Accepted Manuscript

Original article

Unsymmetrically substituted benzimidazolium based Silver(I)-N-Heterocyclic

carbene complexes: Synthesis, Characterization and In vitro Anticancer study
against Human Breast Cancer and Colon cancer

Aqsa Habib, Mansoureh Nazrai Vishkaei, Muhammad Adnan Iqbal, Haq Nawaz
Bhatti, M.B. Khadeer Ahmed, A.M.S. Abdul Majid

PII: S1319-6103(19)30032-8
DOI: https://doi.org/10.1016/j.jscs.2019.03.002
Reference: JSCS 1041

To appear in: Journal of Saudi Chemical Society

Received Date: 24 January 2019

Revised Date: 10 March 2019
Accepted Date: 13 March 2019

Please cite this article as: A. Habib, M.N. Vishkaei, M.A. Iqbal, H.N. Bhatti, M.B.K. Ahmed, M.S.A. Majid,
Unsymmetrically substituted benzimidazolium based Silver(I)-N-Heterocyclic carbene complexes: Synthesis,
Characterization and In vitro Anticancer study against Human Breast Cancer and Colon cancer, Journal of Saudi
Chemical Society (2019), doi: https://doi.org/10.1016/j.jscs.2019.03.002

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Unsymmetrically substituted benzimidazolium based Silver(I)-N-Heterocyclic carbene
complexes: Synthesis, Characterization and In vitro Anticancer study against Human
Breast Cancer and Colon cancer

Aqsa Habib [a][b], Mansoureh Nazrai Vishkaei[c] Muhammad Adnan Iqbal[b] †, Haq Nawaz
Bhatti [a] *, , M.B. Khadeer Ahmed [c], A.M.S. Abdul Majid[c]
[a]
Environmental Chemistry Laboratory, Department of Chemistry, University of Agriculture Faisalabad-38000,
Punjab, Pakistan
[b]
Organometallic & Coordination Chemistry Laboratory, Department of Chemistry, University of Agriculture
Faisalabad-38040, Punjab, Pakistan
[c]
EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia,
11800 USM, Penang, Malaysia

Corresponding Authors:
*
Prof. Haq Nawaz Bhatti
Email: hnbhatti2005@yahoo.com

†Dr. Muhammad Adnan Iqbal

Email: adnan.iqbal@uaf.edu.pk
ABSTRACT
The promising biomedical applications of silver complexes stimulated the researchers to test
these compounds against cancer. The present research work was designed to achieve this
goal. In this work, a series of 5-methyl benzimidazole based N-Heterocyclic carbene ligands
and respective silver(I) complexes were synthesized and tested on cancer cell lines to assess
their anticancer activity. Unsymmetrically substituted benzimidazole was found unique in its
reactivity and generation of a single product during NHC ligand formation was only possible
after two successive alkylations with same alkyl halide. The corresponding Ag(I)-NHC
adducts were obtained by in situ deprotonation of the NHC ligands. Synthesized compounds
were characterized by various physcio-chemical and spectroscopic methods. Single crystal X-
ray diffraction study of complex 7 revealed its mononuclear structure. Preliminary in vitro
anticancer study of azolium salts and respective Ag(I)-NHC complexes against human breast
cancer (MDA-MB-231), colon cancer (HCT-116) and normal endothelial cells (EA.hy926)
cells revealed that all the compounds are more cytotoxic to cancer cells than normal cells and
the complexes are relatively more potent compared to the corresponding NHC ligands. It was
found that increased chain length and presence of methyl substituent on benzimidazole ring
enhance the biopotency of Ag(I)-NHC complexes. The synthesized compounds were further
studied for pro-apoptotic mechanism of action via Rhodamin 123 test. The tested compounds
were found to induce apoptosis via extrinsic mitochondrial pathway.
Key words: Metallodrugs, unsymmetrical, deprotonation, cell lines

1. INTRODUCTION
Metallodrugs have been continuously drawing the attention of researchers as
promising bioactive agents to combat against cancer since last century [1] out of which,
cisplatin (platinum based) has become most popular [2-4]. Although, cisplatin is very
efficient anticancer agent, its use is not free of shortcomings: it is effective only against few
types of cancer and its extensive use results in severe side-effects, for example bone marrow
suppression, nausea and kidney toxicity. Due to its high potency, it causes unbearable pain in
patients, and finally they have to stop the treatment [5]. These issues have stimulated the
researchers to introduce new anticancer metallodrugs free of above mentioned limitations.
Recently, organometallic silver complexes have gained importance as promising substitute
and are anticipated to surpass the flaws of platinum based anticancer drugs [6]. A variety of
silver forms i.e silver coordination complexes, silver nanoparticles and silver organometallic
compounds have been introduced since last few years due to remarkable biological especially
anticancer potential of silver [7-14]. Numerous Ag(I)-NHC complexes synthesized from
azolium salts having un-substituted benzene ring have been tested against various cancer cell
lines i.e renal cancer (Caki-1), breast cancer (MB157), cervical cancer (HeLa), ovarian cancer
(OVCAR-3) [15-20]. It has been investigated that anticancer potential of Ag(I)-NHC
complexes is predominantly controlled by their lipophilic or hydrophobic character which in
turn depends on electronic or steric factor of substituents [21]. Previous reports have revealed
that the main target sites of Ag(I)-NHC complexes are mitochondrial membrane of infected
cells where they promote the apoptosis unlike other metal-coordination complexes which
target DNA of infected cells [22-25]. The characteristic anticancer features of the Ag(I)–
NHC complexes discussed herein further highlight the worth of exploring modern synthetic
routes in organometallic chemistry for drug development. Previously, researchers have
investigated the anticancer activity of Ag(I)-NHC complexes derived from symmetrical (5,6
disubstituted) benzimidazole [26]. Their study revealed that the presence of substituents has
significant impact on synthesis and activity of silver complexes. This article describes further
extension of previous study. Herein we report the synthesis and in vitro anticancer potential
of interesting class of silver complexes derived from unsymmetrical (i.e 5-methyl)
benzimidazole. We found that unsymmetrical benzimidazole is unique in its reactivity and
generation of a single product during formation of NHC ligand (for synthesis of silver
complex) is only possible if two successive alkylations proceed with same alkyl halide.

2. EXPERIMENTAL
2.1. Materials and Instruments
Analytical grade starting materials were obtained from commercial sources and were used as
such without further purification. 5-methyl benzimidazole, alkyl halides (1-bromopropane, 1-
bromobutane, 1-bromopentane) and Ag2O were purchased from Sigma Aldrich. For
anticancer study, culture media ATCC was obtained from Germany. TECAN Multi-mode
microplate reader was obtained from USA. Phosphate buffered saline (PBS),
penicillin/streptomycin (PS) solution, MTT reagent, 5-fluorouracil, tamoxifen and betulinic
acid were purchased from Sigma-Aldrich, Germany. Human breast cancer (MDA-MB-231),
colon cancer (HCT-116) and human endothelial normal cells (EA.hy926) were collected from
American type culture collection (Rockvill, MD, USA). Rhodamine 123 stains were
purchased from Immunochemistry Technologies (Minnesota, USA).

The melting points of all compounds were assessed by using a Stuart Scientific SMP-1 (UK)
instrument. The solvents were evaporated using EYELA 1L Rotary Evaporator N-1001V-
WD. FTIR spectra of the compounds were recorded in ALPHA-P, compact FTIR
spectrometer equipped with a universal attenuated total reflectance (UATR) accessory, which
allows the observation of the electromagnetic spectrum over 4000−250 cm−1 region. 1H,
13
C{1H}, 19F and 31
P{1H} NMR spectra were recorded on Bruker Avance-300 and Avance-
400 or on a Varian Inova-300 spectrometer in CDCl3, acetone-d6, C6D6 and DMSO-d6 as
solvents at chemical shifts (δ) 0-12 ppm for 1H NMR, 0-200 ppm for 13C NMR, -200-0 ppm
for both 19F and 31P NMR studies. Chemical shifts are reported in parts per million relative to
tetramethylsilane and coupling constants (J) are given in Hertz. The 1H and 13
C{1H} NMR
19
spectra were referenced using the residual solvent signal as internal standard, while F and
31
P{1H} spectra were referenced to CFCl3 and H3PO4 (85%), respectively. The N-alkylation
reactions were monitored by Agilent Technologies, 7890A GC/MS. Elemental study of the
compounds for C, H and N analysis was carried out using EA1108 (CHNS), Fisons
instrument. ESI mass spectra were recorded using an ESI-POS-DI-TOF 6224 spectrometer.
The crystallographic data was collected on a Bruker APEX II equipped with an Incoatec
 I/muS Microsource and graphite-monochromated Mo Kα radiation (λ=0.71073 Å ). Cell
refinement and data reduction were done using SAINT [27]. An empirical absorption
correction, based on the multiple measurements of equivalent reflections was applied using
the program SADABS [28]. The space group was confirmed by XPREP routine [29] in the
program SHELXTL [30]. The structures were solved by direct methods and refined by full-
matrix least-squares and difference Fourier techniques with SHELX-97 [31]. All non-
hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms
were set in calculated positions and refined as riding atoms with a common thermal
parameter.

2.2. Synthesis of preligands (1-3)

2.2.1. 5-methyl-1-propyl-1H-benzo[d]imidazole (1)

Stirred a mixture of 5-methyl benzimidazole (2.64 g, 20mmoles), 1-bromopropane (1.95 mL,

20 mmoles) and KOH (1.68 g, 30 mmoles) in DMSO (20 mL) for 4 hours at room
temperature and monitored the reaction by GC-MS. Upon completion of reaction, added the
reaction mixture in 200 mL ice cold distilled water to remove DMSO. Suddenly turbidity
appeared, extracted with a mixture of ether and hexane (25 ml, 1:1) and washed with cold
water thrice to remove DMSO. Collected organic layer containing product and added some
MgSO4 to absorb residual water molecules. After evaporation of ether and hexane, oily
product obtained. Yield 2.1 g (60%). GC-MS, m/z, (%): ([C11 H14N2]+ 174.24 (100). FTIR
(ATR, ν, Cm-1): 2963, 2874 (Caliph-H str), 1567, 1496 (C=Nbenzimid str), 1257 (Carom-Nbenzimid),
806, 745, 606 (C-Hoop ben). 1H NMR (400MHz, DMSO-d6,  ppm) 0.83 (6H, m, 2×CH3), 1.7
(4H, m, 2×CH2), 2.5 (6H, t, 2×CH3-Ar), 4.16 (4H, d.t, R-CH2-N, J=2.7 Hz), 7.0 (2H, q, Ar-
H), 7.4 (4H, m, Ar-H), 8.1 (2H, d, N-CH-N). Anal. Calcd. for C11H14N2 : C 75.8, H 8.1, N
16.0. Found: C 75.7, H 8.2, N 16.1.

2.2.2. 5-methyl-1-butyl-1H-benzo[d]imidazole (2)

This synthesis followed the procedure for synthesis of 1 but using 1-bromo butane (2.74 g, 20
mmoles). Oily product obtained in Yield 2.14 g (57%). GC-MS, m/z, (%): ([C12H16N2]+
188.1 (100). FTIR (ATR, ν, Cm-1): 2957, 2872 (Caliph-H str), 1567 (C=Nbenzimid str), 1200
(Carom-Nbenzimid), 806, 745, 605 (Carom-H oop ben). 1H NMR (400MHz, CDCl3,  ppm) 0.87
(6H, , 2×CH3), 1.26 (4H, m, 2×CH2), 1.74 (4H, m, CH2), 2.44 (6H, d, 2×CH3-Ar, J= 8.24
Hz), 3.99 (4H, d.t, R-CH2-N, J= 7.21 Hz), 7.05 (2H, t, Ar-H, J= 8.01 Hz), 7.11 (1H, s, Ar-H),
7.2 (1H, d, Ar-H, J= 8.19 Hz), 7.56 (1H, s, Ar-H), 7.64 (1H, d, Ar-H, J= 8.22 Hz), 7.74 (2H,
d, N-CH-N, J= 5.97 Hz). 1H-NMR spectrum indicates as if chlorobutane attacked from both
sides after deprotonation of benzimidazolium proton so all peaks appeared double. Anal.
Calcd. for C12H16N2: C 76.5, H 8.5, N 14.8. Found C 76.6, H 8.6, N 14.7.

2.2.3. 5-methyl-1-pentyl-1H-benzo[d]imidazole (3)

This synthesis followed the procedure for synthesis of 1 but using 1-bromo pentane (3.02 g,
20 mmoles). Oily product obtained in Yield 2.5 g (61%). GC-MS, m/z, (%): ([C13H18N2]+
202.3 (100). FTIR (ATR, ν, Cm-1): 2964, 2873 (Caliph-H str), 1491 (C=Nbenzimid str), 1212
(Carom-Nbenzimid), 823, 749, 625 (Carom-H oop ben). 1H NMR (400MHz, CDCl3,  ppm) 0.85
(6H, , 2×CH3), 1.34 (4H, m, 2×CH2), 1.81 (4H, m, CH2), 2.12 (4H, m, CH2), 2.61 (4H, m,
CH2), 2.72 (6H, d, 2×CH3-Ar, J= 8.24 Hz), 4.14 (4H, d.t, R-CH2-N, J= 7.21 Hz), 7.37 (2H, t,
Ar-H, J= 8.01 Hz), 7.11 (4H, m, Ar-H), 7.84 (4H, m, Ar-H). 1H-NMR spectrum indicates as
if bromobutane attacked from both sides after deprotonation of benzimidazolium proton so all
peaks appeared double. Anal. Calcd. for C13H18N2: C 77.2, H 8.9, N 13.8. Found C 77.6, H
8.7, N 13.7.

2.3. Synthesis of NHC ligands (4-6)

2.3.1. 5-methyl-1,3-dipropyl-1H-benzo[d]imidazol-3-ium bromide (4)
Refluxed a mixture of 1 (2 g, 11.5 mmoles) and bromopropane (5 mL (excess)) for 24 hours
at 70 oC and monitored the reaction by GC-MS. Upon completion of reaction, white
precipitates of 4 appeared in the reaction flask. Filtered them, washed excessively with ether
and dried at room temperature. Yield 2.4 g (70%); M.P= 120 oC. FTIR (ATR, ν, Cm-1): 3010
(Caliph–Nbenzimi), 2923, 2782 (Caliph-H str), 1464, 1424 (C=Nbenzimid str), 1265, 823, 610 (Carom-
H ben). 1H NMR (400 MHz, CDCl3,  ppm) 1.04 (6H, m, 2×CH3), 2.05 (4H, m, 2×CH2),
2.61 (3H, s, CH3-Ar), 4.43 (4H, m, R-CH2-N), 7.5 (2H, t, Ar-H), 7.42 (2H, d, Ar-H, J =
8.59), 9.15 (1H, s, N-CH-N). 13
C NMR (125.72 MHz, CDCl3,  ppm) 10.98 (CH3), 21.8
(CH3), 22.7, 22.82 (R-CH2), 48.7, 48.9 (N-CH2-R, 2×CH2), 67.0 (Ar-CH3), 112.6, 112.7,
128.8, 129.4, 131.5, 138.0 (Ar-C), 142.0 (N=C=N). Anal. Calcd. for C14H21N2Br: C 46.5, H
5.5, N 7.7. Found C 46.4, H 5.3, N 7.1.2.3.2. 5-methyl-1,3-dibutyl-1H-benzo[d]imidazol-
3-ium bromide (5)

This synthesis followed the procedure for synthesis of 1 but using 1-bromo butane (5 mL
(excess)). White precipitates of 5 were obtained in yield 2.13 g (61%). FTIR (ATR, ν, cm-1):
2935, 2873 (Caliph-H str), 1567, 1457, 1402 (C=Nbenzimid str), 818, 745 (Carom-H ben). 1H NMR
(400 MHz, CDCl3,  ppm) 1.1 (6H, m, 2×CH3), 2.05 (4H, m, 2×CH2), 2.55 (4H, m, 2×CH2),
2.65 (3H, s, CH3-Ar), 4.51 (4H, m, R-CH2-N), 7.67 (2H, t, Ar-H), 7.89 (2H, d, Ar-H, J = 8.53
Hz), 9.75 (1H, s, N-CH-N). 13C NMR (125.72 MHz, CDCl3,  ppm) 10.03 (CH3), 21.8 (CH3),
22.8, 22.8, 22.7 (R-CH2), 40.0, 48.6, 48.7 (N-CH2-R, 2×CH2), 67.9 (Ar-CH3), 112.7, 112.9,
128.8, 129.4, 131.5, 132.0 (Ar-C), 148.1 (N=C=N). Anal. Calcd. for C16H25N2Br: C 59.1, H
7.7, N 8.6. Found C 59.4, H 7.4, N 8.2.

2.3.3. 5-methyl-1,3-dipentyl-1H-benzo[d]imidazol-3-ium bromide (6)

This synthesis followed the procedure for synthesis of 1 but using 1-bromo pentane (5 mL
(excess)). White precipitates of 6 were obtained in yield 1.8 g (56%). FTIR (ATR, ν, cm-1):
2961, 2863 (Caliph-H str), 1558, 1469, 1443 (C=Nbenzimid str), 865, 732 (Carom-H ben). 1H NMR
(400 MHz, CDCl3,  ppm) 1.08 (6H, m, 2×CH3), 1.62 (4H, m, 2×CH2), 2.13 (4H, m, 2×CH2),
2.49 (4H, m, 2×CH2), 2.76 (3H, s, CH3-Ar), 4.49 (4H, m, R-CH2-N), 7.56 (2H, t, Ar-H), 7.67
(2H, d, Ar-H, J = 8.53 Hz), 9.62 (1H, s, N-CH-N). 13
C NMR (125.72 MHz, CDCl3,  ppm)
10.1 (CH3), 21.9 (CH3), 22.8, 22.9 (R-CH2), 40.6, 49.9, 52.6 (N-CH2-R, 2×CH2), 67.9 (Ar-
CH3), 112.6, 112.9, 128.8, 129.4, 131.5, 132.0 (Ar-C), 149.1 (N=C=N). Anal. Calcd. for
C18H29N2Br: C 61.2, H 8.3, N 7.9. Found C 61.4, H 8.4, N 8.1.

2.4. Synthesis of Ag(I)-NHC complexes (7-9)2.4.1. bis (5-methyl-1,3-dipropyl-

benzo[d]imidazol-3-ium silver I) hexafluoro phosphate (7)
Stirred a mixture of 4 (2 g, 6.5 mmoles) and silver oxide (3 g, 13 mmoles) in methanol (120
mL) for 48 hours at room temperature and covered flask with aluminium foil. Silver mirror
formed on the wall of flask. Filtered the black suspension through celite and directly
exchanged halide countere ion with PF6- ions by adding aqueous solution of potassium
hexaflouro phosphate (2.5 g, 13.6 mmoles) into the filtrate at room temperature under
continuous stirring. Immediately, precipittion occurred. Left it to stir for 4 hours, filtered and
dried the precipitates at room temperature. Beige coloured powder of 7 obtained. Single
crystals were grown by slow evaporation of saturated solution in acetonitrile at room
temperature. Yield 2.65 g (57%); M.P 250-260 oC. ESI+, m/z, %): ([C28 H40 Ag N4]+, 539.23,
100). FT-IR (ATR, ν, cm-1): 2935, 2874 (Caliph-H str), 1567, 1456, 1401 (C=Nbenzimid str), 875,
827, 745 (Carom-H ben). 1H NMR (400 MHz, DMSO-d6,  ppm) 0.95 (12H, q, 4×CH3, J
=7.3Hz), 1.95 (8H, m, 4×CH2), 2.5 (6H, s, 2×Ar-CH3), 4.5 (8H, t, N-CH2-R, J =6.4 Hz), 7.3
13
(2H, d, Ar-H, J =8.28), 7.7 (2H, s, Ar-H), 7.76 (2H, d, J =8.28). C NMR (125.72 MHz,
DMSO-d6,  ppm) 11.6 (CH3), 21.5 (CH3), 24.01, 24.09 (R-CH2), 50.4, 50.5 (N-CH2-R,
2×CH2), 112.3, 125.8, 132.06, 134.08, 134.4 (Ar-C), 187.08, 189.05 (C-Ag-C). 19F (470.4
MHz, DMSO-d6,  ppm) -72.2 (d, 6F). 31
P (202.4 MHz, DMSO-d6,  ppm) -145.1 (h, 1P).
Anal. Calcd. for C28H40AgF6N4P: C 49.0, H 5.8, N 8.1 Found: C 48.9, H 5.7, N8.3.

2.4.2. bis (5-methyl-1,3-dibutyl-benzo [d] imidazol-3-ium silver I) hexafluorophosphate

(8)

This synthesis followed the procedure for synthesis of 7 but using compound 5 (1.72 g, 6.5
mmoles). Beige coloured powder of 8 obtained in yield 2.4 g (52%); M.P 261-262 oC. FTIR
(ATR, ν, cm-1): 2936, 2873 (Caliph-H str), 1566, 1460, 1400 (C=Nbenzimid str), 827, 742 (Carom-
H ben). 1H NMR (400 MHz, DMSO-d6,  ppm) 0.97 (12H, q, 4×CH3, J = 7.51Hz), 1.92 (8H,
m, 4×CH2), 2.5 (6H, s, 2×Ar-CH3), 2.98 (8H, m, 4×CH2), 4.5 (8H, t, N-CH2-R, J = 6.4 Hz),
7.3 (2H, d, Ar-H, J = 8.28), 7.7 (2H, s, Ar-H), 7.76 (2H, d, J = 8.28). 13C NMR (125.72 MHz,
DMSO-d6,  ppm) 10.03 (CH3), 21.8 (CH3), 22.8, 22.9, 40.0 (R-CH2), 48.6, 48.7 (N-CH2-R,
19
2×CH2), 67.9, 112.7, 112.9, 131.5, 132.04 (Ar-C), 182.04 (C-Ag-C). F (470.4 MHz,
DMSO-d6,  ppm) -72.2 (d, 6F). 31
P (202.4 MHz, DMSO-d6,  ppm) -145.1 (h, 1P). Anal.
Calcd. for C32H48AgF6N4P: C 51.8, H 6.5, N 7.6; Found: C 52.1, H 6.3, N 7.9.

2.4.3. bis (5-methyl-1,3-dipentyl-benzo [d] imidazol-3-ium silver I) hexafluorophosphate

(9)
This synthesis followed the procedure for synthesis of 7 but using compound 6 (1.54 g, 6.5
mmoles). Beige coloured powder of 9 obtained in yield 2.6 g (51%); M.P 264-266 oC. FTIR
(ATR, ν, cm-1): 2893, 2875 (Caliph-H str), 1566, 1456, 1401 (C=Nbenzimid str), 830, 747 (Carom-
H ben). 1H NMR (400 MHz, DMSO-d6,  ppm) 0.94 (12H, q, 4×CH3, J = 7.51Hz), 1.51 (8H,
m, 4×CH2), 1.92 (8H, m, 4×CH2), 2.51 (6H, s, 2×Ar-CH3), 2.95 (8H, m, 4×CH2), 4.51 (8H, t,
N-CH2-R, J = 6.4 Hz), 7.32 (2H, d, Ar-H, J = 8.28), 7.67 (2H, s, Ar-H), 7.71 (2H, d, J =
8.28). 13
C NMR (125.72 MHz, DMSO-d6,  ppm) 11.0 (CH3), 21.5 (CH3), 24.0, 24.1, 30.5
(R-CH2), 50.4, 50.5 (N-CH2-R, 2×CH2), 112.3, 125.8, 132.0, 134.0, 134.4 (Ar-C), 187.4, 89.4
(C-Ag-C). 19F (470.4 MHz, DMSO-d6,  ppm) -72.2 (d, 6F). 31
P (202.4 MHz, DMSO-d6, 
ppm) -145.1 (h, 1P). Anal. Calcd. for C36H56AgF6N4P: C 66.2, H 8.6, N 8.5; Found: C 66.1,
H 8.3, N 8.7.
 2.5. Anticancer study

2.5.1. Preparation of cell culture

Human breast cancer (MDA-MB-231), colon cancer (HCT-116) and normal endothelial
(EA.hy926) cells were permitted to grow under optimal conditions in incubator. After
reaching 70-80% confluence rate, the cells were selected for cell plating by aspirating the old
medium from the plate subsequently washing the cells thrice with slightly basic (pH 7.4)
sterile phosphate buffered saline (PBS) which was later discarded. Then introduced trypsin,
distributed it evenly on cell surfaces and incubated the cells in 5% CO2 at 37 oC for 1 min,
gently tapped the flasks containing the cells to assist their segregation and observed them
under inverted microscope. The activity of trypsin was suppressed by adding 10% fresh FBS
medium (5 mL). Final concentration 2.5 × 105 cells/mL of cells was achieved after diluting
the medium and then inoculated into wells (100 μL per well). Finally, incubated the plates
containing the cells in 5% CO2 environment at 37 oC.

2.5.2. MTT assay

After seeding the cancer cells (100 μL, 1.5 × 105 cells/mL), the 96-well microtiter plate was
incubated in an incubator having CO2 environment for 24 h to allow the attachment of cells.
The test substance (100 μL) was introduced into each well (containing cells) after making
serial dilutions from stock solution of test substance in medium and again incubated the
plates in 5% CO2 atmosphere at 37 oC for 72 h and then MTT reagent was introduced into
each well and again incubated for 4 h. Then 50 μL DMSO (MTT lysis solution) was
introduced in each well and plates were incubated in CO2 incubator for 5 min and plates were
read at two different wavelengths (570 and 620 nm) by multimode monochromator-based
microplate reader (Tecan Infinite® M200 PRO). To evaluate the effects of test samples on
growth inhibition and cell viability, data were recorded and analyzed. Optical density (OD)
obtained during MTT assay was used to calculate the percentage of growth inhibition which
was further employed to calculate IC50 values to compare the activities of ligands with that of
silver complexes.

2.5.3. Measurement of Selectivity Index (SI):

Selectivity index (SI) is a ratio of IC50 of normal cells to the cancer cells. SI value indicates
more cytotoxicity of a compound towards cancer cells than compared to normal cell. In this
study, human endothelial cells (EA.hy926) were used as the representative cell line for
normal cells. SI values of the test samples were determined using the following formulation,

Selectivity Index = IC50 of EA.hy926 cells / IC50 of cancer cells

2.5.4. Detection of Apoptosis Using Rhodamin 123 Assay

In order to detect the mode of cytotoxicity, Rhodamin 123 assay was conducted [32]. MDA-
MB 231 cells were selected for the study and seeded in 6 well plates. After overnight
attachment, the cells were treated with the compounds for 6 h and then fixed by 4%
paraformaldehyde for 20 min. Tamoxifen (10 µM) and 0.1% DMSO were used as positive
and negative controls, respectively. The rhodamine 123 was added to cells at a final
concentration of 5 μg/mL and incubated for 30 min to stain the mitochondria. The wells then
were photographed using inverted EVOS f1 digital microscope at 20× magnification power
to monitor for fluorescent signals.

2.5.5. Statistical Analysis

Statistical difference between the treatments and the control were evaluated by one-way
analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Differences
were considered significant at p < 0.05, and p < 0.01.

3. RESULTS AND DISCUSSION

3.1. Synthesis

NHC precursors and silver(I)-NHC complexes were successfully synthesized by following a

modified protocol [17, 33]. First step of procedure involved the N-alkylation of 5-methyl
benzimidazole (after its deprotonation in alkaline medium) by reacting its equimolar amount
with alkyl halide. It appeared as if delocalized π-electrons of benzimidazole (after
deprotonation) attack the alkyl group in such a way that it generates two isomeric N-alkylated
products with 50% probability of each (Scheme 1), so second alkylation was done with 1
equivalent of same alkyl halide in 1,4 dioxane under reflux (in order to generate a single
product during formation of NHC ligand). After workup, 4 equivalents of silver oxide was
added in methanolic solution for metallation. For easy handling of end product, halide
counter ions were replaced in situ with hexaflourophosphate ions by adding 2 equivalent
aqueous solution of KPF6 after metallation step. Silver complexes were purified by excessive
washing with water and obtained in powder form. Scheme 1 shows the simple steps of
syntheses, starting from synthesis of N-alkylated benzimidazoles to the silver salts with PF6-
counter ions.

Scheme 1: Synthesis of N-alkylated benzimidazoles (1-3), benzimidazolium salts (4-6) and Ag(I)-NHC
complexes (7-9) where alkyl groups (R1= R2): propyl (for 1,4,7), butyl ( for 2,5,8), pentyl (for 3,6,9) and X
represents halide ion.

3.2. Characterization

3.2.1. FTIR spectroscopy

FTIR spectroscopy provided a preliminary proof of synthesis of all compounds by identifying

some specific patterns of peaks in their FTIR spectra. All pre-ligands (1-3) and ligands (4-6)
showed bands at 3,402–3,424 cm-1 which may be ascribed to Caliph–Nbenzimi stretching
vibrations [33] which in some cases appear broad (See supplementary files Figures S2-S7)
due to overlapping with water signal as residual water molecule with these compounds form
H-O-H---X- interactions (as indicated from their 1H NMR spectra) [33]. Signals in the range
3000-3100 cm-1 can be attributed to C-H stretching vibrations of aromatic ring. The
appearance of signals in the region 2850-3000 cm-1 in spectra of 1-6 are ascribed to Calip-H
stretching vibrations of alkyl chains, however these signals were observed to diminish in their
silver complexes 7-9 due to coordination of Ag(I) ions and electron donating properties of
alkyl groups. Variation in this region is due to different nature of alkyl groups [34]. C=N
stretching vibrations of benzimidazole ring appeared at 1,350–1,500 cm-1 for 1-6 while for
complexes 7-9 these vibrations appeared in specific four fingers (f.fs) pattern [34] which is
an evidence of successful synthesis of these complexes [35] (Figure 1, also see
supplementary files Figures S8-S9). C-H out of plane (oop) bending vibrations (a
characteristic of methyl substitution to the ring) of aromatic ring appeared at 675-900 cm-1 as
sharp signal (see supplementary files Figures S8-S9).

Figure 1: FTIR spectrum of compound 9

3.2.2. NMR studies

NMR studies further helped to provide evidences of synthesis. Synthesis of preligands 1-3
was confirmed by observing specific signals for protons of alkyl groups at chemical shift 0-6
ppm and aromatic protons at 6-8 ppm. 1H NMR spectra of 1-3 indicated as if delocalized π-
electrons of benzimidazole (after deprotonation) attack the alkyl group in such a way that it
generates two isomeric N-alkylated products with 50% probability of each (Figure 2, also
See supplementary files Figures S10-S12). Synthesis of NHC ligands 4-6 was confirmed by
observing changes in chemical shifts of protons of preligands and appearance of new signals
due to one more alkyl group and specific signal for the most deshielded proton and carbon
(NCHN) between 9-12 ppm in 1H NMR and 140-145 ppm in 13C NMR spectra, respectively
[36] ) (See supplementary files Figures S13-S18). The progress of the metallation reaction of
4-6 to form silver complexes (7-9) can be understood by observing proton and carbon
resonances of NCHN in 1H and 13C NMR spectra. The NCHN proton resonance of NHC salt
at 9-12 ppm disappears during metallation with silver [37] which may be an indication of Ag-
13
NHC bonding (Figures 5, S19, S21). C NMR spectra reveal more absolute information
about the nature of silver-NHC bonding. The resonance values of Ccarbene-Ag-Ccarbene in such
silver-NHC complexes fall between 180-200 ppm [38] which appear doublet of doublet in
107 109
most of the complexes due to occurrence of two natural isotopes of silver Ag and Ag
and both are NMR active having nuclear spin of 1/2. Depending on the coupling constants of
each isotope, a complex splitting pattern (doublet of doublet) is expected. The coupling
constants for the Ccarbene-107Ag and Ccarbene-109Ag fall between 180-234 Hz and 204-270 Hz,
respectively [39]. Such splitting patterns (doublets) were observed in NMR spectra of
compounds 7 and 9 at 187-189 ppm (Figures 6 and S21). The absence of this splitting pattern
in some complexes can be attributed to the fluxional behaviour of silver-NHC complexes on
the NMR time scale and the resonances coalesce and eventually appear as singlet as the
dynamic behaviour increases on the NMR time scale [39]. This behaviour is evident in
13
Figure S22 where singlet appeared in C NMR spectrum of compound 8. The presence of
phosphorous and fluorine in complexes having PF6- counter ions was also confirmed by
31 19
observing heptet and doublet at (-144) - (-145) ppm and (-71) - (-73) ppm in P and F
NMR spectra, respectively (See supplementary files Figures S23-S24).

Figure 2: 1H NMR spectrum of compound 3

Figure 3: 1H NMR spectrum of compound 4

Figure 4: 13C NMR spectrum of compound 4

Figure 5: 1H NMR spectrum of compound 9

Figure 6: 13C NMR spectrum of compound 9

3.2.3. Electron spray ionization mass spectroscopy

Electron spray ionization mass spectroscopy further provided confirmation of synthesis.

Positive mode ESI-mass spectrum of complex 7 displayed a peak at m/z 539.23 which
exactly corresponds to molecular weight of cationic species [M]+ i.e [C28H40AgN]+ arising
from the loss of one hexafluoro phosphate ion suggesting a mononuclear structure where Ag+
ion is coordinated by two carbene moieties of NHC ligand. Also, the 1H and 13
C NMR
spectra are in agreement with the ESI-MS result (Figure 7).

Figure 7: ESI-MS spectrum of compound 7

3.2.4. X-ray crystallographic study

X-ray-quality crystals of complex 7 were grown by slow evaporation of its saturated solution
in acetonitrile at room temperature. Single crystals of the compound appeared as colourless
blocks. The pertinent structural parameters (Crystal refinement data, selected bond lengths
and angles) are listed in Table 1 and 2.
XRD crystal data of compound 7 (silver complex of 5-methyl-N,N-di-n-propyl-
benzimidazolium salt) with molecular formula C28H40AgN8F6N4P indicates that it is actually
the expected complex, which crystallizes in triclinic space group P1(2) having one cationic
benzimidazolium core and one hexaflouro phosphate counter anion. Perspective view of
compound 7 is shown in Figure 8. The compound is generated by symmetry since the silver
ion is located on an inversion centre. NHC ligand is coordinated to the metal centre in a
monodendate manner forming a non-linear structure. The P of the PF6- anion is also sitting on
an inversion centre. Propyl group points below the plane for one NHC and above the plane
for the second NHC which indicates that inversion centre is going through the silver centre.
The N1–C1 (1.353(4) Å) and N2–C1 (1.335(3) Å) bond lengths are similar and they are
shorter than N1–C2 (1.387(4) Å) and N2–C7 (1.395(3) Å), due to delocalization of electrons
between the N1–C1–N2 atoms. The internal benzimidazole ring angle (N–C–N) at the
carbene centre is 106.5(2)° for N1-C1-N2 which is consistent with the reported values [36].
The bond angle between benzimidazole ring and propyl moiety was found 125.0(2)° for C1-
N1-C9 and 124.4(2)° for C1-N2-C12. Dihedral angle between benzimidazolium ring and
propyl substituent for N1–C9–C10–C11 -179.8(3)° and N2-C12-C13-C14 -74.1(5)° indicates
that they are not coplanar. N–C and P–F bond distances are in the range 1.345(4)-1.469(4)
and 1.592-1.607 A°, respectively. As the compound is symmetry generated, so the Ag(I) ions
lie in linear coordination geometry at 180o for C1-Ag1-C1. In the crystal, the
hexaflourophosphate anions link the cations with a three-dimensional network via weak
electrostatic intermolecular C---F interaction (3.77-5.1 Å). These bond distances and angles
are comparable with those reported in other similar silver(I)-NHC complexes [36].

Single crystal XRD could not further support the structures of other Ag(I)-NHC complexes as
all attempts to get single crystals remained unsuccessful. However, 1H and 13
C-NMR
provided evidences of their successful synthesis.

Figure 8: ORTEP view of complex 7 (50% probability level).

Table 1. Crystal data and structure refinement details for compound 7

Formula C28H40AgN8F6N4P
Formula weight 685.48
Crystal System Triclinic
Space group P 1 (2)
Unit cell dimensions a (Ao) 7.4637(2)
 b (Ao) 9.8480(3)
c (Ao) 10.9354(4)
 (o) 80.497(2)
 (o) 73.3010(10)
( )
o
88.0460(10)
o3
V (A ) 759.241
R-factor (%) 2.65
Z, Z’ 1, 0
Density (calcd) (g/cm3) 1.499
-1
Abs coeff. (mm ) 6.362
F(000) 352.0
Crystal size (mm) 0.13 x 0.11 x 0.09
Temperature (K) 100
Radiation (A° ) CuKa 1.54178
theta Min, max (o) 4.277, 72.169
Data set -9: 9 ; -12: 12 ; -13: 13
Tot.; Uniq. Data 2894
Nref, Npar 3002, 195
R, wR2, S 0.0265( 2878), 0.0786( 2894),
1.158

Table 2. Bond lengths and bond angles for 7

Bond lengths (Å) Bond angles Dihedral angle
(o) (o)
Ag1-C1 2.081 C1-Ag1-C1 180.0 C2-N1-C1-Ag1 -178.5

N1-C1 1.353(4) C1-N1-C2 110.7(2) C2-N1-C1-N2 0.5(3)

N1-C2 1.387(4) C1-N1-C9 125.0(2) C9-N1-C1-Ag1 0.6

C1-N2 1.345(4) Ag1-C1-N2 127.1 C1-N1-C2-C7 -0.5(3)

N2-C7 1.395(3) N1-C2-C3 132.6(3) C9-N1-C2-C7 -179.6(2)

C2-C3 1.392(4) C4-C5-C6 120.3(3) C2-N1-C9-C10 -79.8(3)

C2-C7 1.395(4) C1-N2-C12 124.2(2) C7-N2-C1-Ag1 178.8

C3-C4 1.377(4) C5-C6-C7 117.4(3) C12-N2-C1-Ag1 3.2

C4-C5 1.406(4) C9-C10-C11 110.9(3) C12-N2-C1-N1 -175.9(2)

C5-C6 1.383(4) C1-N1-C2 110.7(2) C1-N2-C7-C6 -178.4(3)

C6-C7 1.389(4) C1-N2-C12 124.4(2) N1-C2-C3-C4 -177.6(3)

C9-C10 1.513(5) Ag1-C1-N2 127.1 N1-C2-C7-N2 0.4(3)

C12-C13 1.505(5) C2-C3-C4 116.7(3) C3-C2-C7-N2 -177.6(3)

C13-C14 1.520(9) C6-C5-C8 120.5(3) C2-C3-C4-C5 -0.8(5)

N1-C1 1.353(4) C5-C6-C7 117.4(3) C8-C5-C6-C7 178.4(3)

N1-C2 1.387(4) C2-C7-C6 121.8(3) C5-C6-C7-N2 177.6(3)

N2-C7 1.395(3) N2-C7-C6 132.4(3) C2-N1-C1-Ag1 178.5

C2-C3 1.392(4) C4-C5-C8 119.2(3) C9-N1-C1-N2 -179.6(2)

C3-C4 1.377(4) C3-C4-C5 122.6(3) C9-N1-C2-C7 179.6(2)

3.3. Anticancer activity

All the compounds were tested for their antiproliferative study against human breast cancer
(MDA-MB-231) colon cancer (HCT 116) and normal endothelial (EA.hy926) cell lines by
measuring their minimum inhibitory concentration (IC50) values. IC50 values of NHC ligands
4-6 and Ag(I)-NHC complexes 7-9 have been given in Table 3. From the table, it is evident
that all the compounds are far lesser cytotoxic to normal cells (EA.hy926) as compared to
cancer cells (MDA-MB-23 and HCT 116). NHC salts exhibited moderate antiproliferative
behaviour against MDA-MB-231 and HTC-116 cells (with IC50 values in the range
25.51±0.24 -34.21±0.62 μM against MDA-MB-231 and 15.21±0.52 -28.66±0.21 μM against
HCT-116 cells). While Ag(I)-NHC complexes 7-9 exhibited strong cytotoxic activity against
both cell lines (with IC50 values 4.22±0.24 -8.12±0.62 μM against MDA-MB-231 and
10.31±0.31 -7.43±0.23 μM against HCT-116 cells). These values indicate that HCT-116 cells
are more sensitive to NHC salts and less sensitive to Ag(I)-NHC complexes while reverse is
the case for MDA-MB-231 cells. Similar results have been reported by Patil and co-workers,
their compounds having symmetrically substituted (5,6 dimethyl) benzimidazole displayed
strong cytotoxic activites (IC50 10.8±1.9- 24.4±1.8 μM) [26]. It was observed that all the
NHC ligands 4-6 have much less activities compared to respective silver complexes 7-9 due
to coordination of silver to carbene carbon of ligands resulting in enhanced antiproliferative
effect of Ag(I)-NHC complexes due to participation of silver ions in the cell death
mechanism. It is also evident that increase in chain length decreases the activity of ligands
while increases that of silver complexes which is attributed to increased lipophilicity that
supports the passage of silver cations through cell membrane into the cell where it penetrates
into the cell organelles resulting in inhibition of metabolic and respiratory mechanisms.
Hence, increased chain length and presence of methyl substituent on benzimidazole ring
enhance the biopotency of Ag(I)-NHC complexes. Figures 9-10 show dose dependent
antiproliferative effect of synthesized ligands and complexes 4-9 on MDA-MB-231 and
HCT-116 cells. All the compounds displayed dose dependent cytotoxic activites against both
cells lines.

Table 3: IC50 (μM) values of NHC ligands and respective silver complexes against MDA-
MB-231, HCT-116 and EA.hy926 cell lines.

Groups Compounds IC50 Values in µM

MDA-MB-231 HCT-116 EA.hy926
Ligands 4 25.51 ± 0.24 15.21 ± 0.52 76.23 ± 0.23
5 27.30 ± 0.44 20.46 ± 0.73 89.71 ± 1.92
6 34.21 ± 0.62 28.66 ± 0.21 112.47 ± 2.21
Complexes 7 8.12 ± 0.62 10.31 ± 0.31 44.92 ± 0.11
8 6.91 ± 0.40 9.23 ± 0.55 56.21 ± 0.42
9 4.22 ± 0.24 7.43 ± 0.23 32.20 ± 1.08
Positive control Standard drug a 8.20 ± 0.14 5.5 ± 0.34 29.3 ± 1.12

a = Tamoxifen, 5-Fluorouracil and betulinic acid were used as positive controls for MDA-MB-231,
HCT 116 and EA.hy926 cells, respectively.
Figure 9: Dose dependent antiproliferative effect of synthesized ligands (4-6) and complexes (7-9) on MDA-
MB-321 cells.

Figure 10: Dose dependent antiproliferative effect of synthesized ligands (4-6) and complexes (7-9) on HCT-
116 cells.

3.4. Selectivity index (SI)

The selectivity index (SI) which indicates the cytotoxic selectivity of the compound for
cancer cells and its safety towards the normal cells [32], was determined from the ratio of the
IC50 value obtained from the test on normal cell (EA.hy926) versus the IC 50 value for cancer
cell. It is recommended that, the sample with more than 3 SI value will be considered to have
high selectivity towards the particular cell line [32]. SI values of the tested samples for MDA-
MB 231 and HCT 116 cells are shown in Table 4. Among the tested compounds, generally
the complexes revealed higher selectivity for cancer cells than that of their ligands.
Particularly, the compounds 8 and 9 showed significantly (p < 0.01) more pronounced
selectivity index (SI) for both the cancer cells tested.

Table 4: Selectivity Index of the compounds for the tested cancer cells.

Groups Compounds SI = IC50 of EAhy.926/IC50 of cancer cell

MDA-MB-231 HCT-116
Ligands 4 2.98 5.01
5 3.29 4.38
6 3.29 3.92
Complexes 7 5.53 4.36
8 8.13 6.09
9 7.63 4.33
Positive control Standard drug a 3.57 5.33

a = Tamoxifen, 5-Fluorouracil and betulinic acid were used as positive controls for MDA-MB-231,
HCT 116 and EA.hy926 cells, respectively.

3.5. Induction of Apoptosis in MDA-MB-231 cells

Rhodamin 123 is a cationic probe which can be readily absorbed and accumulated in
mitochondria of a live cell [32]. A loss of mitochondrial membrane potential (∆Ψ) is a
marked indication of apoptosis. To investigate whether the apoptosis induced by the
synthesized compounds in MDA-MB-231 cells involved the loss of mitochondrial integrity,
the mitochondrial membrane potential in the breast cancer cells was evaluated by confirming
the uptake of rhodamine 123 by mitochondria. The treated and untreated cells were exposed
to rhodamine 123 and the intensity of rhodamine in the cells was observed. When the
mitochondrial membrane potential decreases, the rhodamine 123 uptake by the cells also
decreases and consequently the flourescent signal reduces exponentially. Results of the
present study showed an obvious intensification of fluorescence in the untreated cells,
whereas the signal in the treated cells reduced significantly. This suggests the loss in
mitochondrial membrane potential. The apoptotic indices after 6 h treatment with the
compounds are given in the Table 5. Among all the tested compounds, the complexes 8 and 9
revealed more pronounced apoptotic effect with 71.91% and 83.16% apoptotic indices,
respectively. This indicates a remarkable reduction in mitochondrial membrane potential in
MDA-MB-231 cell line caused by the complex 8 and 9.

Table 5: Apoptotic Indices of the tested compounds for MDA-MB-231 cells

Compounds % apoptotic index

4 42.83
5 57.05
6 39.33
7 64.36
8 71.91
9 83.16
Tamoxifen 79.11

3.6. Anticancer mechanism of action and structure-activity relationship (SAR)

The mechanism of action of silver complexes during anticancer activity has been studied and
it has been demonstrated that bonding of silver with NHC ligand offers enhanced biopotency
against cancer cells by inhibiting fast release of silver ions in biological system [19, 40].
Literature survey revealed that anticancer phenomenon during interaction of NHC ligands
and their silver complexes with cancer cells may proceed through apoptosis (caspase
independent) by external/internal stress factors which can be indicated from formation of
apoptotic bodies and chromatin condensation in the cytoplasm of cancer cells [33, 34, 41].
Furthermore, it has been reported that silver ions stop the cell functionalities by getting
deposited in the cytosol where they may interact with proteins and enzymes [33, 42, 43].
Thus the outcomes of this study are in agreement with the reported work as black deposits
(might be of silver oxide) in treated cancer cells have been observed which is the evidence
that deposits of silver ions contribute in cytotoxic activity of the tested compounds.

4. Conclusion
In conclusion, a novel series of benzimidazolium based NHC ligands and silver complexes
was synthesized and characterized by various analytical techniques. Unsymmetrically
substituted benzimidazole was found unique in its reactivity and generation of a single
product during NHC ligand formation was only possible after two successive alkylations with
the same alkyl halide. XRD crystal data of compound 7 revealed that NHC ligand is
coordinated to the metal centre in a monodentate manner forming a non-linear structure. All
benzimidazolium salts and their respective Ag(I)-NHC complexes were tested in vitro against
human breast cancer (MDA-MB-231) and colon cancer (HCT-116) cells to study their
anticancer potential. All the compounds are far lesser cytotoxic to normal cells (EA.hy926) as
compared to cancer cells (MDA-MB-23 and HCT 116). NHC salts exhibited moderate
antiproliferative behaviour against MDA-MB-231 and HTC-116 cells. While Ag(I)-NHC
complexes 7-9 exhibited strong cytotoxic activity against both cell lines. All the compounds
displayed dose dependent cytotoxic activites against both cells lines. Mechanistic study
revealed that cancer cell death is governed by apoptosis via mitochondrial pathway. For
further study, pre-clinical trials of synthesized drugs for chemotherapeutic purposes are in
progress.

Acknowledgements
Prof. Haq Nawaz Bhatti and Dr. Muhammad Adnan Iqbal would like to appreciate Higher
Education Commission, Pakistan for support in the frame of startup research grant Vide
Letter No. 21-1085/SRGP/R&D/HEC/2016 to establish Organometallic and Coordination
Chemistry Laboratory at University of Agriculture, Faisalabad where major part of this
research was accomplished and IRSIP Vide Letter No. 1-8/HEC/HRD/2017/6940 which was
carried out at University de Montreal, Canada. Aqsa Habib expresses her deep gratitude to
anonymous Prof. Zargarian Davit and Dr. Loic Mangin, University de Montreal for their
valuable contribution to prepare and improve the structure, quality and clarity of
the manuscript.

Authors Contribution
The research work described in this manuscript was conducted by Aqsa Habib, under the
supervision of Prof. Haq Nawaz Bhatti and supreme co-supervision of Dr. Muhammad
Adnan Iqbal. Anticancer studies were carried out under the guidance of Mansoureh N.
Vishkaei. The useful suggestions for interpretation of these results were provided by Dr.
M.B. Khadeer Ahmed and Dr. A.M.S. Abdul Majid.

Additional information
The manuscript includes supplementary material as a separate file. CCDC 1892737 contains
the supplementary crystallographic data for complex 7 included in this paper. This data can
be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif

Conflict of Interests
The authors declare no conflicts of interest

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Graphical Abstract
Unsymmetrically substituted benzimidazole is unique in its reactivity and generates two
isomeric N-alkylated products after first alkylation, so its second alkylation is necessarily
done with same alkyl halide in order to ensure the formation of single product during ligand
synthesis.