Synthesis, characterization and anticancer activity of allyl substituted N-Heterocyclic carbene silver(I) complexes

Article abstract of DOI:10.1016/j.molstruc.2018.10.094

Metal N-heterocyclic carbene (NHC) complexes have attracted considerable attention in biological fields for their potential applications in cancer and antimicrobial therapies. In this study, four new benzimidazole-based N-heterocyclic carbene salts (1a-d) and their silver (I) complexes (2a-d) were synthesized. All new compounds were characterized by elemental analysis, FT-IR, ¹H NMR and, ¹³C NMR spectroscopy. Additionally, single crystal structural studies for complex 2d show that the benzene rings (C9–C14) and the central benzimidazole ring system make dihedral angles of 83.58(13)°. The Ag–Cl and Ag???C single bond lengths are 2.3267(8) and 2.087(3) ?, respectively. The C–Ag–Cl bond angle is 175.20(7)°. The prop-1-ene moiety attached at the second N-atom of benzimidazole is disordered at two set of sites with an occupancy ratio of 0.592(6): 0.408(6). There is one intramolecular hydrogen bond interaction between C22A-H22A … N2. The salts and Ag-complexes were further evaluated for their in vitro anticancer activities against DU-145 human prostate cancer cells, MCF-7, MDA-MB-231 human breast cancer cells and L-929 non-cancer cell for 24 h, 48 h and 72 h using the MTT assay. The Ag(I)–NHC complexes (2a-d) showed a dose and time-dependent cytotoxic activity against all cell lines. The IC₅₀s for all Ag(I)–NHC complexes lower than 1 μM for 72 h time points on cancer cells. The results showed that complex 2d exhibited the highest activity against all cancer cell lines studied. Further, the complexes had relatively higher cytotoxicity to cancer cells than to non-cancer cell lines.

Full text of DOI:10.1016/j.molstruc.2018.10.094

Accepted Manuscript
Synthesis, characterization and anticancer activity of allyl substituted N-Heterocyclic
carbene silver(I) complexes
Neslihan Şahin, Serap Şahin-Bölükbaşı, Muhammed Nawaz Tahir, Cengiz Arıcı,
Esranur Çevik, Nevin Gürbüz, İsmail Özdemir, Brian S. Cummings
PII:
S0022-2860(18)31298-5
DOI:
https://doi.org/10.1016/j.molstruc.2018.10.094
MOLSTR 25823
Reference:
To appear in: Journal of Molecular Structure
Received Date: 7 September 2018
Revised Date: 24 October 2018
Accepted Date: 31 October 2018
Please cite this article as: N. Şahin, S. Şahin-Bölükbaşı, M.N. Tahir, C. Arıcı, E. Çevik, N. Gürbüz,
İ. Özdemir, B.S. Cummings, Synthesis, characterization and anticancer activity of allyl substituted
N-Heterocyclic carbene silver(I) complexes, Journal of Molecular Structure (2018), doi: https://
doi.org/10.1016/j.molstruc.2018.10.094.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, Characterization and Anticancer Activity of Allyl
Substituted N-Heterocyclic Carbene Silver(I) Complexes
Neslihan Şahina,b,c,*, Serap Şahin-Bölükbaşıd,e, Muhammed Nawaz Tahirf, Cengiz Arıcıg,
Esranur Çevikd, Nevin Gürbüzb,c, İsmail Özdemirb,c, Brian S Cummingse,h
aDepartment of Basic Education, Faculty of Education, University of Cumhuriyet, 58140, Sivas,
Turkey,
bDepartment of Chemistry, Faculty of Science and Art, University of İnönü, 44280 Malatya, Turkey
cCatalysis Research and Application Center, University of İnönü, 44280, Malatya, Turkey
dDepartment of Biochemistry, Faculty of Pharmacy, University of Cumhuriyet, 58140, Sivas, Turkey
eDepartment. of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA
fDepartment 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
Metal N-heterocyclic carbene (NHC) complexes have attracted considerable attention in
biological fields for their potential applications in cancer and antimicrobial therapies. In this
study, four new benzimidazole-based N-heterocyclic carbene salts (1a-d) and their silver (I)
complexes (2a-d) were synthesized. All new compounds were characterized by elemental
13
analysis, FT-IR, 1H NMR and, C NMR spectroscopy. Additionally, single crystal structural
studies for complex 2d show that the benzene rings (C9-C14) and the central benzimidazole
ring system make dihedral angles of 83.58(13)°. The Ag-Cl and Ag-C single bond lengths are
2.3267(8) and 2.087(3) Å, respectively. The C-Ag-Cl bond angle is 175.20(7)°. The prop-1-
ene moiety attached at the second N-atom of benzimidazole is disordered at two set of sites
with an occupancy ratio of 0.592(6): 0.408(6). There is one intramolecular hydrogen bond
interaction between C22A-H22A...N2. The salts and Ag-complexes were further evaluated for
their in vitro anticancer activities against DU-145 human prostate cancer cells, MCF-7, MDA-
MB-231 human breast cancer cells and L-929 non-cancer cell for 24 h, 48 h and 72 h using
the MTT assay. The Ag(I)-NHC complexes (2a-d) showed a dose and time-dependent
cytotoxic activity against all cell lines. The IC50s for all Ag(I)-NHC complexes lower than 1
ꢀM for 72 h time points on cancer cells. The results showed that complex 2d exhibited the
highest activity against all cancer cell lines studied. Further, the complexes had relatively
higher cytotoxicity to cancer cells than to non-cancer cell lines.
Keywords: N-Heterocyclic carbene; silver complex; benzimidazole-2-ylidene; anticancer
activity.
1

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1. Introduction
The heterocyclic substituent, including ligands, have constituted the basis of organometallic
and coordination chemistry. N-Heterocyclic Carbenes (NHCs) are a well-known class of
organometallic ligands that are strong σ-donating and weak π-accepting when connected with
metal ions. Since the first NHC was isolated and characterized by Arduengo in 1991, NHCs
have attracted interest due to their all-purpose features as ligands for diverse metallic
complexes [1].
Metal-NHC complexes have been popularly investigated due to their noteworthy applications
as catalysis [2-5] and potential biological properties [6-8]. These compounds are prepared by
the different process such as via free carbene, in situ and using transmetallation methods, but
the more commonly used process is deprotonation of azolium precursors with a simple metal
salt or strong bases, such as sodium hydride or potassium tert-butoxide [9,10].
For years metallic silver and silver compounds have been applied to a variety of applications
like water purification, wound management, eye-drops, anti-infective coatings in medical
devices and in burn treatment as they have potent antimicrobial properties [11-16]. One of the
most commonly used compounds of silver is silver(I) sulfazine, which is used to treat
bacterial infections caused by severe burns [17]. Silver has low toxicity as compared to other
transition metals.
In the family of metal-NHCs, Ag-NHCs have received significant attention because of their
medicinal applications such as antibacterial activity [18] or anticancer activity [19]. Due to
the high stability of Ag-NHC complexes and slowly release of silver ions to the diseased area
from Ag-NHC complexes, the Ag-NHC complexes exhibit high efficacy influence against
various types of cancer cell lines. Ag-NHC complexes are synthesized by the reaction of free
NHC and silver salt or silver base, mostly Ag2O [20]. A number of new Ag-NHC complexes
were also synthesized after initial reports of cytotoxic properties of Ag-NHC complexes
against cervical cancer (HeLa), breast cancer (MCF-7), and colon adenocarcinoma (HCT 116)
cells [21].
Within this manuscript, we present the synthesis and characterization of four new allyl
substituted NHC salts and their silver(I) complexes. All the new synthesized compounds (1a-
d, 2a-d) were tested as anticancer agents against three human cancer cell lines (DU-145,
MCF-7, MDA-MB-231) and non-cancer cells (L-929) for 24 h, 48 h and 72 h by MTT assays.
2

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2. Experimental
2.1. Materials and Methods
All synthesis of benzimidazolium salts and Ag-NHC complexes were prepared 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 [22]. All Ag2O
reactions were carried out in the absence of light. 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 (FT-IR)
spectra were obtained in the range 400–4000 cm−1 on a Perkin Elmer Spectrum 100 FT-IR.
13
1H NMR and C NMR spectra were recorded using a Bruker As 400 Mercury spectrometer
operating at 400 MHz (1H), 100 MHz (13C) in CDCl3 with tetramethylsilane as the internal
1
reference. H peaks were labeled as singlet (s), doublet (d), triplet (t), quartet (q), quintet
(quint.) and multiplet (m). Chemical shifts and coupling constants were reported in ppm and
in Hz, respectively. The intensity data were collected using a Bruker Kappa ApexII CCD
diffractometer [23], using Mo-Kα radiation (λ= 0.71073 Å). The structure was solved by direct
methods using the solution program SHELXS97 [24] and then refined with full-matrix least-
square methods based on F2 SHELXL2014/6 [25]. All non-hydrogen atoms were first refined
isotropically and then with anisotropic atomic displacement parameters by the full matrix
least square methods. 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.( Sigma-Aldrich Chemie GmbH , Steinheim, Deutschland). L-
929 (non-cancer cells from the adipose tissue of mice) were purchased from ECACC
(European Collection of Animal Cell Culture, Salisbury, U.K.). All absorbance values were
measured with a microplate reader (Bio-Tek, Epocch, USA) at 570nm.
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2.2. 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).
The benzimidazolium salts (1a-d) and Ag-NHC (2a-d) compounds were prepared under
argon gas atmosphere. The unsymmetrically N1, N3-substituted benzimidazolium salts (1a-d)
and respective Ag(I)-NHC complexes (2a-d) were synthesized as described in our previous
study [26].
2.3. General procedure for the preparation of 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
dropwise to obtained a solution that was heated for 24 h at 60 C. Then, the THF was
removed under the vacuum. Dichloromethane (50 mL) was added upon to solid. The mixture
was filtered and the obtained clear solution was concentrated under vacuum. Then the
solution was distilled to 1-allyl benzimidazole. The 1-allyl benzimidazole (1 mmol) and alkyl
0
halide (1 mmol) were stirred in DMF (5 mL) for 24 h at 80 C. White product was
precipitated. Following the completion of the process the solution was filtered, the solid was
rinsed with diethylether and dried under vacuum. Crude product was recrystallized from
dichloromethane/diethylether.
1-(Allyl)-3-(4-isopropylbenzyl)benzimidazolium chloride, 1a
°
1
Yield: 88%; m.p. 153-155 C; H NMR (400 MHz, CDCl3) δ (ppm): 1.19 (m, 6H,
CH2C6H4(CH(CH3)2)-4), 2.62-2.63 (m, 1H, CH2C6H4(CH(CH3)2)-4),5.35 (d, 2H,
NCH2CHCH2, J = 8 Hz), 5.45-5.50 (m, 2H, NCH2CHCH2), 5.84 (s, 2H,
CH2C6H4(CH(CH3)2)-4), 6.08-6.16 (m, 1H, NCH2CHCH2), 7.21-7.23, 7.44-7.46, 7.56-7.58,
7.65-7.67 and 7.70-7.72 (m, 8H, NC6H4Nand CH2C6H4(CH(CH3)2)-4), 11.85 (s, 1H,
NCHN).13C NMR (100 MHz, CDCl3) δ (ppm):23.8 (CH2C6H4(CH(CH3)2)-4), 33.8
(CH2C6H4(CH(CH3)2)-4),50.2
(CH2C6H4(CH(CH3)2)-4), 51.3 (NCH2CHCH2),113.7
(NCH2CHCH2),121.8 (NCH2CHCH2),113.9, 127.0, 127.1,127.4,129.7,128.4,130.0,131.3,
131.5 and 150.1 (CH2C6H4(CH(CH3)2)-4 and NC6H4N), 143.6 (NCHN); FT-IR ν(CN): 1547
cm-1; % Anal. Calcd for C20H23N2Cl: C, 73.49; H, 7.09; N: 8.57; Found: C, 73.47; H, 7.08;
N, 8.54.
4

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1-(Allyl)-3-(4-ter-butylbenzyl)benzimidazoliumbromide, 1b
°
1
Yield: 86%; m.p.196-198 C; H NMR (400 MHz, CDCl3) δ (ppm):1.26 (s, 9H,
CH2C6H4(C(CH3)3-4), 5.34 (d, 2H, NCH2CHCH2, J = 4 Hz), 5.45-5.53 (m, 2H,
NCH2CHCH2),5.85 (s, 2H, CH2C6H4(C(CH3)3)-4),6.11-6.23 (m, 1H, NCH2CHCH2),7.36-
7.38, 7.48-7.50, 7.57-7.58 and 7.69-7.75 (m, 8H, NC6H4Nand CH2C6H4(C(CH3)3)-4,11.55 (s,
1H, NCHN).13C NMR (100 MHz, CDCl3) δ (ppm): 31.2 (CH2C6H4(C(CH3)3-4), 34.6
(CH2C6H4(C(CH3)3-4), 50.2 (CH2C6H4(C(CH3)3-4), 51.2 (NCH2CHCH2), 113.7
(NCH2CHCH2), 122.0 (NCH2CHCH2),113.9, 126.3, 127.1, 127.2, 128.2, 129.6, 129.7, 131.3,
131.4 and 152.4 (CH2C6H4(C(CH3)3-4 and NC6H4N), 142.7 (NCHN); ν(CN): 1551 cm-1; %
Anal. Calcd for C21H25N2Br: C, 65.46; H, 6.54; N: 7.27; Found: C, 65.49; H, 6.57; N, 7.30.
1-(Allyl)-3-(2,4,6-trimethylbenzyl)benzimidazolium chloride, 1c
°
1
Yield: 89%; m.p. 223-225 C; H NMR (400 MHz, CDCl3) δ (ppm): 2.25 and 2.32 (s, 9H,
CH2C6H2(CH3)3-2,4,6),5.32-5.37 (m, 4H, NCH2CHCH2), 5.79 (s, 2H, CH2C6H2(CH3)3-2,4,6),
5.99-6.06 (m, 1H, NCH2CHCH2), 6.86 (s, 2H, CH2C6H2(CH3)3-2,4,6), 7.19 (d, 1H, NC6H4N,
J= 8 Hz), 7.40 (t, 1H, NC6H4N, J= 8 Hz), 7.50 (t, 1H, NC6H4N, J= 6 Hz), 7.65 (d, 1H,
13
NC6H4N, J= 8 Hz), 11.36 (s, 1H, NCHN). C NMR (100 MHz, CDCl3) δ (ppm):20.2 and
21.0 (CH2C6H2(CH3)3-2,4,6), 47.2 (CH2C6H2(CH3)3-2,4,6),50.1 (NCH2CHCH2),113.6
(NCH2CHCH2),121.2 (NCH2CHCH2),113.7, 125.0, 127.0, 127.2, 129.9, 130.1, 131.4, 131.5,
137.9 and 139.7 (CH2C6H2(CH3)3-2,4,6 and NC6H4N),143.6 (NCHN); ν(CN): 1554 cm-1; %
Anal. Calcd for C20H23N2Cl: C, 73.49; H, 7.09; N: 8.57; Found: C, 73.46; H, 7.08; N, 8.55.
1-(Allyl)-3-(2,3,4,5,6-pentamethylbenzyl)benzimidazolium chloride, 1d
°
1
Yield: 88%; m.p. 205-206 C, H NMR (400 MHz, CDCl3) δ (ppm): 2.18, 2.21 and 2.23 (s,
15H, (CH2C6(CH3)5-2,3,4,5,6), 5.29-5.38 (m, 4H, NCH2CHCH2), 5.77 (s, 2H, (CH2C6(CH3)5-
2,3,4,5,6), 5.96-6.04 (m, 1H, NCH2CHCH2), 7.31 (d, 1H, NC6H4N, J= 8 Hz),7.42-7.53 (m,
2H, NC6H4N), 7.64 (d, 1H, NC6H4N, J= 8 Hz), 10.91 (s, 1H, NCHN). 13C NMR (100 MHz,
CDCl3) δ (ppm): 17.0,17.2 and 17.3, (CH2C6(CH3)5-2,3,4,5,6), 48.9 (CH2C6(CH3)5-2,3,4,5,6),
50.2 (NCH2CHCH2), 113.6 (NCH2CHCH2), 121.0 (NCH2CHCH2),113.7, 124.8, 127.0,
127.2, 130.2, 131.5, 131.7, 133.6, 134.0 and 137.4 (NC6H4N and CH2C6(CH3)5-2,3,4,5,6),
143.3 (NCHN); ν(CN): 1558 cm-1; % Anal. Calcd for C22H27N2Cl: C, 74.45; H, 7.67; N: 7.89.
Found: C, 74.44; H, 7.65; N, 7.86.
5

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2.4. General procedure for preparation of Ag-NHC complexes, 2a-d
A solution of 1 mmol of Ag2O and 0.5 mmol of the corresponding benzimidazolium salt (1a-
d) 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.
Chloro[1-allyl-3-(4-isopropylbenzyl)benzimidazole-2-ylidene]silver(I), 2a
°
1
Yield: 83%; m.p. 159-160 C; H NMR (400 MHz, CDCl3) δ (ppm): 1.19-1.21 (m, 6H,
CH2C6H4(CH(CH3)2)-4), 2.83-2.90 (m, 1H, CH2C6H4(CH(CH3)2)-4), 5.08 (d, 2H,
NCH2CHCH2, J = 4 Hz), 5.22-5.35 (m, 2H, NCH2CHCH2), 5.59 (s, 2H, CH2C6H4(CH(CH3)2)-
4), 5.97-6.07 (m, 1H, NCH2CHCH2), 7.16-7.22, 7.28-7.40 and 7.45-7.49 (m, 8H, NC6H4N
and CH2C6H4(CH(CH3)2)-4). 13C NMR (100 MHz, CDCl3)
δ
(ppm): 23.8
(CH2C6H4(CH(CH3)2)-4), 33.8 (CH2C6H4(CH(CH3)2)-4), 52.0 (CH2C6H4(CH(CH3)2)-4), 53.3
(NCH2CHCH2), 112.2 (NCH2CHCH2), 119.3 (NCH2CHCH2), 112.0, 124.3, 127.1, 127.2,
128.4, 131.8, 133.8, 134.0 and 149.3 (CH2C6H4(CH(CH3)2)-4 and NC6H4N), 189.0 (Ccarbene
-
Ag); ν(CN): 1395 cm-1; % Anal. Calcd for C20H22N2AgCl: C, 55.38; H, 5.11; N: 6.46; Found:
C, 55.37; H, 5.09; N, 6.44; LC-MS: 687.2 [AgL2]+
Bromo[1-allyl-3-(4-ter-butylbenzyl)benzimidazole-2-ylidene]silver(I), 2b
°
1
Yield: 80%, m.p. 199-200 C; H NMR (400 MHz, CDCl3) δ (ppm):1.27 (s, 9H,
CH2C6H4(C(CH3)3-4), 5.11-5.13 (m, 2H, NCH2CHCH2), 5.22-5.32 (m, 2H, NCH2CHCH2),
5.63 (s, 2H, CH2C6H4(C(CH3)3)-4), 5.98-6.07 (m, 1H, NCH2CHCH2), 7.24-7.27, 7.31-7.33,
13
7.36-7.41 and 7.44-7.49 (m, 8H, NC6H4N and CH2C6H4(C(CH3)3)-4. C NMR (100 MHz,
CDCl3)
δ
(ppm):31.2 (CH2C6H4(C(CH3)3-4), 34.7 (CH2C6H4(C(CH3)3-4), 51.2
(CH2C6H4(C(CH3)3-4), 53.0 (NCH2CHCH2), 111.8 (NCH2CHCH2), 119.1 (NCH2CHCH2),
112.1, 121.8, 124.0, 125.9, 127.1, 128.2, 132.1, 133.9, 134.1and 151.4 (CH2C6H4(C(CH3)3-4
and NC6H4N), 191.2 (Ccarbene-Ag); ν(CN): 1391 cm-1; % Anal. Calcd for C21H24N2AgBr: C,
51.24; H, 4.91; N: 5.69; Found: C, 51.22; H, 4.89; N, 5.67; LC-MS: 716.3 [AgL2]+.
Chloro[1-allyl-3-(2,4,6-trimethylbenzyl)benzimidazole-2-ylidene]silver(I), 2c
°
1
Yield: 81%; m.p. 205-206 C; H NMR (400 MHz, CDCl3) δ (ppm): 2.24and 2.35 (s, 9H,
CH2C6H2(CH3)3-2,4,6),5.03-5.01 (m, 2H, NCH2CHCH2), 5.16 (d, 1H, NCH2CHCH2, J= 16
Hz), 5.29 (d, 1H, NCH2CHCH2,J = 12 Hz), 5.49 (s, 2H, CH2C6H2(CH3)3-2,4,6), 5.99-5.90 (m,
6

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1H, NCH2CHCH2), 6.99 (s, 2H, CH2C6H2(CH3)3-2,4,6), 7.27-7.40 and 7.45-7.47 (m, 4H,
NC6H4N). 13C NMR (100 MHz, CDCl3) δ (ppm): 20.4 and 21.2 (CH2C6H2(CH3)3-2,4,6), 47.8
(CH2C6H2(CH3)3-2,4,6), 52.6 (NCH2CHCH2), 111.7 (NCH2CHCH2), 119.0 (NCH2CHCH2),
111.9, 124.2, 124.3, 126.5, 130.3, 131.8, 133.8, 134.3, 137.5 and 139.6 (CH2C6H2(CH3)3-2,4,6
and (NC6H4N), Ccarbene-Ag: not observed; ν(CN): 1388 cm-1; % Anal. Calcd for C20H22N2AgCl:
C, 55.38; H, 5.11; N: 6.46; Found: C, 55.36; H, 5.10; N, 6.44; LC-MS: 687.2 [AgL2]+.
Chloro[1-allyl-3-(2,3,4,5,6-pentamethylbenzyl)benzimidazole-2-ylidene]silver(I), 2d
°
1
Yield: 76%; m.p. 218-219 C, H NMR (400 MHz, CDCl3) δ (ppm): 2.35, 2.22 and 2.31 (s,
15H, (CH2C6(CH3)5-2,3,4,5,6), 5.02 (s, 2H, NCH2CHCH2), 5.16 (d, 1H, NCH2CHCH2, J =
17.2 Hz), 5.28 (d, 1H, NCH2CHCH2, J = 10 Hz), 5.50 (s, 2H, CH2C6(CH3)5-2,3,4,5,6), 5.95
(quint, 1H, NCH2CHCH2, J = 6.54 Hz), 7.44-7.52 (m, 4H, NC6H4N). 13C NMR (100 MHz,
CDCl3) δ (ppm):17.2 and 17.4 (CH2C6(CH3)5-2,3,4,5,6), 47.7 (CH2C6(CH3)5-2,3,4,5,6), 52.9
(NCH2CHCH2), 111.4, 112.0, 118.8, 124.1, 124.4, 126.6, 132.0,133.0, 133.8, 134.3, 134.5 and
137.4 (NC6H4N, NCH2CHCH2 and CH2C6(CH3)5-2,3,4,5,6); ν(CN): 1387 cm-1; % Anal. Calcd
for C22H26N2AgCl: C, 57.22; H, 5.68; N: 6.07; Found: C, 57.20; H, 5.66; N, 6.05; LC-MS:
743.4 [AgL2]+.
2.5. Cell viability (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 37 ºC humidified air condition. Cells were passaged at 70–80%
confluence and the media were changed every two/three days. The anticancer activity of the
salts and complexes were evaluated using the MTT assay [27]. Cells including DU-145,
MCF-7, MDA-MB-231, and L-929 were seeded in 96-well plates at a volume of 100 ꢀL per
well (1x105 cell/well) and incubated for 24 h at 37 °C in 5% CO2. Then the cells were further
incubated with various concentrations of the salts and Ag-complexes (1, 5, 10 and 20 ꢀM) for
24 h, 48 h, and 72 h. Media without cells were used as a blank and the control wells contained
cells with media and 0.5% DMSO. After the incubation period, 10 mL of MTT solution (5
mg/mL in PBS, pH 7.2) was added to each well. Samples were further incubated for 2 h at 37
°C in 5% CO2. The medium was replaced with DMSO (100 ꢀL/well) to dissolve the formazan
crystals. Cytotoxicity curves and IC50 (µM) concentrations (defined as the concentration of
drug that decreases MTT staining by 50% as compared to non-treated control cells), were
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fitted using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Each experiment
was repeated at least three times and all data expressed as mean ± SEM.
3. Result and discussion
3.1. Chemistry
N-heterocyclic carbene salts and Ag-NHC complexes were synthesized as shown and their
chemical structures are summarized in Scheme 1 and Scheme 2. N-heterocyclic carbene salts
were synthesized by reaction of allyl substituted benzimidazolium precursor with different
alkyl halides in DMF at 80 0C. Collapsed products were crystallized in
dichloromethane/diethyl ether for purification. Synthesized NHC salts were reacted with
Ag2O in dichloromethane at room temperature under the dark condition and their Ag(I)-NHC
complexes were obtained. Crude products were purified by crystallization in
dichloromethane/diethyl ether. The structure of all new compounds characterized by
elemental analysis, FT-IR, 1H NMR and, 13C NMR spectroscopy. And for compound 2d was
made X-ray crystallography studies.
H
N
N
N
+
N
N
THF
DMF
X-
+ NaH
+
+
Cl
R-X
N
R
1a-d
N
+
N
N
+
N
N
+
N
N
+
N
Cl-
Cl-
Cl-
Br-
1c
1d
1b
1a
Scheme 1. Synthesis of 1-allyl substituted N-heterocyclic carbene salts (1a-d)
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Scheme 2. Synthesis of Ag-NHC complexes (2a-d)
The FT-IR spectra for all NHC salts (1a-d) showed the aliphatic and aromatic C-H stretching
vibrational bands at around 2867–2997 cm-1 and 3015-3185 cm-1, respectively. C=C
stretching vibration modes of all compounds were seen at around 1609-1680 cm-1.
Benzimidazole ring C=N vibrations of benzimidazolium salts (1a-d) were assigned at around
1545-1556 cm-1. These vibrations have shown a shift in the Ag-NHC complex (2a-d) at
around 1387-1394 cm-1. This negative shift is because of the electropositive metal center
which pulls electron density towards itself and as a result C=N vibrations shifts to the lesser
energy region in the complex.
NMR spectra of all the compounds were analyzed in d-CDCl3. In the 1H NMR spectra, acidic
protons (NCHN) for benzimidazolium salts (1a-d) were seen at 11.85, 11.55, 11.36 and
10.91 ppm, respectively, as a characteristic sharp singlet. Formation of Ag-NHC complexes
(2a-d) were evidenced by the disappearance of the acidic proton peak. For salts (1a-d),
aromatic protons appeared in the range 7.19-7.75 ppm. Allylic N-CH2 protons at 1a and 1b
salts came to at 5.35 and 5.34 ppm as the doublets. For 1a and 1, protons at the terminal -CH2
and -CH- protons on allyl group were observed at 5.54-5.50 and 6.08-6.16 ppm as multiplet
for 1a and at 5.45-5.53 and 6.11-6.23 ppm as the multiplets for 1b. N-CH2 protons and
terminal -CH2 protons of allyl group belonging to 1c and 1d salts came to together as
multiplet at 5.32-5.37 and 5.29-5.38 ppm, respectively. The –CH protons of allyl groups at 1c
and 1d salts were detected at 5.99-6.09 and 5.96-6.04 ppm as a multiplet. Benzylic -CH2
protons at benzimidazolium salts (1a-d) were observed at 5.84, 5.85, 5.79 and 5.77 ppm as a
sharp singlet, respectively. Methyl protons of benzimidazolium salts (1a-d) appeared at 1.19
ppm as a multiplet, at 1.26 ppm as a singlet, at 2.25 and 2.32 ppm as two singlets and at 2.18,
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13
2.21 and 2.23 ppm as three singlets, respectively. In the C NMR spectra, aromatic carbons
on benzimidazolium salts (1a-d) were detected in the range at 113.7-152.4 ppm. NCHN
carben carbon on salts (1a-d) signaled at 143.6, 142.7, 143.6 and 143.3 ppm, respectively.
Terminal -CH2 carbons of allyl group on 1a and 1b gave a peak at 113. 7 ppm and the same
carbons on 1c and 1d gave a peak at 113.6 ppm. For 1a-d salts, -NCH2 and –CH- carbons on
allyl groups appeared in the range at 50.1-51.3 ppm and 121.0-122.0 ppm, respectively.
Benzylic carbons of benzyl groups were observed at 50.2, 50.2, 47.2 and 48.9 ppm for 1a-d,
respectively. Observed peaks in the range at 17.0-31.2 ppm were attributed to methyl carbons
1
on the salts. H NMR spectra and 13C NMR spectra of benzimidazolium salts (1a-d) are
13
compatible with each other. After complexation, 1H NMR and C NMR spectra of Ag-NHC
complexes were observed to almost close of spectra corresponding benzimidazolium salts,
except for two characteristic peaks. The first of them was the disappearance of an acidic
proton peak at spectra of Ag-NHC complexes which confirmed that the Ag-NHC complexes
(2a-d) were synthesized. The second one was NCN carben resonance on the Ag-NHC
complexes (2a-d) which shifted much downfield region compared to the corresponding the
benzimidazolium salts (1a-d). Carben peaks of Ag-NHC complexes 2a and 2b were observed
at 189.0 and 191.2 ppm slightly and was not observed for 2c and 2d. These values, and the
lack of the carben peak are in agreement with reported data for similar Ag-NHC
complexes.[26]
3.2. X-ray crystallography for 2d
Crystal data and collection details of chloro[1-allyl-3-(2,3,4,5,6-pentamethylbenzyl)
benzimidazole-2-ylidene]silver(I), 2d, (Figure 1) are given in Table 1. The relevant
crystallographic for 2d has been deposited at Cambridge Crystallographic Data Center
(CCDC) with No. 1588742. See the Supplementary Materials for more details on complete
NMR, FT-IR, LC-MS data and X-ray crystallography studies.
Table 1. The crystal data and collection details of 2d
Crystal data of chloro[1-allyl-3-(2,3,4,5,6-pentamethylbenzyl)benzimidazole-2-ylidene]silver(I)
Chemical formula
C22H26AgClN2
Mr
461.77
Crystal system, space group
Temperature (K)
a, b, c (Å)
Monoclinic, P21/c
296
10.9722 (15), 14.5472 (19), 13.1839 (19)
99.309 (4)
2076.6 (5)
β (°)
V (Å3)
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Z
4
Radiation type
Mo Kα
µ (mm-1)
1.11
Crystal size (mm)
Tmin, Tmax
0.44 × 0.40 × 0.37
0.570, 0.746
12120, 4011, 3350
No. of measured, independent and
observed [I > 2σ(I)] reflections
Rint
0.034
(sin θ/λ)max (Å-1)
R[F2 > 2σ(F2)], wR(F2), S
No. of reflections
No. of parameters
No. of restraints
H-atom treatment
∆ρmax, ∆ρmin (e Å-3)
0.617
0.031, 0.087, 1.04
4011
241
8
H-atom parameters constrained
0.34, -0.36
Figure 1: PLATON drawing of the 2d compound with the atomic numbering scheme.
Displacement ellipsoids are drawn at 50 % probability level. The H-atoms are shown as small
circles of arbitrary radii.
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3.3. In vitro anticancer effects of NHC salts and Ag complexes
The cytotoxicity of the salts (1a-d) and Ag-complexes (2a-d) in DU-145 human prostate
cancer cells, MCF-7, MDA-MB-231 breast cancer cells, and L-929 normal adipose cells were
done by assessing their ability to alter 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) staining over a period of 24 h, 48 h and 72 h.[27] The cytotoxicity of the
complexes was found to be concentration- and time-dependent (Figure 2-5).
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Figure 2. Cell proliferation was determined by MTT assay. DU-145 cells treated with 1, 5, 10 and 20 µM of salts (1a-d) and Ag-NHC
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).
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Figure 3. Cell proliferation was determined by MTT staining. 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).
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Figure 4. Cell proliferation was determined by MTT staining. 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).
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Figure 5. Cell proliferation was determined by MTT staining. L-929 cells treated with 1, 5, 10 and 20µM 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).
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The data were used to calculate IC50 values. These values ranged up 1 to 20 µM, suggesting
that Ag(I)-NHC complexes exhibited anticancer activity against all cell lines to a different
degree (Table 2). As shown in Table 2, the IC50 values varied among different complexes and
appeared to be cell-line dependent. Complexes 2a-d show marked potencies compared with
the respective the salts 1a-d. The results indicated that these complexes have potent activity
against the three cancer cell lines studied, and the efficacy of complex 2d is much higher than
that of complexes 2a, 2b and 2c.
In general, on the IC50 followed the order of 2d>
2c>2a>2b>.
Table 2. Inhibition of cell viability (IC50 ± SEM, µM) of the free salts (1a-d) and Ag-
complexes (2a-d) on DU-145 (prostate carcinoma), MCF-7, MDA-MB-231(breast
carcinoma) and L-929 (non-cancer cell lines)a after 48 h of incubation
Compounds
DU-145
MCF-7
MDA-MB-231
L-929a
48 h (IC50, µMb)
4.96 ± 0.35
3.62 ± 0.18
4.91 ± 0.20
2.99 ± 0.16
1.69 ± 0.21
2.09 ± 0.43
1.41 ± 0.55
1.21 ± 0.23
6.17 ± 0.84
2.61 ± 0.18
5.10 ± 0.29
1.15 ± 0.48
2.14 ± 0.13
2.19 ± 0.22
<1
10.4 ± 0.33
3.85 ± 0.31
10.8 ± 0.17
6.18 ± 0.58
1.10 ± 0.19
1.57 ± 0.19
<1
>20
15.62 ± 0.33
1a
1b
1c
1d
2a
2b
2c
2d
>20
14.7 ± 0.40
14.2 ± 0.22
8.93 ± 0.12
4.67 ± 0.11
3.56 ± 0.20
<1
<1
aNon-cancer cells, b Cytotixicity as IC50 for each cell lines is in average of three independent experiments after
treatment for 48 h (± SEM).
Compared with healthy cells, it was found that 2a is 8.4 fold, 6.63-fold and more than 12.9-
fold more toxic, 2b is 1.88-fold, 4-fold and more than 5.68 fold more cytotoxic to DU-145,
MCF-7, and MDA-MB-231 cancer cells, respectively. 2c is more than 4.67-fold more
cytotoxic to MCF-7 and MDA-MB-231 cancer cells and more than 3.31-fold to DU-145
cells. 2d is more than 3.56-fold more cytotoxic to MCF-7 and MDA-MB-231 cancer
cells more than 2.94-fold on DU-145 cells, for 48 h.
Complexes 2c and 2d, with a -CH3 substituent group on the benzene ring, were more
cytotoxic than the other complexes. The efficacy of 2d was higher than 2c, which may be
attributed to the increased -CH3 substituent group. Moreover, the additional -CH3 group in the
benzene ring can also contribute to the lipophilicity of the complexes. The higher efficacy of
complex 2c and 2d may also be related to their faster rate of hydrolysis. The cytotoxicities of
2a were higher than 2b, which may be attributed to the isopropyl benzyl substituent on the
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benzene ring more than the tert-butyl benzyl substituent on the benzene ring. All Ag-
complexes exhibited increased efficacy against MCF-7 cells than cisplatin (IC50 values; 3.19
ꢀM) after 48 h [28]. This correlates to the reports that the silver in these complexes play
important role in the antitumor activity of these complexes. In addition, the IC50 value of salts
and Ag-complexes for L-929 mouse adipose fibroblasts (non-cancer cells) were all above the
IC50 value of the cancer cell lines tested, which suggests that the salts and Ag-complexes act
very specifically on cancer cells. Interestingly, all of the complexes exhibit a certain degree of
selectivity towards cancer cells.
4. Conclusion
In the present work, four benzimidazole-based N-heterocyclic carbene and their Ag(I)-NHC
complexes have been designed, synthesized and characterized. The assessment of efficacy
using in vitro studies indicate that these complexes inhibit cancer cell growth in at least three
different cells (DU-145, MCF-7 and MDA-MB-231) especially 2c and 2d. Notably, these
complexes showed lower efficacy against non-cancer derived from mouse adipose (L-929
cells). The findings of the present work conclude that the isopropyl benzyl substituents on
the benzene ring increased the efficacy of these complexes to a greater degree than tert-butyl
benzyl substituents on the benzene ring. In addition, the additional CH3 group in the benzene
ring can also contribute to the anticancer activity of these complexes. These results will direct
the focus of our future research program to modifications of these derivatives, which may be
necessary to further improve efficacy.
Appendix A. Supplementary data
CCDC 1588742 contain the supplementary crystallographic data for the compounds
reported in this article. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures.
Acknowledgments
The authors thank the Scientific and Technological Research of Turkey (TÜBITAK-BIDEB-
2218), the National Research Fellowship Programme for grants to N. Ş.
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Highlights
-N-heterocyclic carbene ligands (NHC) and their Ag(I)-NHC complexes have been
synthesized.
-All the ligands and complexes were characterized by spectroscopic methods.
-All compounds have been applied for the anticancer activity on MCF-7, MDA-MB-231
breast cancer and DU-145 prostate cancer cells.
-The Ag(I)-NHC complexes showed a dose and time-dependent cytotoxic activity against all
cell lines.