Novel Silver-NHC complexes: Synthesis and anticancer properties

Article abstract of DOI:10.1016/j.jorganchem.2019.04.018

The aim of this study was to present the synthesis, characterization and anticancer activities of two novel benzimidazole-based NHC salts (1a-b) and their silver(I) complexes (2a-b) with methylbenzyl and isopropylbenzyl chains. All compounds were prepared using Schlenk techniques in an inert atmosphere. The carbene complexes were prepared with the interaction of carbene precursors and Ag₂O. All new compounds were characterized by elemental analysis, LC-MS, FT-IR, ¹H NMR, and ¹³C NMR spectroscopic techniques. The anticancer activities of the salts and complexes were determined by cell proliferation analysis using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human prostate cancer cells (DU-145) and human breast cancer cells (MCF-7, MDA-MB-231). L-929, mouse adipose tissue fibroblasts were used as model normal cells. The cells were plated at a cell density of 1 × 10⁵ cells in 96-well plates and treated with different concentrations (1–20 μM) of salts and complexes during 24, 48 and 72 h. The MTT assay results indicated that the salts (1a-b) have lower anticancer activity on cancer cells than their silver(I) complexes (2a-b). It was also observed that benzimidazole salts (1a-b) and their Ag-NHC complexes (2a-b) displayed lower anticancer activities on L-929 normal cells than on cancer cells. These results highlight that the anticancer activities of N-heterocyclic carbene-silver complexes vary according to the structure of the silver complex and cell line type.

Full text of DOI:10.1016/j.jorganchem.2019.04.018

Accepted Manuscript
Novel Silver-NHC complexes: Synthesis and anticancer properties
Serap Şahin-Bölükbaşı, Neslihan Şahin
PII:
S0022-328X(19)30157-3
DOI:
https://doi.org/10.1016/j.jorganchem.2019.04.018
JOM 20768
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 28 January 2019
Revised Date: 16 April 2019
Accepted Date: 19 April 2019
Please cite this article as: S. Şahin-Bölükbaşı, N. Şahin, Novel Silver-NHC complexes: Synthesis
and anticancer properties, Journal of Organometallic Chemistry (2019), doi: https://doi.org/10.1016/
j.jorganchem.2019.04.018.
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Novel Silver-NHC Complexes: Synthesis and Anticancer Properties
Serap Şahin-Bölükbaşıa,*, Neslihan Şahinb,c,d
aDepartment of Biochemistry, Faculty of Pharmacy, Sivas Cumhuriyet University, 58140,
Sivas, Turkey
bDepartment of Basic Education, Faculty of Education, Sivas Cumhuriyet University, 58140,
Sivas, Turkey,
cDepartment of Chemistry, Faculty of Science and Art, İnönü University, 44280 Malatya,
Turkey
dCatalysis Research and Application Center, İnönü University, 44280, Malatya, Turkey
Abstract
The aim of this study was to present the synthesis, characterization and anticancer activities of
two novel benzimidazole-based NHC salts (1a-b) and their silver(I) complexes (2a-b) with
methylbenzyl and isopropylbenzyl chains. All compounds were prepared using Schlenk
techniques in an inert atmosphere. The carbene complexes were prepared with the interaction
of carbene precursors and Ag2O. All new compounds were characterized by elemental
1
13
analysis, LC-MS, FT-IR, H NMR, and C NMR spectroscopic techniques. The anticancer
activities of the salts and complexes were determined by cell proliferation analysis using (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human prostate
cancer cells (DU-145) and human breast cancer cells (MCF-7, MDA-MB-231). L-929,
mouse adipose tissue fibroblasts were used as model normal cells. The cells were plated at a
cell density of 1x105 cells in 96-well plates and treated with different concentrations (1-20
µM) of salts and complexes during 24, 48 and 72 hours. The MTT assay results indicated that
the salts (1a-b) have lower anticancer activity on cancer cells than their silver(I) complexes
(2a-b). It was also observed that benzimidazole salts (1a-b) and their Ag-NHC complexes
(2a-b) displayed lower anticancer activities on L-929 normal cells than on cancer cells. These
results highlight that the anticancer activities of N-heterocyclic carbene-silver complexes vary
according to the structure of the silver complex and cell line type.
Keywords: N-Heterocyclic carbene; Benzimidazole-2-ylidene; Silver; Prostate cancer; Breast
cancer; Anticancer activity
1. Introduction
Cancer is one of the major causes of death worldwide and deaths due to cancer are
increasing. According to global cancer statistics (GLOBOCAN 2018), there will be an
estimated 18.1 million new cancer cases and 9.6 million cancer deaths in 2018 [1]. In both

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sexes combined, lung cancer is the most commonly diagnosed cancer, followed by breast,
prostate, colorectal, stomach and liver cancer. Prostate cancer is the second most commonly
occurring cancer in men and there will be approximately 1.3 million new cases in 2018.
Breast cancer is the most commonly occurring cancer in women and there will be over 2
million new cases in 2018. Therefore, treatment for cancer is one of the most researched
areas.
Cancer treatment varies according to the type of cancer that develops. Different strategies
have been developed for treating various types of cancers, including drug treatment,
radiotherapy, and surgical treatment. Drug treatment includes hormonal therapy,
chemotherapy, and targeted therapy. In chemotherapy, different types of drugs are used to
treat cancer patients in combination with surgery and/or radiotherapy [2]. Cis-platin, which
was discovered by Rosenberg et al, was the first example of metal-based anticancer drugs [3].
Cis-platin and its analogs are used mostly for the medical treatment of cancer patients [4-6].
However, platinum-based anticancer drug cis-platin has several side effects, such as vomiting,
nephrotoxicity, hair loss, neurotoxicity, diarrhea, ototoxicity and the development of intrinsic
and acquired resistance in some cancer cells, and these are considered to be serious health
problems [7]. These side-effects may result in the patient being reluctant to progress with
treatment [8]. Therefore, it is important to design and synthesize transition metal-containing
new anti-cancer agents that are chemically different from those currently available, and which
have a broad spectrum and low cytotoxicity.
N-Heterocyclic Carbenes (NHCs) have become a well-known class of organometallic ligands.
Recently, several metal N-heterocyclic carbene (M-NHC) complexes have been widely
investigated in medicinal chemistry due to their noteworthy biological properties [9-18]. In
biological systems, the selection of metals is also an important issue. Silver salts have
historically been used for the purification of drinking water, as antimicrobial agents, for
prevention of eye infections in newborns, and in wound healing [19-20]. The low toxicity of
silver salts for humans has also attracted researchers to further investigate their in vitro and in
vivo biological properties, including antimicrobial and anticancer features [21-25]. A number
of new silver(I)-NHC complexes were synthesized and screened against several carcinoma
cell lines after the first report of cytotoxic activities of silver(I)-NHC complex on breast
cancer (MCF-7), cervical cancer (HeLa), and colon adenocarcinoma (HCT 116) [26-32].
Although the imidazole structure is very frequently used in organometallic chemistry,
benzimidazole derivatives have been rarely used, especially for biological applications [33-

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34]. In organic chemistry, a number of organic derivatives of benzimidazole have shown
important therapeutic activities including anti-cancer activity [35-36]. The structural
similarity of benzimidazole derivatives with naturally occurring nucleotides makes them
biologically important structures [35]. For those reasons, benzimidazole and silver were used
to synthesize organometallic compounds in this study.
The aim of this study was to design two novel NHC salts and their Ag(I) complexes (Scheme
1) and investigate their anticancer activities. All salts and complex structures were
1
13
characterized by elemental analysis, LC-MS, FT-IR, H NMR, and C NMR spectroscopy.
The anticancer activities of the compounds were examined on DU-145 (HTB-81, human
prostate carcinoma), MCF-7 (HTB-22, human breast adenocarcinoma), MDA-MB-231 (HTB-
26, human breast adenocarcinoma) cell lines with the MTT assay. L-929, mouse adipose
tissue fibroblast cells were used as model normal cells.
2. Experimental
2.1. Materials and Methods
The synthesis of benzimidazolium salts and Ag(I)-NHC complexes were carried out under
argon in flame-dried glassware using standard Schlenk line techniques. Chemicals and
solvents were purchased from Sigma Aldrich Co. (Dorset, UK). The solvents used were
purified by distillation over the drying agents indicated and were transferred under Argon.
Elemental analyses were performed by İnönü University Scientific and Technology Center.
Melting points were determined using Electrothermal 9100 melting point detection apparatus
Fourier transform infrared (FTIR) spectra were obtained in the range 400–4000 cm−1 on
1
Perkin Elmer Spectrum 100 FT-IR. H NMR and 13C NMR spectra were recorded using a
Bruker Avance III spectrometer operating at 400 MHz (1H), 100 MHz (13C) in CDCl3 with
1
tetramethylsilane (TMS) as the internal reference. H peaks are labeled as singlet (s), doublet
(d), multiplet (m) and heptet (hept.). Chemical shifts and coupling constants are reported in
ppm and in Hz, respectively.
2.2. General procedure for the preparation of benzimidazolium salts, (1a-b)
Benzimidazole (10 mmol) was added to a solution of NaH (10 mmol) in dry THF (30 mL)
and the mixture was stirred for 1 h at room temperature. 2-methyl-2-propenyl chloride (10.1
0
mmol) was added dropwise to the obtained solution and heated for 24 h at 60 C. Then, the
solvent was removed under the vacuum. Dichloromethane (50 mL) was added to solid. The
mixture was filtered and the obtained clear solution was concentrated under vacuum. The last
solution was distilled and 1-(2-methyl-2-propenyl) benzimidazole was obtained. The 1-(2-

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methyl-2-propenyl) benzimidazole (1 mmol) and alkyl halide (1 mmol) were stirred in DMF
0
(5 mL) for 24 h at 80 C. White product was collapsed. After the solution was filtered, solid
was rinsed out with diethylether and dried under vacuum. The crude product was
recrystallized from dichloromethane/diethylether [37].
2.2.1. 1-(2-Methyl-2-propenyl)-3-(4-methylbenzyl)benzimidazolium chloride, 1a
1
Yield: 82%, m.p. 195-197 °C. FT-IR ν(CN): 1551 cm-1. H NMR (400 MHz, CDCl3) δ (ppm):
1.72 (s, 3H, NCH2C(CH3)CH2), 2.23 (s, 3H, CH2C6H4(CH3)-4), 4.90 (s, 1H,
NCH2C(CH3)CH2), 5.04 (s, 1H, NCH2C(CH3)CH2), 5.21 (s, 2H, NCH2C(CH3)CH2), 5.78 (s,
2H, CH2C6H4(CH3)-4), 7.09 (d, 2H, Ar-H, J = 8 Hz), 7.31 (d, 2H, Ar-H, J = 8 Hz), 7.45-7.51,
7.53-7.55 and 7.59-7.61 (m, 4H, Ar-H), 11.74 (s, 1H, NCHN). 13C NMR (100 MHz, CDCl3) δ
(ppm): 19.7 (NCH2C(CH3)CH2),
21.2 (CH2C6H4(CH3)-4), 51.4 (CH2C6H4(CH3)-4), 53.6
(NCH2C(CH3)CH2), 113.7 (NCH2C(CH3)CH2), 139.2 (NCH2C(CH3)CH2), 113.9, 116.1,
127.1, 128.2, 128.3, 129.8, 130.0, 131.2, 131.7 and 137.5 (Ar-C), 144.0 (NCHN). % Anal.
Calcd for C19H21N2Cl: C, 72.95; H, 6.77; N: 8.95. Found: C, 72.83; H, 6.68; N, 8.87.
2.2.2. 1-(2-Methyl-2-propenyl)-3-(4-isopropylbenzyl)benzimidazolium chloride, 1b
1
Yield: 91%, m.p. 158-160 °C. FT-IR ν(CN): 1558 cm-1. H NMR (400 MHz, CDCl3) δ (ppm):
1.20 (d, 6H, CH2C6H4(CH(CH3)2)-4), 1.79 (s, 3H, NCH2C(CH3)CH2), 2.87 (hept., 1H,
CH2C6H4(CH(CH3)2)-4, J = 8 Hz), 4.98 (s, 1H, NCH2C(CH3)CH2), 5.12 (s, 1H,
NCH2C(CH3)CH2), 5.30 (s, 2H, NCH2C(CH3)CH2), 5.87 (s, 2H, CH2C6H4(CH(CH3)2)-4),
7.22 (d, 2H, Ar-H, J = 8 Hz), 7.43 (d, 2H, Ar-H, J = 8 Hz), 7.54-7.57, 7.64-7.70 (m, 4H, Ar-
H), 11.78 (s, 1H, NCHN). 13C NMR (100 MHz, CDCl3) δ (ppm): 19.8 (NCH2C(CH3)CH2),
23.8 (CH2C6H4(CH(CH3)2)-4), 33.8 (CH2C6H4(CH(CH3)2)-4), 51.3 (CH2C6H4(CH(CH3)2)-4),
53.6 (NCH2C(CH3)CH2), 113.7 (NCH2C(CH3)CH2), 137.5 (NCH2C(CH3)CH2), 113.8, 116.1,
127.1, 127.4, 128.3, 128.4, 130.2, 131.3, 131.7 and 150.1 (Ar-C), 144.0 (NCHN). % Anal.
Calcd for C21H25N2Cl: C, 73.99; H, 7.39; N: 8.22. Found: C, 73.86; H, 7.26; N, 8.17.
2.3. General procedure for preparation of Ag(I)-NHC complexes, (2a-b)
A solution of 1 mmol of Ag2O and 0.5 mmol of benzimidazolium salts (1a-b) in
dichloromethane (25 mL) were stirred at room temperature for 24 h under the dark condition.
After that, the mixture was filtered through celite. The clear filtrate was evaporated under
vacuum to afford the crude product. The crude product was recrystallized from
dichloromethane/diethylether.

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2.3.1.
Chloro[1-(2-Methyl-2-propenyl)-3-(4-methylbenzyl)benzimidazole-2-ylidene]silver(I), 2a
1
Yield: 83%, m.p. 198-200 °C. FT-IR ν(CN): 1391 cm-1. H NMR (400 MHz, CDCl3) δ (ppm):
1.67 (s, 3H, NCH2C(CH3)CH2), 2.23 (s, 3H, CH2C6H4(CH3)-4), 4.76 (s, 1H,
NCH2C(CH3)CH2), 4.92 (s, 2H, NCH2C(CH3)CH2), 4.96 (s, 1H, NCH2C(CH3)CH2), 5.51 (s,
13
2H, CH2C6H4(CH3)-4), 7.04-7.11, 7.23-7.30 and 7.37-7.39 (m, 8H, Ar-H). C NMR (100
MHz, CDCl3) δ (ppm): 20.1 (NCH2C(CH3)CH2), 21.2 (CH2C6H4(CH3)-4),
53.4
(CH2C6H4(CH3)-4), 55.6 (NCH2C(CH3)CH2), 112.1 (NCH2C(CH3)CH2), 139.1
(NCH2C(CH3)CH2), 112.2, 114.6, 124.3, 127.1, 127.2, 129.8, 131.8, 133.7, 134.1 and 138.4
(Ar-C), 189.1 (Ccarbene-Ag). % Anal. Calcd for C19H20N2ClAg: C, 54.37; H, 4.80; N: 6.67.
Found: C, 54.22; H, 4.71; N, 6.54; LC–MS: 659.3 [AgL2]+.
2.3.2.
Chloro[1-(2-Methyl-2-propenyl)-3-(4-isopropylbenzyl)benzimidazole-2-ylidene]silver(I),
2b
1
Yield: 90%, m.p. 163-165 °C. FT-IR ν(CN): 1387 cm-1. H NMR (400 MHz, CDCl3) δ (ppm):
1.22 (d, 6H, CH2C6H4(CH(CH3)2)-4), 1.75 (s, 3H, NCH2C(CH3)CH2), 2.87 (hept., 1H,
CH2C6H4(CH(CH3)2)-4, J = 8 Hz), 4.84 (s, 1H, NCH2C(CH3)CH2), 5.00 (s, 2H,
NCH2C(CH3)CH2), 5.04 (s, 1H, NCH2C(CH3)CH2), 5.59 (s, 2H, CH2C6H4(CH(CH3)2)-4),
13
7.16-7.22 and 7.30-7.40 (m, 7H, Ar-H), 7.46 (d, 1H, Ar-H, J = 8 Hz). C NMR (100 MHz,
CDCl3)
δ
(ppm): 20.1 (NCH2C(CH3)CH2), 23.9 (CH2C6H4(CH(CH3)2)-4), 33.8
(CH2C6H4(CH(CH3)2)-4), 53.3 (CH2C6H4(CH(CH3)2)-4), 55.6 (NCH2C(CH3)CH2), 112.0
(NCH2C(CH3)CH2), 139.1 (NCH2C(CH3)CH2), 112.2, 114.6, 116.7, 120.2, 124.3, 127.1,
127.2, 132.1, 133.7, 134.0 and 149.3 (Ar-C), no peak (Ccarbene-Ag). % Anal. Calcd for
C21H24N2ClAg: C, 56.33; H, 5.40; N: 6.26. Found: C, 56.21; H, 5.32; N, 6.17; LC–MS: 715.4
[AgL2]+.
2.4. In-vitro anticancer studies
2.4.1. Cell culture;
The cells lines DU-145 (HTB-81, human prostate carcinoma), MCF-7 (HTB-22, human
breast adenocarcinoma), MDA-MB-231 (HTB-26, human breast adenocarcinoma), Eagles
Minimum Essential Medium (EMEM, 30-2003), RPMI-1640 (30-2001), fetal bovine serum
(FBS, 30-2020) and penicillin and streptomycin (30-2300) were purchased from American

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Type Culture Collection (ATTC, Manassas, VA). Dulbecco's Modified Eagle's Medium
(DMEM, D6429) and Trypsin-EDTA solution (T-3924) were purchased from Sigma Aldrich.
L-929 (normal cells mouse adipose tissue fibroblast) were purchased from ECACC (European
Collection of Animal Cell Culture, Salisbury, U.K.). All absorbance values were measured
with a microplate reader (BioTek, Epoch, USA) at 570 nm.
2.4.2. Anticancer properties, (MTT) assay;
DU-145 prostate cancer cell lines grown in EMEM medium, MCF-7 and MDA-MB-231
breast cancer cell lines were grown in DMEM medium and L-929 cell lines grown in RPMI-
1640 medium with L-glutamine supplemented with 10 % FBS and 1% penicillin/streptomycin
solution under 5% CO2 at 37ºC humidified air condition. Anticancer activity of the salts (1a-
b) and complexes (2a-b) was evaluated using the MTT assay [38]. L-929, mouse adipose
tissue fibroblast was used as model normal cells to determine the anticancer activity of salts
and complexes against normal cells. DU-145, MCF-7, MDA-MB-23, and L-929 cells were
seeded in 96-well plate at a volume of 100 µL per well (1x105 cell/well) and incubated for 24
h at 37 °C in 5% CO2. Then the cells were further incubated with four different concentrations
(1, 5, 10 and 20 µM) of salts and complexes, for 24 h, 48 h and 72 h. 0.5% dimethyl sulfoxide
in media (DMSO) and nutrient media without cells were used were used as negative control
and a blank, respectively. After 24 h, 48 h, and 72 h treatment 10 mL of MTT solution (5
mg/mL in PBS, pH 7.2) was added to each well and samples were further incubated for 2 h at
37 °C in 5% CO2. The medium was aspirated and added 100 µL/well DMSO to dissolve the
formazan crystals. Absorbance values were measured with a microplate reader (Epoch, USA)
at 570nm. Cytotoxicity curves and IC50 (µM) concentrations (defined as a concentration of
drug that decreases the cell viability by 50% compared to non-treated control cells), were
fitted by GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Selectivity index
was calculated by comparing the IC50 values of the test sample in normal and cancer cell
lines.
2.5. Statistical analysis
Each experiment was repeated at least three separate experiment and three replicates (n=9).
All data expressed as mean ± SEM. Data were analyzed using one-way analysis of variance
and differences were considered significant at p < 0.0001. The IC50 were determined by
statistical software, GraphPad Prism7 (GraphPad Software, San Diego, CA, USA).
3. Result and Discussion
3.1. Synthesis and characterization of benzimidazolium salts and Ag(I)-NHC complexes

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Nitrogen-containing heterocyclic salts have attracted attention in the fields of coordination
chemistry due to their metal complexes show high reactivity as catalyst and biologically
agents. Benzimidazolium salts (1a-b) were synthesized by reaction of 2-methyl-2-propenyl
substituted benzimidazolium precursor with different alkyl halides as shown Scheme 1.
Scheme 1.
Ag(I)-NHC complexes (2a-b) were obtained from reacting Ag2O with corresponding
benzimidazolium salts (1a-b) in dichloromethane at room temperature under dark condition.
Synthesis of the Ag(I)-NHC complexes (2a-b) is summarized in Scheme 2. Benzimidazolium
salts (1a-b) and Ag(I)-NHC complexes (2a-b) were characterized by elemental analysis, LC-
1
MS, FT-IR, H NMR and 13C NMR spectroscopy (Figure S1-S10). Ag(I)-NHC complexes
(1a-b) are air and moisture stable in the solid state but unstable to light and soluble in
dimethylformamide, dimethylsulfoxide and halogenated solvents such as chloroform,
dichloromethane but insoluble in petroleum ether, diethyl ether. NMR spectra of all the
compounds were analyzed in d-CDCl3.
Scheme 2.
The benzimidazolium salts (1a-b) have an acidic NCHN proton which came to 11.74 and
1
11.78 ppm as a characteristic sharp singlet for 1a and 1b respectively, in the H NMR
spectrum. When benzimidazolium salts form Ag(I)-NHC complexes, they lose an acidic
proton. Therefore, in the 1H NMR spectrum Ag(I)-NHC complexes (1a-b), the disappearance
13
of an acidic proton is evidence of formation complex. In the C NMR spectra of 1a-b, the
characteristic signals of carbon (NCHN) were seen as singlets at 144.0 ppm for both
benzimidazolium salts. For complex 2a, a signal of carbon (NCN) was seen to have shifted
greatly downfield region compared to the corresponding benzimidazolium salt 1a and was
observed at 189.1 ppm as slight. No signal of carbon (NCN) of complex 2b was observed.
These values and the lack of the carben peak are in agreement with reported data for similar
Ag(I)-NHC complexes [45]. At the same time, formation of the Ag(I)-NHC complexes (2a-b)
was proven by IR spectra, which showed CN bond vibrations at 1391 and 1387, respectively,
and these vibrations for benzimidazolium salts (1a-b) were 1551 and 1558 cm-1, respectively.
However LC–MS spectrometry can be used in order to clarify the structures of silver(I)–NHC
complexes in the solution in the absence of crystallographic data. LC–MS spectrometry of the

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prepared complexes showed that maximal peak intensities for each complex were attributable
to [Ag(NHC)2]+ as the molecular ion, which is not uncommon for these types of complexes.
3.2. In vitro anticancer properties of benzimidazolium salts and Ag(I)-NHC complexes
The anticancer activities of novel benzimidazole-based NHC salts (1a-b) and their silver(I)
complexes (2a-b) in prostate cancer DU-145 cells, breast cancer MCF-7, MDA-MB-231
cells, and normal fibroblasts L-929 cells were investigated after exposing the cells to the
compounds for 24, 48 and 72 hours, using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) cell viability assay [46]. The percentage inhibition of cell
proliferation was determined at different concentrations of the test compounds (1a-b) and
(2a-b) ranging from 1 to 20 µM (Figure 1-4).
Figure 1-4
The anticancer efficacy parameters, in terms of IC50 values after 24 h, 48 h and 72 h of
incubation time, are demonstrated in Table 1. Tests were made to determine whether the salts
by themselves could exert any anticancer activity on cancer cells. The MTT assay results
indicated that the salts had an inhibitory effect on cancer cells in a time- and dose-dependent
manner except for 1a on DU-145 cell line. 1a had no anticancer effects on DU-145 cells at all
time points, on MCF-7 cells at 24 h and on MDA-MB-231 cells at 24 and 48 hours, at least at
doses equal to 20 µM.
Table 1
The benzimidazole salts (1a-b) displayed the following IC50 values against the DU-145,
MCF-7 and MDA-MB-231 cell lines; >20, 12.9 ± 0.28, >20, 5.83 ± 0.14, 9.91 ± 0.11, 8.56 ±
0.18 µM respectively for 48 h. As 1b was determined with lower IC50 values than 1a for
cancer cells, the anticancer activity of 1b was higher than that of 1a. All the complexes
showed higher anticancer activity compared to their benzimidazolium salts. Similarly, IC50
values demonstrated that the anticancer activity of 2a was lower than that of 2b. This feature
was attributed to the different chemical properties (such as lipophilicities, steric and
electronic) of the ligands around the benzimidazole nucleus. The differences in these ligands
enable various inhibition activities in the complexes. Isopropyl substituted complex, 2b, was
more active than methyl substituted complex, 2a. The isopropyl group is bulkier and could
interfere with the uptake mechanism across cell or organelle membranes. The Ag-NHC
complexes (2a-b) displayed the following IC50 values against the DU-145, MCF-7 and MDA-

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MB-231 cell lines; 4.04 ± 0.11, 8.25 ± 0.10, 8.06 ± 0.21, 2.08 ± 0.11, 1.20 ± 0.19, 1.10 ± 0.17
µM respectively for 48 h. Increasing doses of the salts and complexes were also tested on
normal cells, L-929. The MTT assay results indicated that the salts displayed no dose- and
time-dependent anticancer activity, even when tested at various concentrations and times on
L-929 cells. The IC50 values of the benzimidazole salts (1a-b) and their Ag-NHC complexes
(2a-b) on L-929 normal cells were determined; >20, >20, 14.0 ± 0.30, 3.81 ± 0.22 and >20,
>20, 6.10 ± 0.10, 2. 80 ± 0.23µM respectively, at 48 h. As the complexes (2a-b) had larger
IC50 values for normal cell lines, these compounds are more effective toward breast cancer
cells and prostate cancer cells. Calculated selectivity index values for salts (1a-b) were 1,
1.55, 1, 3.43, 2.08, 2.33, and for Ag-NHC complexes (2a-b), they were 3.46, 1.70, 1.73, 1.83,
3.18, 3.46 on DU-145, MCF-7 and MDA-MB-231 cell lines respectively, for 48 h.
While the majority of organometallic pharmaceutical research has been focused on platinum
and gold, NHC-silver complexes have become an important class of anticancer agents in
medicinal applications. Tacke et al. synthesized 6 different cyanobenzyl-NHC-silver
complexes and determined their antibacterial and anticancer activity [13]. All 6 NHC–silver
complexes were tested to determine their cytotoxic activity on human renal-cancer cell line
Caki-1 using MTT in vitro tests. The IC50 values of NHC-silver complexes 4a–f were found to
be 6.2 (±1.0), 7.7 (±1.6), 1.2 (±0.6), 10.8 (±1.9), 24.2 (±1.8) and 13.6 (±1.0) µM, respectively.
From that study, compound 4c was determined to be approximately three-times more
cytotoxic (1.2 ±0.6 µM) than cisplatin (3.3 µM). The current study results indicated that
compound 2a showed similar activity as 4c against DU-145 and as compound 2b showed
against MCF-7 and MDA-MB-231 cell lines. It has been previously reported that researchers
synthesized seven novel non-symmetrically p-Benzyl-Substituted imidazole/benzimidazole
NHC- Silver(I) Acetate Complexes and evaluated their in vitro biological activities [14].
Cytotoxic activities of the seven complexes were determined against human renal cancer cell
line Caki-1. The IC50 values, of complexes were found to be 25 (±1), 15 (±2), 5.4 (±0.8), 16
(±2), 7.1 (±1), 20 (±4), and 14 (±1) µM, respectively. The complex 2c, gave the smallest IC50
value of 5.4 (±0.8) µM indicating the highest anticancer activity. Tacke et al, also tested in
vitro and in vivo cytotoxic activity of the anticancer drug candidate (1-methyl-3-(p-
cyanobenzyl)benzimidazole-2-ylidene)silver(I)acetate (SBC1) against UKF-NB-3 and UKF-
NB-6 (neuroblastoma) HCT8 (colon), and paclitaxel-resistant cell line PC-3 (prostate) [24].
They found that the compound was effective in vitro against platinum-resistant cell lines
human neuroblastoma cells, UKF-NB-3, human colon carcinoma cell line HCT8 as well as
paclitaxel-resistant cell line human prostate cancer cell line PC-3. From the first in vivo

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experiments using non-tumor bearing mice, the toxicity of SBC1 was also demonstrated by
Tacke et al, [24]. They first determined the MTD value. For this purpose, in three groups of
two mice, each was treated with single doses of 25, 50 and 100 mg/kg/d of SBC1. The first
two groups of mice showed a body weight loss of 1 or 2%, respectively, while the value
extended to 5% in the group receiving 100 mg/kg/d of SBC1. The highest dose led to one
toxic death within one day after treatment, while the other mice recovered and the body
weight loss was found to be reversible. After these results, SBC1 at 25 and 50 mg/kg/d, in
four injections were administered to two cohorts of eight CAKI-1 tumor-bearing NMRI:nu/nu
mice, while a further cohort was treated with solvent only. It was observed that these two
dosages of SBC1 showed borderline toxicity leading to mortality and body weight loss, while
there was no significant tumor growth reduction or influence on blood parameters compared
to the solvent-treated control group. It was concluded that SBC1 is a potent cytotoxic and
resistance-breaking anticancer agent, which shows very little selectivity in vivo. Youngs et al
were the first to report the anticancer activity of Ag(I)-NHC compounds with a series of 4,5-
dicholorimidazole based Ag(I)-NHC compounds [25]. These were evaluated for their in vitro
antitumor activity against the cancer cell lines OVCAR-3 (ovarian), MB157 (breast), and
HeLa (cervical). The silver compounds were determined to be the most active against MB157
but compounds 6–8 were ineffective against the HeLa cell line (>200 µM). They were also
tested in an in vivo xenograft model developed utilizing OVCAR-3 and 6. According to the
pathological studies, significant tumor cell death was observed while no ill-effects to the
major organs of the mice were detected. According to the results of the Tacke and
Youngsstudies, the structure of the compound affects the in vivo cytotoxic activity of the
compound.
4. Conclusions
In this study, two new benzimidazolium salts and their Ag(I)-NHC complexes were
1
synthesized, characterized by elemental analysis, LC-MS, FT-IR, H NMR, and 13C NMR
spectroscopy and anticancer activities were evaluated. The in vitro anticancer activities of
novel benzimidazole-based NHC salts (1a-b) and their silver(I) complexes (2a-b) against DU-
145 prostate cancer cells, MCF-7, MDA-MB-231breast cancer cells, and L-929 normal
fibroblasts cells were demonstrated after exposing the cells to the compounds for 24, 48 and
72 hours, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell
viability assay. L-929, mouse adipose tissue fibroblasts were used as model normal cells. The
MTT assay results showed that the compounds had an inhibitory effect on cancer cells in a

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time- and dose-dependent manner. The results also indicated that the salts (1a-b) have lower
anticancer activity on cancer cells than their silver(I) complexes (2a-b). The benzimidazole
salts (1a-b) and their Ag-NHC complexes (2a-b) were seen to display lower anticancer
activities on L-929 normal cells than cancer cells. The isopropyl substituted complex, 2b, was
more active than the methyl substituted complex, 2a. These results showed that the in vitro
and in vivo anticancer activities of N-heterocyclic carbene-silver complexes vary according to
the structure of the silver complex and cell line type.
Acknowledgments
The authors thank the Scientific and Technological Research of Turkey (TÜBITAK-BIDEB),
the National Research Fellowship Programme for grants to N. Ş. Anticancer activity studies
were done at Sivas Cumhuriyet University Advanced Technology Application and Research
Center (CUTAM).
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Table 1. IC50 (µM) values of salts (1a-d) and complexes (2a-d) against the selected human
cancer cells and normal cellsa in vitro after 24 h, 48 h and 72 h of incubation
Ligands (IC50, μM)b
Complexes (IC50, μM)b
Cell lines
DU-145
Time
1a
>20
1b
2a
2b
19.2 ± 0.2
5.8 ± 0.1
3.2 ± 0.2
13.9 ± 0.2
9.9 ± 0.1
< 1
20.0 ± 0.8
4.0 ± 0.1
1.2 ± 0.4
9.6 ± 0.1
8.2 ± 0.1
< 1
3.0 ±0.1
2.1 ± 0.1
< 1
24h
48 h
72h
24h
48 h
72h
24h
48 h
72h
24h
48 h
72h
>20
>20
>20
1.8 ± 0.2
1.2 ± 0.2
< 1
MCF-7
12.9 ± 0.2
4.1 ± 0.2
>20
12.9 ± 0.2
8.5 ± 0.2
3.8 ± 0.2
>20
10.4 ± 0.2
8.1 ± 0.2
2.2 ± 0.2
17.9 ± 0.5
14.0 ± 0.3
6.1 ± 0.1
1.8 ± 0.7
1.1 ± 0.2
< 1
MDA-MB-231
L-929a
>20
7.5 ± 0.2
>20
5.9 ± 0.1
3.8 ± 0.2
2.8 ± 0.2
>20
>20
>20
>20
a Normal cells,.bCell viability after treatment for 24 h, 48 h and 72 h were determined by MTT staining as described in the
Experimental section. Each IC50 value represents the mean ± SEM of three independent experiments.

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Scheme 1. Synthesis of benzimidazolium salts (1a-b)
Scheme 2. Synthesis of Ag(I)-NHC complexes (2a-b)

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Figure 1
.
The dose and time-dependence of anticancer activities of ligands (1a-b) and Ag-NHC complexes (2a-b) for DU-145 cells. Control cells
were treated with DMSO. Data are representative of the mean of three separate experiments done in triplicate and are reported at the SEM
(* p<0.0001 vs control).

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Figure 2
.
The dose and time-dependence of anticancer activities of ligands (1a-b) and Ag-NHC complexes (2a-b) for MCF-7 cells. Control cells
were treated with DMSO. Data are representative of the mean of three separate experiments done in triplicate and are reported at the ±SEM
(* p<0.0001 vs control).

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Figure 3
.
The dose and time-dependence of anticancer activities of ligands (1a-b) and Ag-NHC complexes (2a-b) for MDA-MB-231 cells.
Control cells were treated with DMSO. Data are representative of the mean of three separate experiments done in triplicate and are reported at
the ±SEM (* p<0.0001 vs control).

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Figure 4. The dose and time-dependence of anticancer activities of ligands (1a-b) and Ag-NHC complexes (2a-b) for L-929 cells. Control cells
were treated with DMSO. Data are representative of the mean of three separate experiments done in triplicate and are reported at the ±SEM
(* p<0.0001 vs control).

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Highlights
•
•
•
Novel Silver-NHC Complexes
Elemental analysis, LC-MS, FT-IR, 1H NMR, and 13C NMR techniques.
Anticancer activities on DU-145 prostate cancer cells and MCF-7, MDA-MB-231
breast cancer cells and L-929 normal cells.
•
Comparison of activity according to the structure of complexes and cell line types