Synthesis, Structure, and Anticancer Activity of Symmetrical and Non-symmetrical Silver(I)-N-Heterocyclic Carbene Complexes

Article abstract of DOI:10.1007/s12010-019-03186-9

Synthesis and anticancer studies of three symmetrically and non-symmetrically substituted silver(I)-N-Heterocyclic carbene complexes of type [(NHC)₂-Ag]PF₆ (7–9) and their respective (ligands) benzimidazolium salts (4–6) are describe

Full text of DOI:10.1007/s12010-019-03186-9

Applied Biochemistry and Biotechnology
https://doi.org/10.1007/s12010-019-03186-9
Synthesis, Structure, and Anticancer Activity
of Symmetrical and Non-symmetrical
Silver(I)-N-Heterocyclic Carbene Complexes
Muhammad Atif1 & Haq Nawaz Bhatti1 & Rosenani A. Haque5
&
Muhammad Adnan Iqbal1,2 & Mohammad B. Ahamed Khadeer4
&
Amin Malik Shah Abdul Majid3
Received: 10 September 2019 /Accepted: 11 November 2019/
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Synthesis and anticancer studies of three symmetrically and non-symmetrically substituted
silver(I)-N-Heterocyclic carbene complexes of type [(NHC)2-Ag]PF6 (7–9) and their respec-
tive (ligands) benzimidazolium salts (4–6) are described herein. Compound 5 and Ag-NHC-
complex 7 were characterized by the single crystal X-ray diffraction technique. Structural
studies for 7 showed that the silver(I) center has linear C-Ag-C coordination geometry
(180.00(10)o). Other azolium and Ag-NHC analogues were confirmed by H1 and C13-NMR
spectroscopy. The synthesized analogues were biologically characterized for in vitro anticancer
activity against three cancer cell lines including human colorectal cancer (HCT 116), breast
cancer (MCF-7), and erythromyeloblastoid leukemia (K-562) cell lines and in terms of in vivo
acute oral toxicity (IAOT) in view of agility and body weight of female rats. In vitro anticancer
activity showed the values of IC50 in range 0.31–17.9 μM in case of K-562 and HCT-116
cancer cell lines and 15.1–35.2 μM in case of MCF-7 while taking commercially known
anticancer agents 5-fluorouracil, tamoxifen, and betulinic acid which have IC50 values 5.2, 5.5,
and 17.0 μM, respectively. In vivo study revealed vigor and agility of all test animals which
explores the biocompatibility and non-toxicity of the test analogues.
.
.
.
.
.
Keywords In vivo acute oral toxicity (IAOT) MCF-7 K-562 HCT 116 Ag-NHC Single
crystal X-ray diffraction
Introduction
With the breakthrough of tremendous research trend in the development of newer and newer
ancillary ligands using N-Heterocyclic compounds as an alternative to pre-existing classes of
* Muhammad Adnan Iqbal
adnan.iqbal@uaf.edu.pk; adnan_chem38@yahoo.com
Extended author information available on the last page of the article

Applied Biochemistry and Biotechnology
ligand analogues of especially cyclopentadienyl, phosphines, and others, the synthetic progress
in development of N-Heterocyclic carbene (NHC) analogues has gained momentum [1, 2].
Because NHCs stand parallel to phosphines for their exceptional biological activities along
with their promising importance in reaction catalysis [3, 4], the superior importance is often
attributed to their better electron-density distributions which endow stability to their metal–
carbon bond in their coordination with either soft or hard metals [5]. Also, 6π-electronic
delocalization in NHCs is believed to be more pronounced; hence, these are the more versatile
classes of ligands in modern organometallic chemistry compared to phosphines, silylenes, and
germylenes. In addition, NHCs possess excellent σ-donor and generally weak π-acceptor
properties, which makes them ligands of choice for transition and inner transition metals [6].
NHC precursors are considered significantly simpler to synthesize even in dry and moist
environment. Although they are not realized with closed-shell ions, yet they have potential to
form stable metal–ligand back bonding [7]. On the basis of this capability, a variety of
transition metal-NHC complexes have been successfully used as catalysts in numerous
processes, such as C-C coupling, olefin metathesis and polymerization, and olefin hydroge-
nation, among others [8–10].
Like other metal-NHC complexes, silver(I)-NHC complexes are also under constant
research consideration, but they have gained greater research trend due to their fascinat-
ing output in their applications especially in biological avenues. It is largely due to their
potential therapeutic and pharmacological applications [11–17]. Based on this approach,
a plethora of novel methods has arisen over the past decade, combining various types of
NHCs with Ag(I) leading to a large number of versatile and useful compounds in
synthetic organometallic and medicinal chemistry. Particularly, medicinal and pharma-
cological avenues, organometallic chemistry offers unique properties of Ag(I) in the
designing of new metallodrugs [18]. Silver(I)-NHC complexes have demonstrated abil-
ities for the inhibition of fast and uncontrolled cell proliferation by means of inhibiting
cellular transcription. Consequently, these complexes can specifically bind or cleave
DNA and have a potential as DNA targeted chemotherapeutics [19]. Hence, appearance
of many exciting biological applications of numerous silver(I)-NHC complexes spanning
from anticancer to antimicrobial and antifungal activities, the focus is gradually shifted
on the design and 3D structure of these complexes to arrest cell proliferation. In view of
their promising biological activities, newer series of NHCs are being synthesized while
varying the nature of substituents. In the present work, the effect of symmetrical and
non-symmetrical substitution on silver(I)-NHC complexes and their respective azolium
salts in terms of their cytotoxicities to HCT-116, MCF-7, and K-562 cancer cell lines has
been discussed briefly.
Experimental
Materials
Commercial suppliers for the provision of analytical grade chemicals and solvents were hired
and all of the chemicals were utilized without any purification treatment. For the synthesis of
silver complexes, silver oxide (Ag2O), benzyl bromide, n-propyl bromide, n-heptyl bromide,
benzimidazole and 5-fluorouracil, tamoxifen and betulinic acid (standards used for anticancer
assays), and 3-[4,5-yl]-2,5-dephenyltetrazolium bromide for the MTT assay were purchased

Applied Biochemistry and Biotechnology
from Sigma–Aldrich for the assessment of their bioactivities. Cancer cell lines were purchased
from AATCC, USA. The reagent for cell culturing was purchased from Gibco, USA.
Methods
FT-IR spectra of the synthesized analogues were obtained from Perkin Elmer 2000 system
spectrometer. NMR spectra were also obtained using deuterated dimethyl sulfoxide (d6-
DMSO) or d3-CD3CN d3-CD3CN at 500 MHz on Bruker 300 wherein ascend spectrometers
was used at ambient temperature. It was standardized with tetramethylsilane as an internal
reference. The synthesized compounds were also analyzed for elemental analysis in terms of
carbon, hydrogen, and nitrogen estimations. All the compounds were analyzed for elemental
C, H, and N using a Series II microanalyzer of PerkinElmer 2400. For X-ray crystallography,
Oxford Diffraction Gemini diffractometer was used. PLATON and SHELXTL software were
employed to get molecular calculations and to refine structure and molecular graphics. For this
purpose, isotopic and non-isotopic displacement parameters were applied for hydrogen and
non-hydrogen atoms, respectively.
N-Alkylation of Benzimidazole
N-alkylation techniques were employed for the production of N-propylbenzimidazole 1, N-
heptylbenzimidazole 2, and N-benzylbenzimidazole 3 in dimethylsulfoxide (DMSO) along
with 1.5 equivalent sodium hydroxide to react benzimidazole and their respective alkyl
bromides [20] as shown in Scheme-I.
Synthesis of Benzimidazolium Salts
All three benzimidazolium salts (4–6) were synthesized by stirring N-alkylated benzimidazoles
(1–3) and alkyl bromides in 1,4-dioxane for at least 24 h as elaborated further:
(a). Synthesis of 1,3-Bis(Propyl) Benzimidazolium Bromide (4)
For this purpose, N-propylbenzimidazole (1.6 g, 10 mmol) was treated with n-propyl bromide
(1.23 g, 10 mmol) in 1,4-dioxane (50 mL) and was stirred for 24 h under reflux, and a pale
yellow oily product was obtained by decantation of the solvent. For purification, the product
was repeatedly washed with 1,4-dioxane and also diethyl ether, and then it was stored in
1
vacuum desiccator. Yield: 73.1%. M.P.: 246 °C, H NMR (500 MHz, DMSO-d6): δ 0.90 (t,
6H, 2 × CH3, J = 7.5 Hz), 1.95 (sext, 4H, 2 × CH2), 4.55 (t, 4H, 2 × N-CH2-R, J = 7.5 Hz), 7.66
(sext, 2H, Ar-H), 8.16 (sext, 2H, Ar-H), 10.21 (s, 1H, NCHN).13C NMR (125.7 MHz, DMSO-
d6): δ 10.6 (CH3), 22.0 (CH2), 48.1 (N-CH2-R), 113.7, 126.4, 131.0 (Ar-C), 142.1 (NCN).
(b). Synthesis of 1-Propyl-3-Heptylbenzimidazolium Bromide (5)
Compound 5 was synthesized in same way as that of 4; only N-heptylbenzimidazole 2 (1.79 g,
10 mmol) instead of 1 was used. The product was obtained as an off-white solid, filtered, and
washed three times with 1,4-dioxane to consequently store in a vacuum desiccator. The
colorless crystals of bromide salt 5 were obtained by recrystallization from methanol/diethyl
ether. Yield: 91.1%, M.P: 233 °C.1H NMR (500 MHz, DMSO-d6): δ 0.72 (t, 3H, 1 × CH3, J =

Applied Biochemistry and Biotechnology
7.5 Hz), 0.87 (t, 3H, 1 × CH3, J = 7.5 Hz), 1.14 (br.m, 4H, 2 × CH2), 1.25 (br.m, 4H, 2 × CH2),
1.90 (sept, 4H, N-CH2-R) 7.57 (sext, 2H, Ar-H), 7.87 (sext, 2H, Ar-H), 9.58 (s, 1H,
NCHN).13C NMR (125.7 MHz, DMSO- d6): δ 10.9, 11.2 (2 × CH3), 14.1(2 × CH2),
17.9(2 × CH2), 20.3(2 × CH2), 20.7(2 × CH2), 23.2(2 × CH2), 24.4 (2 × CH2), 40.4, 58.5 (2 ×
N-CH2-R), 105.1, 123.4 (Ar-C), 133.5 (NCHN). FT-IR (KBr disc) in cm−1: 3435 (Caliph
benzimi), 3162, 3044 (C-Harom), 2960, 2935, 2873 (C-Haliph), 1050, ~1200 cm−1 (Carom
-
-
N
Nbenzimi). Anal. Calcd for C17H27N2Br: C, 60.2; H, 8.0; N, 8.3%. Found: C, 59.8; H, 8.2; N,
7.9%.
(c). Synthesis of 1-Propyl-3-Benzylbenzimidazolium Bromide (6)
This compound 6 was prepared in a manner similar to for 5, but N-benzylbenzimidazole 3
(1.72 g, 10 mmol) instead of 2 was employed. In this case, product obtained in form of pale
yellow solid was filtered and washed and stored in the same way as in the case of earlier
analogues. Yield: 83.4%, M.P.: 202.3 °C.1H NMR (500 MHz, DMSO-d6): δ 0.87 (t, 6H, 2 ×
CH3, J = 7.5 Hz), 1.91 (sext, 2H, 1 × CH2), 4.55 (t, 2H, 1 × N-CH2-R, J = 7.5 Hz), 5.97 (s, 2H,
N-CH2-Ar), 7.28 (t, 3H, Ar-H, J = 3.5 Hz), 7.53–7.59 (m, 2H, Ar-H), 7.66 (d, 2H, Ar-H, J =
7.5 Hz), 8.06 (d, 1H, Ar-H, J = 1.5 Hz), 8.13 (d, 1H, Ar-H, J = 1.5 Hz), 10.85 (s, 1H,
NCHN).13C NMR (125.7 MHz, DMSO- d6): δ 10.5 (CH3), 21.9 (CH2), 48.1 (N-CH2-R),
49.7 (N-CH2-Ar), 113.7, 126.4, 128.3, 128.4, 128.7, 130.6, 131.1, 134.2 (Ar-C), 142.6
(NCN).FT-IR (KBr disc) in cm−1: 3405 (Caliph-Nbenzimi), 3144, 3032 (C-Harom), 2960, ~2965
(C-Haliph), 1040, ~1200 cm−1 (Carom-Nbenzimi). Anal. Calcd for C17H19N2Br: C, 61.6; H, 5.8; N,
8.5%. Found: C, 59.5; H, 6.1; N, 8.8%.
Synthesis of Ag(I)-NHC Complexes
Synthesis of Bis-[1,3-Bis(Propyl)Benzimidazol-2-Ylidene]Silver(I) Hexafluorophosphate (7)
Synthesized azolium salt 4 (0.284 g, 1 mmol) was treated with silver oxide (0.14 g,
0.6 mmol) in methanol (25 mL). The suspension was stirred for 48 h wherein reaction
flask was remained in dark during the reaction. The resulting mixture was then filtered
through a pad of Celite, and the resulting colorless solution was added KPF6 (0.227 g,
1.2 mmol) and further stirred for 4 h to precipitate an off-white solid. Isolation by
filtration yields hexafluorophosphate silver complex 7. Yield: 55.9%, M.P.: 244-
246 °C.1H NMR (300 MHz, CD3CN-d3): δ 0.99 (t, 6H, 2 × CH3, J = 7.5 Hz), 2.02 (sext,
4H, 2 × CH2), 4.48 (t, 4H, 2 × N-CH2-R, J = 7.5 Hz), 7.47 (sext, 2H, Ar-H), 7.71 (sext,
2H, Ar-H).13C NMR (125.7 MHz, DMSO- d6): δ 11.7 (s, 2 × CH3), 24.7 (2 × CH2), 51.7
(N-CH2-R), 113.0, 125.2, 134.7 (Ar-C), 188–190 (C-Ag, J107Ag-C = 182.5 Hz, J109Ag-C
=
206.0 Hz).FT-IR (KBr disc) in cm−1: ~3400 (Caliph-Nbenzimi), 3130, 3030 (C-Harom), 2930,
~2960 (C-Haliph), ~1045, 1235 cm−1 (Carom-Nbenzimi). Anal. Calcd for C26H36N4PF6Ag: C,
46.5; H, 5.5; N, 8.5%. Found: C, 46.2; H, 5.7; N, 8.7%.
Synthesis of Bis-[1-Propyl-3-Heptylbenzimidazol-2-Ylidene]Silver(I) Hexafluorophosphate
(8)
This compound was prepared in a manner analogous to that for 7, only with 5 (0.34 g, 1 mmol)
instead of 3.Yield: 58.1%, M.P.: 252-254 °C.1H NMR (500 MHz, CD3CN-d3): δ 0.71 (t, 3H,

Applied Biochemistry and Biotechnology
1 × CH3, J = 7.0 Hz), 0.91 (t, 3H, 1 × CH3, J = 7.5 Hz), 1.11 (br.m, 4H, 2 × CH2), 1.27 (br.m,
4H, 2 × CH2), 1.98 (sept, 4H, N-CH2-R, J = 7.0 Hz) 7.42–7.61 (sext, 2H, Ar-H), 7.93 (sext,
2H, Ar-H).13C NMR (125.7 MHz, DMSO-d6): δ 10.7, 11.6(2 × CH3), 14.9(2 × CH2), 17.6
(2 × CH2), 20.7 (2 × CH2), 20.9 (2 × CH2), 24.6 (2 × CH2), 26.1(2 × CH2), 43.0, 59.0 (2 × N-
CH2-R), 114.7, 125.8 (Ar-C), 183.0 (NCN-Ag). FT-IR (KBr disc) in cm−1: 3405 (Caliph
benzimi), 3160, ~3045 (C-Harom), ~2965, 2948 (C-Haliph), 1060, 1253 cm−1 (Carom-Nbenzimi).
-
N
Anal. Calcd for C34H52N4PF6Ag: C, 53.1; H, 6.8; N, 7.3%. Found: C, 53.6; H, 6.5; N, 7.7%.
Synthesis of Bis-[1-Propyl-3-Benzylbenzimidazol-2-Ylidene]Silver(I) Hexafluorophosphate
(9)
This compound was prepared in a manner analogous to that for 7, only with 6 (0.33 g, 1 mmol)
instead of 3.Yield: 61.0%, M.P.: 242-243 °C.1H NMR (500 MHz, CD3CN-d3): δ 0.94 (t, 6H,
2 × CH3, J = 7.5 Hz), 1.97 (sext, 2H, 1 × CH2), 4.44 (t, 2H, 1 × N-CH2-R, J = 7.0 Hz), 5.66 (s,
2H, N-CH2-Ar), 7.32 (br.m, 4H, Ar-H), 7.41–7.50 (t of t, 3H, Ar-H, J = 2.7 Hz), 7.58 (d, 2H,
Ar-H, J = 8.0 Hz), 7.74 (d, 1H, Ar-H, J = 8.5 Hz).13C NMR (125.7 MHz, DMSO- d6): δ 11.6
(2 × CH3), 24.5 (2 × CH2), 51.8 (1 × N-CH2-R), 53.3 (s, 1 × N-CH2-Ar), 113.1 (d, Ar-C, J =
2.5 Hz), 125.3, 128.2, 129.3, 130.0, 134.8, 137.2 (Ar-C), 189–191 (C-Ag, J107Ag-C = 183.8 Hz,
J
109Ag-C = 210.7 Hz).FT-IR (KBr disc) in cm−1: 3432 (Caliph-Nbenzimi), 3176, ~3040 (C-Harom),
~2935, 2902 (C-Haliph), 1074, ~1250 cm−1 (Carom-Nbenzimi). Anal. Calcd for C34H36N4PF6Ag:
C, 54.2; H, 4.8; N, 7.4%. Found: C, 54.7; H, 4.5; N, 7.7%.
In Vitro Anticancer Activity
Preparation of Cell Culture
The proliferation studies of three silver(I)-NHC complexes and their respective
benzimidazolium salts were accomplished by MTT assay using three different human cancer
cell lines including HCT 116, MCF-7, and K-562. Firstly, cancer cells were grown under
previously reported optimal incubator conditions. When they reached 70–80% confluence,
they were employed for cell plating purposes. After aspirating old medium present on cell
plate, the cells were washed three times at pH 7.4 by sterile phosphate-buffered saline (PBS).
Then PBS was also discarded by washing, and trypsin was added in such way that it was
evenly distributed onto cell surfaces. After adding trypsin, cells were incubated for 1 minute
under 5% CO2 at 37 °C. Then, the cell flasks were tapped to promote their cell segregation
which was monitored by inverted microscope. Trypsin was inactivated by 5 mL freshly
prepared 10% fetal bovine serum (FBS) media. The grown cells were counted to dilute to
2.5 × 105 cells/mL, and they were inoculated into wells (100 μL cells/well) to incubate at
37 °C with under 5% CO2 [21].
MTT Assay
After incubating inoculated cancer cells overnight, cell attachment was assured; after diluting
the test compounds with media as per desired concentration, the plates were again incubated
for 72 h while keeping temperature and CO2 atmosphere unchanged. A 20 μL of MTT reagent,
namely, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], was added in indi-
vidual well, and they were again incubated for 4 h. Then, 50 μL of MTT lysis solution

Applied Biochemistry and Biotechnology
(DMSO) was added 5 mL in each well, and they were again incubated for 5 min under CO2
environment. Finally, ELISA microplate reader (Ascent Multiskan) was used to count the
number of plate at 570 and 620 nm. Recorded data was used for the assessment of cell viability
under influence of test compounds. The percentage growth inhibition was estimated on the
basis of optical density (O.D.) as per Eq. (1) [22].
½O:DðcontrolÞ−O:D ðsurvivedފ
%of growth inhibition ¼
 100
ð1Þ
O:D ðcontrolÞ
In Vivo Acute Oral Toxicity Tests
Selection, Preparation, and Grouping of Animals
Mostly rodents like rats are used for acute oral toxicity dose methods where the females are
most sensitive compared to male rats. These female rats were obtained from Animal Research
and Service Centre (ARASC) USM Main Campus under approval of grant number USM/
Animals Ethics Approval/2012/(76) (364). Female rats were selected randomly, were marked
for identification, and were kept in cages for at least 5 days in animal transient room. Animals
were acclimatized to the laboratory conditions prior to the start dosing for acute oral toxicity
sighting study. All the animals were nulliparous and nonpregnant and their weight was 210 g
10. Initially, female rats were divided into 7 groups. The first three were marked with
benzimidazolium salts 4, 5, and 6 and next three with silver-carbene complexes 7, 8, and 9,
respectively. Finally, the last group was treated for vehicle containing water and minute
quantity of tween 80.
Preparation of Doses
As these test compounds were synthetic, so solubility of these substances was enhanced by
using tween 80 in limited doses to avoid toxicity originated from it as is obvious toxicological
properties of vehicle except water should be known. 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 preparation should
be known and acceptable.
Acute Oral Toxicity Tests
The study was aimed at selecting appropriate dose of active compounds in view of biosafety.
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 it was assumed that experimental dose was likely to
produce some toxicity on the bases of data obtained from in vivo and in vitro studies of
structurally related compounds [23, 24]. A duration of at least 24 h was set between one dose
to the next one for each animal, and it was observed for 14 days consecutively.

Applied Biochemistry and Biotechnology
Results and Discussion
Synthetic Strategy
Both symmetrically and non-symmetrically substituted benzimidazolium salts, used as NHC
pro-ligands, were prepared with slight modifications using previously reported procedure for
the preparation of similar heterocyclic systems. 1-Propyl-3-alkyl/alkyl-aryl benzimidazolium
bromides (4, alkyl = n-propyl; 5, alkyl = n-heptyl; and 6, alkyl-aryl = benzyl) were synthesized
from benzimidazole by stepwise N-alkylation with 1-bromopropane followed by 1-
bromoalkane in sequence [25]. The reaction of 1-propylbenzimidazole with 1-bromopropane,
1-bromoheptane, or benzyl bromide in 1,4-dioxane under reflux for 24 h affords the corre-
sponding bromide salts 4, 5, or 6, respectively, in good yield. The reaction of two equivalents
of 4, 5, or 6 with Ag2O in methanol yields the new Ag(I)- or 7, 8, 9. The reaction mixture was
subjected to stirring for 48 h at room temperature to afford the NHC-silver or mercury
complexes as off-white or beige solids in acceptable yields. Silver bromide complexes were
converted into their hexafluorophosphate counterparts by a metathesis reaction with KPF6 in
methanol, for ease of handling and purification as the complexes in their halide forms appeared
as thick yellowish fluid (Scheme 1). Both bromide salts and the complexes are stable to air and
moisture and soluble in common polar organic solvents such as (m)ethanol, acetonitrile, and
dichloromethane; sparingly soluble in hexane and benzene; and completely insoluble in
petroleum ether and diethyl ether. However, corresponding Ag(I) complexes showed a negtive
shift in their vibration bands indicating coordination of the C2-carbon to Ag center.
Spectral Characterizations
FTIR spectra of compounds 4–6 evidenced two sharp bands with medium intensity at around
1050 and 1200 cm−1, attributed to stretching vibrations of C-N modules of benzimidazole ring
as well as at the substitutions. These vibrations suffered a negative shift in the corresponding
complex spectra indicating the coordination of the C2-carbon to Ag center. This observation is
consistent with the previous reports. Apart from this, spectra also showed corresponding
vibrational bands for aliphatic and aromatic C-H modules at around 2900 cm−1, which is
consistent with our previous observations. Additionally, the results obtained from the elemen-
tal analysis are consistent with the proposed structures of the salts and complexes [20].
The benzimidazolium salts and their silver complexes were fully characterized by 1H, 13
NMR, and FTIR spectroscopy and elemental analysis. In the H NMR spectrum of 4–6,
C
1
resonances for the benzimidazole, the propyl, and the propyl/heptyl/benzyl protons are
Scheme 1 Synthesis of N-alkylatedazole (1–3), azolium salts (4–6) and Ag-NHC complexes (7–9).

Applied Biochemistry and Biotechnology
observed as expected. The spectra show a characteristic singlet at the downfield region in the
range 9.58–10.85 ppm corresponding to the resonance of benzimidazolium C2 proton
(NCHN), which is attributed to formation of desired salts. Additionally, two multiplets
centered in the range 7.65–8.17 ppm and a triplet, a multiplet, and a second triplet in the
range0.73–0.90, 1.90–1.95, and 4.55–4.70 ppm were observed corresponding to the reso-
nances of benzimidazole and propyl protons, respectively. In the case of compounds 5 and 6,
additional resonances corresponding to heptyl and benzyl protons were observed, respectively.
These observations are consistent with the chemical shifts of analogues benzimidazolium salts.
Upon silver complex formation, the 1H NMR signals for the resonance of benzimidazolium C2
proton completely disappeared, confirming the coordination of the benzimidazol-2-ylidene
group to silver to form new carbine complexes 7–12 in pure form [26].
In the 13C NMR spectra of compounds 4–6, the benzimidazolium ring and alkyl carbon
signals appear in the range 113–143 and 4.95–59 ppm, respectively, which are consistent with
the carbon chemical shifts of known benzimidazolium salts having similar substitutions.
The13C NMR resonances of the carbene carbon nuclei for 4–6 appeared at 142.1, 133.59,
and 142.64, respectively. Expectedly, these carbene carbon resonances in silver (7–9) 13C
NMR spectrum of silver complexes 7 and 8; the carbene carbon resonance appeared as two
doublets centered at δ 189.12 and 189.7, respectively. This is due to the presence of 13C-109Ag
and 13C-107Ag coupling of 1J(C-109Ag) (182.5 for 7 and 183.87 Hz for 8) and 1J(C-107Ag) (206
for 7 and 210.75 Hz for 8), respectively. Further, these signals are consistent with the absence
of NHC-exchange processes at the Ag centers, and the coupling constants are in well
agreement with the reported results [27, 28].
Single Crystal X-Ray Diffraction Studies
To confirm the unambiguous molecular structures, single crystals of salt 5 and complex 7 were
obtained by ether diffusion method, and the solid-state molecular structures were determined
by single crystal X-ray diffraction method. Single crystals of salts 5 and complexes suitable for
X-ray diffraction analysis were grown from either slow evaporation of methanolic solution (for
salt 5) or slow diffusion of diethyl ether into a complex solution in acetonitrile (for complexes)
at ambient temperature. A view of the salt 5 is shown in Fig. 1. Salt 5 crystallizes in the triclinic
space group P-1 containing three benzimidazolium cations, three bromide anions, and three
water molecules in an asymmetric unit. In the five-membered ring (NCNCC) of salt 5 and
substitutions attached, the bond angles and distances are in good agreement with the published
similar molecules [29]. In the extended structure, bromide ions and water molecules link the
benzimidazolium cations into three dimensional networks through C-H…Br (2.539(3) Å), O-
H…Br (2.523(6) Å), and H-O…H-C (3.317(7) Å) hydrogen bonds thats' further detail is
tabulated in Tables 1, 2 and 3.
The molecular structure of symmetric silver complex 7 is depicted in Fig. 2. Complex 7
crystallizes in the monoclinic space group P2(1)/having half an equivalent of complex cation
and anion in an asymmetric unit. Complex consists of the two benzimidazolium NHC units
coordinating to the silver atom; the coordination at the silver atom carbene described as linear
with C1–Ag2–C1A bond angle of 180(10), having the carbene carbon–silver bond distances,
C1-Ag1 and C1A-Ag, is 2.0837(17) and 2.0838(17) Å, respectively. The internal ring angles
of both the benzimidazole rings at the carbene center (C-N-C) are 106.41(15)°. These bond
distances and angles are similar to those observed in other NHC silver complexes. Both the
benzimidazolium rings are almost parallel to each other with the negligible dihedral angle;

Applied Biochemistry and Biotechnology
Fig. 1 Molecular structure of the bromide salt 5 with displacement ellipsoids drawn at 50%probability
perhaps, this arises from the symmetric nature of the ligand. In the extended structure,
hexafluorophosphate anions link the complex cations into three-dimensional networks through
C-H…F (3.076(3) Å) hydrogen bonds.
In Vitro Anticancer Activity
Silver complexes have been known to serve as antimicrobials since a long time. In particular,
silver-NHC complexes are effective against a series of human cancer cell lines such as, HCT
116, HL 60, Caki-1, OVCAR-3, MB157, MCF-7, among others [30]. The specific activity of
silver-NHC complexes depends on their bioavailability and compatibility. Further, the delivery
materials and methods, solubility in biological fluids and ionization of the complex, and the
presence of biological polymeric ligands determine the extent of activity and toxicity. Recent-
ly, we have reported a series of silver-NHC complexes enabled as promising anticancer agents
against human-derived cancer cell lines such as HCT 116 and HL 60 [31]. In continuation of
our research on the structural diversity, stability, and solubility of silver-NHC complexes and
the search for new potential anticancer agents, we examined reported benzimidazolium salts 4–
6 and corresponding silver complexes 7–9 for their potential anticancer activities against HCT
116, MCF-7, and K-562 cancer cell lines by MTT assay method. Percentage inhibition of cell
proliferation was assessed at different concentration levels of salts 4–6 and complexes 7–9
ranging from 1.56 to 100 μM against each cell line. IC50 values of the compounds tested are
tabulated in Tables 1.
Figure 3 depicts the effect of concentration of salts/silver complexes against HCT 116 cell
line after 48 h of incubation time. It is clear from these graphs that cytotoxicity of the tested
compounds increased with the increase in concentration that proved that the compounds have
dose-dependent antiproliferative effect. Cells from control group showed high confluent

Applied Biochemistry and Biotechnology
Fig. 2 Molecular structure of the Ag(I) complex 7with displacement ellipsoids drawn at 50%probability
growth with compactly proliferating HCT 116 cells after 48 h of incubation. However, 5-
fluorouracil-treated cells showed decreased viability with the IC50 value of 5.2 μM. These cells
show apoptotic characteristic features wherein nuclear condensation and cell membrane
blebbing were much clear.
Treatment with the symmetric benzimidazolium salt 4 showed moderate inhibitory effect
on HCT 116 cell proliferation with IC50value of 33.7 μM. The cell picture revealed the
affected cellular morphology of the treated cells, and the intercellular junctions were discon-
nected leading to appear cells less intact. Surprisingly, cells treated corresponding silver
complex 7 showed the strong cytotoxic effect of the complex with IC50value of 0.31 μM. It
is to justify that complex 7 inhibited the proliferation in almost the cells which rendered the
cells losing their pseudopodial-shaped extensions and viability wherein cellular debris
remained in the growth medium. Further, treatments with the non-symmetrical
benzimidazolium salts 5 and 6 showed lesser cytotoxicity with IC50values of 61.0and
57.5 μM, respectively, compared to the symmetric salt. HCT 116 cells treated with 5 and 6
showed no/less significant inhibitory effect on the cellular proliferation. Conversely, the cells
treated with their respective silver complexes 8 and 9 exhibited promising cytotoxicity with
IC50values of 15.1 and 1.99 μM, respectively, as the cell population was reduced exceptionally
within 48 h of treatment, which causes severe cell death. Images of the control HCT116 cell
line, cells treated with standard 5-FU, and benzimidazolium salts 4–6 and their respective
silver complexes 7–9 after 48 h of incubation are depicted in Fig. 4.
Figures 5 and 6 display dose-dependent antiproliferative effects of the distinct salts and
silver complexes studied toward MCF-7and K-563 cancer cells, respectively. Again, it was
verified that silver complexes were particularly effective against both the cell lines, while
benzimidazolium salts displayed a rather weak antiproliferative activity.

Applied Biochemistry and Biotechnology
Table 1 Crystal data and structure refinement details of benzimidazolium salt 5 and Ag-NHC complexes 7
5 (Salt)
7 (Ag complex)
Formula
Formula weight
C7H27N2Br.H2O
339.31
C26H36AgF6N4P
657.43
Crystal system
Space group
Triclinic
P1
Monoclinic
P21/c
Unit cell dimensions
a (Å)
b (Å)
9.8666(2)
13.9114(2)
21.4815(3)
105.410(1)
91.392(1)
101.592(1)
2775.10(8)
3
8.1529(1)
16.3288(2)
11.0793(1)
90
108.696(1)
90
1397.13(3)
2
c (Å)
α (o)
β (o)
γ (o)
V (Å3)
Z
Density (calcd) (gm/cm3)
1.281
2.224
1124
1.563
0.842
672
Abs coeff (mm−1
F(000)
)
Crystal size (mm)
Temperature (K)
Radiation (Å)
θ Min, max (o)
Dataset
0.13 × 0.24 × 0.29
100
MoKa 0.71073
1.0, 29.0
−13: 13; −18: 18; −29: 25
43,865
0.041
0.13 × 0.18 × 0.26
100
MoKa 0.71073
2.3, 35.1
−13: 13; −25: 26; −17: 13
23,301
0.027
Tot.; Uniq. data
R (int)
Nref, Npar
R, wR2, S
14,597, 574
0.0514, 0.1728, 1.02
6067, 177
0.0353, 0.0824, 1.11
Salt 4 displayed little effect on viability and moderate inhibition in cell proliferation for
MCF-7 cancer cell line, yieldingIC50 value of 41.3 μM. The corresponding silver complex 7,
however, showed promising activity with the IC50 value of 15.1 μM, as the doubling time of
cell population was significantly reduced within 48 h of treatment.
Conversely, benzimidazolium salts 5 and 6 displayed considerable cytotoxic effect with the
IC50values 20.3 and 35.2 μM on the proliferation of MCF-7 cells, respectively. Further,
population of the cells reduced significantly and significant effect on cellular morphology
was also noted (Fig. 7). Corresponding silver complexes 8 and 9 showed improved cytotox-
icity yielding IC50values of 16.1 and 35.2 μM, respectively, on proliferation of MCF 7 cells,
which are much lesser than the standard tamoxifen (IC50 = 5.5 μM) used.
Finally, all the tested compounds displayed better activity against K-562 cell line compared
to MCF-7cell line. Salts 4 and 5 displayed little or no effect cell growth as well as on viability
Table 2 Selected bond lengths (Å) and angles (o) of 5
C8B-N1B
1.387(5)
N1B-C1B
1.398(5)
C1B-C2B
1.399(5)
N1B-C7B
C7B-N2B
N2B-C11B
C8B-N1B-C1B
N1B-C1B-C2B
C8B-C9B-C10B
C1B-C2B-C3B
1.334(5)
1.338(5)
1.471(5)
126.1(3)
131.0(4)
113.2(4)
115.9(3)
N2B-C6B
C1B-C6B
1.384(5)
1.391(5)
C2B-C3B
O1W-H1W1
1.386(6)
0.8400
N1B-C7B-N2B
C7B-N1B-C1B
C7B-N2B-C6B
C11B-N2B-C6B
109.4(3)
108.6(3)
109.0(3)
126.2(3)

Applied Biochemistry and Biotechnology
Table 3 Selected bond lengths (Å) and angles (o) of silver complex 7
Ag1-C1
2.0839(18)
C2-C7
1.398(3)
N2-C11
1.465(2)
C1-N2
N1-C1
C2-N1
1.355(2)
1.356(2)
1.393(2)
C2-C3
C7-C6
N2-C7
1.393(3)
1.394(3)
1.393(2)
C11-C12
C12-C13
P1-F1
1.525(3)
1.528(3)
1.6000(14)
N1-C8
1.467(2)
C1-Ag1-C1A
C13-C12-C11
C12-C11-N2
N2-C1-Ag1
C11-N2-C7
180.00(10)
111.01(16)
112.53(15)
129.11(13)
124.97(15)
C8-N1-C2
N2-C7-C2
F1-P1-F2
F1-P1-F1A
N1-C2-C7
125.58(15)
106.11(15)
90.32(7)
180.00
106.09(15)
for K-562 cell line yielding IC50 values of 84.0 and > 200 μM, respectively, while correspond-
ing complexes 7and 8 demonstrated potent antiproliferative effect (IC50 = 7.0and 17.9 μM) as
shown in Fig. 8. The activity was more pronounced in the case of complex 7 than the standard
reference betulinic acid (IC50 = 17.0 μM) used. Interestingly, salt 6 demonstrated moderate
cytotoxic effect on K562 cell proliferation as it showed IC50value 24.0 μM. It can be seen
clearly that the population of cells reduced significantly after 48 h of treatment. However,
silver complex 9 showed promising inhibition the cells with IC50value of 10.7 μM.
The cell picture (Fig. 5) elaborates the drastic reduction of cancer cells. Thus, a selectivity
of anticancer activity was observed, related to the conformational characteristics of the
compounds tested versus the kind of cancer cells studied.
Possible Mechanism of Action
Chromatin condensation, formation of apoptotic bodies, and membrane blebbing were mon-
itored to analyze apoptosis wherein HCT 116 cells treated with test compounds are elaborated
(Fig. 4B, F, H). These results are in accordance with the previous findings where the silver(I)-
NHC complexes induced caspase-independent apoptotic cellular death via mitochondrial
apoptosis-inducing factor [32]. However, several studies justified silver cations deposition in
cytosol to render the cellular defunct while interacting with rate-limiting enzymes and proteins
involved in metabolic pathways [33]. The present findings revealed clear signs of black silver
deposits in the cytoplasm in the infected cells (Fig. 5B, F, H) to confirm silver deposition-
based cytotoxicity. Hence, it is speculated that the activity decreased by increasing the chain
length of complexes (7–9). This phenomenon is in accordance with the previous reports on
Fig. 3 MTT assay result of salts (4–6) in (a) and Ag–NHC complexes (7–9) in (b) and vs. the HCT 116 cell line

Applied Biochemistry and Biotechnology
Fig. 4 Images of the control HCT116 cells (A), cells treated with 5-FU (B), symmetric salt 4 (C) and complex 7
(D), and non-symmetric salts 5, 6 (E, G) and complexes 8, 9 (F, H) after 72 h of incubation. FiF
similar mononuclear Ag(I)-NHC complexes summarized by Gautier and co-workers [34].
Whereas for dinuclear Ag(I)-NHC complexes, the cytotoxicity increases nonpolar character.
This might be due to the rotation of NHC units bonded to a single metal ion in a mononuclear

Applied Biochemistry and Biotechnology
Fig. 5 MTT assay result of salts (4–6) in (a) and Ag–NHC complexes (7–9) in (b) and vs. MCF-7 cell line
silver NHC complex, whereas these rotations are not possible in dinuclear silver complexes
and structure remains rigid.
In Vivo Acute Oral Toxicity Studies
Acute toxicity is actually the assessment of adverse effects posed by drug candidates occurring
in an animal within a short duration of administration of either a single or multiple doses given
to the test animal. In the current study, “fixed dose method” was adopted to determine the
sighting study dose according to the reported procedure [35]. All test compounds were
evaluated for sighting study using 300 mg/kg dose. Since the synthesized compounds were
not soluble in saline, so each compound (75 mg 5/210 g 10 of animal weight) was mixed
with 300 μL of tween 80 in order to make the compound soluble, and total volume was marked
up to 2 mL with saline. All the experimental animals after initial dose were observed after
20 min and then after 1 hour and after that periodically at least once a day until 14th day. During
this time, body weight and moribund symptoms of the animals were checked periodically. No
significant weight gain or moribund symptoms were observed during the aforementioned time.
Furthermore, in the present study, all animals were survived and remained healthy and agile.
Therefore, it is concluded that 300 mg/kg body weight of these selected benzimidazolium salts
and their silver(I)-NHC complexes can be the starting dose for main study.
Fig. 6 MTT assay result of salts (4–6) in (a) and Ag–NHC complexes (7–9) in (b) and vs. K-562 cell line

Applied Biochemistry and Biotechnology
Fig. 7 Images of the control MCF-7 cells (A), cells treated with tamoxifen (B), symmetric salt 4 (C) and complex
7 (D), and non-symmetric salts 5, 6 (E, G) and complexes 8, 9 (F, H) after 72 h of incubation
Conclusion
In short, high-yielding symmetrically and non-symmetrically substituted benzimidazolium
salts and their silver–carbene complexes of the types [(NHC)2-Ag]PF6 are reported herein.
They were also studied to explore their anticancer potential against three human cancer cell
lines which showed that silver complexes are potent anticancer agents; particularly, higher

Applied Biochemistry and Biotechnology
Fig. 8 Images of the control K-562 cells (A), cells treated with betulinic acid (B), symmetric salt 4 (C) and
complex 7 (D), and non-symmetric salts 5, 6 (E, G) and complexes 8, 9 (F, H) after 72 h of incubation
cytotoxic potential was demonstrated by complex 7 for its IC50 value 0.31 μM which is tenfold
lower than that of standard used while benzimidazolium salts exhibited lower activities. It is
speculated from the available data that the increased antiproliferative effects of silver com-
plexes are associated with cellular apoptosis and cell cycle arrest, as analogous carbene
complexes do exert similar activity. It persuades for the preparation of other transition
metal–carbene complexes, and it is also underway by authors.

Applied Biochemistry and Biotechnology
Acknowledgments The authors are thankful to Higher Education Commission (HEC) of Pakistan for providing
grant National Research Programme for Universities No. NRPU-8396 & NRPU-8198. They are also very much
obliged to Rosenani A. Haque, School of Chemical Sciences as well as to Mohamed B. Khadeer Ahamed and
Amin M. S. Abdul Majid of EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences of
Universiti Sains Malaysia, 11800 USM, Penang, Malaysia for their technical support and proof-reading of the
manuscript.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
Appendix A Supplementary Material
CCDC 916242 and 916239 contain the supplementary crystallographic data. The supplemen-
tary crystallographic data for salt 5 and carbene complex 7 is available free of charge at CCDC
916242 and 916239 of Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ,UK; fax (+44) 1223–336-033 or e-mail deposit@ccdc.cam.ac.uk or using link http://
www.ccdc.cam.ac.uk/conts/retrieving.html. Moreover, the effects of increasing amounts of test
compound on the percentage inhibition of cell proliferation are also available.
References
1. Gardiner, M. G., & Ho, C. C. (2018). Recent advances in bidentate bis (N-heterocyclic carbene) transition
metal complexes and their applications in metal-mediated reactions. Coordination Chemistry Reviews, 375,
373–388.
2. El-Kady, D. S., Abd Rabou, A. A., Tantawy, M. A., Abdel-Rahman, A. A.-H., Abdel-Megeed, A. A.-S.,
AbdElhalim, M. M., & Elmegeed, G. A. (2019). Synthesis and evaluation of novel cholestanoheterocyclic
steroids as anticancer agents. Applied Biochemistry and Biotechnology, 188(3), 635–662. https://doi.
org/10.1007/s12010-018-02943-6.
3. Kaloğlu, N., Kaloğlu, M., Özdemir, İ., Günal, S., & Özdemir, İ. (2017). Silver–N-heterocyclic carbene
complexes: synthesis, characterization, and antimicrobial properties. Journal of the Chinese Chemical
Society, 64(4), 420–426.
4. Xu, L., Hong, M., Wang, Y., Li, M., Li, H., Nair, M. P. N., & Li, C.-Z. (2016). Tunable synthesis solid or
hollow Au–Ag nanostructure, assembled with GO and comparative study of their catalytic properties.
Science Bulletin, 61(19), 1525–1535. https://doi.org/10.1007/s11434-016-1165-0.
5. Hopkinson MN., Richter C., Schedler M., Glorius F. (2014). An overview of N-heterocyclic carbenes.
Nature 510(7506):485.
6. Meng, G., Kakalis, L., Nolan, S. P., & Szostak, M. (2019). A simple 1H NMR method for determining the
σ-donor properties of N-heterocyclic carbenes. Tetrahedron Letters, 60(4), 378–381.
7. Munz, D. (2018). Pushing electrons▪ which carbene ligand for which application? Organometallics, 37(3),
275–289.
8. Wang, M. H., & Scheidt, K. A. (2016). Cooperative catalysis and activation with N-heterocyclic carbenes.
Angewandte Chemie International Edition, 55(48), 14912–14922.
9. Pudlarz, A. M., Ranoszek-Soliwoda, K., Czechowska, E., Tomaszewska, E., Celichowski, G., Grobelny, J.,
& Szemraj, J. (2019). A study of the activity of recombinant Mn-superoxide dismutase in the presence of
gold and silver nanoparticles. Applied Biochemistry and Biotechnology, 187(4), 1551–1568. https://doi.
org/10.1007/s12010-018-2896-y.
10. Pudlarz, A. M., Czechowska, E., Ranoszek-Soliwoda, K., Tomaszewska, E., Celichowski, G., Grobelny, J.,
& Szemraj, J. (2018). Immobilization of recombinant human catalase on gold and silver nanoparticles.
Applied Biochemistry and Biotechnology, 185(3), 717–735. https://doi.org/10.1007/s12010-017-2682-2.
11. Mercs L., & Albrecht M. (2010). Beyond catalysis: N-heterocyclic carbene complexes as components
for medicinal, luminescent, and functional materials applications. Chemical Society Reviews. 39(6):
1903–12.

Applied Biochemistry and Biotechnology
12. Medici, S., Peana, M., Crisponi, G., Nurchi, V. M., Lachowicz, J. I., Remelli, M., & Zoroddu, M. A. (2016).
Silver coordination compounds: a new horizon in medicine. Coordination Chemistry Reviews, 327-328,
349–359. https://doi.org/10.1016/j.ccr.2016.05.015.
13. Soleymani-Babadi, S., Beheshti, A., Bahrani-Pour, M., Mayer, P., Motamedi, H., Trzybiński, D., &
Wozniak, K. (2019). Synthesis, structural characterization, photophysical properties, and antibacterial
assessment of silver(I)-thione coordination polymers based on a competition between nitrate anion and
coanions CF3SO3–, ClO4–, BF4–, PF6–, and SbF6. Crystal Growth & Design, 19(9), 4934–4948.
https://doi.org/10.1021/acs.cgd.9b00038.
14. Habib, A., Iqbal, M. A., Bhatti, H. N., & Shahid, M. (2019). Effect of ring substitution on synthesis of
benzimidazolium salts and their silver(I) complexes: characterization, electrochemical studies and evalua-
tion of anticancer potential. Transition Metal Chemistry. https://doi.org/10.1007/s11243-019-00321-7.
15. Habib, A., Iqbal, M. A., & Bhatti, H. N. (2019). Polynuclear Ag(I)-N-heterocyclic carbene complexes:
Synthesis, electrochemical and in vitro anticancer study against human breast cancer and colon cancer.
Journal of Coordination Chemistry, 1–15. https://doi.org/10.1080/00958972.2019.1632837.
16. Habib, A., MN, V., Iqbal, M. A., Bhatti, H. N., MBK, A., & AMSA, M. (2019). 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. https://doi.org/10.1016/j.jscs.2019.03.002.
17. Noor u, H., Islam, S., Zia, M., William, K., Abbas Fakhar, I., Umar Muhammad, I., Iqbal Muhammad, A.,
& Mannan, A. (2018). Anticancer, antimicrobial and antioxidant potential of sterically tuned bis-N-
heterocyclic salts. Zeitschrift für Naturforschung C, 74. https://doi.org/10.1515/znc-2018-0095.
18. Habib, A., Iqbal, M. A., & Bhatti, H. N. (2019). Polynuclear Ag (I)-N-heterocyclic carbene complexes:
Synthesis, electrochemical and in vitro anticancer study against human breast cancer and colon cancer.
Journal of Coordination Chemistry, 1–15.
19. Varna, D., Zainuddin, D. I., Hatzidimitriou, A. G., Psomas, G., Pantazaki, A. A., Papi, R., Angaridis, P., &
Aslanidis, P. (2019). Homoleptic and heteroleptic silver(I) complexes bearing diphosphane and thioamide
ligands: Synthesis, structures, DNA interactions and antibacterial activity studies. Materials Science and
Engineering: C, 99, 450–459.
20. Iqbal, M. A., Haque, R. A., Ahamed, M. B. K., Majid, A. A., & Al-Rawi, S. S. (2013). Synthesis and
anticancer activity of para-xylyl linked bis-benzimidazolium salts and respective Ag (I) N-heterocyclic
carbene complexes. Medicinal Chemistry Research, 22(5), 2455–2466.
21. Haque, R. A., Iqbal, M. A., Budagumpi, S., Khadeer Ahamed, M. B., Abdul Majid, A. M., & Hasanudin, N.
(2013). Binuclear meta-xylyl-linked Ag (I)-N-heterocyclic carbene complexes of N-alkyl/aryl-alkyl-
substituted bis-benzimidazolium salts: Synthesis, crystal structures and in vitro anticancer studies. Applied
Organometallic Chemistry, 27(4), 214–223.
22. Asif, M., Iqbal, M. A., Hussein, M. A., Oon, C. E., Haque, R. A., Ahamed, M. B. K., Majid, A. S. A., &
Majid, A. M. S. A. (2016). Human colon cancer targeted pro-apoptotic, anti-metastatic and cytostatic effects
of binuclear silver(I)–N-heterocyclic carbene (NHC) complexes. European Journal of Medicinal Chemistry,
108, 177–187.
23. Fatima, T., Haque, R. A., Razali, M. R., Ahmad, A., Iqbal, M. A., Asif, M., Ahamed, M. B. K., & Abdul
Majid, A. M. S. (2016). Synthesis, crystal structure, in vitro anticancer and in vivo acute oral toxicity studies
of tetramethylene linked bis-benzimidazolium salts and their respective dinuclear Ag(I)–NHC complexes.
Journal of Coordination Chemistry, 69(22), 3367–3383. https://doi.org/10.1080/00958972.2016.1230670.
24. Haque, R. A., Hasanudin, N., Iqbal, M. A., Ahmad, A., Hashim, S., Abdul Majid, A., & Ahamed, M. B. K.
(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. https://doi.org/10.1080/00958972.2013.831406.
25. Liu, Q.-X., Yin, L.-N., & Feng, J.-C. (2007). New N-heterocyclic carbene silver (I) and mercury (II) 2-D
supramolecular layers by the π–π stacking interactions. Journal of Organometallic Chemistry, 692(17),
3655–3663.
26. Liu, Q.-X., Zhao, X.-J., Wu, X.-M., Guo, J.-H., & Wang, X.-G. (2007). New mercury (II) and silver (I)
complexes containing NHC metallacrown ethers with the π–π stacking interactions. Journal of
Organometallic Chemistry, 692(25), 5671–5679.
27. Baker, M. V., Brown, D. H., Haque, R. A., Skelton, B. W., & White, A. H. (2004). Dinuclear N-heterocyclic carbene
complexes of silver (I), derived from imidazolium-linked cyclophanes. Dalton Transactions, 21, 3756–3764.
28. Hahn, F. E., Radloff, C., Pape, T., & Hepp, A. (2008). Synthesis of silver(I) and gold(I) complexes with
cyclic tetra-and hexacarbene ligands. Chemistry–A European Journal, 14(35), 10900–10904.
29. Liu, Q.-X., Yu, J., Zhao, X.-J., Liu, S.-W., Yang, X.-Q., Li, K.-Y., & Wang, X.-G. (2011). Mercury (II),
copper (II) and silver (I) complexes with ether or diether functionalized bis-NHC ligands: Synthesis and
structural studies. CrystEngComm, 13(12), 4086–4096.

Applied Biochemistry and Biotechnology
30. Kankala, S., Thota, N., Björkling, F., Taylor, M. K., Vadde, R., & Balusu, R. (2019). Silver carbene
complexes: An emerging class of anticancer agents. Drug Development Research, 80(2), 188–199.
31. Iqbal, M. A., Haque, R. A., Budagumpi, S., Ahamed, M. B. K., & Majid, A. M. A. (2013). Short metal–
metal separations and in vitro anticancer studies of a new dinuclear silver (I)-N-heterocyclic carbene
complex of para-xylyl-linked bis-benzimidazolium salt. Inorganic Chemistry Communications, 28, 64–69.
32. Teyssot, M. L., Jarrousse, A. S., Chevry, A., De Haze, A., Beaudoin, C., Manin, M., Nolan, S. P., Díez-
González, S., Morel, L., & Gautier, A. (2009). Toxicity of copper (I)–NHC complexes against human tumor
cells: Induction of cell cycle arrest, apoptosis, and DNA cleavage. Chemistry–A European Journal, 15(2),
314–318.
33. Haque, R. A., Hasanudin, N., Iqbal, M. A., Ahmad, A., Hashim, S., Abdul Majid, A., & Ahamed, M. B. K.
(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.
34. Gautier, A., & Cisnetti, F. (2012). Advances in metal–carbene complexes as potent anti-cancer agents.
Metallomics, 4(1), 23–32.
35. Walum, E. (1998). Acute oral toxicity. Environmental Health Perspectives, 106(suppl 2), 497–503.
Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
Affiliations
Muhammad Atif1 & Haq Nawaz Bhatti1 & Rosenani A. Haque5 & Muhammad Adnan
Iqbal1,2 & Mohammad B. Ahamed Khadeer4 & Amin Malik Shah Abdul Majid3
1
Department of Chemistry, University of Agriculture Faisalabad-38040, Faisalabad, Pakistan
2
Organometallic & Coordination Chemistry, Laboratory, Department of Chemistry, University of
Agriculture, Faisalabad 38040, Pakistan
3
Department of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia,
11800 Minden, Pulau Penang, Malaysia
4
EMAN Biodiscoveries Sdn. Bhd., A1-4, Lot 5, Persiaran 2/1, Kedah Halal Park, Kawasan Perindustrian
Sungai Petani, 08000 Sungai Petani, Kedah, Malaysia
5
School of Chemical Science, Universiti Sains Malaysia, 11800 Penang, Malaysia