Synthesis, characterization and anti-proliferative activity of propylene linked bis-benzimidazolium salts and their respective dinuclear Silver(I)-N-heterocyclic carbene complexes

Article abstract of DOI:10.1016/j.jorganchem.2017.04.011

A new series of propylene linked bis-benzimidazolium salts, 1–4 were reacted with Ag₂O to facilitate the formation of their respective dinuclear Ag(I)-N-heterocyclic carbene (NHC) complexes, 5–8 respectively. All compounds were fully characterized by elemental analyses, ¹H NMR, ¹³C NMR and FTIR spectroscopy techniques. The molecular structures of compounds 1 and 6 were elucidated through single crystal X-ray diffraction analyses. Both salts and complexes were tested for anti-cancer potential against breast cancer (MCF-7) cell line. The IC₅₀ values are in the range of 7?±?1–18?±?3?μM show that the complexes 5–8 possess moderate antiproliferative effect while their respective salts 1–4 are found to be inactive against MCF-7?cell line.

Full text of DOI:10.1016/j.jorganchem.2017.04.011

Accepted Manuscript
Synthesis, characterization and anti-proliferative activity of propylene linked bis-
benzimidazolium salts and their respective dinuclear Silver(I)-N-heterocyclic carbene
complexes
Sunusi Y. Hussaini, Rosenani A. Haque, Patrick O. Asekunowo, A.M.S. Abdul Majid,
M. Taleb Agha, Mohd. R. Razali
PII:
S0022-328X(17)30238-3
10.1016/j.jorganchem.2017.04.011
JOM 19898
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 9 March 2017
Revised Date: 10 April 2017
Accepted Date: 12 April 2017
Please cite this article as: S.Y. Hussaini, R.A. Haque, P.O. Asekunowo, A.M.S. Abdul Majid, M. Taleb
Agha, M.R. Razali, Synthesis, characterization and anti-proliferative activity of propylene linked bis-
benzimidazolium salts and their respective dinuclear Silver(I)-N-heterocyclic carbene complexes,
Journal of Organometallic Chemistry (2017), doi: 10.1016/j.jorganchem.2017.04.011.
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ACCEPTED MANUSCRIPT
Graphical Abstract
-
2PF6
N
N
N
N
N
N
N
N
R
R
BrCH2CH2CH2Cl
reflux / 24 h
1. Ag2O in MeOH / 36 h
2. KPF6 in MeOH / 3 h
R
R
DMSO
Br-R
Ag
Ag
KOH, stir.
N
NH
N
N
N
N
N
N
R
R
R
Br-
Cl-
KPF6 in MeOH / 3 h
-
2PF6
R = ethyl (5), propyl (6), butyl (7) or pentyl (8)
N
N
N
N
R
R
R = ethyl (1), propyl (2), butyl (3) or pentyl (4)

ACCEPTED MANUSCRIPT
Synthesis, characterization and anti-proliferative activity of propylene linked bis-
benzimidazolium salts and their respective dinuclear Silver(I)-N-heterocyclic carbene
complexes
Sunusi Y. Hussaini,a,c Rosenani A. Haque,a Patrick O. Asekunowo,a A.M.S. Abdul Majid,b
Taleb Agha M.b and Mohd. R. Razalia‡
aSchool of Chemical Sciences, Universiti Sains Malaysia, Malaysia.
bEMAN Research and Testing Laboratory, School of Pharmacy, Universiti Sains Malaysia, Malaysia.
cDepartment of Chemistry, Kano University of Science and Technology Wudil, Kano, Nigeria.
‡Correspondence email: mohd.rizal@usm.my
Abstract
A new series of propylene linked bis-benzimidazolium salts, 1-4 were reacted with Ag2O to
facilitate the formation of their respective dinuclear Ag(I)-N-heterocyclic carbene (NHC)
complexes, 5-8 respectively. All compounds were fully characterized by elemental analyses,
1H-NMR, 13C-NMR and FTIR spectroscopy techniques. The molecular structures of
compounds 1 and 6 were elucidated through single crystal X-ray diffraction analyses. Both
salts and complexes were tested for anti-cancer potential against breast cancer (MCF-7) cell
line. The IC50 values are in the range of 7±1-18±3 µM show that the complexes 5-8 possess
moderate antiproliferative effect while their respective salts 1-4 are found to be inactive
against MCF-7 cell line.
Keywords: Dinuclear Ag(I)-NHC complexes; Anti-cancer activity; N-heterocyclic carbene.
1

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1. Introduction
N-heterocyclic carbenes (NHCs) are generally originated from the so-called persistent
carbenes which are stabilized by two π-donor nitrogen to formed stable compounds of
divalent carbon carbene atom [1]. The NHCs are known as a strong σ-donating and weak π-
accepting when connected with metal ions [2-4]. Metal-NHC complexes are accessible by
different routes, but the most common route is deprotonation of azolium precursors with
simple metal salt or coordination compounds (neutral, cation and anion) in the presence of
strong base, such as potassium tert-butoxide [5]. From literature, synthesized metal-NHC
complexes adopt different structural diversity have been investigated for various applications
due to their high and ease of derivatization. These thus allow the metal-NHC complexes to be
explored for transmetalation, catalysis and drugs developments [6-9].
The newly emerging interest of medicinal and transmetalation applications of Ag(I)-
NHC complexes led an extensive investigations due to their ease of synthesis, ease of
handling and stability towards air and moisture. These properties influenced the highly
abundance of Ag(I)-NHC in literature that concentrated in synthesis and applications in
bioorganometallic as well as transmetalation reactions [8, 10, 11]. The biological applications
of Ag(I)-NHC complexes are primarily based on the silver element and its salts. The ionic
silver and silver salts compounds were used as antimicrobial agents and used to prevent
infections in wound dressing treatment [12,13]. Meanwhile, the Ag(I)-NHC complexes show
high effect against a different type of cancer cell line due to high stability and slow release of
silver ions from Ag(I)-NHC complexes to the affected area over a period of time; eventually
the cytoplasm constituents leak and cause the death of cells [14].
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The higher nuclearity of Ag(I)-NHC complexes with multidentate benz/imidazolium-based
NHC ligands are postulated to be more stable compared with mononuclear Ag(I)-NHCs
complexes of monodentate ligands [15]. The later complexes are also documented to be more
active and betterment biological applications in which two Ag(I) ions are slowly released for
better activity in high nuclearity Ag(I) complexes [16]. Furthermore, the multidentate ligands
precursor can also be anionic tethered NHCs which able to compensate the charges of the
coordinated metal [17]. Bis-benzi/imidazolium salts bearing numerous spacers or linkers
between the two benz/imidazolium moieties are linked, for examples by alkylene [18],
xylylene [19] or pyridyl [20]. These spacers provide an extra stability to their respective
dinuclear Ag(I)-NHC complexes in which the scaffolds formed behaving as an agents in
biological and medicinal applications [21-23]. Several works were reported on synthesis of
linked bis-benz/imidazolium salts and their respective dinuclear Ag(I)-NHC complexes with
potential activities against various types of cancer cell lines and showed effective results in
their activity due to the present of two Ag(I) ions [24]. Herein, our main focus is to develop
the benzimidazole-based dinuclear Ag(I)-NHC complexes of propylene linker with various
alkyl group as substituents to investigate their efficacy of anti-proliferative differences against
breast cancer cell line (MCF-7).
2. Experimental
2.1 Material and Measurements
All chemicals and solvents were used as received. The NMR spectra were recorded on
Bruker 500 MHz spectrometer at ambient temperature in either DMSO-d6 or acetonitrile-d3
using TMS as an internal standard. FT-IR spectra were recorded on Perkin Elmer 200 system
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spectrometer. Elemental analysis was carried out on Perkin Elmer Series II, 2400 micro-
analyzer. Melting points were assessed using Stuart Scientific SMP-1 (UK) instrument.
2.2 Syntheses
2.2.1. Synthesis of 1,1-propylenebis(3-ethylbenzimidazolium)bis(hexafluorophosphate) (1)
The mixture of benzimidazole (1.00 g, 8.46 mmol) and KOH (0.95 g, 17.00 mmol) in
DMSO (30 mL) was stirred for 30 minutes at room temperature.1-bromoethane (0.93 g, 8.46
mmol) was added dropwise and the mixture was stirred at room temperature for 3 h. The
solution mixture was then poured into water (250 mL) and extracted with chloroform (4 x 25
mL). The product was filtered through four plies of Whatman filter papers and clear solution
of N-ethylbenzimidazole was obtained. The mixture of N-ethylbenzimidazole (1.50 g, 10.30
mmol) and 1-bromo-3-chloropropane (0.81 g, 5.20 mmol) in acetonitrile (30 mL) was
O
refluxed at 80 C for 24 h and then the solvent was evaporated under reduced pressure to
yield a white crystalline solid of 1,1-propylenebis(3-ethylbenzimidazolium)bromidechloride
salt which was converted to its hexafluorophophate salt by metathesis using KPF6 (0.95 g,
5.20 mmol) in methanol (20 mL). The mixture was stirred at room temperature for 3 h, which
was then solvent was removed under reduced pressure. The obtained white powder was
washed with distilled water (3 x10 mL). The product was recrystallized from acetonitrile by
o
1
ether diffusion. Yield: 2.19 g (67%), mp: 183-186 C. H NMR (500 MHz, DMSO-d6): 1.58
(6H, t, J = 7.5 Hz, 2 x CH3); 2.66 (2H, m, NCH2CH2CH2N); 4.52 (4H, q, 2 x NCH2-CH3);
4.67 (4H, t, J =7.0 Hz, 2 x NCH2CH2); 7.74 (2H, m, Ar-H); 8.11(8H, m, 2 x Ar-H); 9.73 (2H,
s, 2 x NCHN).
13C {1H} NMR (125 MHz, DMSO-d6): 13.98 (CH3), 27.93
(NCH2CH2CH2N); 42.13 (NCH2CH3); 43.92 (NCH2CH2); 113.66, 126.63, 130.98, 131.12
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(benzimidazole Ar-C); 141.88 (NCHN). FTIR (KBr disc) in cm-1, 3168, 3117 (C-Harom);
2984, 2889 (C-Halph); 1577 (C=N); 1290 (C-N). Anal. Calc. for C21H26F12N4P2: C, 40.39; H,
4.17; N, 8.97%. Found: C, 40.35; H, 3.93; N, 8.98 %
2.2.2. Synthesis of 1,1-propylenebis(3-propylbenzimidazolium)bis(hexafluorophosphate) (2)
The procedure is similar to that in 2.2.1 but using 1-bromopropane (1.05 g, 8.46 mmol)
instead of 1-bromoethane. Yield:1.62 g (80%), mp: 185-188 oC. 1H NMR (500 MHz, DMSO-
d6): 0.97 (6H, t, J = 7.0 Hz, 2 x CH3); 1.96 (4H, m, 2 x CH2CH3); 2.64 ( 2H, m,
NCH2CH2CH2N); 4.46 (4H, t, J = 7.5 Hz, 2 x NCH2-R); 4.66 (4H, t, J =7.5 Hz, 2 x
NCH2CH2); 7.74 (2H, m, Ar-H); 8.15 (8H, m, 2 x Ar-H) 9.75 (2H, s, 2 x NCHN). 13C {1H}
NMR (125 MHz, DMSO-d6): 10.68 (CH3); 22.02 (CH2CH2CH3); 27.95 (NCH2CH2CH2N );
43.96 (NCH2-R); 48.17 (NCH2CH2); 113.77, 126.62, 131.09, 131.19 (benzimidazole Ar-C);
142.17 (NCHN). FTIR (KBr disc) in cm-1, 3162, 3094 (C-Harom); 2974, 2885 (C-Halph); 1574
(C=N); 1265 (C-N). Anal. Calc. for C23H30F12N4P2: C, 42.34; H, 4.60; N, 8.59%. Found: C,
42.25; H, 4.56; N, 8.57%
2.2.3. Synthesis of 1,1-propylenebis(3-butylbenzimidazolium)bis(hexafluorophosphate) (3)
The procedure is similar to that in 2.2.1 but using 1-bromobutane (1.16 g, 8.46 mmol)
instead of 1-bromoethane Yield: 2.13 g (77%), mp: 187-189 oC. 1H NMR (500 MHz, DMSO-
d6) 0.96 (6H, t, J = 7.5 Hz, 2 x CH3); 1.40 (4H, m, 2 x CH2CH3); 1.91 (4H, m, 2 x
CH2CH2CH3); 2.63 ( 2H, m, NCH2CH2CH2N); 4.50 (4H, t, J = 7.5 Hz, 2 x NCH2-R); 4.67
(4H, t, J = 7.5 Hz, 2 x NCH2CH2); 7.74 (4H, m,2 x Ar-H); 8.13 (4H, m, 2 x Ar-H); 9.81 (2H,
s, 2 x NCHN). 13C {1H} NMR (125 MHz, DMSO-d6): 13.36 (CH3); 19.07 (CH2CH3); 27.94
(CH2CH2CH3); 30.53 (NCH2CH2CH2N ); 43.95 (NCH2-R); 46.50 (NCH2CH2); 113.54,
5

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126.61, 131.09, 131.16 (benzimidazole Ar-C); 142.13 (NCHN). FTIR (KBr disc) in cm-1,
3160, 3103 (C-Harom); 2964, 2939 (C-Halph); 1570 (C=N), 1298 (C-N). Anal. Calc. for
C25H34F12N4P2: C, 44.12; H, 5.00; N, 8.24%. Found: C, 43.67; H, 4.58; N, 8.22%
2.2.4. Synthesis of 1,1-propylenebis(3-pentylbenzimidazolium)bis(hexafluorophosphate) (4)
The procedure is similar to that in 2.2.1 but using 1-bromopentane (1.28 g, 8.46 mmol)
o
1
instead of 1-bromoethane. Yield: 2.24 g (79%), mp: 190-192 C. H NMR (500 MHz,
DMSO-d6): 0.90 (6H, t, J = 7.0 Hz, 2 x CH3); 1.36 (8H, m, 4 x CH2); 1.93 (4H, m, 2 x
NCH2CH2); 2.64 (2H, m, NCH2CH2CH2N ); 4.64 (4H, t, J = 7.5 Hz, 2 x NCH2-R); 4.66 (4H,
t, J = 7.0 Hz, 2 x NCH2CH2); 7.74 (2H, m, 2 x Ar-H); 8.13 (2H, m, 2 x Ar-H); 9.74 (2H, s, 2
x NCHN). 13C {1H} NMR (125 MHz, DMSO-d6): 13.70 (CH3); 21.58 (CH2CH3); 27.85
(CH2CH2CH3); 27.93 (NCH2CH2); 28.20 (NCH2CH2CH2N); 43.95 (NCH2-R);
46.71(NCH2CH2); 113.53, 126.61, 131.07, 131.16 (benzimidazole Ar-C); 142.11 (NCHN).
FTIR (KBr disc) in cm-1, 3160, 3102 (C-Harom); 2965, 2937 (C-Halph); 1568 (C=N), 1265 (C-
N). Anal. Calc. for C27H38F12N4P2: C, 45.77; H, 5.37; N, 7.91 %. Found: C, 45.55; H, 4.97;
N, 7.87 %.
2.2.5.
Synthesis
of
1,1-propylenebis(3-ethylbenzimidazol-2-ylidene)disilver(I)
bis(hexafluorophosphate) (5)
Compound 1 (0.50 g, 1.60 mmol) was dissolved in methanol (50 mL) along with Ag2O
(0.76 g, 3.30 mmol) with exclusion of light by covering using aluminum foil. The mixture
was stirred for 36 h at room temperature and then filtered. The obtained clear solution was
converted to its hexafluorophosphate by presence of KPF6 (0.60 g, 3.20 mmol) in a mixture of
methanol/water (40 mL). The white precipitate appeared was collected and washed with distill
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water (3 x 5 mL) to facilitate the formation of grey powder after air-dried. Product of grey
powder was further recrystallized from acetonitrile to give white crystalline solid. Yield: 0.64
o
1
g (35%). mp: 211-213 C. H NMR (500 MHz, acetonitrile-d3): 1.41 (6H, t, J = 7.5 Hz, 2 x
CH3); 2.76 (2H, m, NCH2CH2CH2N); 4.52 (4H, q, 2 x NCH2-CH3); 4.81 (4H, t, J = 7.0 Hz 2
x NCH2CH2); 7.45 (2H, m, 2 x Ar-H); 8.13 (8H, m, 2 x Ar-H). 13C {1H} NMR (125 MHz,
acetonitrile-d3): 14.96 (CH3), 28.27 (NCH2CH2CH2N); 46.08 (NCH2CH3); 56.87
(NCH2CH2); 110.95, 123.11, 131.72, 132.05 (benzimidazole Ar-C); 187.05 (C-Ag). FTIR
(KBr disc) in cm-1, 3624, 3406 (C-Harom); 2990, 2880 (C-Halph); 1465 (C=N); 1168 (C-N); 750
(P-F). Anal. Calc. for C42H48Ag2F12N8P2: C, 41.39; H, 4.10; N, 9.01%. Found: C, 41.32; H,
4.11; N, 8.93 %.
2.2.6
Synthesis
of
1,1-propylenebis(3-propylbenzimidazol-2-ylidene)disilver(I)
bis(hexafluorophosphate) (6)
The procedure is similar to that in 2.2.5 but using compound 2 (0.50 g, 1.60 mmol)
instead of compound 1 to obtain 6 as white crystalline solid. Single crystals appropriate for
single crystal X-Ray diffraction studies were obtained by diffusion of diethyl ether into the
O
1
salt solution in acetonitrile at room temperature. Yield: 1.02 g (52%) mp: 218-220 C . H
NMR (500 MHz, acetonitrile-d3): 0.86 (6H, t, J = 7.0 Hz, 2 x CH3); 1.87 (4H, m, 2 x
CH2CH3); 2.94 ( 2H, m, NCH2CH2CH2N); 4.34 (4H, t, J = 7.5 Hz, 2 x NCH2-R); 4.75 (4H, t,
13
J = 6.5 Hz 2 x NCH2CH2); 7.48 (8H, m, Ar-H); 7.67 (8H, m, 2 x Ar-H). C{1H} NMR (125
MHz, acetonitrile-d3): 12.58 (CH3); 19.52 (CH2CH2CH3); 30.35 (NCH2CH2CH2N); 50.81
(NCH2-R); 57.57 (NCH2CH2); 118.59, 132.92, 133.06, 133.39 (benzimidazole Ar-C); 187.74
(Ag-C,d). FTIR (KBr disc) in cm-1, 3056 (C-Harom); 2969, 2942 (C-Halph); 1481(C=N); 1182
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(C-N); 749 (P-F). Anal. Calc. for C46H56Ag2F12N4P2: C, 45.04; H, 4.57; N, 9.13%. Found: C,
44.93; H, 4.49; N, 9.18%
2.2.7
Synthesis
of
1,1-propylenebis(3-butylbenzimidazol-2-ylidene)disilver(I)
bis(hexafluorophosphate) (7)
The procedure is similar to that in 2.2.5 but using compound 3 (0.50 g, 1.50 mmol)
instead of compound 1 to obtain 7 as white crystalline solid. Yield: 0.97g (51%) mp: 219-223
oC. 1H NMR (500 MHz, acetonitrile-d3): 0.79 (6H, t, J = 7.5 Hz, 2 x CH3); 0.99 (4H, m, 2 x
CH2CH3); 1.41 (4H, m, 2 x CH2CH2CH3); 2.82 ( 2H, m, NCH2CH2CH2N); 4.56 (4H, t, J =
7.0 Hz, 2 x NCH2-R); 4.72 (4H, t, J = 7.0 Hz, 2 x NCH2CH2); 7.52 (4H, m, 2 x Ar-H); 7.71
(4H, m, 2 x Ar-H). 13C {1H} NMR (125 MHz, acetonitrile-d3): 12.58 (CH3); 19.52
(CH2CH3); 29.63 (CH2CH2CH3); 30.52 (NCH2CH2CH2N); 50.81 (NCH2-R); 57.57
(NCH2CH2); 111.83, 118.59, 132.64, 133.39 (benzimidazole Ar-C); 188.37 ( C-Ag). FTIR
(KBr disc) in cm-1, 3061 (C-Harom); 2961, 2874 (C-Halph); 1481 (C=N), 1182 (C-N); 747 (P-F).
Anal. Calc. for C50H64Ag2F12N8P2: C, 46.80; H, 4.99; N, 8.74%. Found: C, 47.54; H, 4.89; N,
8.56%.
2.2.8.
Synthesis
of
1,1-propylenebis(3-pentylbenzimidazol-2-ylidene)disilver(I)
bis(hexafluorophosphate) (8)
The procedure is similar to that in 2.2.5 but using compound 4 (0.50 g, 1.40 mmol)
instead of compound 1 to obtain 8 as white crystalline solid. Yield: 0.89g (48%) mp: 220-223
oC. 1H NMR (500 MHz, acetonitrile-d3): 0.72 (6H, t, J = 7.0 Hz, 2 x CH3); 1.21 (8H, m, 4 x
CH2); 1.82 (4H, m, 2 x NCH2CH2); 2.95 ( 2H, m, NCH2CH2CH2N ); 4.29 (4H, t, J = 7.5 Hz,
2 x NCH2-R); 4.69 (4H, t, J = 7.0 Hz, 2 x NCH2CH2); 7.52 (2H, m, 2 x Ar-H); 7.71 (2H, m, 2
x Ar-H). 13C {1H} NMR (125 MHz, acetonitrile-d3): 12.90 (CH3); 21.78 (CH2CH3); 27.95
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(CH2CH2CH3); 28.29 (NCH2CH2); 29.61 (NCH2CH2CH2N); 48.96 (NCH2-R); 49.04
(NCH2CH2); 111.66, 124.21, 132.75, 133.49 (benzimidazole Ar-C); 187.46 (C-Ag,d). FTIR
(KBr disc) in cm-1, 3431 (C-Harom); 2956, 2861 (C-Halph); 1466 (C=N), 1118 (C-N); 743 (P-F).
Anal. Calc. for C54H72Ag2F12N8P2: C, 48.44; H, 5.38; N, 8.37%. Found: C, 48.77; H, 5.13; N,
8.10%.
2.3 Crystal structure determination
Single crystal X-ray diffraction data for 1 and 6 were collected on APEXII Duo CCD area-
detector diffractometer operating at 50 kV and 30 mA using Mo Kα radiation (λ = 0.71073 Å)
at 297 K. Data collection and reduction were performed using the APEX2 and SAINT
software. The SADABS software was used for absorption correction. Solution was obtained by
direct methods using SHELXS-2014 followed by successive refinements using full matrix least
squares method against F2 using SHELXL-2014 [25]. All non-hydrogen atoms were refined
anisotropically whereas hydrogen atoms were refined isotropically. In complex 6, the propyl
chains are disordered over two positions and were refined with 50:50 occupancies. The PF6
molecules in the lattice in compound 6 are also disordered over two positions of equal weight.
Crystal data and structure refinement details are tabulated in Table 1.
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Table 1: Crystal data and structure refinement details of 1 and 6.
1
6
Formula
Formula weight
Crystal system
Space group
a(Å)
C21H25F12N4P2
623.39
C46H56Ag2F12N8P2
1226.67
Monoclinic
C2/c
Triclinic
P-1
20.2339(18)
9.2233(7)
13.7804(10)
90(1)
13.0807(10)
13.1888(10)
17.0316(13)
97.903(3)
104.192(3)
93.580(3)
2807.4(4)
2
b(Å)
c(Å)
α(o)
β(o)
γ(o)
94.138(2)
90(1)
V(Å3)
2565.0(4)
4
Z
Dc
1.614
1.451
F(000)
1268
1240
Tot.; Uniq. Data
Rint
69514, 3154
0.031
92749, 12867
0.074
Nref, Npar
R, wR2, S
3154, 177
0.0380, 0.1006, 1.05
12867, 866
0.0761, 0.2636, 1.07
2.4 In Vitro Anticancer Activity
2.4.1 Preparation of cell culture
The MCF-7 cells were developed under optimal incubator conditions. Cells with the
confluence of 70 – 80% were selected for cell plating purposes. The cells were washed using
sterile phosphate buffered saline (PBS) (pH 7.4) three times. After washing the PBS was
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completely separated, than trypsin was added and distributed evenly onto cell surfaces. The
o
cells were incubated at 37 C in 5% CO2 for a minute. The flasks containing the cells were
gently tapped to aid cell segregation and observed under an inverted microscope. Trypsin
activity was inhibited by adding 5mL of fresh complete media (10% PBS). Cells were counted
and diluted to final concentration of 2.5 x 105 cells/mL and inoculated the cells were incubated
at 37 oC with an internal atmosphere of 5% CO2.
2.4.2 MTT Assay
Measurement of cell viability using the MTT assay was performed in 96-well plate using
the human breast cancer cell line (MCF-7). MTT reagent was freshly prepared at 5 mg in sterile
PBS, filtered through a 0.2 µm syringe filter, and further diluted in fresh culture medium to
obtain a final concentration of 500 µg/mL. The 1 × 104 cells in 100 µL media were seeded in 96-
wells plate, after 24 h the cells attached to the wells. Samples were added to each well within 100
µL media at concentrations of 100 µg/mL. The old culture medium was aspirated carefully using
a vacuum pump, and 200 µL of culture medium containing MTT reagent were added and
incubated for 3–5 h. At the end of the incubation, the supernatant was aspirated carefully and the
water-insoluble formazan salt was solubilized in 200 µL of DMSO per well. After 10 minutes of
incubation at 37 °C, optical density (OD) of the violet color was measured using a microplate
reader (Thermolab Systems 354, Finland) at a primary wavelength of 570 nm and a reference
wavelength of 650 nm. The percentage of viable cells at each concentration was calculated using
the equation:
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% cell inhibition = [1-((Abs Sample – Abs Blank) / (Abs Control – Abs Blank))] × 100
where Abs Sample is the absorbance of treated wells, Abs Blank is the absorbance of cell free
wells and Abs Control is the absorbance of treated wells with DMSO (1% v/v). Percent cell
inhibition was plotted against the concentrations of the sample to calculate this value.
3.0 Results and discussion
3.1 Synthesis
The bis-(benzimidazolium) salts (1-4) possessing propylene linker were prepared from
benzimidazole by stepwise alkylation reaction in the presence of KOH in DMSO for 3h at
room temperature in which the N-alkylbenzimidazole was obtained [26]. The N-
alkylbenzimidazole was refluxed with 1-bromo-3-chloropropane in 2:1 whereas after the
completion, the solvent was evaporated and the solid formed washed with diethyl ether and
dried at ambient temperature. The bromidechloride ion salts obtained were subsequently
-
converted to PF6 ions (Scheme 1); the later ionic compounds are known to be more stable in
air and moisture condition which is used for further analysis and anticancer activity. All
dinuclear Ag(I)-NHC complexes (5-8) were synthesized via in situ deprotonation reaction
between their respective salt and Ag2O in 1:2 molar ratio where Ag2O behaving as base to
deprotanate the acidic proton of carbene in the salts. The dinuclear Ag(I)-NHC complexes
were obtained in a good yield and the complexes were undergo metathesis reaction by added
the methanolic solution of KPF6 (Scheme 1) [27-29].
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DMSO
Br-R
KOH, stir.
N
NH
N
N
R
BrCH2CH2CH2Cl
reflux / 24 h
-
2PF6
KPF6 in MeOH / 3 h
N
N
N
N
N
N
N
N
R
R
R
R
Br-
Cl-
R = ethyl (1), propyl (2), butyl (3) or pentyl (4)
1. Ag2O in MeOH / 36 h
2. KPF6 in MeOH / 3 h
-
2PF6
N
N
N
N
N
N
N
N
R
R
R
R
Ag
Ag
R = ethyl (5), propyl (6), butyl (7) or pentyl (8)
Scheme 1: Synthesis of bis-(benzimidazolium) salts and their respective dinuclear Ag(I)-NHC
complexes.
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3.2 FT-IR Analysis
The FT-IR spectra of salts and their respective Ag(I) complexes can be used as
preliminary studies of successful synthesis[30]. The intensity bands can be seen as medium
intensity around 3000-3650 cm-1 is assigned for v(C-Harom) while the bands at 2800-2900cm-1
are corresponding for v(C-Halph) in both salts and their respective complexes. A sharp and
strong intensity bands in the range of 1560-1580 cm-1 as demonstrated in all salts indicating the
presence of v(C=N) vibrations that attributed to the benzimidazolium ring. Upon the
complexation with silver(I) ions, the aforementioned bands are shifted to the range of 1460-
1490 cm-1 and this observation occurs in all complexes [31].
3.3 1H- and 13C- NMR analysis
The formation of bis(benzimidazolium) salts and their respective dinuclear Ag(I)-NHC
complexes in each step was traced by 1H- and 13C-NMR analysis over the scan range of δ 0-12
1
and δ 0-200 ppm respectively. The H-NMR spectra shows a sharp singlet peak at region of δ
9.73 to 9.81 ppm ascribed to the benzimidazolium rings (NCHN) acidic proton, in accord with
our previous reports [32, 33]. The formation of complexes was confirmed by completely
disappearance of acidic (NCHN) proton in all complexes [34]. All other peaks remain the same
with no other significant changes for both salts and complexes; peaks resonance between δ 7.40
to 8.20 ppm was assigned to benzimidazolium ring protons while the peaks resonance range
from δ 4.46 to 4.67 ppm and δ 27.60 to 2.95 ppm are assigned to CH2-N and CCH2C
(propylene bridge) protons respectively. The presence of peaks from δ 0.60 to 1.95 ppm are
assigned to CH2CH3 protons [35].
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The 13C NMR spectra were further used to evaluate the characteristic of salts and Ag(I)-
NHC complexes. The spectra of salts displayed peaks at range of δ 140-142 ppm ascribed to
the benzimidazole ring carbon (NCHN) [36]. However, upon complexation with Ag(I), the
characteristic of parent doublet peak at the range of δ 187-188 ppm are observed for C-Ag
bonds with the average coupling constant of 187.7 Hz [37, 38]. All other peaks remain the
same with no other significant changes for both salts and complexes; peaks resonance between
δ 110.0 to 135.0 ppm was assigned to benzimidazolium ring carbon, the peaks range from δ
43.0 to 58.0 ppm and the peaks in the region of δ 27.0 to 30.0 ppm are assigned to CH2-N and
CCH2C (propylene bridge) respectively.
3.4 Crystal Structures
Compound 1 crystallizes in monoclinic space group C2/c with half of the molecule in the
asymmetric unit (Figure 1). The presence of two hexafluorophosphate anions in the lattice thus
balances the charge of the molecule. The N1-C1-N2 bond angle in this compound being
110.49(12)o, agreeable with those reported for similar type of ligand [38-40].
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Figure 1: Structure of salt 1 with the ellipsoids shown at 50% probability. The
i
hexafluorophosphate anions in the lattice omitted for clarity. Symmetry element used: = -x, y,
½-z.
Complex 6 crystalline in triclinic space group P-1 with the entire molecule in the
asymmetric unit (Figure 2(a)). The metal complex is balanced by the presence of two
hexafluorophosphate anions. The two Ag(I) ions are bridged by two bis-NHC ligand that display
a µ-ĸ1(C)Ag:ĸ1(C`)Ag` bridging mode. Both Ag(I) anions display a distorted linear geometry
with the angle of C1-Ag1-C37 and C14-Ag2-C24 is 173.2(3)o and 173.9(3)o, respectively (Table
2). The bond distance between the Ag(I) ions and the carbene carbon is in the range of 2.088(6) –
2.095(7) Å, and agreeable with those reported before [41]. The structure of this complex is
somewhat similar to that reported complex having similar ligand architecture, however with the
allyl group attached to the nitrogen atom [41], which leads to the similar molecular arrangement.
The face-to-face π-π supramolecular interactions are observed to play an important role in
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stabilizing the crystal structure of 6. Among them is the interaction between two adjacent
benzimidazolium rings with the separation being 3.416(5) Å (Figure 2(b)).
(a)
(b)
Figure 2: (a) Structure of complex 6 with the ellipsoids shown at 50% probability.
Hydrogen atoms and hexafluorophosphate anions in the lattice omitted for clarity. (b) The face-
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to-face π-π interactions between adjacent dinuclear complexes in crystal structure of 6.
Symmetry elements used: i= 1-x, 2-y, 1-z; ii= -x, 1-y, 1-z.
Table 2: Selected bond lengths [Å] and angles [o] for 6.
Ag1-C1
2.088(6)
2.090(7)
Ag1-C24
Ag1-C37
2.095(7)
2.095(6)
Ag1-C14
C1-Ag1-C37
C14-Ag2-C24
N1-C1-N2
173.2(2)
N3-C14-N4
N5-C24-N6
N7-C37-N8
105.8(6)
173.9(2)
105.9(5)
106.5(6)
105.9(6)
3.4 In vitro anticancer study
Preliminary screen of proligands 1-4 and their respective dinuclear Ag(I)-NHC
complexes, 5-8, respectively were evaluated using MTT assay on cancer cell line, MCF-7. The
comparative studies of dinuclear Ag(I)-NHC complexes and their respective proligands indicates
that complexes exhibited good inhibition against MCF-7, while their corresponding proligands
showed no inhibition. The antiproliferative potencies of the test complexes shows clearly that all
complexes (5-8) inhibited the proliferation of MCF-7 cells in a dose-dependent manner parallel
to standard drug used in this study, Tamoxifen (Figure 3). All complexes exhibited almost
similar IC50 in the range of 7±1 to 18±3 µM (Table 3). However, complex 6 appeared to have
enhanced cytotoxicity effect more than complexes 5 and 8, while complex 7 is less active
compared with the aforementioned complexes. In overall, all new complexes are comparable to
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or less potent than standard drug (IC50 = 11±2 µM, Tamoxifen). The present results are
consistent with other reported studies in the literature having linked bis-benzimidazole based
dinuclear Ag(I)-NHC complexes against various cancer cell line [41-42].
Figure 3. Dose-dependent antiproliferative effect of dinuclear Ag(I)–NHC complexes (5-8) on
breast cancer (MCF-7) cell line.
Table 3. The IC50 values of tested complexes against MCF-7 cell line.
Complex
IC50(µM)
MCF-7
SD±
9
7
1
1
3
5
6
7
18
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11
2
2
8
Tamoxifen
11
Different Ag(I) complexes have been studied with high efficacy in their biological
potential [43-44], and reported mechanism reaction studies of these complexes indicates that
available Ag+ released might bind and interact with the proteins involved in cell wall and finally
disrupt the cell functions [45]. This study suggested that incorporation of Ag(I)-NHC bond plays
a vital role in antiproliferative effects due to their strong σ-donor and weak π-accepting of NHC
ligands [46]. Furthermore, the activity of Ag+ ions are varies depends on structure, ionization,
solubility and stability of Ag(I)-NHC complexes [47]. However, both propylene linker and alkyl
carbon chain length substituents enhances the stability of complexes by improving the
lipophilicity of complexes. This stabilities then alleviate the transport of Ag+ into the cell
membrane and subsequently into the cell organelles where Ag+ may possibly impart the toxicity
effect by inhibiting cellular respiration and metabolism of biomolecules.
4.0 Conclusions
In conclusion, a series of new propylene linked bis-benzimidazolium salts and their
1
respective dinuclear Ag(I)-NHC complexes were synthesized and characterized by FTIR, H-
NMR, 13C-NMR and elemental analyses. Single crystal X-ray diffraction study of dinuclear
Ag(I) complexes reveals that the Ag(I) ions display a distorted linear geometry with significant
π-π interactions stabilized the entire crystal packing. All compounds were screened for their
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possible cytotoxicity on breast cancer, MCF-7 and the tested compounds show dose-dependent
cytotoxicity against breast cancer. From the anticancer results its shows that the dinuclear Ag(I)-
NHC complexes are active while their respective salts are not active.
5.0 Supplementary material
Crystallographic data for the structure in this work has been deposited at the Cambridge
Crystallographic Data center, 1527934 and 1527935 for 1 and 6, respectively. Copy of these
material can be obtained from the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK
(Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk/deposit).
6.0 Acknowledgements
The author thanks Universiti Sains Malaysia for the RUI Grant (1001/PKimia/811346). Sunusi
Y. Hussaini thanks Kano University of Science and Technology, Wudil, Nigerian for the study
sponsorship.
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