Effect of ring substitution on synthesis of benzimidazolium salts and their silver(I) complexes: characterization, electrochemical studies and evaluation of anticancer potential

Article abstract of DOI:10.1007/s11243-019-00321-7

Abstract: A series of bidendate 5,6-dimethyl benzimidazolium-based N-heterocyclic carbene (NHC) proligands and their silver complexes were synthesized. The synthetic approaches to the proligands were constrained by the methyl substituents, which impose a significant impact on the reactivity according to their sigma electron-donating abilities. The corresponding silver(I) complexes were obtained by in situ deprotonation of the NHCs and characterized by physicochemical and spectroscopic methods. In addition, a single-crystal X-ray diffraction study of complex C5 revealed its dinuclear structure. Irreversibility of redox events was observed in electrochemical studies of these complexes. In vitro anticancer studies of the azolium salts and their silver(I) complexes against human breast cancer (MDA-MB-231), colon cancer (HCT-116) and normal endothelial (EA.hy926) cells revealed that all the compounds are more cytotoxic to cancer cells than to normal cells and the complexes are more potent than their corresponding NHC proligands. Increased chain length, the presence of methyl substituents on the benzimidazole ring and aryl linker and two silver centres all enhance the biopotency of these complexes. Graphical abstract: Substituted benzimidazolium-based N-heterocyclic carbene (NHC) proligands and silver complexes are advantageous anticancer tools for medicinal chemistry.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s11243-019-00321-7

Transition Metal Chemistry
https://doi.org/10.1007/s11243-019-00321-7
Efect of ring substitution on synthesis of benzimidazolium salts
and their silver(I) complexes: characterization, electrochemical studies
and evaluation of anticancer potential
Aqsa Habib1 · Muhammad Adnan Iqbal1,2 · Haq Nawaz Bhatti1 · Muhammad Shahid3
Received: 11 January 2019 / Accepted: 4 April 2019
© Springer Nature Switzerland AG 2019
Abstract
A series of bidendate 5,6-dimethyl benzimidazolium-based N-heterocyclic carbene (NHC) proligands and their silver com-
plexes were synthesized. The synthetic approaches to the proligands were constrained by the methyl substituents, which
impose a signifcant impact on the reactivity according to their sigma electron-donating abilities. The corresponding silver(I)
complexes were obtained by in situ deprotonation of the NHCs and characterized by physicochemical and spectroscopic
methods. In addition, a single-crystal X-ray difraction study of complex C5 revealed its dinuclear structure. Irreversibility
of redox events was observed in electrochemical studies of these complexes. In vitro anticancer studies of the azolium salts
and their silver(I) complexes against human breast cancer (MDA-MB-231), colon cancer (HCT-116) and normal endothelial
(EA.hy926) cells revealed that all the compounds are more cytotoxic to cancer cells than to normal cells and the complexes
are more potent than their corresponding NHC proligands. Increased chain length, the presence of methyl substituents on
the benzimidazole ring and aryl linker and two silver centres all enhance the biopotency of these complexes.
Introduction
strategy involves the in situ deprotonation of the azolium salt
in the presence of a basic anion generated from a transition
metal precursor [4]. A variety of silver(I) complexes have
been synthesized by this method [5–10]. Numerous silver
complexes with anticancer potential against various cancer
cell lines have been reported [6, 8, 9]. The strong antican-
cer activity of M-NHC (M = Au, Cu, Ag, Pd) complexes
(aforded from azolium salts having an unsubstituted ben-
zene ring) against various cancer cell lines including breast
cancer (MB157), renal cancer (Caki-1), ovarian cancer
(OVCAR-3) and cervical cancer (HeLa) has been reported
[11, 12]. Iqbal and co-workers reported the anticancer poten-
tial of dinuclear silver-meta/para-xylyl-linked bis-benzimi-
dazolium complexes (having short Ag–Ag distances) against
human leukaemia (HL-60) and colon (HCT-116) cancer cell
lines [8, 12]. Their study revealed that metal–metal interac-
tions in both gold(I) and silver(I) complexes (shorter than
the sum of their van der Waals radii) are strong enough to
dominate the repulsive forces between cationic metal cen-
tres, thus leading to substantial cationic NHC networks [13,
14].
In contemporary medicinal and material sciences, N-hetero-
cyclic carbenes (NHCs) have proved to be valuable species
for the design of a broad range of coordination complexes
with medicinal and catalytic applications by virtue of their
strong σ-donor and weak π-acceptor features and compara-
tively facile synthesis [1–3]. NHC transition metal com-
plexes can be obtained by various approaches. The simplest
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11243-019-00321-7) contains
supplementary material, which is available to authorized users.
* Muhammad Adnan Iqbal
adnan.iqbal@uaf.edu.pk
* Haq Nawaz Bhatti
hnbhatti2005@yahoo.com
1
Department of Chemistry, University of Agriculture
Faisalabad, Punjab 38040, Pakistan
2
Organometallic and Coordination Chemistry Laboratory,
The anticancer potential of silver(I) complexes is strongly
infuenced by their lipophilicity/hydrophilicity which in
turn is controlled by the steric and electronic character of
the substituents. In contrast to other metal coordination
Department of Chemistry, University of Agriculture
Faisalabad, Punjab 38040, Pakistan
3
Department of Biochemistry, University of Agriculture
Faisalabad, Punjab 38040, Pakistan
Vol.:(0123456789)
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Transition Metal Chemistry
complexes which generally target the DNA of cancer cells,
Ag/Au–NHC complexes modulate the mitochondria-induced
apoptosis by directly targeting the infected cell’s mitochon-
drial membrane [15–18]. A variety of drugs are already
available for treatment of cancer, but most of these drugs
have severe side efects. Hence, current research is targeted
towards the discovery of new metallodrugs with little or no
side efects. Previously, we have investigated the anticancer
potential of silver(I) complexes derived from unsymmetri-
cal (5-substituted) benzimidazoles [19]. The study revealed
that the presence of substituents has a signifcant impact
on synthesis and activity of silver complexes. This article
describes a further extension of our previous study. We
report the synthesis, characterization, electrochemical and
anticancer properties of a series of benzene ring disubsti-
tuted benzimidazolium-based silver(I) complexes.
counterions were replaced in situ with hexafuorophosphate
by adding two equivalents of aqueous KPF6. All the silver
complexes were purifed by washing with water and obtained
in powder form. Scheme 1 shows the overall synthesis.
Characterization
FTIR spectra of all the species in Scheme 1 were recorded.
A strong band at 3402–3424 cm−1 assigned to Caliph–Nbenzim
stretching vibrations was observed in the spectra of com-
pounds a and b and also of the proligands (L1–L6), which
in some cases appeared broad (see supplementary fles Fig-
ures S3–S10) due to overlap with residual water involved in
H–O–H–X− interactions (as indicated from their 1H NMR
spectra) [7]. A specifc pattern of peaks at 1350–1500 cm−1
in the spectra of a and b and L1–L6 can be ascribed to
C=N stretching vibrations of the benzimidazole ring,
which are less intense for L1–L6 due to the presence of
one more electron-donating alkyl group. These bands appear
as a strong and specifc “four fngers” pattern for the sil-
ver complexes (C1–C6) where conjugation is absent due to
Ag(I)–NHC coordination [10, 20] (see supplementary fles
Figures S11–S16). A very sharp signal at 900–675 cm−1 can
be ascribed to C–H out of plane (oop) bending vibrations of
the aromatic rings, which is a characteristic of methyl substi-
tution to the ring (see supplementary fles Figures S11–S16).
NMR studies provided further evidence for the success-
ful synthesis of all the compounds. Synthesis of the NHC
Results and discussion
NHC precursors and their dinuclear silver(I) complexes were
synthesized by following a modifed literature protocol [7,
9]. This involved the N-alkylation of 5,6-dimethyl benzimi-
dazole by reaction with one equivalent of alkyl halide fol-
lowed by second alkylation with half an equivalent of alkyl
halide under refux for in situ generation of the free carbene,
to which two equivalents of silver oxide were added for met-
allation. For easy handling of the end product, the halide
Scheme 1 Synthesis of
N-alkylated benzimidazoles
(a–b), bis-benzimidazolium
salts (L1–L6) and Ag(I)–
NHC complexes (C1–C6). (a
R1 =propyl; b R1 =butyl; L1,
C1, R1 =propyl, R2 =butyl;
L2, C2, R1 =propyl, R2 =pen-
tyl; L3, C3, R1 =propyl,
R2 =m-xylyl; L4, C4,
R1 =butyl, R2 =butyl; L5, C5,
R1 =butyl, R2 =pentyl; L1, C1,
R1 =butyl, R2 =m-xylyl)
1 3

Transition Metal Chemistry
Table 1 IC50 (μM) values of NHC proligands and their silver com-
proligands (L1–L6) was confrmed by changes in the chemi-
cal shifts compared to a and b (Figures S16–S17) together
with the presence of new signals arising from one more
alkyl group and the distinctive signal for the most deshielded
proton and carbon (NCHN) between 9 and 12 ppm in the
1H NMR and 140–145 ppm in 13C NMR spectra, respec-
tively [21] (see supplementary files Figures S19–S30).
Similarly, the synthesis of the Ag(I) complexes was con-
plexes against MDA-MB-231, HCT-116 and EA.hy926 cell lines
Compound
IC50 (µM)
MDA-MB-231
HCT-116
EA.hy926
L1
20.51 0.34
24.30 0.44
31.21 0.52
23.40 0.35
27.60 0.54
33.15 0.51
9.12 0.21
7.91 0.32
5.22 1.14
8.14 0.91
7.42 0.73
4.41 0.24
7.50 0.12
18.41 0.45
21.45 0.54
27.56 0.31
20.89 0.62
25.25 0.53
29.43 0.22
12.34 0.24
10.23 0.87
8.45 1.02
11.67 0.93
10.24 0.82
7.67 0.51
5.5 0.34
66.25 1.30
78.74 1.21
89.12 1.45
82.14 1.67
94.23 2.10
105.14 2.14
54.32 1.56
42.21 1.42
35.20 1.08
49.16 2.13
51.20 2.04
45.46 1.14
29.31 1.12
L2
L3
1
frmed by changes in both the H and 13C NMR spectra
L4
L5
together with the loss of the signal for the most deshielded
proton (NCHN) and shift of the carbon (NCHN) signal to
180–190 ppm (see supplementary fles Figures S31–S42)
[22]. Two-dimensional (HSQC) 1H–13C correlation NMR
spectra were used to help assign the 1H and 13C signals [23]
(see supplementary fles Figures S43–S48). Furthermore,
the presence of phosphorous and fuorine in the complexes
L6
C1
C2
C3
C4
C5
−
C6
having PF6 counter ions was confrmed by the observed
Positive controla
heptet and doublet from −144 to −145 ppm and from −71
to −73 ppm in the 31P and 19F NMR spectra, respectively
(see supplementary fles Figures S49–S50).
aTamoxifen, 5-fuorouracil and betulinic acid were used as positive
controls for MDA-MB-231, HCT 116 and EA.hy926 cells, respec-
tively
Electron spray ionization mass spectroscopy provided fur-
ther confrmation of successful synthesis of the complexes.
ESI–MS of complexes C2, C3 and C6 displayed peaks at
m/z 1247.42, 1315.39 and 1371.45, respectively, which cor-
respond to the molecular weights of the [M]+ cations for
these compounds [C58H80Ag2N8PF6]+, [C64H76Ag2N8PF6]+
and [C68H84Ag2F6N8P]+, respectively. This indicates dinu-
clear structures in which each Ag+ ion is coordinated by
two carbene moieties of two bis-NHC ligands. ESI–MS
spectra of the compounds are given in supplementary fles
Figures S51–S53.
and bond angles [e.g. N2–C5–N1 = 107.5° and
N3–C19–N4=106.0(5)°] between similar atoms within two
halves of the molecule are the result of twinning. The inter-
nal benzimidazole ring angle (N–C–N) at the carbene centre
is 107.1(5)° for N1–C5–N2 and 106.0(5)° for N3–C19–N4;
these are consistent with reported values [21]. The bond
angle between the benzimidazole ring and pentyl linker is
124.5(5)° for C5–N2–C14 and 128.9(2)° for C18–N3–C19,
while the dihedral angle N2–C14–C15–C16 between the
benzimidazolium ring and pentyl substituent is 179.8(5)°
which indicates that they are coplanar. The N–C and P–F
bond distances are in the range of 1.341(7)–1.478(9) and
1.592(4)–1.606(4) Å, respectively. The Ag(I) centres have
a nonlinear coordination geometry (due to twisting) at
173.6(2)° for C5–Ag1–C19. In the crystal, the hexafuoro-
phosphate anions link the cations into a three-dimensional
network via weak electrostatic intermolecular C–F interac-
tions (3.71–5.7 Å). These bond distances and angles are
comparable with those reported in similar silver(I) NHC
complexes [21].
X‑ray crystallographic studies
X-ray quality crystals of compound C5 were grown by slow
evaporation of a saturated solution in acetonitrile at room
temperature. Single crystals of the compound were obtained
as colourless blocks. Crystal refnement data are presented
in the experimental part, while selected bond lengths and
angles are listed in the supplementary data fle (Table 1).
Complex C5 crystallizes in triclinic space group P-1(2)
having one cationic NHC ligand and two hexafuorophos-
phate counter anions in the unit cell. The bidentate NHC
ligand is coordinated to two metal centres in a nonlinear
dinuclear structure. The 5,6-dimethyl benzimidazolyl units
are located on either side of the central pentyl core. A per-
spective view of C5 is shown in Fig. 1. The structure is
twinned and symmetry-generated such that each dimer occu-
pies an inversion centre and the two NHC ligands attached to
one silver centre are not coplanar, but twisted by 60°. There
is no possibility of Ag–Ag interactions since the Ag–Ag
distance is greater than 8 Å. The slight diferences in bond
lengths [e.g. N1–C5=1.361(8) Å and N4–C19=1.363(7) Å]
All attempts to gain single crystal of the other complexes
proved unsuccessful. However, 1H, 13C NMR, 2D (HSQC)
NMR as well as ESI–MS studies provided strong evidence
for the successful synthesis of these complexes.
Cyclic voltammetry and chemical oxidation
experiments
In cyclic voltammetry experiments, these complexes showed
similar redox behaviour in line with their common metal ion,
1 3

Transition Metal Chemistry
Fig. 1 ORTEP view of C5 (50%
probability level). The other
PF6− counter anion is omitted
for clarity
i.e. Ag+. Therefore, the electrochemistry of complex C6 will
be discussed as representative.
Anticancer studies
Cyclic voltammograms for complex C6 are shown in
Fig. 2a–c. Three scan cycles were performed, and the
direction of the scans did not affect the voltammograms.
An irreversible redox process was observed for the com-
plex in both the reduction and oxidation cycles [24]. It
can be seen from Fig. 2a that during the reduction half
cycle (− 1.2 to 1.4 V), an irreversible one-electron reduc-
tion process Ag(I)/Ag(0) occurs at Epc − 750 mV (at scan
rates of 50 and 100 mV/s) and the irreversibility indi-
cates that the reduced complex is unstable and decom-
poses before the Ag(0)/Ag(I) oxidation. The presence
of a very small bump at 250 mV may be attributed to a
small impurity which appeared after the initial scans. The
irreversibility of the reduction event was confirmed from
three scan cycles (Fig. 2b). During the oxidation half
cycles, oxidation events were observed at Epa 950, 1600
and 1750 mV which may be most simply attributed to
Ag(0)/Ag(I) redox event of species other than the original
silver complex, i.e. during reduction, the silver complex
decomposed and the oxidation occurred in the decom-
posed species (Fig. 2c). These oxidation processes cannot
be attributed to Ag(II)/Ag(I) as it has been reported at
E1/2 > 2500 mV depending on the pH of the solution [25].
Over the last few years, several groups have tested a number
of structurally diverse silver(I) complexes against various
cancer cell lines [9, 26–28]. All of these silver complexes
were based on NHC ligands containing unsubstituted benzi-
midazole rings. However, to the best of our knowledge, the
anticancer potential of silver(I) complexes with NHC ligands
containing disubstituted benzimidazole rings has not yet
been reported. We have therefore explored the activities of
the present complexes against human breast cancer (MDA-
MB-231), colon cancer (HCT 116) and normal endothelial
(EA.hy926) cell lines by obtaining minimum inhibitory con-
centration (IC50) values.
The IC50 values of the free NHC proligands (L1–L6)
and their Ag(I) complexes (C1–C6) are given in Table 1.
All of these compounds are less cytotoxic to normal cells
(EA.hy926) compared to cancer cells (MDA-MB-23 and
HCT 116). The free NHC salts exhibited pronounced anti-
proliferative behaviour against MDA-MB-231 and HTC-
116 cells (with IC50 values in the range 20.51–33.15 μM
against MDA-MB-231 and 18.41–29.43 μM against HCT-
116 cells). Similarly, their Ag(I) complexes exhibited
strong cytotoxic activities against both cancer cell lines
(with IC50 values 4.41–9.12 μM against MDA-MB-231
1 3

Transition Metal Chemistry
Fig. 2 Cyclic voltammograms
for complex C6 indicating
various events of oxidation and
reduction. The measurements
were taken at 298 K on acetoni-
trile solutions containing 0.1 M
of [Bu4N] [PF6] and 1 mM of
the complex
and 7.67–12.34 μM against HCT-116 cells). All of the
NHC proligands L1–L6 are less active than their silver
complexes C1–C6. Hence, it seems that coordination of
silver to these NHCs results in enhanced antiproliferative
efects, perhaps due to participation of silver ions in the
cell death mechanism. It is also evident that the aryl group
linker decreases the cytotoxicity of proligands L3 and L6
but increases that of silver complexes C3 and C6. This
efect can be attributed to increased lipophilicity aiding
the passage of silver cations through the cell membrane
and into the cell organelles, resulting in inhibition of
metabolic and respiratory mechanisms. Hence, increased
chain length, presence of methyl substituents on the ben-
zimidazole ring and aryl linker and two silver centres all
enhance the biopotencies of these complexes. Figures 3, 4,
5 and 6 show the dose-dependent antiproliferative efects
of the proligands L1-L6 and complexes C1–C6 on MDA-
MB-231 and HCT-116 cells. All of these compounds
1 3

Transition Metal Chemistry
Fig. 3 Dose-dependent antipro-
liferative efects of proligands
L1–L6 on HCT-116 cells
Fig. 4 Dose-dependent antipro-
liferative efects of complexes
C1–C6 on HCT-116 cells
displayed dose-dependent cytotoxic activities against both
cancer cells lines.
particular, complex C6 showed high SI values against both
cancer cell lines.
Mechanism of action and structure–activity
relationships
Selectivity index
The selectivity index (SI) provides an indication of the cyto-
toxic selectivity of a compound against cancer cells com-
pared to normal cells [29]. The SI values were calculated
from the ratio of the IC50 values for normal cells (EA.hy926)
versus those for cancer cells. A compound with SI>3 is con-
sidered highly selective for that cell line [29]. SI values for
the present compounds are shown in Table 2. The complexes
displayed greater selectivities than the free proligands. In
It is known that, in general, free silver is inert towards can-
cer cells; however, in its cationic form its activity depends
on its bioavailability. Thus, solubility, delivery approaches
and ionization of the silver sources all are important fac-
tors that infuence the cytotoxicities of silver compounds
[30]. In this study, we have found that complexation of
Ag(I) with NHC ligands gives higher anticancer activity
than the NHC salts alone. It has been established that the
1 3

Transition Metal Chemistry
Fig. 5 Dose-dependent antipro-
liferative efects of proligands
L1–L6 on MDA-MB-231 cells
Fig. 6 Dose-dependent antipro-
liferative efects of complexes
C1–C6 on MDA-MB-231 cells
anticancer potential of drugs is generally decreased by
fast release of silver cations [27]. To overcome this prob-
lem, silver(I) complexes are of interest. Due to their weak
π-accepting and strong σ-donating features, NHC ligands
are slow to release silver ions [27, 31]. Previous stud-
ies have shown that anticancer properties of silver NHC
complexes against human colon cancer (HCT 116) and
breast cancer (MDA-MB-231) cells may proceed through
apoptosis (caspase independent) by external/internal stress
factors [32], which can be verifed from the observation
of apoptotic bodies and chromatin condensation in the
cytoplasm of cancer cells [6, 7, 10]. Additionally, it has
been shown that silver ions hinder cell functions by get-
ting deposited in the cytosol, where they may interact with
proteins and enzymes [7, 30, 33]. Thus, the outcomes of
this study are in agreement with previously reported work
in which black deposits (which may be of silver oxide) in
treated cancer cells have been observed [34]. Lipophilicity
depends on alkyl chain length and infuences the ability
of compounds to penetrate cell membranes, interact with
cell organelles and disturb their functions. Hence, it may
be inferred that longer side chains, the presence of aryl
linker and greater number of silver centres all enhance the
biopotencies of silver(I)-complexes.
1 3

Transition Metal Chemistry
Table 2 Selectivity index values of the compounds towards the tested
Avance-400 or Varian Inova-300 spectrometers in CDCl3,
cancer cells
acetone-d6, C6D6 and DMSO-d6 as solvents at chemical shift
1
(δ) ranges of 0–12 ppm for H NMR, 0–200 ppm for 13C
Compound
SI
NMR and −200 to 0 ppm for both 19F and 31P NMR studies.
Chemical shifts are reported in ppm relative to tetramethyl-
silane, and coupling constants (J) are given in Hertz. The
1H and 13C{1H} NMR spectra were referenced using the
residual solvent signal as internal standard, while 19F and
31P{1H} spectra were referenced to CFCl3 and H3PO4 (85%),
respectively. Spectral assignments were made with the help
of 1H–13C heteronuclear single quantum correlation (HSQC)
experiments. The N-alkylation reactions were monitored by
chromatography using an Agilent Technologies 7890A GC/
MS. C, H, N elemental analyses were carried out using a
Fisons EA1108 instrument. ESI mass spectra were recorded
with an ESI-POS-DI-TOF 6224 spectrometer.
MDA-MB-231
HCT-116
L1
L2
L3
L4
L5
L6
C1
C2
C3
C4
C5
C6
3.23
3.24
2.86
3.50
3.41
3.17
5.95
5.33
6.74
6.0
3.59
3.67
3.23
3.93
3.73
3.57
4.40
4.12
4.16
4.21
5.0
6.9
10.3
3.90
5.9
5.32
Electrochemical measurements were taken with a BAS
Epsilon potentiostat. Crystallographic data for complex C5
were collected on a Bruker APEX II difractometer equipped
with an Incoatec I\muS Microsource and graphite-mono-
chromated Mo Kα radiation (λ=0.71073 Å). Cell refnement
and data reduction were done using SAINT software [35].
An empirical absorption correction, based on the multiple
measurements of equivalent refections, was applied using
the SADABS program [36]. The space group was confrmed
by the XPREP routine [37] in the program SHELXTL [38].
The structure was solved by direct methods and refned by
full-matrix least-squares and diference Fourier techniques
with SHELX-97 [39]. The crystal appeared as a two-com-
ponent twin. The HKLF4 refection fle was generated by
TWINABS to treat the twinning. All non-hydrogen atoms
were refined with anisotropic displacement parameters.
Hydrogen atoms were set in calculated positions and refned
as riding atoms with a common thermal parameter.
Positive controla
aTamoxifen, 5-fuorouracil and betulinic acid were used as positive
controls for MDA-MB-231, HCT 116 and EA.hy926 cells, respec-
tively
Experimental
Materials and methods
Analytical-grade starting materials were obtained from
commercial sources and used without further purifcation.
5,6-Dimethyl benzimidazole, 1-bromopropane, 1-bro-
mobutane, 1,5-dibromopentane, 1,3-dichloro metaxylene,
1,4-dibromobutane and Ag2O were purchased from Sigma-
Aldrich. A TECAN multimode microplate reader was
obtained from the USA. Phosphate-bufered saline (PBS),
penicillin/streptomycin (PS) solution, 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent,
5-fuorouracil, tamoxifen and betulinic acid were purchased
from Sigma-Aldrich. Human breast cancer (MDA-MB-231),
colon cancer (HCT-116) and human endothelial normal cells
(EA.hy926) were obtained from the American type culture
collection (Rockville, MD, USA). The cells were kept in
RPMI 1640 media supplemented with 10% heat-inactivated
foetal bovine serum (HIFBS) and 1% phosphatidylserine
(PS) and cultured in a 5% CO2 humidifed atmosphere at
37 °C.
Preparation of 5,6‑dimethyl‑1‑propyl‑1H‑benzo[d]
imidazole (a)
A solution of 5,6-dimethyl benzimidazole (2.92 g,
20 mmol), 1-bromopropane (1.92 mL, 20 mmol) and KOH
(1.68 g, 30 mmol) in DMSO (20 mL) was stirred for 3 h
at room temperature, while monitoring the reaction by
GC–MS. Upon completion of the reaction, the mixture was
poured into ice-cold distilled water (200 mL). The white
precipitates were fltered of, washed with water and dried.
Yield 3.31 g (87%); M.P 40–43 °C. FTIR (cm−1): 3376(m)
(Caliph–Nbenzimid), 3077(m), 3037(w) (Carom–H str), 2964(m),
2932(m), 2873(m) (Caliph–H str), 1361(s), 1330(s), 1292(s)
(Carom–Carom str), 880(s), 864(s), 838(s), 823(s), 625(s)
(Carom–H oop ben). 1H NMR (400 MHz, CDCl3, δ ppm) 0.95
(3H, t, 1×CH3, J=14 Hz), 1.91 (2H, m, 1×CH2), 2.32 (3H,
s, 1×CH3–Ar), 2.41 (3H, s, CH3–Ar), 4.07 (2H, t, R-CH2–N,
J=4 Hz), 7.17 (1H, s, Ar–H), 7.58 (1H, s, Ar–H), 7.78 (1H,
Melting points were obtained with a Stuart Scientifc
SMP-1 (UK) instrument. The solvents were evaporated
using an EYELA 1L Rotary Evaporator N-1001V-WD.
FTIR spectra of the compounds were recorded with between
4000 and 250 cm−1 with an ALPHA-P compact FTIR spec-
trometer equipped with a universal attenuated total refec-
1
tance (UATR) accessory. H, 13C{1H}, HSQC, 19F and
31P{1H} NMR spectra were recorded on Bruker Avance-300,
1 3

Transition Metal Chemistry
s, N–CH–N). GC–MS, m/z, (%): ([C12 H16N2]+ 188.2 (100).
Anal. Calcd. for C12H16N2: C 76.6, H 8.6, N 14.9. Found C
77.1, H 8.65, N 14.2%.
(8H, m, 1×CH2), 2.42 (12H, d, 2×CH3–Ar, J=4.42), 4.41
(8H, m, R-CH2–N), 7.83–7.91 (4H, s, Ar–H), 9.72 (2H, s,
N–CH–N). 13C NMR (125.72 MHz, CDCl3, δ ppm) 11.1
(CH3), 20.3, 20.6, 22.7, 22.8, 28.1, (R-CH2) 46.7, 48.9
(Ar–CH3, 2 × CH3), 112.5, 113.3 (N–CH2-R, 2 × CH2),
129.7, 129.8, 137.2, 137.4 (Ar–C), 141.0 (N=C=N). Anal.
Calcd. for C29H42Br2N4: C 57.4, H 6.9, N 9.2. Found C 57.9,
H 6.6, N 8.9%.
Preparation of 5,6‑dimethyl‑1‑butyl‑1H‑benzo[d]
imidazole (b)
The synthesis of b followed the above procedure, but
using 1-bromobutane (2.84 mL, 20 mmol). Compound b
was obtained as a white solid. Yield 3.64 g (90%); M.P
50–52 °C. FTIR (cm−1): 2961(s), 2933(s), 2863(s) (Caliph–H
str), 1558(w), 1493(w), 1468(w), 1328(s) (C=Nbenzimid str),
881(s), 842(s), 732(S), 62(S) (Carom–H oop ben). 1H NMR
(400 MHz, CDCl3, δ ppm) 0.97 (3H, t, 1×CH3, J=7.46 Hz),
1.37 (2H, m, 1×CH2), 1.86 (2H, m, 1×CH2), 2.39 (3H, s,
1×CH3–Ar), 2.42 (3H, s, CH3–Ar), 4.13 (2H, t, R-CH2–N,
J=7 Hz), 7.17 (1H, s, Ar–H), 7.58 (1H, s, Ar–H), 7.78 (1H,
s, N–CH–N). GC–MS, m/z, (%): ([C13 H18N2]+ 202.3 (100).
Anal. Calcd. for C13H18N2: C 77.18, H 8.97, N 13.85. Found
C 77.62, H 8.65, N 13.18%.
Preparation of L3
This synthesis followed the same procedure for as for L1,
but using 1,3-dichloro metaxylene (0.927 g, 5.3 mmol).
L3 was obtained as a white solid. Yield 3.5 g (76%).
M.P = 145–150 °C. FTIR (cm−1) 2935(w), 2873(w)
(Caliph–H str), 1564(m), 1456(w), 1400(m), 1359(w)
(C=Nbenzimid str), 862(s), 743(s), 622(w) (Carom–H oop ben).
1H NMR (400 MHz, CDCl3, δ ppm) 0.92 (6H, t, 2 ×CH3,
J=7.45 Hz), 1.92 (4H, m, 2×CH2), 2.29 (6H, s, R-CH3),
2.40 (6H, s, R-CH3), 4.41 (4H, t, N–CH2, J=7.7 Hz), 5.61
(4H, s, Ar–CH2–N), 7.42 (3H, t, Ar–H, J = 3.5 Hz), 7.55
(1H, s, Ar–H), 7.58 (2H, s, Ar–H), 7.90 (2H, s, Ar–H),
9.74 (2H, s, N–CH–N). 13C NMR (125.72 MHz, CDCl3,
δ ppm) 11.1 (CH3), 20.3 (2 × Ar–CH3), 22.4 (R-CH2-R),
48.6 (CH2–N), 49.9 (N–CH2–Ar), 113.4, 113.7, 128.6,
129.8, 130.2, 130.2, 135.4, 136.8, 136.9 (Ar–C), 141.6
(N=C=N). 19F (470.4 MHz, CDCl3, δ ppm) −71.7 (d, 6F).
31P (202.4 MHz, CDCl3, δ ppm) − 144.6 (h, 1P). Anal.
Calcd. for C32H40Cl2N4: C 69.7, H 7.3, N 10.2. Found C
69.1, H 7.1, N 10.3%.
Preparation of L1
A mixture of compound a (2 g, 10.6 mmol) and 1,4-dibro-
mobutane (0.6 mL, 5.3 mmol) was refuxed in 1,4 diox-
ane (30 mL) at 100 °C until the reaction was complete (as
monitored by GC–MS). After 4 h, white precipitates of
L1 appeared; these were fltered of, washed with water
(3 × 5 mL), and dried at ambient temperature. Yield 3.5 g
(75%); M.P 170–173 °C. FTIR (cm−1): 2940(w), 2874(w)
(Caliph–H str), 1569(w), 1456(w), 1399(w) (C=Nbenzimid
str), 829(s), 741(s) (Carom–H oop ben). 1H NMR (400 MHz,
DMSO-d6, δ ppm) 1.02 (6H, t, 2×CH3, J=7.43 Hz), 2.07
(8H, m, 4×CH2), 2.50 (12H, t, 4×Ar–CH3, J=10.62 Hz),
4.57 (4H, m, R-CH2–N), 4.71 (4H, t, J=5.96 Hz, R-CH2–N),
7.88 (4H, d, Ar–H, J=24.58), 7.73 (2H, s, Ar–H), 9.3 (2H,
s, N–CH–N). 13C NMR (125.72 MHz, acetone-d6, δ ppm)
10.12 (CH3), 19.5, 22.3, 25.8 (R-CH2), 46.6, 48.6 (Ar–CH3,
2×CH3), 113.1, 113.2 (N–CH2-R, 2×CH2), 130.1, 130.2,
137.2, 137.2 (Ar–C), 140.2 (N=C=N). Anal. Calcd. for
C28H40Br2N4: C 56.7, H 6.8, N 9.4. Found C 56.9, H 6.6,
N 9.2.
Preparation of L4
A mixture of compound b (1.5 mL, 4.9 mmol) and
1,4-dibromobutane (5 mL) was refuxed without solvent at
100 °C, and the progress of the reaction was monitored by
GC–MS. After 24 h, a white solid of L4 was obtained. This
was fltered of, washed with copious amounts of ether, and
dried at room temperature. Yield 2.75 g (75%); M.P 120 °C.
FTIR (cm−1): 3433(s), 3015(s) (Caliph–Nbenzimid), 2956(s),
2872(s) (Caliph–H str), 1563(s), 1455(s) (C=Nbenzimid str),
1223(m), 1145(m) (Carom–Nbenzimid), 852(m), 775(m), 636(s)
1
(Carom–H oop ben). H NMR (400 MHz, CDCl3, δ ppm)
Preparation of L2
0.97 (6H, t, 2 × CH3, J=7.57 Hz), 1.41 (4H, m, 2×CH2),
2.07 (8H, m, 4×CH2), 2.15 (12H, d, 2×Ar–CH3), 4.26 (4H,
t, 2 × R-CH2–N, J = 7.57 Hz), 4.71 (4H, t, 2 × R-CH2–N,
J=11.36 Hz), 7.82 (2H, s, Ar–H), 7.92 (2H, s, Ar–H), 9.31
(2H, s, N–CH–N). 13C NMR (125.72 MHz, CDCl3, δ ppm)
13.4 (CH3), 19.8, 20.7, 26.6, 2, 29.1, 31.2, 44.2 (R-CH2)
46.5, 47.3 (Ar–CH3, 2×CH3), 112.7 (N–CH2-R, 2×CH2),
129.7, 129.8, 137.3, 137.4 (Ar–C), 142.4 (N=C=N). Anal.
Calcd. for C30H44Br2N4: C 58.1, H 7.2, N 9.1. Found C 58.1,
H 7.1, N 9.3%.
This synthesis followed the same procedure as for L1,
but using 1,5-dibromopentane (0.75 mL, 5.3 mmol). L2
was obtained as a white solid. Yield 3.6 g (75%); M.P
100–101 °C. FTIR (cm−1): 3483(s) (Caliph–Nbenzimid str),
2934(s) (Caliph–H str), 1561(s), 1485(s), 1359(s) (C=Nbenzimid
str), 1261(m), 1212(m) (Carom=Carom str), 898(s), 640(m)
(Carom–H oop ben).1H NMR (400 MHz, CDCl3, δ ppm)
0.89 (6H, t, 2×CH3, J=7.25 Hz), 1.38 (2H, m, CH2), 1.93
1 3

Transition Metal Chemistry
the wall of fask. The black suspension was fltered through
Celite, and a solution of KPF6 (0.85 g, 5 mmol) was added
to the fltrate at room temperature under continuous stirring.
The mixture was stirred for 4 h, and the precipitates formed
were fltered of and dried at room temperature. Complex
C1 was obtained as a beige powder. All eforts to obtain
single crystals were unsuccessful. Yield 1.74 g (57%); M.P
260–262 °C. FTIR (cm−1): 2934(w), 2874(w) (Caliph–H
str), 1559(s), 1496(m), 1401(m) (C=Nbenzimid str), 876(m),
824(s), 749(m) (Carom–H ben). 1H NMR (400 MHz, DMSO-
d6, δ ppm) 0.83 (12H, t, 4×CH3, J=7.53 Hz), 1.49 (8H, m,
4×CH2), 1.81 (8H, m, 4×CH2), 2.41 (24H, d, 4×Ar–CH3,
J = 7.35 Hz), 4.54 (8H, t, 4 × N–CH2, J = 7.65 Hz), 4.45
(8H, t, 4 × N–CH2, J = 7.01 Hz) 7.56 (8H, s, Ar–H). 13C
NMR (125.72 MHz, DMSO-d6, δ ppm) 11.5 (CH3), 20.3,
(R-CH2), 23.4, 24.5 (Ar–CH3, 2 × CH3), 30.5, 48.9, 50.3,
(R-CH2) 112.4, 112.9 (N–CH2-R, 2 × CH2), 132.1, 132.2,
132.2, 132.9, 133.1, 133.9 (Ar–C), 184.3, 185.7 (d, N=C=N,
J = 15.9 Hz), 186.7, 187.0 (d, N=C=N, J = 15.9 Hz).
19F (470.4 MHz, DMSO-d6, δ ppm) − 71.01 (d, 6F). 31P
(202.4 MHz, DMSO-d6, δ ppm) − 145.2 (h, 1P). Anal.
Calcd. for C56H76Ag2F12N8P2: C 49.2, H 5.6, N 8.2. Found
C 48.9, H 5.2, N 7.9%.
Preparation of L5
A mixture of compound b (1.51 g, 7.5 mmol) and
1,5-dibromopentane (0.5 mL, 3.7 mmol) in 1,4 dioxane
(30 mL) was refuxed at 100 °C, and the progress of the
reaction was monitored by GC–MS. After 24 h, the sticky
material was allowed to settle, decanted, washed with
fresh 1,4-dioxane and dried until a shiny crystalline mate-
rial L5 was obtained. Yield 3.5 g (75%); M.P 75 °C. FTIR
(cm−1): 3386(s) (Caliph–Nbenzimid), 2934(s), 2871(s) (Caliph–H
str), 1559(s), 1486(s), 1454(s), 1362(m) (C=Nbenzimid str),
1205(s) (Carom–Nbenzimid), 853(m), 684(s) (Carom–H oop ben).
1H NMR (400 MHz, CDCl3, δ ppm) 1.02 (6H, t, 2 ×CH3,
J=8.07 Hz), 1.48 (4H, m, 2×CH2), 1.76 (2H, m, R-CH2-R),
2.05 (4H, m, 2 × CH2), 2.24 (4H, q, 2 × CH2), 2.48 (12H,
d, 4 × CH3–Ar, J = 12.10 Hz), 4.51 (4H, t, J = 7.5 Hz,
R-CH2–N), 4.70 (4H, t, J= 7.5 Hz, R-CH2–N), 7.42 (2H,
s, Ar–H), 7.71 (2H, s, Ar–H), 11.11 (2H, s, N–CH–N). 13
C
NMR (125.72 MHz, CDCl3, δ ppm) 13.5 (CH3), 19.8, 20.5,
20.7, 28.1, 31.3, (R-CH2) 46.7, 47.5 (Ar–CH3, 2 × CH3),
112.5, 113.3 (N–CH2-R, 2×CH2), 129.8, 129.9, 137.2, 137.4
(Ar–C), 141.1 (N=C=N). Anal. Calcd. for C30H44Br2N4: C
58.1, H 7.2, N 9.1. Found C 58.3, H 7.3, N 9.2%.
Preparation of C2
Preparation of L6
This synthesis followed the same procedure as for C1, but
using L2 (1.6 g, 2.5 mmol). Complex C2 was obtained
as a beige powder. All efforts to get single crystals
failed. Yield 1.74 g (52%); M.P 250 °C. ESI+, m/z, %):
[C58H80Ag2N8PF6]+ 1247.43, 56). FTIR (cm−1): 2937(w),
2875(w) (Caliph–H str), 1566(w), 1456(m), 1401(m)
(C=Nbenzimid str), 1030(m) (Carom–Nbenzimid), 830(s), 743(s)
The synthesis of L6 followed the same procedure as for L5,
but using 1,3-dichloro metaxylene (0.65 g, 3.7 mmol). The
product was obtained as a white solid. Yield 3.7 g (78%);
M.P = 170 °C. FTIR (cm−1): 3381(s) (Caliph–Nbenzimid),
2957(s), 2873(s) (Caliph–H str), 1557(s), 1485(s), 1425(m),
1388(m) (C=Nbenzimid str), 1143(m) (Carom–Nbenzimid),
952(w), 750(s), 665(s) (Carom–H oop ben). 1H NMR
(400 MHz, CDCl3, δ ppm) 1.03 (6H, t, 2×CH3, J=7.40 Hz),
1.5 (4H, m, 2×CH2), 2.11 (4H, m, 2×CH2), 2.50 (12H, d,
2 × R-CH3, J = 13.7 Hz), 4.60 (4H, t, N–CH2, J = 7.4 Hz),
5.81 (4H, s, Ar–CH2–N), 7.31 (1H, s, Ar–H), 7.42 (3H,
t, Ar–H, J = 8.46 Hz), 7.55 (2H, d–d, Ar–H, J =7.47 Hz),
7.74 (2H, s, Ar–H), 11.61 (2H, s, N–CH–N). 13C NMR
(125.72 MHz, CDCl3, δ ppm) 13.5 (CH3), 19.8, 20.7
(Ar–CH3), 20.8, 31.2 (R-CH2-R), 47.4 (CH2–N), 50.4
(N–CH2–Ar), 112.5, 113.6, 129.2, 129.6, 130, 134.4, 137.3,
137.8 (Ar–C), 141.9 (N=C=N). 19F (470.4 MHz, DMSO-d6,
δ ppm) −71.8 (d, 6F). 31P (202.4 MHz, DMSO-d6, δ ppm)
−146.3 (h, 1P). Anal. Calcd. for C34H44 Cl2N4: C 70.4, H
7.6, N 9.6. Found C 70.1, H 7.2, N 9.9%.
1
(Carom–H oop ben). H NMR (400 MHz, DMSO-d6, δ
ppm) 0.78 (12H, t, 4 × CH3, J = 7.39 Hz), 1.44 (4H, m,
2×CH2), 1.75 (8H, m, 4×CH2), 1.96 (8H, m, CH2), 2.37
(24H, d, 4×Ar–CH3, J=7.53 Hz), 4.31 (8H, t, 4×N–CH2,
J = 7.53 Hz), 4.45 (8H, t, 4 × N–CH2, J = 7.01 Hz) 7.51
(8H, s, Ar–H). 13C NMR (125.72 MHz, DMSO-d6, δ
ppm) 11.5 (CH3), 20.3, (R-CH2), 23.9, 24.4 (Ar–CH3,
2×CH3), 30.5, 48.9, 50.3, (R-CH2) 112.4, 112.5 (N–CH2-R,
2×CH2), 132.1, 132.9, 132.2, 132.3, 133.7, 133.7 (Ar–C),
184.5, 185.9, 186.1, 187.9 (d, N=C=N, J = 13.93 Hz).
19F (470.4 MHz, DMSO-d6, δ ppm) − 71.01 (d, 6F). 31P
(202.4 MHz, DMSO-d6, δ ppm) − 145.2 (h, 1P). Anal.
Calcd. for C58H80Ag2F12N8P2: C 49.9, H 5.7, N 8.1. Found
C 49.4, H 5.6, N 7.9%.
Preparation of C1
Preparation of C3
Solid L1 (1.5 g, 2.5 mmol) was added to a suspension of
silver oxide (1.2 g, 5 mmol) in methanol (50 mL) in a fask
covered with aluminium foil, and the mixture was stirred
for 48 h at room temperature. A silver mirror formed on
This synthesis followed the same procedure as for C1,
but using L3 (1 g, 1.8 mmol) and silver oxide (0.89 g,
3.6 mmol). Complex C3 was obtained as a beige powder.
1 3

Transition Metal Chemistry
All eforts to get single crystals failed. Yield 1.58 g (60%);
M.P 260–262 °C. ESI+, m/z, %): ([C64H76Ag2N8PF6]+
1315.39, 50). FTIR (cm−1): 2938(w), 2874(w) (Caliph–H
str), 1568(m), 1456(m), 1400(m), 1359(m) (C=Nbenzimid
str), 830(s), 741(s) (Carom–H oop ben). 1H NMR (400 MHz,
DMSO-d6, δ ppm) 0.90 (12H, t, 4×CH3, J=7.41 Hz), 1.87
(8H, m, 4 × CH2), 2.20 (12H, s, 4 × R-CH3), 2.41 (12H, s,
4×R-CH3), 4.42 (8H, t, 4×N–CH2, J=7.1 Hz), 5.50 (8H,
s, 4×Ar–CH2–N), 6.81 (2H, s, Ar–H), 7.07 (4H, d, Ar–H,
J=7.57 Hz), 7.17 (2H, d, Ar–H, J=7.32 Hz), 7.32 (4H, s,
Ar–H), 7.63 (4H, s, Ar–H). 13C NMR (125.72 MHz, CDCl3,
δ ppm) 11.6 (CH3), 20.2 (2×Ar–CH3), 20.3 (R-CH2-R), 50.5
(CH2–N), 51.3 (N–CH2–Ar), 112.3, 112.5, 124.5, 126.3,
129.6, 132.2, 132.3, 132.4, 133.7, 133.8, 137.7 (Ar–C),
187.0, 189.0 (d, Ag–C–Ag, J = 195.7 Hz) (N=C=N).
19F (470.4 MHz, DMSO-d6, δ ppm) − 70.5 (d, 6F). 31P
(202.4 MHz, DMSO-d6, δ ppm) − 143.8 (h, 1P). Anal.
Calcd. for C64H76Ag2F12N8P2: C 52.5, H 5.2, N 7.6; Found:
C 51.8, H 5.3, N 7.5%.
NMR (400 MHz, DMSO-d6, δ ppm) 0.80 (12H, t, 4×CH3,
J=7.66), 1.24 (8H, m, 4×CH2), 1.43 (4H, m, 2×CH2), 1.75
(8H, t, 4×CH2, J=7.15 Hz), 1.96 (8H, m, 4×CH2), 2.37
(24H, d, 2×Ar–CH3, J=8.17 Hz), 4.34 (8H, t, 4×N–CH2,
J = 7.15), 4.45 (8H, t, 4 × N–CH2, J = 7.15) 7.50 (8H, s,
Ar–H). 13C NMR (125.72 MHz, CDCl3, δ ppm) 13.9 (CH3),
20.2, 20.3 (R-CH2), 24.5 (Ar–CH3, 2 × CH3), 30.4, 32.7
(R-CH2), 48.7, 48.9 (N–CH2-R, 2 × CH2), 112.5, 132.2,
133.7 (Ar–C), 186.1, 188.1 (s, N=C=N, J= 191.18 Hz).
19F (470.4 MHz, DMSO-d6, δ ppm) − 71.1 (d, 6F). 31P
(202.4 MHz, DMSO-d6, δ ppm) − 143.1 (h, 1P). Anal.
Calcd. for C62H88Ag2F12N8P2: C 51.3, H 6.1, N 7.7; Found:
C 51.2; H 6.2; N 7.8%.
Crystal data: C62H88Ag2F12N8P2; Mol. wt. 1451.08;
colourless block, space group: P-1 (2); Crystal size (mm):
0.2×0.18×0.16; crystal system: triclinic; Z=0.5; a=8.5272
(2), b= 12.7654 (3), c = 14.8616 (3) Å; α = 85.717 (1)°,
β=74.708(1)°, γ=86.197(1)°; V=1554.27 (6) Å3; μ (Mo
Kα) = 6.251 mm−I; dcalcd. = 1.550 g/cm−3); F(000) = 748;
Nref = 6147; Npar = 394; R(refections) = 0.0439(5792);
wR2(refections)=0.1635(5910); S=1.082; T=100 K.
Preparation of C4
This synthesis followed the same procedure as for C1,
but using L4 (1.5 g, 2.5 mmol) and silver oxide (1.2 g,
5 mmol). Complex C4 was obtained as a beige powder. All
eforts to get single crystals failed. Yield 1.91 g (57%); M.P
175–178 °C. FTIR (cm−1): 2928(w), 2871(w) (Caliph–H
str), 1455(w), 1403(m), 1360(m) (C=Nbenzimid str), 873(m),
830(s), 736(s) (Carom–H ben). 1H NMR (400 MHz, DMSO-
d6, δ ppm) 0.85 (12H, t, 4 × CH3, J = 7.53), 1.27 (8H, m,
4×CH2), 1.78 (8H, t, 4 × CH2, J= 7.75 Hz), 1.83 (8H, m,
4 × CH2), 2.45 (24H, d, 2 × Ar–CH3, J = 8.47 Hz), 4.41
(8H, t, 4 × N–CH2, J = 7.15 Hz), 4.43 (8H, t, 4 × N–CH2,
J=7.53 Hz) 7.50 (8H, s, Ar–H). 13C NMR (125.72 MHz,
CDCl3, δ ppm) 11.4 (CH3), 20.3, 23.9 (R-CH2), 24.4
(Ar–CH3, 2×CH3), 30.5, 38.5 (R-CH2), 48.9, 50.3 (N–CH2-
R, 2 × CH2), 112.4, 112.5, 132.1, 132.2, 132.3, 133.7,
133.8 (Ar–C), 183.1, 184.4 (s, N=C=N, J= 190.18 Hz).
19F (470.4 MHz, DMSO-d6, δ ppm) − 71.1 (d, 6F). 31P
(202.4 MHz, DMSO-d6, δ ppm) − 143.1 (h, 1P). Anal.
Calcd. for C60H84Ag2F12N8P2: C 50.6, H 5.9, N 7.8; Found:
C 51.0; H 6.1; N 7.5%.
Preparation of C6
This synthesis followed the same procedure as for synthe-
sis of C1, but using L6 (1.2 g, 5.1 mmol) and silver oxide
(1.2 g, 5.1 mmol). The complex C6 was obtained as a beige
powder. All eforts to get single crystals failed. Yield 2.24 g
(59%); M.P 260 °C. ESI+, m/z, %: ([C64H76Ag2N8PF6]+
1371.45, 50). FTIR (cm−1): 2959(m), 2875(w) (Caliph–H
str), 1559(w), 1445(m), 1402(s), 1358(m) (C=Nbenzimid str),
832(s) (Carom–H oop ben). 1H NMR (400 MHz, DMSO-d6,
δ ppm) 0.90 (12H, t, 4 × CH3, J = 7.58 Hz), 1.32 (8H, m,
4×CH2), 1.84 (8H, m, 4×CH2), 2.21 (12H, s, 4×R-CH3),
2.41 (12H, s, 4 × R-CH3), 4.40 (8H, t, 4 × N–CH2,
J=7.2 Hz), 5.51 (8H, s, 4×Ar–CH2–N), 7.01 (4H, t, Ar–H,
J = 10.37 Hz), 7.12 (1H, t, Ar–H, J= 7.07 Hz), 7.31 (3H,
s, Ar–H), 7.60 (4H, s, Ar–H), 8.31 (4H, s, Ar–H). 13C
NMR (125.72 MHz, DMSO-d6, δ ppm) 14.0 (CH3), 20.1,
20.3 (CH2), 30.9, 32.6 (2×Ar–CH3), 46.9, 48.9 (CH2–N),
49.9, 51.3 (N–CH2–Ar), 112.4, 113.7, 125.0, 126.4, 129.6,
130.37, 132.4, 133.6, 136.9, 137.9, 141.7 (Ar–C), 187.0,
188.9 (s, Ag–C–Ag). 19F (470.4 MHz, DMSO-d6, δ ppm)
−70.6 (d, 6F). 31P (202.4 MHz, DMSO-d6, δ ppm) −141.9
(h, 1P). Anal. Calcd. for C68H84Ag2F12N8P2: C 44.6, H 4.7,
N 5.8; Found: C 44.7; H 4.8; N 5.9%.
Preparation of C5
This synthesis followed the same procedure as for C1,
but using L5 (1.5 g, 2.4 mmol) and silver oxide (1.14 g,
4.8 mmol). Complex C5 was obtained as a beige powder.
Single crystals were grown by slow evaporation of satu-
rated solution in acetonitrile at room temperature. Yield
1.81 g (52%); M.P 170 °C. FTIR (cm−1): 2934(m), 2872(m)
(Caliph–H str), 1561(m), 1465(m), 1398(m) (C=Nbenzimid str)
1163(m) (Carom–Nbenzimid), 830(s), 750(s) (Carom–H ben). 1H
Cyclic voltammetry experiments
Electrochemical measurements of the Ag(I)–NHC com-
plexes were taken using a BAS Epsilon potentiostat having
a three-electrode standard system. Tetrabutyl ammonium
hexafuorophosphate [nBu4N][PF6] (1 mM in CH3CN) was
1 3

Transition Metal Chemistry
used as a supporting electrolyte. Ag/AgCl was used as refer-
ence, platinum disc as working and platinum wire as counter
electrodes. The solutions were bubbled with nitrogen before
each experiment. The electrochemical measurements were
taken in acetonitrile with potentials referred to the Ag/
AgCl (0.1 mol dm−3) reference electrode (El/2 (FeCp+2 /
FeCp2)=+0.43 V) at a scan rate of 50 and 100 mV/s under
N2 at room temperature.
followed by Tukey’s multiple comparison test. Diferences
were considered signifcant at p<0.05, and p<0.01.
Conclusion
In summary, a series of bidendate benzimidazolium-based
NHC ligands and their silver complexes were synthesized and
characterized by various analytical techniques. Electrochemi-
cal studies of the complexes revealed that during the reduc-
tion half cycle, an irreversible one-electron reduction process
(Ag(I)/Ag(0) produces unstable species which decomposes
before the Ag(0)/Ag(I) oxidation event. Both the free azolium
salts and their silver complexes were less cytotoxic to normal
cells compared to cancer cells, and the silver complexes were
more potent than the corresponding NHC precursors (salts).
The IC50 values of the NHC salts decreased with increasing
terminal chain length, but increased for the respective com-
plexes. Complex C6 exhibited the highest selectivity towards
cancer cell lines. Overall, increased chain length, presence
of methyl substituents on benzimidazole ring and aryl linker
and two silver centres enhance the biopotency of Ag(I)–NHC
complexes. Further studies of these complexes as potential
chemotherapeutic agents are in progress.
Anticancer studies
Preparation of cell cultures
Human breast cancer (MDA-MB-231), colon cancer (HCT-
116) and normal endothelial (EA.hy926) cells were permit-
ted to grow under optimal conditions in an incubator. After
reaching 70–80% confuence rate, the cells were prepared
for plating by aspirating the old medium from the plate and
washing the cells thrice with slightly basic (pH 7.4) sterile
PBS, which was then discarded. Trypsin was then distrib-
uted evenly on the cell surfaces and the cells were incubated
in 5% CO2 at 37 °C for 1 min, gently tapping the fasks
containing the cells to assist their segregation. Cells were
then observed with an inverted microscope. The activity of
trypsin was suppressed by adding 10% fresh HIFBS medium
(5 mL). A fnal concentration (2.5 × 105cells/mL) of cells
was achieved by diluting with medium and the cultures were
inoculated into the wells (100 μL/well). Finally, the plates
containing the cells were incubated in 5% CO2 at 37 °C.
Additional information
CCDC 1890988 contains the supplementary crystallographic
data for complex C5 included in this paper. These data can be
obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgements Aqsa Habib would like to thank Higher Educa-
tion Commission (HEC), Pakistan, for fnancial support during Inter-
national Research Support Initiative Program (IRSIP) (1-8/HEC/
HRD/2017/6940). She would also like to appreciate the eforts of
Prof. Davit Zargarian (University de Montreal, Canada) for facilitat-
ing during research work in his laboratory during IRSIP period and
Dr. Loic Mangin (University de Montreal, Canada) for solving crystal
structure and helpful discussion for the preparation of this manuscript.
Dr. Muhammad Adnan Iqbal is thankful to HEC for awarding research
grant No. NRPU-8396 under National Research Program for Universi-
ties (NRPU).
MTT assays
After seeding the cells (100 μL, 1.5 × 105 cells/mL), the
96-well microtiter plate was incubated in a CO2 environment
for 24 h to allow for cellular attachment. The test substance
(100 μL) was introduced into each well after making serial
dilutions from stock solution with medium, and the plates
were further incubated in 5% CO2 atmosphere at 37 °C for
72 h. MTT reagent was then introduced into each well, and
incubation was continued for 4 h. Then 50 μL DMSO was
introduced in each well, and plates were incubated in CO2
for 5 min and then assayed at two diferent wavelengths
(570 and 620 nm) with a multimode monochromator-based
microplate reader (Tecan Infnite® M200 PRO). Optical den-
sity (OD) obtained during MTT assays was used to calcu-
late the percentage of growth inhibition, which was further
employed to calculate IC50 values.
Compliance with ethical standards
Conflict of interest The authors declare no conficts of interest.
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