Novel N-heterocyclic carbene silver(I) complexes: Synthesis, structural characterization, and anticancer activity

Article abstract of DOI:10.1016/j.ica.2018.11.044

In this study, we synthesized four novel unsymmetrically substituted NHC ligands (1a-d) and their Ag(I) complexes (2a-d). All new compounds were characterized using elemental analysis, FT-IR, ¹H NMR, and ¹³C NMR spectroscopy and X-ray crystallography. The molecular structure of complex 2d was elucidated through single crystal X-ray diffraction analyses. Single crystal structural studies for complex 2d show that the benzene rings (C9–C14) and the central benzimidazole ring system make dihedral angles of 85.65(11)°. The Ag–Cl and Ag–C single bond lengths are 2.3553(7) and 2.096(2) ? respectively. The C–Ag–Cl bond angle is 168.27(7)°. Both salts and complexes were tested for their anti-cancer potential against three human cancer cell lines (DU-145, MCF-7, and MDA-MB-231) and non-cancer cells adipose from mouse (L-929) for 24 h, 48 h and 72 h using the MTT assays. However, the Ag(I)–NHC complexes (2a-d) showed a dose and time-dependent cytotoxic activity against all cell lines. MDA-MB-231 human breast carcinoma cells were the most sensitive to the Ag(I)–NHC complex displaying IC₅₀ lower than 1 μM all time points. Further, the IC₅₀s for Ag(I)–NHC were higher in non-cancer cells, suggesting that complexes possessed noteworthy selectivity for human cancer cells.

Full text of DOI:10.1016/j.ica.2018.11.044

Accepted Manuscript
Research paper
Novel N-Heterocyclic Carbene Silver(I) Complexes: Synthesis, Structural
Characterization, and Anticancer Activity
Serap Şahin-Bölükbaş ı, Neslihan Şahin, Muhammed Nawaz Tahir, Cengiz
Arıcı, Esranur Çevik, Nevin Gürbüz, İsmail Özdemir, Brian S. Cummings
PII:
S0020-1693(18)31029-6
https://doi.org/10.1016/j.ica.2018.11.044
ICA 18659
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 July 2018
24 November 2018
27 November 2018
Please cite this article as: S. Şahin-Bölükbaş ı, N. Şahin, M. Nawaz Tahir, C. Arıcı, E. Çevik, N. Gürbüz, I. Özdemir,
B.S. Cummings, Novel N-Heterocyclic Carbene Silver(I) Complexes: Synthesis, Structural Characterization, and
Anticancer Activity, Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/j.ica.2018.11.044
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Novel N-Heterocyclic Carbene Silver(I) Complexes: Synthesis,
Structural Characterization, and Anticancer Activity
Serap Şahin-Bölükbaşıa,b*, Neslihan Şahinc,d,e, Muhammed Nawaz Tahirf, Cengiz Arıcıg,
Esranur Çevika, Nevin Gürbüzd,e, İsmail Özdemird,e, Brian S Cummingsb,h
a Department of Biochemistry, Faculty of Pharmacy, University of Cumhuriyet, 58140, Sivas, Turkey
bDept. of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA
cDepartment of Basic Education, Faculty of Education, University of Cumhuriyet, 58140, Sivas,
Turkey,
dDepartment of Chemistry, Faculty of Science and Art, University of İnönü, 44280 Malatya, Turkey
eCatalysis Research and Application Center, University of İnönü, 44280, Malatya, Turkey
f Department of Physics, University of Sargodha, Sargodha, Pakistan
gThe Scientific And Technological Research Council of Turkey, 06100, Ankara, Turkey
hInterdisciplinary Toxicology Program, University of Georgia, Athens, GA
Abstract
In this study, we synthesized four novel unsymmetrically substituted NHC ligands (1a-d) and
their Ag(I) complexes (2a-d). All new compounds were characterized using elemental
1
analysis, FT-IR, H NMR, and 13C NMR spectroscopy and X-ray crystallography. The
molecular structure of complex 2d was elucidated through single crystal X-ray diffraction
analyses. Single crystal structural studies for complex 2d show that the benzene rings (C9-
C14) and the central benzimidazole ring system make dihedral angles of 85.65(11). The Ag-
Cl and Ag-C single bond lengths are 2.3553(7) and 2.096(2) Å, respectively. The C-Ag-Cl
bond angle is 168.27(7). . Both salts and complexes were tested for their anti-cancer
potential against three human cancer cell lines (DU-145, MCF-7, and MDA-MB-231) and
non-cancer cells adipose from mouse (L-929) for 24h, 48h and 72 h using the MTT assays.
However, the Ag(I)-NHC complexes (2a-d) showed a dose and time-dependent cytotoxic
activity against all cell lines. MDA-MB-231 human breast carcinoma cells were the most
sensitive to the Ag(I)-NHC complex displaying IC50 lower than 1 μM all time points. Further,
the IC50s for Ag(I)-NHC were higher in non-cancer cells, suggesting that complexes
possessed noteworthy selectivity for human cancer cells.
Keywords: N-Heterocyclic carbene; silver complex; benzimidazole-2-ylidene; anticancer
activity.
1. INTRODUCTION
Cancer is a major public health problem worldwide and is the second leading cause of death.
According to GLOBOCAN 2012, an estimated 14.1 million new cancer cases and 8.2 million
cancer-related deaths occurred in 2012, compared with 12.7 million and 7.6 million,
respectively, in 2008 [1]. One of the primary approaches to cancer treatment is
chemotherapy. Unfortunately, chemotherapy is not always effective and is associated with

severe side effects [2]. Cisplatin was on of the first metal-based anticancer drugs, also called
metallotherapeutic drugs. Cisplatin spearheaded thousands of studies seeking to identify
novel metal-based anticancer therapeutics. A primary goal of these studies is the
identification of compounds with increased efficacy, but reduced side effect.
Heterocyclic azoles, such as imidazole, triazole, and especially benzimidazole have
significant biological activities [3-7]. Especially, N-heterocyclic carbenes (NHC) derived
from the imidazole or benzimidazole core which have considerable antibacterial or anticancer
activities [8-13]. NHCs are also a well-known class of organometallic ligands which are a
strong σ-donating and weak π-accepting when connected with metal ions. Metal-NHC
complexes have exhibited an interesting propensity to bind DNA by means of non‐ covalent
interactions, which is believed to partially mediate their anti‐ proliferative activity, and to
cleave DNA strands [14-17]. In addition to their antimicrobial and anticancer activity, metal-
NHC complexes have also been intensely investigated due to their noteworthy applications in
catalysis [18-21].
Silver has lower toxicity as compared to other transition metals. One reason for this could be
the slower the rate of release of silver ions, from the metallodrug complexes, which also
enhanced their biological activity. Among the many developed anticancer agents consisting
of organometallic compounds, those based on silver–NHC complexes have attracted much
attention because of their biological properties such as antimicrobial, anticancer, and
antibacterial activities [13, 22-27]. Ag-NHC complexes have been suggested that as
antimicrobial agents to prevent infections, in wound care antiseptics and in water purification
[28,29]. Metallic silver was used as a treatment for gonorrhea before the development of
antibiotics, while silver nitrate was commonly used as an antimicrobial compound before the
discovery of penicillin in ancient times. [30]. Silver nitrate is still being used as a preventive
drug against the development of neonatal conjunctivitis in infants.
With the above in mind, we report the synthesis and characterization of four new
benzimidazolium salts and their Ag(I) complexes. All the new synthesized compounds (1a-d,
2a-d) were tested as potential anticancer agents against three human cancer cell lines (DU-
145, MCF-7, MDA-MB-231) and non-cancer cells adipose from mouse (L-929) for 24 h, 48
h and 72 h by MTT assay [31].
2. EXPERIMENTAL
2.1. Materials and Methods

All manipulations were carried out under argon in flame-dried glassware using
standard Schlenk line techniques. The silver (I) oxide Ag2O, benzimidazole, and allyl
bromide were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Steinheim,
Germany). The solvents were dried by conventional methods and were distilled immediately
prior to use and were transferred under Argon [32]. Elemental analyses were performed by
Inönü University Scientific and Technology Center (Malatya/Turkey). Microlab. Melting
points determined using Electrothermal 9100 melting point detection apparatus. Fourier
transform infrared (FTIR) spectra were obtained in the range 400–4000 cm−1 on Perkin
1
13
Elmer Spectrum 100 FT-IR. H NMR and C NMR spectra were recorded using a Varian
As 400 Mercury spectrometer operating at 400 MHz (1H), 100 MHz (13C) in CDCl3. with
1
tetramethylsilane as the internal reference. H peaks are labeled as singlet (s), doublet (d),
triplet (t), quartet (q), and multiplet (m). Chemical shifts and coupling constants are reported
in ppm and in Hz, respectively. The intensity data were collected using a Bruker Kappa
ApexII CCD diffractometer using Mo-Kα radiation (λ= 0.71073 Å) [33]. The structure was
solved by direct methods using the solution program SHELXS97, and then refined with full-
matrix least-square methods based on F2 SHELXL2014/6 [34, 35]. All the measurements
were taken at room temperature for freshly prepared solutions
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), F-12K
Medium (30-2004), 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 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 (non-cancer cells adipose from mouse) were purchased
from ECACC (European Collection of Animal Cell Culture, Salisbury, U.K.). All absorbance
values were measured with a microplate reader (Epoch, USA) at 570 nm.
2.2 General procedure for the benzimidazolium salts, 1a-d
Benzimidazole (10 mmol) was added to a solution of NaH (10 mmol) in dry THF (30 mL),
the mixture was stirred for 1 h at room temperature. Allyl bromide (10.1 mmol) was added
0
slowly to the solution and heated for 24 h at 60 C. All solvent was evaporated under the
vacuum. After that, dichloromethane (50 mL) was added to solid and the solution was

filtered. The obtained clear solution was distilled. 1-Allyl benzimidazole was obtained. The
1-allyl benzimidazole (1 mmol) and alkyl halide (1 mmol) were stirred in DMF (5 mL) for 24
0
h at 80 C. White product was precipitated. The solution was filtered, solid was rinsed out
with diethylether and dried under vacuum. The crude product was recrystallized from
dichloromethane/diethylether [36].
1-(Allyl)-3-(2-methylbenzyl)benzimidazolium chloride, 1a
o
1
Yield: 92%; mp 176-177 C; H NMR (400 MHz, CDCl3) δ (ppm): 2.35 (s, 3H,
CH2C6H4(CH3)-2), 5.30-5.31 (m, 2H, NCH2CHCH2), 5.39-5.43 (m, 2H, NCH2CHCH2) (5.84
(s, 2H, CH2C6H4(CH3)-2), 6.02-6.12 (m, 1H, NCH2CHCH2), 7.05-7.20 (m, 4H,
CH2C6H4(CH3)-2), 7.36 (d, 1H, NC6H4N, J = 8 Hz), 7.44 (t, 1H, NC6H4N, J = 6 Hz), 7.52 (t,
1H, NC6H4N, J = 6 Hz), 7.66 (d, 1H, NC6H4N, J = 8 Hz), 11.80 (s, 1H, NCHN). 13C NMR
(100 MHz, CDCl3) δ (ppm): 19.6 (CH2C6H4(CH3)-2), 50.0 (CH2C6H4(CH3)-2), 50.2
(NCH2CHCH2), 113.7 (NCH2CHCH2), 121.7 (NCH2CHCH2), 113.9, 126.8, 127.1, 127.2,
128.2, 129.3, 129.7, 131.3 and 136.5 (CH2C6H4(CH3)-2 and NC6H4N), 144.3 (NCHN); ν(CN)
:
1553 cm-1; % Anal. Calcd for C18H19N2Cl: C, 72.35; H, 6.41; N: 9.38; Found: C, 72.33; H,
6.39; N, 9.37.
1-(Allyl)-3-(3-methylbenzyl)benzimidazolium chloride, 1b
o
1
Yield: 91%; mp 133-134 C; H NMR (400 MHz, CDCl3) δ (ppm): 2.32 (s, 3H,
CH2C6H4(CH3)-3), 5.36 (d, 2H, NCH2CHCH2, J = 8 Hz), 5.51-5.54 (m, 2H, NCH2CHCH2),
5.96 (s, 2H, CH2C6H4(CH3)-3), 6.09-6.19 (m, 1H, NCH2CHCH2),
7.14 (d, 1H,
CH2C6H4(CH3)-3, J = 8 Hz), 7.23-7.30 (m, 3H, CH2C6H4(CH3)-3), 7.52-7.62 (m, 3H,
NC6H4N), 7.72 (d, 1H, NC6H4N, J = 8 Hz), 11.68 (s, 1H, NCHN). 13C NMR (100 MHz,
CDCl3) δ (ppm): 21.3 (CH2C6H4(CH3)-3), 50.2 (CH2C6H4(CH3)-3), 51.6 (NCH2CHCH2),
113.7 (NCH2CHCH2), 121.7 (NCH2CHCH2), 113.9, 125.3, 127.0, 127.1, 128.9, 129.2,
129.8, 130.0, 131.4, 132.7 and 139.3 (CH2C6H4(CH3)-3 and NC6H4N), 143.6 (NCHN); ν(CN)
:
1561 cm-1; % Anal. Calcd for C18H19N2Cl: C, 72.35; H, 6.41; N: 9.38; Found: C, 72.32; H,
6.39; N, 9.36.
1-(Allyl)-3-(4-methylbenzyl)benzimidazolium chloride, 1c
o
1
Yield: 89%; mp 119-120 C; H NMR (400 MHz, CDCl3) δ (ppm): 2.30 (s, 3H,
CH2C6H4(CH3)-4), 5.34 (d, 2H, NCH2CHCH2, J = 4 Hz), 5.44-5.50 (m, 2H, NCH2CHCH2),
5.86 (s, 2H, CH2C6H4(CH3)-4), 6.07-6.17 (m, 1H, NCH2CHCH2), 7.15 (d, 2H,
CH2C6H4(CH3)-4, J = 8 Hz), 7.42 (d, 2H, CH2C6H4(CH3)-4, J = 8 Hz), 7.52-7.60 (m, 2H,

NC6H4N), 7.66 (d, 1H, NC6H4N, J = 8 Hz), 7.73 (d, 1H, NC6H4N, J = 8 Hz), 11.79 (s, 1H,
NCHN). 13C NMR (100 MHz, CDCl3) δ (ppm): 21.2 (CH2C6H4(CH3)-4), 50.1
(CH2C6H4(CH3)-4), 51.3 (NCH2CHCH2), 113.7 (NCH2CHCH2), 121.7 (NCH2CHCH2),
113.9, 127.1, 128.4, 129.7, 129.8, 130.0, 131.5 and 139.1 (CH2C6H4(CH3)-4 and NC6H4N),
143.5 (NCHN); ν(CN): 1558 cm-1; % Anal. Calcd for C18H19N2Cl: C, 72.35; H, 6.41; N: 9.38;
Found: C, 72.33; H, 6.39; N, 9.36.
1-(Allyl)-3-(2-chlorobenzyl)benzimidazolium
chloride,
1d
o
1
Yield: 89%; mp 122-123 C; H NMR (400 MHz, CDCl3) δ (ppm): 5.38 (d, 2H,
NCH2CHCH2, J = 4 Hz), 5.45-5.50 (m, 2H, NCH2CHCH2), 6.04 (s, 2H, CH2C6H4Cl-2), 6.11-
6.18 (m, 1H, NCH2CHCH2), 7.31-7.35, 7.41-7.44, 7.54-7.62 and 7.66-7.74 (m, 8H,
CH2C6H4Cl-2), 11.79 (s, 1H, NCHN). 13C NMR (100 MHz, CDCl3) δ (ppm): 48.6
(CH2C6H4Cl-2), 50.3 (NCH2CHCH2), 113.7 (NCH2CHCH2), 121.7 (NCH2CHCH2), 113.8,
127.2, 127.3, 128.1, 129.7, 130.1, 130.3, 130.9, 131.2, 131.3, 131.4 and 133.6 (CH2C6H4Cl-2
and NC6H4N), 144.3 (NCHN); ν(CN): 1558 cm-1; % Anal. Calcd for C17H16N2Cl2: C, 63.96; H,
5.05; N: 8.78; Found: C, 63.98; H, 5.06; N, 8.80.
2.3 General procedure for preparation of Ag(I)-NHC complexes, 2a-d
A solution of benzimidazolium salt (0.5 mmol) (1a–d) and Ag2O (1.0 mmol) in
dichloromethane (15 mL) was stirred at room temperature for 24 h in the dark condition and
the suspension was passed filtered through celite. The solvent removed under vacuum. The
crude product was recrystallized from dichloromethane/diethyl ether.
Chloro[1-allyl-3-(2-methylbenzyl)benzimidazole-2-ylidene]silver(I), 2a
Yield: 86%; mp: 176-178 °C; 1H NMR (400 MHz, CDCl3) δ: 2.39 (s, 3H, CH2C6H4(CH3)-2),
5.10 (d, 2H, J = 4 Hz, NCH2CHCH2), 5.24 (d, 1H, J = 16 Hz, NCH2CHCH2), 5.34 (d, 1H, J =
8 Hz, NCH2CHCH2), 5.62 (s, 2H, CH2C6H4(CH3)-2), 5.98-6.10 (m, 1H, NCH2CHCH2), 6.74
(d, 1H, J = 8 Hz, NC6H4N), 7.06-7.13 (m, 1H, CH2C6H4(CH3)-2), 7.22-7.24 (m, 3H,
CH2C6H4(CH3)-2, 7.36-7.40 and 7.28-7.32 (m, 2H, NC6H4N), 7.50 (d, 1H, J = 8 Hz,
NC6H4N). 13C{H}-NMR (100 MHz,CDCl3) δ : 19.6 (CH2C6H4(CH3)-2),
51.4
(NCH2CHCH2), 52.1 (CH2C6H4(CH3)-2), 111.9 (NCH2CHCH2), 119.2 (NCH2CHCH2),
112.1, 124.3, 126.4, 126.6, 126.8, 128.4, 131.0, 131.8, 132.7, 133.8, 134.0 and 135.4
-1
(NC6H4N and (CH2C6H4(CH3)-2), 189.9 (Ccarbene-Ag); υ(CN): 1387 cm ; Anal. Calcd for
C18H18AgClN2: C, 53.29; H, 4.47; N: 6.91. Found: C, 53.22; H, 4.78; N, 6.95; Found: C,
53.26; H, 4.44; N: 6.89; LC–MS: 631.1 [AgL2]+.

Chloro[1-allyl-3-(3-methylbenzyl)benzimidazole-2-ylidene]silver(I), 2b
o
1
Yield: 78%; mp 157-158 C; H NMR (400 MHz, CDCl3) δ (ppm): 2.31 (s, 3H,
CH2C6H4(CH3)-3), 5.07-5.10 (m, 2H, NCH2CHCH2), 5.24 (d, 1H, NCH2CHCH2, J = 16 Hz),
5.34 (d, 1H, NCH2CHCH2, J = 8 Hz), 5.58 (s, 2H, CH2C6H4(CH3)-3), 5.98-6.08 (m, 1H,
NCH2CHCH2), 7.04-7.12 and 7.19-7.23 (m, 4H, CH2C6H4(CH3)-3),7.30-7.39 and 7.46-7.48
13
(m, 4H, NC6H4N). C NMR (100 MHz, CDCl3) δ (ppm): 21.4 (CH2C6H4(CH3)-3), 52.1
(CH2C6H4(CH3)-3), 53.5 (NCH2CHCH2), 112.0 (NCH2CHCH2), 119.3 (NCH2CHCH2),
112.2, 124.3, 124.4, 127.8, 129.0, 131.8, 133.8, 134.0 and 139.0 (CH2C6H4(CH3)-3 and
NC6H4N), Ccarbene-Ag: not observed; ν(CN): 1391 cm-1; % Anal. Calcd for C18H18N2AgCl: C,
53.29; H, 4.47; N: 6.91; Found: C, 53.27; H, 4.43; N: 6.89; LC–MS: 631.1 [AgL2]+.
Chloro[1-allyl-3-(4-methylbenzyl)benzimidazole-2-ylidene]silver(I), 2c
o
Yield: 79; mp 153-154 C; 1H NMR (400 MHz, CDCl3) δ (ppm): 2.31 (s, 3H,
CH2C6H4(CH3)-4), 5.06-5.09 (m, 2H, NCH2CHCH2), 5.21-5.34 (m, 2H, NCH2CHCH2), 5.58
(s, 2H, CH2C6H4(CH3)-4), 5.98--6.05 (m, 1H, NCH2CHCH2), 7.12-7.24, 7.27-7.37 and 7.45-
7.47 7.15 (m, 8H, CH2C6H4(CH3)-4 and NC6H4N). 13C NMR (100 MHz, CDCl3) δ (ppm):
21.1 (CH2C6H4(CH3)-4), 52.1
(CH2C6H4(CH3)-4), 53.4 (NCH2CHCH2), 111.9
(NCH2CHCH2), 119.3 (NCH2CHCH2), 112.2, 119.3, 124.3, 127.2, 129.8, 131.8, 133.8 and
138.4 (CH2C6H4(CH3)-4 and NC6H4N), 188.8 (Ccarbene-Ag); ν(CN): 1395 cm-1; % Anal. Calcd
for C18H18N2AgCl: C, 53.29; H, 4.47; N: 6.91; Found: C, 53.26; H, 4.42; N: 6.88; LC–MS:
631.1 [AgL2]+.
Chloro[1-allyl-3-(2-chlorobenzyl)benzimidazole-2-ylidene]silver(I), 2d
o
1
Yield: 83%; mp 143-144 C; H NMR (400 MHz, CDCl3) δ (ppm): 5.11-5.13 (m, 2H,
NCH2CHCH2), 5.23-5.36 (m, 2H, NCH2CHCH2), 5.73 (s, 2H, CH2C6H4Cl-2), 5.99-6.07 (m,
1H, NCH2CHCH2), 6.92-6.94, 7.15-71.9, 7.26-7.28, 7.34-7.42 and 7.46-7.51 (m, 8H,
CH2C6H4Cl-2). 13C NMR (100 MHz, CDCl3) δ (ppm): 50.5 (CH2C6H4Cl-2), 52.2
(NCH2CHCH2), 112.0 (NCH2CHCH2), 119.4 (NCH2CHCH2), 112.1, 124.6, 127.5, 128.3,
129.9, 130.1, 131.7, 132.3, 132.9 and 133.8 (CH2C6H4Cl-2 and NC6H4N), 189.7 (Ccarbene-Ag);
ν(CN): 1387 cm-1; % Anal. Calcd for C17H15N2 AgCl2: C, 47.92; H, 3.55; N: 6.57; Found: C,
47.90; H, 3.57; N, 6.59; LC–MS: 671.1 [AgL2]+.
2.4. Cell viability (MTT) assay

DU-145 cells were grown in EMEM media containing 10% (v/v) FBS, and 100 Units/ml
penicillin and 100 μg/ml streptomycin in a 37°C humidified incubator with 5% CO2. MCF-7
cells, MDA-MB-231, and L-929 cells were grown under the same conditions except that
DMEM and RPMI-1640 media were used, respectively. Cells were passaged at 70–80%
confluence, about twice a week by trypsinization. The passages of all cells lines were no
more than 30 during all experiments. The cytotoxic effect of newly synthesized ligands and
complexes were determined using MTT [31]. A stock solution of the tested compounds was
prepared in DMSO and diluted in complete culture medium such that the maximum DMSO
content did not exceed 0.5% (v/v). Exponentially growing DU-145, MCF-7, MDA-MB-231,
and L-929 cells were seeded into 96-well plates at a density of 1 x 105 cells/well and allowed
to attach for 24h before treatment. Then the cells were treated with various concentration (1–
20 μM) of compounds in 5% CO2, at 37°C for 24h, 48 h, and 72 h. The control wells
contained cells with media and 0.5% DMSO (final concentration of DMSO) and were kept
the same in this study. At the end of the exposure period, the cells were subjected to
assessment of viability by adopting the MTT assay. 10 μL/well MTT (5 mg/mL, dissolved in
phosphate-buffered saline) was added and the cells were incubated for 2 h at 37°C in 5%
CO2. After removal of the medium and MTT, the purple-blue precipitated crystals were
dissolved in 100 μL of DMSO (Sigma, St. Louis, MO). The absorbance was read at 570 nm
with Biotek plate reader (Biotek. GA). Data were is based on the means from at least three
independent experiments, each comprising three replicates per concentration. The anticancer
effects of all compounds were determined in the concentration range of 1-20 μM, the dose-
response curves were fitted by means of GraphPad Prism 7 (GraphPad Software, San
Diego,CA, USA) and the IC50 values were calculated. Graphs indicating the cytotoxic effects
of 1a-d, and 2a-d on DU-145, MCF-7, MDA-MB-231, and L-929 cells.
2.5. Statistical analysis
All experiments were carried out in triplicates and results are expressed as means ± 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. RESULTS AND DISCUSSIONS
3.1. Chemistry
Benzimidazolium salts were synthesized as shown in Scheme 1. Benzimidazolium salts were
obtained from the reaction of allyl substituted benzimidazolium precursors with alkyl halides

0
in THF at 80 C. Collapsed products were crystallized in dichloromethane/diethylether for
purification.
<<Scheme 1 is here>>
Ag(I)-NHC complexes were synthesized from reaction benzimidazolium salts with Ag2O in
dichloromethane at room temperature under dark condition as shown in Scheme 2. Crude
products were purified by crystallization in dichloromethane/diethylether [36]. The structures
1
of all new compounds were characterized by elemental analysis using FT-IR, H NMR, and
13C NMR spectroscopy. The structure for compound 2d was assessed using X-ray
crystallography.
<<Scheme 2 is here>>
FT-IR spectra demonstrated, values in the range at 2872-2985 and 3011-3149 cm-1 belonging
to the aliphatic and aromatic C-H stretching vibrational bands, respectively, for all
compounds. C=C stretching vibration modes of all compounds were observed at around
1591-1667 cm-1. Benzimidazole ring C=N vibrations of benzimidazolium salts (1a-d) showed
peaks at around 1547-1559 cm- 1. Benzimidazole ring C=N vibrations of Ag(I)-NHC
complexes (2a-d) were seen at around 1385-1396 cm-1. After complexation, C=N vibrations
of Ag(I)-NHC complexes shifted to the lesser energy region. This negative shift is because of
the electropositive metal center which pulls electron density towards itself.
NMR spectra of all the compounds were analyzed in d-CDCl3. In the 1H NMR spectra, acidic
proton (NCHN) for benzimidazolium salts (1a-d) were seen at 11.80, 11.68, 11.79 and 11.79
ppm, respectively, as a characteristic sharp singlet. Benzimidazolium salts lost their acidic
proton when they formed silver complexes. This was shown by the disappearance of the
1
acidic proton during H NMR. Aromatic protons of benzimidazolium salts (1a-b) were
detected in the range of 7.05-7.74 ppm. -CH- protons of allyl group on all benzimidazolium
salts where in the range of 6.02-6.19 ppm and were observed as multiplets. -NCH2 protons
allyl group on the benzimidazolium salts (1a-b) appeared at 5.39-5.43 as mutiplet, 5.36, 5.34
and 5.38 as a singlet, respectively. Terminal -CH2 protons of allyl group gave peaks in the
range at 5.30-5.54 ppm as multiplets. Benzylic -CH2 protons at benzimidazolium salts (1a-d)
were observed at 5.84, 5.96, 5.86 and 6.04 ppm as a sharp singlet. Methyl protons of
13
benzimidazolium salts (1a-c) signaled at 2.35, 2.32 and 2.30 ppm as singlets. The C NMR
spectra showed aromatic carbons of benzimidazolium salts (1a-d) in the range of 113.8-139.3
ppm. NCHN carbons on salts (1a-d) were observed at 144.3, 143.6, 143.5 and 144.3 ppm,

respectively. Terminal -CH2 and –CH- carbons of the allyl group of all benzimidazolium
salts gave peaks at 113. 7 and 121.7 ppm, respectively. For 1a-d salts, -NCH2 carbons on
allyl groups appeared at 50.2, 51.6, 51.3 and 50.3 ppm, respectively. Observed peaks at 50.0,
50.2, 50.1 and 48.6 ppm were attributed to benzylic carbons of benzyl groups (1a-b),
respectively. Methyl carbons on the benzyl group of salts (1a-c) were observed at 19.6, 21.3
1
and 21.2 ppm, respectively. H NMR spectra and 13C NMR spectra of benzimidazolium salts
(1a-d) were compatible with each other.
1
13
It was observed that the H NMR and C NMR spectra of Ag(I)-NHC complexes detected
peaks in the same regions of spectra corresponding to benzimidazolium salts, except for two
characteristic peaks. The first of these corresponded to the disappearance of acidic proton
peaks in the spectra of Ag(I)-NHC complexes, which confirmed that Ag(I)-NHC complexes
(2a-d) were synthesized. The second of these, NCN carben resonance of the Ag(I)-NHC
complexes (2a-d), which shifted to a downfield region as compared to the corresponding
benzimidazolium salts (1a-d). Carben peaks of Ag(I)-NHC complexes 2a, 2c and 2d were
observed at 189.9, 188.8 and 189.7 ppm but were somewhat small, as these peak was not
observed for 2b. The lower values, or the absence of these peaks, are in agreement with
reported data for similar Ag(I)-NHC complexes [37].
3.2. X-ray crystallography for 2d
The details of crystalization of complex 2d are given below (Figure 1). All non-hydrogen
atoms were refined isotropically first and then with anisotropic atomic displacement
parameters by full matrix least square methods. All the hydrogen atoms bonded to carbon
atoms were placed geometrically. All hydrogen atoms were refined as riding with Ueq(H)=1.2
Uiso(C). The Empirical Formula = C17H15AgCl2N2; Molecular Weight= 426.08 g.mol-1;
Crystal System = Triclinic; Space Group= P-1 system; UnitCell Parameters, a= 9.0290(7), b=
9.9785(9), c= 10.3038(9) Å; α = 94.964(3)o, β = 100.682(3)°,  = 114.349(3)o; Cell Volume=
817.22(12) Å3, Z= 2, d = 1.732 g.cm-3, μ= 1.557 mm-1. F(000)= 424; T= 296(2) K. The
Crystal Size (0.43 × 0.38 × 0.36 mm). The number of measured reflections are 20104 with
3503 independent reflections. The observed [I > 2σ(I)] reflections are 3325. A final
refinement on F2, with 3503 unique intensities and 199 parameters. The value of weight
parameter is w=0.063 and goodness of fit S = 1.09. Some selected parameters are given in
Table 1. The revelant crystallographic for 2d has been deposited at Cambridge
Crystallographic Data Center (CCDC) with No. 1821504. The See the Supplementary

Materials for more details on the experimental methods, as well as complete NMR, FT-IR,
LC-MS data and X-ray crystallography studies
<<Table 1. is here>>
<<Figure 1 is here>>
3.3. MTT Assays results
The anticancer potential of four newly synthesized benzimidazolium salts (1a-d) and their
Ag(I)-NHC complexes (2a-d) were evaluated using the MTT assay on DU-145, MCF-7,
and MDA-MB-231 cancer cells, as well as L-929 non-cancer cells. MTT staining assesses
the ability of cells to convert a soluble yellow tetrazolium salt (MTT: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into insoluble purple formazan
crystals, which is facilitated by mitochondrial dehydrogenase enzymes [31]. The effects of
these benzimidazolium compounds on MTT were evaluated after 24, 48, and 72 h of
exposure. Table 2 list the IC50 values of compounds for 48 h. (See the Supplementary Data,
Table S1, for 24 h and 72 h IC50 values).
<<Table 2 is here>>
The IC50 values of all salts were > 20 µM at 24 h but decreased to 3.85 for some cells after to
48 h and to 1.86 after 72 h. MDA-MB-231 cells were the most susceptible to 1b and 1c salts
which displayed IC50 values of 5.17 and 3.85 µM after 48 h respectively. Notably, the IC50
values of 1c for 48 h and 72 h were lower than those of 1a, 1b, and 1d, suggesting higher
antiproliferative activity than 1a, 1b and 1d. The silver complexes (2a-d) decreased MTT
staining in all cells, as compared to controls. Similar to the salts, a decrease in MTT staining
were concentration and time-dependent. The silver complexes (2a-d) showed better
antiproliferation activity with IC50 (concentration of the test compound to achieve 50%
inhibition) ranging from <1 to 18.22 µM, <1 to 12.61 µM and <1 to 9.25 µM for 24, 48 and
72 h respectively. The MDA-MB-231 cells were again the most susceptible to 2a-d
complexes with lower IC50 values, lower than1 µM.
<<Figure 2, 3, 4 and 5 are here>>
Figure 2, 3, 4 and 5 shows the dose-dependent antiproliferative effect of synthetic (1a-d)
salts having chloride counter anions and synthetic complexes (2a-d) having silver

counteranions on DU-145, MCF-7, MDA-MB-231, and L-929 cells, respectively. A
comparison of cytotoxicity between the cell lines identifies compounds as selective
anticancer agents. 1a salt did not display any activity toward any cell line tested even at a
concentration of 20 µM. Whereas 1d salts displayed similar activity in all the cell lines
tested. This suggests that 1d salts would display potentially poor selectivity in vivo. In
contrast, 1b salts displayed activity only against MDA-MB-231 cells. 1c salts exert
differences in cytotoxicity among all cell lines. Complexes 2c and 2d showed the
greatest differences in activity between all cell line tested, 2a and 2b only displayed
activity against DU-145 cells.
M. Napoli et. al. were synthesized, characterized and evaluated the antibacterial activity of
five new silver complexes having bidentate N-heterocyclic carbene ligands [13]. They were
used four ligands neutral, having an alcohol group on alkyl substituent of one of the two
nitrogen atoms of the heterocycle [NHCeOH]. The last ligand having a alkoxide, is mono-
anionic [NHCeO]. They were evaluated antibacterial activity for all the reported compounds,
using a standard assay against a Gram negative (Escherichia coli) and a Gram positive
(Bacillus subtilis) strain. The results of the study showed that all samples were not cytotoxic,
whereas antibacterial activity, tested on E. coli and B. subtilis, complexes 1 and 10 have a
MIC of 5 mg/ml. Thus, the complexes 1 and 10 have suggests that they can be very
promising candidates for use as antibacterial compounds. The complexes 2 and 3 kill E. coli
cells at a concentration of 5 mg/ml, but a higher concentration was necessary to inhibit the B.
subtilis growth (50 mg/ml). M. Napoli et. al. reported that the synthesis and characterization
of titanocenes compounds containing dialkyl ether groups appended to the Cp ligands, having
different leaving groups on the metal [24]. All the compounds have been fully characterized
by NMR, FT-IR and elemental analysis. Their cytotoxic activities have been evaluated on
human breast cancer (MCF-7), human embryonic kidney (HEK-293) and murine macrophage
(J774.A1) cells. IC50 value of the complex 3, which is the most-active complex, determined
that 57 µM for MCF-7 cells. They were also reported that complex 2 more cytotoxic than
complex 7 on MCF-7 cells. Complexes 2 and 7 have the same basic structure, differing only
in the type of metal cation: in complex 2 it is titanium and in the complex 7 it is zirconium.
BK. Rana et. al have synthesized and characterized novel Au(I)− and Au(III)−NHC
complexes [25]. In addition to the characterization of the complexes 2 and 3, their
cytotoxicities were tested against human cancer cell lines, including HCT 116, HepG2, A549,
and MCF7, and compared to that displayed by cisplatin. They were determined that Gold(I),
complex 2 was more potent as an anticancer agent than 3 or cisplatin in four cell lines. IC50

values of complex 2, 3 and cisplatin found to be 4.7 ± 0.8 µm, 6.2 ± 0 1.4 µm and 9.4 ±
1.0µm for MCF-7 cells, respectively. J. Dinda et. al. reported that synthesized and
characterized using various spectroscopic techniques; starting ligand,1-methyl-2-pyridin-2-
yl-2H-imidazo[1,5-a]pyridin-4-ylium chloride (1 HCl), three novel complexes [Ag(1)Cl] (2),
[Au(1)Cl] (3) and [Au(1)Cl3] (4) [26]. They have been studies cytotoxic activities of
complexes 2, 3, and 4 against HepG2 (human hepatocellular carcinoma), HCT 116 (human
colorectal carcinoma), A549 (human lung adenocarcinoma), and MCF-7 (human breast
adenocarcinoma) cells. They were reported that complex 3 exhibited a cytotoxicity that was
similar to that of cisplatin towards all the four cancer cell lines tested. D. Lacopetta et. al.
prepared a new four carbene complexes of silver and gold derived from 4,5- dichloro-1H-
imidazole and 4,5-diphenyl-1H-imidazole [22]. They were stable to light and water, which
makes them viable candidates for use as chemotherapeutic. They were reported that the
synthesized complexes possess good antitumor activity MDAMB-231, HeLa and
ISHIKAWA. Also, complexes showed better cytotoxic profile than cisplatin. The IC50 values
of the most-active compound (AuL4)were found to be (3.98±0.4) µm for MCF-7 cells and
(6.02±0.7) µm for MDA-MB-231 cells. Wang et. el. reported that amino–NHC metal
complex analogs inhibited the growth of MCF-7 and MDA-MB-231 cancer-cell lines. IC50
values of the most-active complexes reported that 6.47±0.07 µM and 4.50± 0.07 µm for
MCF-7 cells and MDA-MB-23, respectively [38]. Liu et al. prepared a new series of NHC–
gold halide complexes derived from 4,5-diarylimidazoles. Bromo[1,3-diethyl-4,5-bis(2-
fluorophenyl)- 1,3-dihydro-2H-imidazol-2-ylidene]gold(I), showed most antiproliferative
activity on breast cancer cell lines with IC50 values of (0.87±0.07) µm on MCF- 7 and
(3.1±0.5) µm on MDA-MB-231 [39].
All complexes (2a-d) are all more cytotoxic in the cancer cell line (except DU-145 cell
line for 2a and 2b) than the healthy cells (L-929; normal cells adipose from mouse).
Complexes showing the greatest difference in cytotoxicity between cancer cells and
normal cells. Complex 2a; more than 5-fold more cytotoxic toward the MCF-7 and
MDA-MB-231 cancer cells, 2b; more than 4.68-fold more cytotoxic toward the MCF-7
and MDA-MB-231 cancer cells, 2c; 2.54-fold, 3.46-fold and more than 12 fold more
cytotoxic toward the DU-145, MCF-7 and MDA-MB-231 cancer cells, respectively and
2d; 1.83-fold, 7.48-fold and more than 11 fold more cytotoxic toward the DU-145,
MCF-7 and MDA-MB-231 cancer cells, respectively.
Some studies have indicated that the ancillary ligands play an important role in biological

properties of compounds, thus differences of ancillary ligands in complexes may create
some difference in the biological activities [40,41]. Although pure metals are biologically
inactive, metal cations have biological activity. It was observed that the activity of the silver
cation was increased by binding the silver cation to different ligands which have
biologically compatible properties [42-45].
It has been previously reported that imidazole-based ortho-, meta-, and para-xylyl linked
salts and their respective Ag(I)-NHC complexes display in vitro anticancer activity
against the HCT 116 cell line and that the allyl-substituted para-xylyl linked dinuclear
Ag(I)-NHC complex had higher cytotoxicity [44]. Another study also showed that the
benzimidazole-based para-xylyl linked iso-propyl-substituted complex demonstrated
higher cytotoxicity whereas the ortho- as well as the meta-xylyl linked complexes
demonstrated comparatively moderate activities [37]. Similarly, our results showed that
para-methylbenzyl substituted complexes more cytotoxic than, -ortho and –meta linked
complexes. This activity could be attributed to the cavity and size of the molecule. The
molecules with para substitution were assumed to have a bigger cavity and larger size,
as compared to the ortho and meta units which increases the exposure of N-
heterocyclic carbene (NHC) rings.
4. CONCLUSIONS
In conclusion, we synthesized four novel unsymmetrically substituted NHC ligands (1a-d)
and their Ag(I) complexes (2a-d). Ag(I)-NHC complexes were found to display
relatively high activity against cancer cell growth, as compared their respective
ligands. These results supports the conclusion that silver cations are critical for the anti-
cancer activity of these novel compounds. The higher cytotoxicity of Ag(I)-NHC
complexes might be due to the deposition of silver cations in the membrane of cells
and the organelles and thus may cause toxicity by interfering with cellular respiration
and metabolism of biomolecules. The findings of the present work support the
conclusion that the para-methyl linked compounds are more active compared to the
complexes with ortho- and meta- compartments. In addition, the ortho-methyl
substituted compound 2a have more cytotoxic activity than the ortho-chloro substituted
compound 2d.
Acknowledgments

The authors thank the Scientific and Technological Research of Turkey (TÜBITAK-BIDEB),
the National Research Fellow-ship Programme for grants to N. Ş. This project was supported
in part with funds from the National Institutes of Health (NIH); National Institute of
Biomedical Imaging and Bioengineering NIBIB (EB0160100 to B.S.C.) and a Department of
Defense Prostate Cancer Research Program Idea Development Award (PC150431
GRANT11996600) to B.S.C.
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Figure 1: PLATON drawing of the 2d compound with the atomic numbering scheme.
Displacement ellipsoids are drawn at the 50 % probability level. The H-atoms are shown as
small circles of arbitrary radii.

Figure 2. Effect of salts and complexes on MTT staining in DU-145 cells. DU-145 cells were treated with 1, 5, 10 and 20 µM of salts (1a-d) and
Ag-complexes (2a-d) for 24, 48 and 72 h. Controls were cells 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).

Figure 3. Cytotoxicity as determined by the MTT assay. MCF-7 cells treated with 1, 5, 10 and 20 µM of salts (1a-d) and Ag-complexes (2a-d)
for 24, 48 and 72 h. DMSO treated cells were used as vehicle control. Data are representative of the mean of three separate experiments done in
triplicate (± SEM). (* p < 0.0001 vs control).

Figure 4. Cytotoxicity as determined by the MTT assay. MDA-MB-231 cells treated with 1, 5, 10 and 20 µM of salts (1a-d) and Ag-complexes
(2a-d) for 24, 48 and 72 h. DMSO treated cells were used as vehicle control. Data are representative of the mean of three separate experiments
done in triplicate (± SEM). (* p < 0.0001 vs control).

Figure 5. Cytotoxicity aas determined by the MTT assay. L-929 cells treated with 1, 5, 10 and 20 µM of salts (1a-d) and Ag-complexes (2a-d)
for 24, 48 and 72 h. DMSO treated cells were used as vehicle control. Data are representative of the mean of three separate experiments done in
triplicate (± SEM). (* p < 0.0001 vs control)

Scheme 1. Synthesis of benzimidazolium salts (1a-b)
Scheme 2. Synthesis of Ag-NHC complexes (2a-d)
Table 1. Selected geometric parameters (Å, °)
Ag1 C1 2.096(2)
N1 C1 1.356(3)
N2 C1 1.347(3)
Ag1 Cl1 2.3554(7)
N1 C7 1.390(3)
N2 C2 1.394(3)
Cl2 C10 1.742(3)
N1 C8 1.465(3)

C1 Ag1 Cl1 168.28(7)
C1 N1 C8 124.89(19)
C1 N2 C15 124.5(2)
N2 C1 N1 105.97(19)
N1 C1 Ag1 125.40(16)
C1 N1 C7 111.10(18)
C1 N2 C2 111.12(18)
C2 N2 C15 124.26(19)
N2 C1 Ag1 128.60(17)
Table 2. Cytotoxic activities of ligands (1a-d) and complexes (2a-d) in selected human
cancer and normal cellsa in vitro (IC50, µMb) after 48 h of incubation
Compounds
DU-145
MCF-7
MDA-MB-231
48 h
L-929a
>20
>20
>20
>20
13.61 ± 0.91
>20
>20
5.17 ± 0.65
3.85 ± 0.71
>20
>20
>20
1a
1b
1c
1d
2a
2b
2c
2d
10.03 ± 0.40
20.00 ± 0.57
6.02 ± 0.30
5.16 ± 0.33
4.97 ± 0.25
6.52 ± 0.18
>20
<1
<1
<1
<1
>20
<1
<1
5.08 ± 0.20
4.68 ± 0.15
12.61 ± 0.21
11.97 ± 0.24
3.64 ± 0.16
1.60 ± 0.17
Non-cancer cells,.bCell viability after treatment for 48h was determined by MTT staining as described in the
a
Experimental section. Each IC50 value represents the mean ± SEM of three independent experiments.

Highlights


Novel N-Heterocyclic Carbene Silver(I) Complexes
Structural characterization by elemental analysis, FT-IR, 1H NMR and 13C NMR and
X-ray crystallography techniques.


Comparison of anticancer activities of ligands and Carbene Silver(I) complexes on
prostate and breast cancer cells and L-929 non-cancer cells.
Selectivity for human cancer cells and non-cancer cells