.2623(14)
90
122.437(7)
90
2372.2(5)
2
1.768
β (°)
γ (°)
V (Å3)
Z
Density (Calcd) (gm/cm3)
1.691
0.276
696
Abs. Coeff. (mm−1
F (000)
)
0.990
1256
Crystal size (mm)
Temperature (K)
Radiation (Å)
θ min, max (°)
Dataset
0.17 × 0.24 × 0.39
100
MoKα 0.71073
2.0, 32.7
−12 : 12; −17 : 17; −23 : 22
31,413
0.027
0.20 × 0.26 × 0.27
100
MoKα 0.71073
2.1, 32.5
−18 : 18; −24 : 24; −20 : 21
56,227
0.051
Tot.; Uniq. Data
R(int)
Nref, Npar
R, wR2, S
9782, 452
0.0548, 0.1636, 1.04
8580, 334
0.0358, 0.0970, 1.09
Table 2. Selected bond lengths (Å) and angles (°) of C1.
C4–C5
C4–N2
C1–N2
C1–N1
N1–C23
1.513(3)
1.462(3)
1.353(3)
1.356(4)
1.448(3)
C23–N4
N4–C12
C12–N5
N5–C15
C15–C16
1.450(3)
1.356(3)
1.355(3)
1.461(4)
1.471(3)
C14–C13
C2–C3
N4–C13
N2–C3
F1–P1
1.348(3)
1.346(4)
1.388(3)
1.387(3)
1.600(3)
Ag1–C1
Ag1–C12A
2.101(2)
2.097(2)
C5–C4–N2
C4–N2–C3
N2–C1–N1
N2–C4–C5
N1–C23–N4
N4–C12–N5
N5–C15–C16
111.41(19)
123.89(18)
104.0(2)
111.41(19)
111.16(17)
104.02(19)
114.2(2)
N5–C14–C13
C14–C13–N4
C13–N4–C23
N1–C2–C3
F1–P1–F2
106.8(2)
106.2(2)
124.22(19)
106.0(2)
179.12(13)
90.86(13)
F1–P1–F3
C1–Ag1–C12A
165.71(8)
Comparison of bond distances and angles between ligand and dinuclear complex is shown
in figure 2. The two silver cations are connected to the carbene through C1–Ag1–C12A and
C1A–Ag1A–C12 (165.78°) bridges. In previous reports, Cx–Ag–Cy angles were 171–178°
[39, 40, 43, 44]. However, in the current study, a bent angle (165.78°) indicates that the
Ag–Ag interaction influences the Cx–Ag–Cy linearity of bonding. The crystal packing (fig-
ure S5) shows that cationic and anionic components of C1 are connected via C–H–F hydro-
gen bonding in a 3-D network.

3220
R.A. Haque et al.
Figure 1. ORTEP of (a) bis-benzimidazolium salt L1 and (b) Ag(I)-NHC complex C1 with displacement ellip-
soids drawn at 50% probability.
In vitro anticancer activity
Effect of L1 and C1 on proliferation of K562 cell line. A variety of heterocyclic
compounds based on imidazole and benzimidazole are being studied as anticancer agents
[29–31, 36, 65–71]. This is subject of recent reviews [29, 31, 70–72]. Thus, antiprolifera-
tive potencies of L1 and C1 were evaluated using MTS assay on cancer cell line, K562.
The antiproliferation test showed a dose-dependent effect of C1 on K562 cells whereas
L1 proved to be inactive. Figure 3 illustrates the antiproliferative effect of test compounds.
The graph clearly shows that C1 inhibited the proliferation of K562 cells in a dose-depen-
dent manner parallel to 5-FU. In our previous reports, with benzimidazole, both the ligands

Dinuclear Ag(I)-N-heterocyclic carbenes
3221
Figure 2. Comparison of selected bond angles and bond lengths between L1 and C1. All labeled values were
selected from X-ray crystallographic data. The figure clearly shows that after bonding with Ag, the bond angle at
carbene carbon (NCN) shrinks from 107.7° to 104.2° whereas the bond angles at both nitrogens expands 2–3°, that
is, from 109.2° to 111.6°. This shows that the charge density drifted from imidazolium ring (ligand) to metal ion
which causes deviation in ring shape. This phenomenon is further supported by significant changes in the C–N
bond lengths as shown.
and xylyl linked dinuclear complexes showed anticancer activity against various cancer cell
lines [43, 44, 49, 53], indicating relatively higher potential of benzimidazole compared to
imidazole against cancer [68, 69]. However, the metal complexes of both of these moieties
(imidazole and benzimidazole) show significant anticancer activity [21, 26, 27, 53, 73].
Figure 3. Dose-dependent antiproliferative effect of L1 and C1 on leukemia cells (K562).

3222
R.A. Haque et al.
Figure S6 shows the cell images of tested compounds and standard drug (5-FU) against
K562 cells. Figure S6B clearly shows L1 has trivial effect (IC50 > 200 μM) on cancer cells
whereas the silver complex had cytotoxicity (IC50 = 43.7 μM), see figure S6C. However,
both the new compounds are comparable to or less potent than standard drug
(IC50 = 35.9 μM, 5-FU).
Effect of bis-benzimidazolium salts (L2–L4) and respective silver complexes (C2–C4) on
proliferation of K562 cell line. For comparison, benzimidazole-based bis-salts and dinu-
clear complexes were tested against the same cell line (K562). Figure S7 shows the cell
images of L2–L4 and C2–C4 against leukemia (K562). This is evident from the graph
(figure 4) and cell images (figure S7) that all the p-xylyl linked bis-benzimidazolium salts
(L2–L4, IC50 = 8.9–34.9 μM) and silver complexes (C2–C4, IC50 = 3.37–6.50 μM) are
many fold active than imidazole-based compounds, L1 (IC50 = > 200 μM) and C1
(IC50 = 43.7 μM) including standard drug (5-FU, IC50 = 35.9 μM), see table 3. These
results support our previous reports [38–41, 43, 44, 52] on the significance of bezimidaz-
ole-based silver NHC complexes compared to imidazole-based ones [42].
Two reports [42, 52] containing synthesis and anticancer potential of N-iPr/-allyl substi-
tuted xylyl (ortho/meta/para) linked imidazolium/benzimidazolium salts and respective
Figure 4. Dose-dependent antiproliferative effect of L2–L4 and C1–C4 on leukemia cells (K562).
Table 3. IC50 values of tested samples against K562 cell line.
Compound
Ligands
R
Compounds
IC50 (μM) K562
–
L1
L2
L3
L4
C1
C2
C3
C4
5-FU
>200
34.9
8.9
octyl
nonyl
decyl
–
octyl
nonyl
decyl
–
9.0
Ag(I)–NHC complexes
43.7
6.57
3.67
3.37
35.9
Standard drug

Dinuclear Ag(I)-N-heterocyclic carbenes
3223
silver NHC complexes show that the para-xylyl linked complexes are active compared to
complexes with ortho and meta. Bigger cavity size for these dinuclear complexes is better
for the anticancer potential [42]. To further confirm this observation, C1 (scheme 1), with
smaller cavity size, was compared with para-xylyl linked complexes (C2–C4, scheme S1),
having bigger cavity size; C2–C4 were many fold more active than C1. These results sup-
port our argument of cavity size as mentioned above. This phenomenon can also be
observed from a recent report [71] where imidazole-based para-xylyl linked dinuclear com-
plexes showed much better antiproliferation activity compared to mononuclear silver NHC
complexes against adenocarcinomas (MCF 7 and DLD1). The exact reasons of this behav-
ior are not clear at this stage, however, but a detailed mechanistic study is in progress.
Mechanism of action and structure activity relationship
In the last decade, various silver(I) complexes have been studied for their antimicrobial
potential [74–77]; these complexes release Ag+ that bind with the bacterial cell surfaces and
interact with the proteins involved in cell wall synthesis to disrupt the cell functions [78].
The scientific community believes that anticancer mechanism of silver(I) complexes is also
monitored by release of silver ions and its binding with the proteins and DNA [79]. How-
ever, for antimicrobial potential of silver(I) complexes, the magnitude of activity was found
to be related with the ease of the ligand displacement [79, 80]; for example, silver com-
plexes with weak Ag–O or Ag–N bonds have a broader spectrum in their antimicrobial
activities [81, 82]. In contrast, for anticancer potential of such silver(I) complexes, rapid
release of Ag+ lose their effect abruptly [30]. This drawback was compensated by introduc-
ing Ag-NHC complexes since N-heterocyclic carbenes (NHCs) are strong σ-donating and
weak π-accepting ligands and release Ag+ more slowly [30, 83–86].
Relatively higher anticancer potential of bis-benzimidazolium salts and dinuclear
Ag(I)-NHC complexes in comparison with imidazole-based compounds might be due to
association of benzene with the cellular system [86]. Researchers established a concept
that benzene metabolites covalently interact with cellular macromolecules and produce
toxicity [86–89]. Perhaps, this is the reason that bis-benzimidazolium salts and respective
dinuclear complexes are relatively active compared to similar imidazole-based com-
pounds [42]; however, this is a preliminary justification in favor of benzimidazole on the
basis of literature. Further work is necessary to assess the actual reason of benzimidazole
advantage over imidazole-based chemical moieties.
This research shows that human leukemia cells (K562) treated with silver complexes
reveal characteristic features of apoptosis, evidenced through the membrane blebbing, chro-
matin condensation, and formation of apoptotic bodies (figures S6D and S7D, F, H). Our
results support previous findings where silver(I)-NHC complexes induced caspase indepen-
dent apoptotic cellular death via mitochondrial apoptosis-inducing factor [26]. Several stud-
ies have proved that silver cations deposit in the cytosol and render the cellular process
defunct by interacting with rate-limiting enzymes and proteins that are crucial for essential
biochemical pathways [25, 42, 90]. Thus, the present study reveals clear signs of black sil-
ver deposits in the cytoplasm of the affected cells (figures S6D and S7D, F, H), confirming
that cytotoxic efficacy of the complexes is contributed by deposition of silver.
Increase in chain length increases the lipophilicity of complexes [36, 43] that probably
further facilitates Ag(I)-NHC system to cross the cell wall barriers to interact with DNA
and interrupt its function. Benzimidazole-based silver(I)-NHC complexes have already

3224
R.A. Haque et al.
shown strong DNA binding [37]. Table 3 clearly shows that with increase in chain length
of N-alkyl substitution (octyl to decyl), the cytotoxicity of L2–L4 and C2–C4 increased.
Hence, the proper tuning of N-alkyl chain lengths, extension of benzene ring systems,
and proper cavity size of such dinuclear Ag(I)-NHC complexes make them biologically
valuable.
Preliminary in vivo acute oral toxicity (sighting study)
Acute toxicity is the observation of adverse effects occurring in an animal within a short
time of administration of either a single dose of chemical or multiple doses given to the ani-
mal [91]. Acute toxicity test can provide better information about the biological properties
of new chemical compounds than any other single test [92]. There are several methods to
determine acute toxicity of a newly synthesized compound subject to the level of biological
information required from it [91]. In the current study, “fixed-dose method” was adopted to
determine the toxic dose by using sighting study [93]. The sighting study allows selection
of the appropriate starting dose for the main in vivo study. In this method, the test substance
is given orally among one of four fixed dose levels (5, 50, 300, and 2000 mg/kg) to ani-
mals. The purpose is to select a dose that produces signs of toxicity but no mortality [94].
The selection of starting dose depends upon the in vivo and in vitro data available in the lit-
erature for compounds having similar structure to the compound(s) undergoing the investi-
gation; however, if such data are not available, 300 mg/kg is recommended as a starting
dose for sighting study [93]. Depending on the results of the first test, either increased dose
(2000 mg/kg) or lower doses (5 or 50 mg/kg) should be tested in case of mortality observed
with 300 mg/kg dose. Figure 5 shows the chart for sighting study.
Initially, L1–L2 and C1–C2 were tested for sighting study using 300 mg/kg dose. Since the
synthesized compounds were not soluble in saline, each compound (75 mg 5/210 g 10 of
animal weight) was mixed with 300 μl of polysorbate 80 (Tween 80) in order to make the
compound soluble and total volume was marked up to 2 mL with saline. To observe any
possible toxicity due to polysorbate 80, 300 μL of it was separately dissolved in saline
(1700 μL) and was orally given to a separate rat. No signs of toxicity due to polysorbate 80
were observed at given concentration.
All the animals after initial dose were observed after 20 min then after one hour and after
that periodically at least once a day until the 14th day. Observation period was not made
rigid as it depends upon the appearance of toxic reactions in animals. However subjected to
the death of animal, the study should be certainly terminated immediately or if animal starts
showing moribund symptoms, animal should be killed humanely. The body weight was also
Figure 5. Sequential steps for sighting study. The green arrows indicate the path followed by the compounds.

Dinuclear Ag(I)-N-heterocyclic carbenes
3225
observed. If weight reduction, moribund symptoms and diarrhea are observed during obser-
vational period then the dose should be labeled as toxic dose. However in the present study,
all animals survived and remained healthy and agile. According to OECD guidelines, the
quantity of test compounds was then increased to 2000 mg/kg of animal body weight and
the same procedure of observation was repeated. No signs of toxicity were observed and
animals remained alive, healthy, and agile. Hence, it is concluded that 2000 mg/kg body
weight of these selected compounds can be the starting dose for main study. All the proto-
cols of the study have been provided in the experimental section.
In vivo studies of Ag(I)-NHC complexes are rare. For example, 1 was tested on the ovar-
ian cancer (OVCAR-3) using nude mice, whereas a high dose of 333 mg/kg caused a major
cell death in the tumors without inducing any toxic effects to the major organs [35]. How-
ever, NHC complexes of Ru, Au, and Pt have been frequently studied using animals [29].
According to the best of our knowledge, this is the first time that dinuclear Ag(I)-NHC
complexes were studied for their acute oral toxicity. The results will be beneficial for future
in vivo studies of silver NHC complexes having similar structures.
Conclusions
Based on the current and previous reports, benzimidazole-based bis-salts as well as respec-
tive dinuclear complexes are many fold more active than similar imidazole-based com-
pounds. Ag(I)-NHC complexes were always relatively active when compared to the
corresponding ligands. This provides evidence that silver cations play a vital role in the
death of cancer cells. The photomicrographs of the cells treated with silver(I)-NHC com-
plexes reveal clear signs of black silver in the cytoplasm of the affected cells, which con-
firms that the cytotoxic efficacy of the complexes is contributed by deposition of silver.
Also, in dinuclear silver(I)-NHC complexes, cavity size and N-substitutions affect the anti-
cancer potential of the compounds. Comparatively, larger cavity size is better for anticancer
potential as well as longer alkyl chain length is better for cytotoxicity of Ag(I)-NHC com-
plexes. The preliminary in vivo acute oral toxicity test results suggest non-toxic behavior in
the biological system. In vivo anticancer studies are in progress and will be reported on
completion.
Supplementary material
CCDC 924662 and CCDC 924661 contain the Supplementary crystallographic data for L1
and C1. This data can be obtained free of charge from http://www.ccdc.cam.ac.uk/conts/
retrieving.html or from the Cambridge Crystallographic Data Center, 12 Union Road,

3226
R.A. Haque et al.
Cambridge CB2 1EZ, UK; Fax: 44-1223-336-033; or E-mail: deposit@ccdc.cam.ac.uk.
Animals were obtained from ARASC USM Main campus under approval of Grant No.
USM/Animals Ethics Approval/2012/(76) (364).
Acknowledgements
RAH thanks Universiti Sains Malaysia (USM) for the Research University (RU) Grant
(1001/PKIMIA/811217) and short-term grant (1001/PKIMIA/823082). NH is grateful to
(IPS) USM for the financial support [fellowship: RU(1001/441/CIPS/AUPE001)] and PRGS
grant (1001/PKIMIA/834080). MAI is grateful to (IPS) USM for financial support [fellow-
ship: USM.IPS/JWT/1/19 (JLD 6): P-KD0030/11(R)].
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