Synthesis, characterization, and investigation of antiproliferative activity of novel Ag (I)-N-Heterocyclic Carbene (NHC) compounds

Article abstract of DOI:10.1016/j.molstruc.2019.126987

The aim of this study was to present the synthesis, characterization, and investigation of the antiproliferative activity of new metal N-Heterocyclic Carbene (NHC) salts (1a-c) and their Ag(I) complexes (2a-c). All synthesized compounds were characterized using elemental analysis, LC-MS, FT-IR, ¹H NMR, and ¹³C NMR spectroscopy techniques. Salts and complexes were tested for antiproliferative activities on human breast and prostate cancer cell lines (MCF-7, MDA-MB-23, DU-145) and L-929 normal cells for 24 h, 48 h and 72 h using MTT assays. The Ag(I)-NHC complexes (2a-c) showed dose and time-dependent cytotoxic activity against all cell lines. MDA-MB-231 and MCF-7 human breast carcinoma cells were the most sensitive to displaying IC₅₀ lower than 1 μM at all time points for 2a and 2b complexes respectively. The IC₅₀s for Ag(I)-NHC were higher in normal cells especially compared to the breast cancer cells, suggesting that complexes possessed noteworthy selectivity for human breast cancer cells. Complex 2a showed high selectivity (≥13-fold) for MDA-MB-231 breast cancer cells at all time points. These results also demonstrated that complex 2b has 4-7-fold selectivity against MCF-7 breast cancer cells.

Full text of DOI:10.1016/j.molstruc.2019.126987

Journal of Molecular Structure 1199 (2020) 126987
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, characterization, and investigation of antiproliferative
activity of novel Ag (I)-N-Heterocyclic Carbene (NHC) compounds
,
c
,
d
a, *
Esranur Çevik-Yıldız a, Neslihan S¸ ahin b
, Serap S¸ ahin-Bolükbas¸ ı
€
a Department of Biochemistry, Faculty of Pharmacy, Sivas Cumhuriyet University, 58140, Sivas, Turkey
b Department of Basic Education, Faculty of Education, Sivas Cumhuriyet University, 58140, Sivas, Turkey
c
_
€
Department of Chemistry, Faculty of Science and Art, Inonü University, 44280, Malatya, Turkey
Catalysis Research and Application Center, Inonü University, 44280, Malatya, Turkey
d
_
€
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was to present the synthesis, characterization, and investigation of the anti-
proliferative activity of new metal N-Heterocyclic Carbene (NHC) salts (1a-c) and their Ag(I) complexes
(2a-c). All synthesized compounds were characterized using elemental analysis, LC-MS, FT-IR, 1H NMR,
and 13C NMR spectroscopy techniques. Salts and complexes were tested for antiproliferative activities on
human breast and prostate cancer cell lines (MCF-7, MDA-MB-23, DU-145) and L-929 normal cells for
24 h, 48 h and 72 h using MTT assays. The Ag(I)-NHC complexes (2a-c) showed dose and time-dependent
cytotoxic activity against all cell lines. MDA-MB-231 and MCF-7 human breast carcinoma cells were the
Received 23 April 2019
Received in revised form
7 August 2019
Accepted 25 August 2019
Available online 26 August 2019
Keywords:
N-Heterocyclic carbene
Silver complex
Antiproliferative activity
Breast cancer
Prostate cancer
most sensitive to displaying IC50 lower than 1 mM at all time points for 2a and 2b complexes respectively.
The IC50s for Ag(I)-NHC were higher in normal cells especially compared to the breast cancer cells,
suggesting that complexes possessed noteworthy selectivity for human breast cancer cells. Complex 2a
showed high selectivity (ꢀ13-fold) for MDA-MB-231 breast cancer cells at all time points. These results
also demonstrated that complex 2b has 4-7-fold selectivity against MCF-7 breast cancer cells.
© 2019 Published by Elsevier B.V.
1. Introduction
activities of silver-NHC complexes [30e34]. Since Ghosh et al. first
reported the cytotoxic properties of silver(I)-NHC complex against
N-Heterocyclic Carbenes (NHCs) have become a well-known
class of organometallic ligands [1e5]. In addition to the catalytic
applications of NHCs, numerous studies have been conducted on
the biological activities of NHC based organometallic compounds
[6e15]. The variability of the biological activities of NHCs greatly
depends on the ring size and structure of the substitutions over the
ring [16e18]. In 1944, Wooley reported that benzimidazole can act
in a similar way to purines [19]. Following that study, it was
demonstrated that benzimidazole derivatives have biological ac-
tivities, such as antihypertensive, anti-inflammatory, antimicrobial,
antiviral, antioxidant, antitumor, lipid modulator, and anticoagu-
lant effects [20e27]. Silver salts have historically been used as
antimicrobial agents for the prevention of eye infections in new-
borns and the purification of drinking water as well as in wound
healing [28,29]. The lower toxicity of silver salts has attracted the
attention of researchers for the investigation of the biological
MCF-7 breast cancer cells, HCT 116 colon adenocarcinoma cells and
HeLa cervical cancer cells, a great number of new silver(I)-NHC
complexes synthesized and investigated against different cancer
cells [35e41]. The structural similarity of benzimidazole derivatives
with naturally occurring nucleotides makes them biologically
important structures [42]. Some studies have shown that a number
of organic derivatives of benzimidazole have important therapeutic
activities including anticancer activity [43e47]. Due to both the
lower toxicity of silver and useful biological activity of benzimid-
azole these two structures were used to synthesize salts and
complexes in this study.
Cancer is one of the most formidable diseases and a major cause
of death worldwide, with an estimated 9.6 million deaths due to
cancer in 2018 [48]. Breast cancer is the most frequently diagnosed
form of cancer in women worldwide, with approximately 2.3
million new cases and 627,000 deaths in 2018. For males, prostate
cancer is the second most common cancer, with approximately 1.3
million new cases in 2018 [48]. Different kinds of treatment pro-
tocols have been developed for cancer treatment; including
radiotherapy, chemotherapy and drug therapy. Treatment protocols
* Corresponding author.
€
E-mail address: wserap@yahoo.com (S. S¸ ahin-Bolükbas¸ ı).
https://doi.org/10.1016/j.molstruc.2019.126987
0022-2860/© 2019 Published by Elsevier B.V.

2
E. Çevik-Yıldız et al. / Journal of Molecular Structure 1199 (2020) 126987
vary depending on the cancer types. Cis-platin, discovered by
Rosenberg et al., was the first example of metal-based anticancer
drugs [49]. Although cis-platin has several side effects, such as hair
loss, neurotoxicity, vomiting, nephrotoxicity, diarrhea and the
development of intrinsic and acquired resistance in some cancer
cells, cis-platin and its analogs are mostly used for the medical
treatment of cancer patients [50e52]. Therefore, it is important to
obtain new anti-cancer agents that are more effective and have low
cytotoxicity.
The aim of this study was to present the synthesis, character-
ization, and investigation of antiproliferative activity of new metal
N-Heterocyclic Carbene (NHC) salts (1a-c) (Scheme 1) and their
Ag(I) complexes (2a-c) (Scheme 2. All synthesized compounds
were characterized using elemental analysis, LC-MS, FT-IR, 1H NMR,
and 13C NMR spectroscopy techniques. Salts and complexes were
tested for antiproliferative activities against human breast cancer
cells (MCF-7, MDA-MB-23), human prostate cancer cells (DU-145)
and L-929 normal cells for 24 h, 48 h and 72 h using MTT assays.
24 h at 80 ꢃC and the salts (1a-c) were precipitated. Following the
completion of the process the solution was filtered, the solids were
rinsed with diethylether and dried under vacuum. Crude products
were recrystallized from dichloromethane/diethylether.
2.3.1. 1-Allyl-3-benzylbenzimidazolium chloride, (1a)
Yield: 83%; m.p. 166-168 ꢃC. FT-IR n(CN): 1551 cmꢁ1 1H NMR
;
(400 MHz, CDCl3)
d
(ppm): 5.26 (d, 2H, NCH2CHCH2, J ¼ 8 Hz),
5.38e5.44 (m, 2H, NCH2CHCH2), 5.84 (s, 2H, CH2C6H5), 6.04 (quint,
1H, NCH2CHCH2, J ¼ 4 Hz), 7.25e7.31 (m, 3H, Ar-H), 7.45e7.57 (m,
5H, Ar-H), 7.65 (d, 1H, Ar-H, J ¼ 8 Hz), 11.90 (s, 1H, NCHN).13C NMR
(100 MHz, CDCl3) d (ppm): 50.2 (CH2C6H5), 51.5 (NCH2CHCH2),121.9
(NCH2CHCH2), 131.3 (NCH2CHCH2), 113.7, 113.9, 127.1, 127.2, 128.4,
129.2, 129.3, 129.6, 131.5, and 132.8 (Ar-C), 143.8 (NCHN). % Anal.
Calcd for C17H17N2Cl: C, 71.70; H, 6.02; N: 9.84; Found: C, 71.62; H,
5.93; N, 9.77.
2.3.2. 1-Allyl-3-(naphthylmethyl)benzimidazolium chloride, (1b)
Yield: 88%; m.p. 173-175 ꢃC. FT-IR n(CN): 1555 cmꢁ1 1H NMR
;
2. Experimental
(400 MHz, CDCl3)
d
(ppm): 5.33 (d, 2H, NCH2CHCH2, J ¼ 4 Hz),
5.42e5.47 (m, 2H, NCH2CHCH2), 6.11 (quint, 1H, NCH2CHCH2,
J ¼ 4 Hz), 6.37 (s, 2H, CH2C10H7), 7.40e7.55 (m, 6H, Ar-H), 7.62 (t, 1H,
Ar-H, J ¼ 8 Hz), 7.69 (d,1H, Ar-H, J ¼ 8 Hz), 7.86 (t, 2H, Ar-H, J ¼ 8 Hz),
8.21 (d, 1H, Ar-H, J ¼ 8 Hz), 11.85 (s, 1H, NCHN).13C NMR (100 MHz,
2.1. Materials and methods
All the synthesis processes of benzimidazolium salts and Ag-
NHC complexes were prepared under argon in flame-dried glass-
ware using standard Schlenk line techniques. The 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 [53]. All Ag2O re-
actions were carried out in the absence of light. Elemental analyses
were performed in Inonü University Scientific and Technology
Center. Melting points were determined using the Electrothermal
9100 melting point detection apparatus. Fourier transform infrared
(FT-IR) spectra were obtained in the range of 400e4000 cmꢁ1 on a
PerkinElmer Spectrum 100 FT-IR. 1H NMR and 13C NMR spectra
were taken using a Bruker As 400 Mercury spectrometer operating
at 400 MHz (1H), 100 MHz (13C) in CDCl3 with tetramethylsilane as
the internal reference. 1H peaks were labeled as singlet (s), doublet
(d), triplet (t), quintet (quint.) and multiplet (m). Chemical shifts
and coupling constants were reported in ppm and in Hz, respec-
tively. LC-MS was performed using LC/MSD SL mass spectrometer
CDCl3)
d (ppm): 49.6 (CH2C10H7), 50.2 (NCH2CHCH2), 121.7
(NCH2CHCH2), 131.5 (NCH2CHCH2), 113.7, 114.0, 122.5, 125.3, 126.5,
127.0, 127.2, 127.3, 127.7, 128.0, 129.2, 129.7, 130.1, 130.7, 131.5, 131.6
and 133.8 (Ar-C), 144.2 (NCHN). % Anal. Calcd for C21H19N2Cl: C,
75.33; H, 5.72; N: 8.37; Found: C, 75.21; H, 5.63; N, 8.32.
_
€
2.4. General procedure for preparation of Ag-NHC complexes, (2a-c)
A solution of 0.5 mmol of Ag2O and 1 mmol of the corresponding
benzimidazolium salt (1a-c) in dichloromethane (25 mL) were
stirred at room temperature for 24 h under dark conditions. The
reaction mixture was then filtered through Celite. The clear filtrate
was evaporated under vacuum to a crude product, which was
recrystallized from dichloromethane/diethyl ether.
2.4.1. Chloro[1-allyl-3-benzylbenzimidazole-2-ylidene]silver(I),
(2a)
(AgilentTechnologies 1100). Samples were introduced on
a
Yield: 81%; m.p. 192-194 ꢃC. FT-IR n(CN): 1395 cmꢁ1 1H NMR
.
continuous flow of 1 mL/min with C18 column (250 ꢂ 4.6 mm) at
(400 MHz, CDCl3)
d
(ppm): 5.09 (d, 2H, NCH2CHCH2, J ¼ 4 Hz), 5.24
25 ꢃC. Nitrogen served both as the nebulizer and dry gas.
(d 1H, NCH2CHCH2, J ¼ 16 Hz), 5.33 (d 1H, NCH2CHCH2, J ¼ 12 Hz),
5.64 (s, 2H, CH2C6H5), 6.02 (quint, 1H, NCH2CHCH2, J ¼ 4 Hz),
7.27e7.39 (m, 8H, Ar-H), 7.46e7.49 (m, 1H, Ar-H). 13C NMR
2.2. Synthesis
(100 MHz, CDCl3)
119.3 (NCH2CHCH2), 131.8 (NCH2CHCH2), 111.9, 112.2, 124.3, 124.4,
127.1, 128.5, 129.1, 133.7, 134.0, 134.9 (Ar-C), 189.1 (slightly, Ccarbene
d (ppm): 52.1 (CH2C6H5), 53.5 (NCH2CHCH2),
Benzimidazolium salts (1a-c) and the respective Ag(I)-NHC
complexes (2a-c) were synthesized according to the previous
study by the current authors [54].1c and bromo salt of 1a have been
synthesized previously in literature [55,56]. In this study, chloro
salt of 1-(allyl)-3-benzylbenzimidazolium (1a) was synthesized.
-
Ag). % Anal. Calcd for C17H16N2AgCl: C, 52.14; H, 4.12; N: 7.15;
Found: C, 52.06; H, 4.03; N, 7.07. LC-MS: 603.2 [AgL2]þ
2.4.2. Chloro[1-allyl-3-naphthylmethylbenzimidazole-2-ylidene]
silver(I), (2b)
2.3. General procedure for the preparation of benzimidazolium
salts, (1a-c)
Yield: 84%, m.p. 203-204 ꢃC. FT-IR n(CN): 1399 cmꢁ1 1H NMR
.
(400 MHz, CDCl3)
d
(ppm): 5.10 (d, 2H, NCH2CHCH2, J ¼ 4 Hz), 5.23
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. Allyl bromide (10.1 mmol) was added dropwise
to obtain a solution that was heated for 24 h at 60 ꢃC. Then, the THF
was removed under vacuum. Dichloromethane (50 mL) was added
to the solid. The mixture was filtered and the obtained clear solu-
tion was concentrated under vacuum. Then the solution was
distilled to 1-allyl benzimidazole. The 1-allyl benzimidazole
(1 mmol) and alkyl halide (1 mmol) were stirred in DMF (5 mL) for
(d 1H, NCH2CHCH2, J ¼ 20 Hz), 5.33 (d 1H, NCH2CHCH2, J ¼ 8 Hz),
6.02 (quint, 1H, NCH2CHCH2, J ¼ 4 Hz), 6.08 (s, 2H, CH2C10H7), 6.97
(d, 1H, Ar-H, J ¼ 4 Hz), 7.25e7.26, 733-7.39 and 7.49e7.60 (m, 7H,
Ar-H), 7.83 (d, 1H, Ar-H, J ¼ 8 Hz), 7.90e7.92 (m, 1H, Ar-H),
8.00e8.03 (m, 1H, Ar-H). 13C NMR (100 MHz, CDCl3)
d (ppm): 51.2
(CH2C10H7), 52.2 (NCH2CHCH2), 122.3 (NCH2CHCH2), 131.8
(NCH2CHCH2), 112.0, 112.2, 119.3, 124.4, 124.9, 125.3, 126.3, 127.1,
129.2, 130.1, 130.5, 133.8, 133.9 and 134.1 (Ar-C), 189.7 (slightly,
Ccarbene-Ag). % Anal. Calcd for C21H18N2AgCl: C, 57.10; H, 4.11; N:

E. Çevik-Yıldız et al. / Journal of Molecular Structure 1199 (2020) 126987
3
6.34; Found: C, 57.04; H, 4.06; N, 6.26. LC-MS: 703.2 [AgL2]þ
means from at least three independent experiments, each
comprising three replicates per concentration. The IC50 values of
compounds were calculated using GraphPad Prism 7 software
(GraphPad Software, San Diego, CA, USA).
2.4.3. Chloro[1-allyl-3-(anthracen-9-yl-methyl)benzimidazole-2-
ylidene]silver(I), (2c)
Yield: 87%; m.p. 205-206 ꢃC. FT-IR n(CN): 1395 cmꢁ1 1H NMR
.
(400 MHz, CDCl3)
d
(ppm): 4.96 (d, 2H, NCH2CHCH2, J ¼ 8 Hz), 5.11
2.6. Statistical analysis
(d, 1H, NCH2CHCH2, J ¼ 16 Hz), 5.24 (d, 1H, NCH2CHCH2, J ¼ 8 Hz),
5.91 (quint, 1H, NCH2CHCH2, J ¼ 4 Hz), 6.40 (s, 2H, CH2C14H9),
7.07e7.15 and 7.28e7.30 (m, 3H, Ar-H), 7.39 (d, 1H, Ar-H, J ¼ 8 Hz),
7.47e7.56 (m, 4H, Ar-H), 8.08 (d, 2H, Ar-H, J ¼ 8 Hz), 0.8.19 (d, 2H,
Ar-H, J ¼ 8 Hz), 8.59 (s, 1H, Ar-H). 13C NMR (100 MHz, CDCl3)
All experiments were carried out in triplicate and the data
represented the average values of three independent measure-
ments with standard error means ( SEM). Data were analyzed
using one-way analysis of variance and differences were considered
significant at *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.
The IC50 were determined using statistics software, GraphPad
Prism7 (GraphPad Software, San Diego, CA, USA).
d
(ppm): 46.5 (CH2C14H9), 52.4 (NCH2CHCH2), 122.9 (NCH2CHCH2),
130.0 (NCH2CHCH2), 111.9, 112.1, 119.0, 123.1, 124.2, 125.3, 127.7,
130.4, 131.1, 131.4, 131.7, 133.9, 134.2 (Ar-C), 189.3 (slightly, Ccarbene
-
Ag). % Anal. Calcd for C25H20N2AgCl: C, 61.06; H, 4.10; N: 5.70;
Found: C, 60.99; H, 4.01; N, 5.62. LC-MS: 804.2 [AgL2]þ
3. Results and discussions
2.5. Antiproliferative activity
3.1. Synthesis and characterization of benzimidazolium salts and
corresponding Ag(I)-NHC complexes
The antiproliferative activity of the newly-synthesized salts and
complexes was determined against MCF-7 (HTB-22, human breast
adenocarcinoma), DU-145 (HTB-81, human prostate carcinoma),
MDA-MB-231 (HTB-26, human breast adenocarcinoma) cancer
cells and L-929 normal cells using the MTT assay [57]. MCF-7 and
MDA-MB-231 cells were grown in DMEM (Sigma, D6429) medium.
DU-145 cancer cells and L-929 normal cells (ECACC, European
Collection of Animal Cell Culture, Salisbury, U.K.) were grown in
EMEM (ATTC, 30-2003) and RPMI-1640 media (ATTC, 30-2001),
respectively. All media contained 10% (v/v) FBS (ATTC, FBS, 30-
The synthetic route for the synthesis of benzimidazolium salts
(1a-c) is shown in Scheme 1. The salts were synthesized with a
reaction of N-allyl benzimidazole with different alkyl halides in
DMF at 80 ꢃC. Collapsed products were crystallized in dichloro-
methane/diethyl ether for purification.
As shown in Scheme 2, synthesized benzimidazolium salts (1a-
c) were reacted with Ag2O in dichloromethane at room tempera-
ture under dark conditions and their Ag(I)-NHC complexes (2a-c)
were obtained. Crude products were purified by crystallization in
dichloromethane/diethyl ether. The structures of all new com-
pounds were characterized by elemental analysis, LC-MS, FT-IR, 1H
NMR and, 13C NMR spectroscopy.
In the elemental analyses, the accepted deviation is 0.4%. For
all compounds (1a-c and 2a-c), deviation of experimental
elemental analysis was < 0.4%. Thus, the experimental elemental
analysis values of all the compounds were compatible with the
theoretical values.
The experimental FT-IR spectra of benzimidazolium salts and
Ag(I)-NHC complexes are given in the Supporting Information
(Figs. S2, S4, S6, S9, S12). The FT-IR spectra of all compounds con-
tained some characteristic bands of the stretching vibrations of the
C]N, C-N, C-H and C]C groups. The aliphatic and aromatic C-H
stretching vibrational bands for all compounds reached a peak at
around 2700e3200 cmꢁ1. Benzimidazole ring CN vibrations of
2020), and 100 Units/ml penicillin and 100 mg/ml streptomycin
(ATTC, 30-2300). Exponentially growing DU-145, MCF-7, MDA-MB-
231, and L-929 cells, in a 37 ꢃC humidified incubator with 5% CO2,
were seeded into 96-well plates at a density of 1 x 105 cells/well
and allowed to attach for 24 h. A stock solution of the tested
compounds was prepared in DMSO and diluted in the complete
culture medium. DMSO concentration did not exceed 1% (v/v). The
cells were treated with 1e20
for 24 h, 48 h, and 72 h. The control wells contained cells and 1%
DMSO. At the end of the exposure period, 10 L MTT solution was
m
M of compounds in 5% CO2, at 37 ꢃC
m
added to each well (5 mg/mL, in PBS) and the cells were incubated
for 2 h at 37 ꢃC in 5% CO2. After removal of the medium, the purple-
blue precipitated crystals were dissolved in 100 mL of DMSO (Sigma,
St. Louis, MO, USA). The absorbance was read at 570 nm with a
Biotek plate reader (Biotek, Epoch, USA). Data were based on the
Scheme 1. Synthesis of benzimidazolium salts (1a-c).

4
E. Çevik-Yıldız et al. / Journal of Molecular Structure 1199 (2020) 126987
Scheme 2. Synthesis of Ag(I)-NHC complexes (2a-c).
benzimidazolium salts (1a-c) were assigned at around 1551-
1555 cmꢁ1. These vibrations show a shift in the Ag(I)-NHC com-
plexes (2a-c) at 1395, 1399 and 1595 cmꢁ1, respectively. This
negative shift is because of the electropositive metal center which
pulls electron density towards itself and as a result, CN vibrations
shift to the lesser energy region in the complex.
show that Ag-complexes have more antiproliferative activity than
their own salts. The IC50 (concentration of the test compound to
achieve 50% inhibition) values of salts (1a-c) and complexes (2a-c)
for 24, 48, and 72 h are listed in Table 1.
The IC50 values of 1a against MDA-MB-231, MCF-7 breast cancer
cells, and L-929 normal cells ranged from 7.07 to >20 mM indicating
NMR spectra of all the compounds were analyzed in d-CDCl3. In
the 1H NMR spectra, acidic protons (NCHN) for benzimidazolium
salts (1a-c) were seen at 11.90, 11.85 and 9.14 ppm [50], respec-
tively, as a characteristic sharp singlet. The formation of Ag(I)-NHC
complexes was evidenced by the disappearance of the acidic proton
peak as seen in 13C NMR spectra (Figs. S1, S3, S5, S8, S11). For all
compounds, aromatic protons appeared in the range
6.97e8.59 ppm. Single protons at CH2CHCH2 reached peaks in the
range 5.91e6.22 ppm as quint for all compounds. In 13C NMR
spectra, while the sharp characteristic signals of the imino carbon
(NCHN) on the 1a-c salts were seen around 140.0 ppm, after
complexation, NCN carben resonance on the 2a-c shifted highly to
the downfield region at around 190.0 ppm as slight peaks
compared to the corresponding benzimidazolium salts. In the
complexes, the resonances for carbene carbon was not detected
exactly, which has also been mentioned in the literature and given
as a reason for the fluxional behavior of NHCs complexes [58e61].
These values are in agreement with reported data for similar
compounds [62,63]. In the LC-MS spectrum of Ag(I)-NHC com-
plexes 2a-c, it was seen that, complexes exist as [Ag(NHC)2]þ in
solution (Figs. S7, S10, S13).
lower antiproliferative activity. Similarly, 1b did not display any
activity toward MDA-MB-231 breast cancer cells and L-929 normal
cells even at a concentration of 20 mM. 1a and 1b were determined
to have time-dependent IC50 values towards DU-145 prostate
cancer cells and MCF-7 breast cancer cells, respectively. These IC50
values indicated high antiproliferative activity. Notably, the IC50
values of 1c were lower than those of 1a, and 1b, suggesting higher
antiproliferative activity than 1a, and 1b. The silver complexes (2a-
c) decreased MTT staining in all cells, compared to the controls. 2a
showed better antiproliferative activity against cancer cells with
IC50 ranging from <1 to 16.3
cinoma cells were the most sensitive to 2a displaying IC50 lower
than 1 M at all time points. These results showed that the IC50
values of 2b differ from <1 to 6.94 M against cancer cells.
Considering 2b, MCF-7 human breast carcinoma cells were the
most sensitive to displaying IC50 lower than 1 M at all time points.
The IC50 values of complex 2c, 2b, and 2a on DU-145 human
prostate cancer cells were found to be range 1.74e3.9 M,
2.11e6.94 M and 2.74e16.3 M at 24, 48 and 72 h respectively.
mM. MDA-MB-231 human breast car-
m
m
m
m
m
m
These results indicated that antiproliferative activity followed the
order of 2c>2b>2a and the increased ring numbers bound on the
benzimidazole ring may be attributed to the increased activities.
The pharmaceutical research on organometallic compounds has
generally been focused on platinum and gold complexes. However
recent reports of lower toxicity and biological activities of silver
have increased interest in Ag-NHC complexes. In a previous study
three series of silver(I) complexes bearing stericallyemodulated
coumarinefunctionalized benzimidazoleebased NHC ligands
were synthesized and characterized (Scheme 3, Scheme 4) [64].
An anticancer activities against human lung cancer cells (H1975
and A549) were also evaluated over a period of 72 h. The results of
that study showed that, all the azolium salts were found to be
inactive, while complexes demonstrated significant activities. The
IC50 values of NHC-silver complexes 13e24 were found to range
3.2. Antiproliferative activity
The antiproliferative activity of synthesized benzimidazolium
salts (1a-c) and their Ag(I)-NHC complexes (2a-c) were determined
using MTT assay against MCF-7, DU-145, and MDA-MB-231 cancer
cells, as well as L-929 non-cancer cells for 24, 48, and 72 h of
exposure. Fig. 1 shows the time and dose-dependent anti-
proliferative activities of salts (1a-c) and complexes (2a-c) against
MCF-7 breast cancer cells.
According to Fig. 1 the antiproliferative activity of the com-
pounds was found to be concentration- and time-dependent
against MCF-7 breast cancer cells. Fig. 1 (Figs. S14, S15, S16) also

E. Çevik-Yıldız et al. / Journal of Molecular Structure 1199 (2020) 126987
5
Fig. 1. Time-dependent antiproliferative activities of salts (1a-c) and complexes (2a-c) on MCF-7 breast cancer cells. Data are representative of the mean of three separate ex-
periments and are reported at the SEM. (***p < 0.0005 vs control, ****p < 0.0001 vs control).
from 9.8 to 19.4
m
M, 8.3 to 17.7 and 7.7e18.3
m
M against H1975 and
complexes have lower IC50 values and showed higher anti-
proliferatife activity than these complexes against MCF-7, MDA-
MB-231 and DU-145 cancer cell line at 72 h. Fatima et al. synthe-
sized and evaluated the cytotoxicity of a group of benzimidazole
compounds with varied alkyl chains of the nitrogen positions of the
ring using the MTT assay on HCT 116 cells [65]. The results showed
that increasing the alkyl chain length to 6 carbons or more marked
A549 lung cancer cells and Hs68 normal cell line, respectively.
Complexes 14, 15, 16 and 18 were found to be cytotoxic against the
human lung cancer cell lines A549 and H1975 with the IC50 value
under 10
13.7 2.70 and 14.5 1.20
and A549, respectively. The current study results indicated that all
mM, while monoeNHC complex 20 had the IC50 of
mM against the cancer cell lines H1975

6
E. Çevik-Yıldız et al. / Journal of Molecular Structure 1199 (2020) 126987
Table 1
Antiproliferative activities of salts (1a-c) and complexes (2a-c) against Prostate Cancer Cells, Breast Cancer Cells, and Normal Cells.
Cell lines
MCF-7
Time
Salts (IC50,
1a
m
M)a
Complexes (IC50
2a
, m
M)a
1b
1c
2b
2c
24 h
48 h
72 h
24 h
48 h
72h
24 h
48 h
72 h
24 h
48 h
72 h
>20
>20
>20
4.70 0.42
<1
>20
>20
5.38 0.25
>20
>20
>20
>20
8.21 0.15
3.67 0.07
1.84 0.08
13.5 0.17
9.33 0.20
4.43 0.16
2.37 0.11
1.76 0.12
1.31 0.50
>20
5.82 0.15
1.12 0.08
<1
16.3 0.23
10.4 0.06
2.74 0.32
<1
<1
<1
13.0 0.41
11.3 0.21
5.75 0.07
<1
<1
<1
2.05 0.72
1.21 0.03
1.02 0.05
3.90 0.09
2.95 0.19
1.74 0.21
1.83 0.03
1.21 0.22
<1
15.8 0.03
>20
8.72 0.60
7.07 0.31
>20
>20
18.8 0.17
>20
DU-145
6.94 0.15
6.10 0.07
2.11 0.05
2.19 0.15
1.60 0.04
1.26 0.02
6.87 0.03
6.70 0.04
4.14 0.05
MDA-MB-231
L-929b
2.89 0.04
2.50 0.03
2.01 0.04
>20
>20
>20
>20
18.7 0.08
18.3 0.20
a
The IC50 value was determined as the concentration causing 50% decrease in cell growth after 24 h, 48 h and 72 h of incubation. The IC50 values were calculated from an
MTT assay. Values are means SEM and represent one of three representative experiments.
b
Normal Cells.
Scheme 3. Synthesis of cationic, biseNHC coordinated silver(I) hexafluorophosphate complexes, 13e18 [64].
Scheme 4. Synthesis of neutral, monoeNHC coordinated silver(I) acetate complexes 19e24 [64].
a significant decrease in IC50 value for both the ligand and the Ag(I)-
NHC complex. While the IC50 values of silver complexes with less
than 6 carbons in the alkyl chain were found to be in the range of
13.2 ( 1.50) to 26.8 ( 2.30), with 6 or more carbons in the alkyl
chain, the range was were found to be range of 0.02 ( 0.02) to 3.9
( 0.62). It has been previously reported that researchers synthe-
sized, characterized and evaluated the in vitro anticancer activity of
para-xylyl linked bis-benzimidazolium salts and respective dinu-
clear AgeNHC complexes against human colorectal cancer (HCT
116) and promyelocytic leukemia (HL-60) cells using the MTT assay
[66]. It was observed that all tested compounds showed dose-
dependent cytotoxic activity against both cell lines. The IC50
values of complexes were found to be in the range of 0.01e18.7
mM
for HCT 116 and 0.7e55.7 M for HL-60 cells. The current study
m
results indicated that complexes have higher cytotoxic activity than
these complexes against HL-60 cells but similar activity against HCT
116 cell lines. Lukevics et al. synthesized bromo-salt of 1-(allyl)-3-
benzylbenzimidazolium (current study 1a) and investigated its

E. Çevik-Yıldız et al. / Journal of Molecular Structure 1199 (2020) 126987
7
in vitro cytotoxic activity against HT-1080 (human fibrosarcoma),
MG-22A (mouse hepatoma), B16 (mouse melanoma), Neuro 2A
(mouse neuroblastoma) and 3T3 normal mouse fibroblast cells
[55]. The IC50 values of bromo salt of 1-(allyl)-3-
MDA-MB-231 human breast cancer cells, whereas the naphthyl-
linked 2b compound has the highest antiproliferative activity on
MCF-7 human breast cancer cells. These results show that the
groups that bind to the benzimidazole ring have a very important
role both in the activity and selectivity of the compounds. The
compounds showed selective behaviour even in different types of
subtypes of the same cancer type. While the MCF-7 human breast
cancer cell line is positive for estrogen receptor (ER), progesterone
receptor (PR), and negative for human epidermal growth factor
receptor (HER2), the MDA-MB-231 7 human breast cancer cell line
is triple negative for the estrogen receptor (ER), progesterone re-
ceptor (PR) and human epidermal growth factor receptor (HER2).
All these results indicated that the antiproliferative activities of the
compounds depend on both the chemical structure of the com-
pounds and the cell line types.
benzylbenzimidazolium were found to be 21, 45 and 29 mg/mL
against HT-1080, MG-22A and B16 cells, respectively. The salt was
also found not to have a cytotoxic effect on Neuro 2A cancer cells
and 3T3 cells. In the current study, the chloro-salt of 1-(allyl)-3-
benzylbenzimidazolium (1a) was synthesized and the IC50 values
of 1a were found to range from 2.76 to 7.82 mg/mL on MCF-7, DU-
145, and MDA-MB-231 cancer cells. These results showed that 1a
did not display any activity toward MDA-MB-231 breast cancer cells
and L-929 normal cells even at a concentration of 7.82 mg/mL,
clearly indicating that the cytotoxic activity of the salt depends on
the type of substituted ion over the benzimidazole ring. It was
found that chloro salt was more cytotoxic than bromo-salt against
cancer cells. It has been previously reported that researchers syn-
thesized 1c (4b in the paper) and evaluated its tyrosinase inhibitory
activity with catechol as substrate [56]. The IC50 value of 4b was
4. Conclusions
In conclusion, new metal N-Heterocyclic Carbene (NHC) salts
(1a-c) and their Ag(I) complexes (2a-c) were synthesized. All syn-
thesized compounds were characterized using elemental analysis,
FT-IR, 1H NMR, and 13C NMR spectroscopy techniques. The anti-
proliferative activity of synthesized benzimidazolium salts (1a-c)
and their Ag(I)-NHC complexes (2a-c) were determined using the
MTT assay against MCF-7, DU-145, and MDA-MB-231 cancer cells,
as well as L-929 non-cancer cells for 24, 48, and 72 h of exposure.
According to the results the antiproliferative activity of the com-
pounds was found to be concentration- and time-dependent except
1a against L-929 normal cells. These results also indicated that Ag-
complexes have more antiproliferative activity than ligands. The
most important findings of this study were that MDA-MB-231 and
MCF-7 human breast carcinoma cells were the most sensitive,
found to be 0.52 mM.
To select the most sensitive cancer cell line, further calculations
were made of the selectivity index (SI) values for complexes (2a-c),
and these are shown in Table 2. Studies have shown that com-
pounds with SI values of 5 and above represent more toxicity to-
wards cancer cells compared to normal cells [34,67]. The results of
the current study showed that 2a was the most selectively toxic
towards MDA-MB-231 breast cancer cells with SI values > 13 at all-
time points. 2a was also determined to be toxic towards MCF-7
breast cancer cells with SI values of 10.1 and > 5.75 at 48 h and
72 h, respectively. 2b showed the most selective toxicity towards
MCF-7 breast cancer cells with SI values > 6 at 24 h and 48 h. As the
SI values of 2c were <5 at all time points, 2c did not show selective
toxicity on any cell lines.
displaying IC50 lower than 1 mM at all time points for 2a and 2b
When the relationship between structure and cytotoxic activity
is examined, the structure which differentiates 2a-c complexes is
the R groups, which are attached to the benzimidazole ring. The R
groups of 2a-c complexes are benzyl, naphthyl, and anthracene,
respectively. Since the 2c complex had lower IC50 values than the
2a and 2b complex on the DU-145 human prostate cancer cell line
for 24, 48 and 72 h, the anthracene group in the 2c complex was
found to play an important role in increased antiproliferative ac-
tivity of the compound on the DU-145 human prostate cancer cell
line. However 2c showed lower antiproliferative activity than 2a
and 2b on MCF-7 and MDA-MB-231 human breast cancer cells. This
suggests that the benzyl and naphthyl groups of 2a and 2b are
responsible for the antiproliferative activities of these compounds
on human breast cancer cells. More importantly, the benzyl-bound
2a compound exhibits the highest antiproliferative activity against
respectively. Furthermore, the IC50s for Ag(I)-NHC were higher in
normal cells especially compared to the breast cancer cells, sug-
gesting that complexes possessed noteworthy selectivity for hu-
man breast cancer cells. 2a showed high selectivity (>13-fold) for
MDA-MB-231 breast cancer cells. The results of this study also
demonstrated that 2b has 4-7-fold selectivity against MCF-7 breast
cancer cells. These results clearly indicate that the IC50 concentra-
tions of 2a and 2b against MDA-MB-231 and MCF-7 were lower
than those of cis-platin which is one of the currently available
chemotherapeutic agents. Therefore, 2a and 2b could be powerful
anticancer drug candidates for MDA-MB-231 and MCF-7 breast
cancer cells, respectively. The detailed mechanism of the cell
apoptosis relationship with anti-apoptotic and pro-apoptotic pro-
teins is currently under further investigation.
Acknowledgments
Table 2
This work was supported by the Technological and Scientific
Research Council of Turkey TÜBITAK -3001(118R045). Anti-
proliferative activity studies were conducted at Sivas Cumhuriyet
University Advanced Technology Application and Research Center
(SCUTAM).
Selectivity Index (SI) of complexes (2a-c).
_
Cell lines
MCF-7
Time
Complexes (IC50
2a
, m
M)a
2b
2c
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
2.23
10.1
>5.75
<1
1.08
2.09
>13
>13
>13
>6.87
>6.70
>4.14
<1
1.09
1.96
3.13
4.18
3.28
1.41
2.06
1.97
<1
Appendix A. Supplementary data
DU-145
<1
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.126987.
1.15
1.66
1.36
>2
MDA-MB-231
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