Effect of lipophilicity of wingtip groups on the anticancer potential of mono N-heterocyclic carbene silver(I) complexes: Synthesis, crystal structures and in vitro anticancer study

Article abstract of DOI:10.1002/aoc.3735

A series of symmetrically n-alkyl-substituted mono benzimidazolium salts with steady increase in n-alkyl chain length have been prepared by stepwise N-alkylation resulting in salts (1–8). The mono N-heterocyclic carbene (NHC)–Ag(I) complexes (9–16) derived from the respective salts were readily accessible by in situ deprotonation using Ag₂O. All the salts and the complexes were characterized using Fourier transform infrared, ¹H NMR, ¹³C NMR and elemental analyses. Furthermore, the structures of salts 3 and 7 and complex 16 were elucidated using X-ray crystallography, which established that this mono NHC–Ag(I) complex has a linear bis-carbene arrangement (C₂–Ag). The proligands and the respective Ag(I) complexes were studied for their in vitro anticancer potential against human colon cancer cell line (HCT-116) using 5-fluorouracil as a standard. From the IC₅₀ values of all the tested compounds, it can be postulated that there is an influential relationship between the increase in chain length of the wingtip n-alkyl groups and the anticancer potential. The proligands 4–8 and their respective complexes 12–16 with long n-alkyl chain lengths (n?=?6–10) showed better IC₅₀ values (0.3–3.9 μM) than the standard drug with the complexes displaying markedly better antiproliferation activity against HCT-116 cell line than the respective proligands and the standard drug (IC₅₀?=?10.2 μM).

Full text of DOI:10.1002/aoc.3735

Received: 21 October 2016
DOI 10.1002/aoc.3735
Revised: 20 November 2016
Accepted: 10 December 2016
F U L L PA P E R
Effect of lipophilicity of wingtip groups on the anticancer potential
of mono N‐heterocyclic carbene silver(I) complexes: Synthesis,
crystal structures and in vitro anticancer study
1
1
1
2
Tabinda Fatima | Rosenani A. Haque | Mohd R. Razali | Ashfaq Ahmad |
3
4
3
Muhammad Asif | Mohamed B. Khadeer Ahamed | A. M. S. Abdul Majid
1
School of Chemical Sciences, Universiti
A series of symmetrically n‐alkyl‐substituted mono benzimidazolium salts with
steady increase in n‐alkyl chain length have been prepared by stepwise N‐alkylation
resulting in salts (1–8). The mono N‐heterocyclic carbene (NHC)–Ag(I) complexes
Sains Malaysia, 11800 USM, Penang,
Malaysia
2
School of Pharmaceutical Sciences,
Universiti Sains Malaysia, 11800 USM,
Penang, Malaysia
(
9–16) derived from the respective salts were readily accessible by in situ deproton-
3
ation using Ag O. All the salts and the complexes were characterized using Fourier
EMAN Research and Testing Laboratory
2
1
13
School of Pharmacy, Universiti Sains
Malaysia, 11800 USM, Penang, Malaysia
transform infrared, H NMR, C NMR and elemental analyses. Furthermore, the
structures of salts 3 and 7 and complex 16 were elucidated using X‐ray crystallog-
raphy, which established that this mono NHC–Ag(I) complex has a linear bis‐
4
EMAN Biodiscoveries Sdn. Bhd., Suite
26, Level 1, EUREKA Complex, Universiti
1
Sains Malaysia (USM), Minden 11800,
Penang, Malaysia
carbene arrangement (C –Ag). The proligands and the respective Ag(I) complexes
2
were studied for their in vitro anticancer potential against human colon cancer cell
line (HCT‐116) using 5‐fluorouracil as a standard. From the IC50 values of all the
tested compounds, it can be postulated that there is an influential relationship
between the increase in chain length of the wingtip n‐alkyl groups and the anticancer
potential. The proligands 4–8 and their respective complexes 12–16 with long
n‐alkyl chain lengths (n = 6–10) showed better IC50 values (0.3–3.9 μM) than the
standard drug with the complexes displaying markedly better antiproliferation
activity against HCT‐116 cell line than the respective proligands and the standard
drug (IC50 = 10.2 μM).
Correspondence
Rosenani A. Haque, School of Chemical
Sciences, Universiti Sains Malaysia, 11800
USM, Penang, Malaysia.
Email: rosenani@usm.my
Funding information
Research University Individual Grant, Grant/
Award Number: RUI/1001/PKIMIA/811346
KEYWORDS
benzimidazolium salts, human colon cancer cell line (HCT‐116), N‐heterocyclic–Ag(I) complexes
[
8,9]
[10,11]
1
| INTRODUCTION
widespread use in transmetallation
and catalysis,
Ag(I)–NHC complexes are gaining importance in the biologi-
cal field where they are studied for their antibacterial
properties and are of great interest in terms of anticancer
Carbenes are strong electrophiles in a free state and become
strong nucleophiles when present in an N‐heterocyclic system.
N‐heterocyclic carbenes (NHCs) are a well‐known class of
ligands with the ability to form complexes with almost all
[
12–14]
activity.
Mono‐ and bis‐imidazolium and benzimidazolium‐
derived Ag(I)–NHC complexes, with functionalized and
non‐functionalized substituents, have been studied for their
[
1]
[2–4]
main group metals as well as transition metals
including
[
5–7]
the rare earth metals.
Ag(I) complexes of NHCs are much
[
14–18]
focused upon among the NHC metal complexes regarding
their synthesis and diverse applications. Apart from their
anticancer potential by various research groups
ing ours,
includ-
[
19–22]
where the Ag(I)–NHC complexes have been
Appl Organometal Chem. 2017;e3735.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.3735

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FATIMA ET AL.
tested against a variety of cancer cell lines and are found to
have some promising anticancer activities. In one study of
the mechanism of cancer cell death by Ag(I)–NHC com-
plexes, it was reported that these complexes induce depolari-
zation of the mitochondrial membrane potential, allowing the
release of mitochondrial proteins and causing early
2.3 | Synthesis of N,N‐n‐
alkylbenzimidazolium bromide
2
.3.1 | Synthesis of N,N‐n‐
propylbenzimidazolium bromide (1)
To
a solution of N‐n‐propylbenzimidazole (1.00 g,
[
23]
apoptosis
and thereby proving them as potential chemo-
5.00 mmol) in 30 ml of dioxane, propyl bromide (0.61 g,
5.00 mmol) was added with stirring and was refluxed at
110 °C for 24 h. On cooling to room temperature the
N,N‐n‐propylbenzimidazolium bromide salt appeared as an oily
layer, the solvent was decanted and the salt was left to crystal-
lize at room temperature in a fume cupboard. The salt was
collected as a colourless crystalline material. Yield 1.06 g
(71%); m.p. 121–125 °C. Anal. Calcd for C H BrN ⋅H O
therapeutic agents.
In the reported cytotoxicity studies addressing the effect
of a steady increase in chain length across the mono‐
imidazolium salts, it was found that cytotoxicity increases
[24]
with an increase in the alkyl chain length.
The bis‐
benzimidazolium salts, on the other hand, are reported to
have lower cytotoxicity with longer alkyl chains, whereas
the corresponding Ag(I)–NHC complexes are found to man-
ifest significant activity with an increase in alkyl chain
13
19
2
2
(%): C, 51.84; H, 6.97; N, 9.30. Found (%): C, 51.75; H,
7.10; N, 9.32. FT‐IR (KBr, ν, cm ): 3132, 3044 (Carom─H),
2934, 2855 (Caliph─H), 1610, 1462, 1427 (Carom═Carom),
−1
[
25]
length. To the best of our knowledge, there are no reported
data on the effect of an increase in chain length of the wingtip
n‐alkyl groups across the mono‐benzimidazolium salts and
their respective mono NHC–Ag(I) complexes.
1
1561 (Carom─N), 1290 (Caliph─N). H NMR (500 MHz,
DMSO‐d , δ, ppm): 0.92 (6H, 2 × CH , t, J = 7.5 Hz),
6
3
1.94–1.99 (4H, 2 × ─CH ─, m), 4.52 (4H, 2 × ─CH ─, t,
2
2
In the work presented here, the anticancer potential of a
series of synthesized mono Ag(I)–NHC complexes and their
respective proligands was studied. The inspiration for this
study comes from the fact that the parent benzimidazole
moiety and the incorporated Ag(I) both have their individual
J = 7.0 Hz), 7.42–7.49 (2H, Ar‐H, m), 7.81–7.88 (2H,
13
Ar‐H, m), 10.01 (1H, NCHN, s). C NMR (125 MHz,
DMSO‐d , δ, ppm): 10.7 (CH ); 11.2, 23.4, 46.4 (CH );
6
3
2
111.5, 112.1, 118.7, 123.9, 133.3 (Ar‐C); 142.1 (NCHN).
[26–28]
biological significance.
The purpose of introducing the
alkyl chain substituents is twofold: firstly, the substituent
does not have its own activity when reaching the target;
secondly, it acts as an excipient that helps the Ag(I)–NHC
complex to pass through the lipophilic cell membrane and
enter the cell where it can produce its anticancer effect.
2.3.2 | Synthesis of N,N‐n‐
butylbenzimidazolium bromide (2)
The procedure was similar to that for compound 1, but using
N‐n‐butylbenzimidazole (0.87 g, 5.00 mmol) instead of N‐n‐
propylbenzimidazole. Salt 2 was collected as a colourless
crystalline solid. Yield 1.20 g (77%); m.p. 132–136 °C. Anal.
Calcd for C H BrN ·H O (%): C, 54.72; H, 7.60; N, 8.51.
15
23
2
2
Found (%): C, 54.70; H, 7.95; N, 8.43. FT‐IR (KBr, ν,
2
2
| EXPERIMENTAL
−1
cm ): 3125, 3033 (Carom─H), 2931, 2870 (Caliph─H),
615, 1461, 1430 (Carom═Carom), 1563 (Carom─N), 1209
1
.1 | Materials and instrumentation
1
(Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.93
6
All the chemicals and solvents were of analytical grade and
were used as received. Furier transform infrared (FT‐IR)
spectra were recorded with a PerkinElmer 2000 spectrometer.
(
6H, 2 × CH , t, J = 7.5 Hz), 1.31–1.39 (4H, 2 × ─CH ─, m),
3
2
1
.89–1.94 (4H, 2 × ─CH ─, m), 4.51 (4H, 2 × ─CH ─N, t,
2
2
J = 7.0 Hz), 7.66–7.72 (2H, Ar‐H, m), 8.10–8.13 (2H,
1
13
H NMR and C NMR spectral analyses were conducted
with a Bruker 500 MHz spectrometer. Elemental analysis
was conducted with PerkinElmer series II 2400
13
Ar‐H, m), 9.89 (1H, NCHN, s). C NMR (125 MHz,
DMSO‐d , δ, ppm): 13.4 (CH ); 19.1, 46.4 (CH ); 113.7,
6
3
2
a
126.5, 131.1 (Ar‐C); 142.1 (NCHN).
microanalyser. Melting points were obtained using a Stuart
Scientific SMP‐1 (UK) instrument.
2
.3.3 | Synthesis of N,N‐n‐
pentylbenzimidazolium bromide (3)
The procedure was similar to that for compound 1, but using
N‐n‐pentylbenzimidazole (1.00 g, 5.50 mmol) instead of N‐n‐
propylbenzimidazole. Salt 3 was collected as a colourless
crystalline solid. Yield 1.45 g (77%); m.p. 122–126 °C. Anal.
Calcd for C H BrN ·H O (%): C, 57.15; H, 8.12; N, 7.84.
2
.2 | Synthesis of N‐alkylbenzimidazoles
N‐Alkylbenzimidazoles were readily accessible in good
yields by reacting benzimidazole and n‐alkyl bromide
[
29]
(
n = 3–10) following a reported literature procedure.
17
27
2
2

FATIMA ET AL.
3 of 14
−
1
Found (%): C, 57.07; H, 8.21; N, 7.83. FT‐IR (KBr, ν, cm ):
3
1
2.3.6 | Synthesis of N,N‐n‐
124, 3037 (Carom─H), 2954, 2859 (Caliph─H), 1614, 1465,
octylbenzimidazolium bromide (6)
1
427 (Carom═Carom), 1568 (Carom─N), 1263 (Caliph─N). H
The procedure was similar to that for compound 1, but using
N‐n‐octylbenzimidazole (1.00 g, 4.50 mmol) instead of N‐n‐
propylbenzimidazole. Salt 6 was collected as a colourless
crystalline solid. Yield 1.60 g (84%); m.p. 110–114 °C. Anal.
NMR (500 MHz, DMSO‐d , δ, ppm): 0.87 (6H, 2 × CH ,
t, J = 7.0 Hz), 1.29–1.34 (8H, 4 × ─CH ─, m), 1.89–1.96
4H, 2 × ─CH ─, m), 4.51 (4H, 2 × ─CH ─N, t,
J = 7.0 Hz), 7.69–7.72 (2H, Ar‐H, m), 8.10–8.14 (2H,
Ar‐H, m), 9.92 (1H, NCHN, s). C NMR (125 MHz,
DMSO‐d , δ, ppm): 13.7 (CH ); 21.5, 27.8, 28.1, 46.6
6
3
2
(
2
2
1
3
Calcd for C23H39BrN ·H O (%): C, 62.59; H, 9.29; N, 6.35.
2 2
Found (%): C, 62.78; H, 9.52; N, 6.42. FT‐IR (KBr, ν, cm
6
3
−1
): 3118, 3022 (Carom─H), 2946, 2847 (Caliph─H), 1613,
(
CH ); 113.7, 126.5, 131.1 (Ar‐C); 142.1 (NCHN).
2
1
466, 1422 (Carom═Carom), 1557 (Carom─N), 1271
1
(Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.84
6
(
6H, 2 × CH , t, J = 7.0 Hz), 1.22–1.31 (20H,
3
1
0 × ─CH ─, m), 1.89–1.92 (4H, 2 × ─CH ─, m), 4.51
2
2
2
.3.4 | Synthesis of N,N‐n‐
(4H, 2 × ─CH ─N, t, J = 7.0 Hz), 7.69–7.72 (2H, Ar‐H,
2
hexylbenzimidazolium bromide (4)
13
m), 8.10–8.14 (2H, Ar‐H, m), 9.92 (1H, NCHN, s).
C
The procedure was similar to that for compound 1, but using
N‐n‐hexylbenzimidazole (1.00 g, 5.00 mmol) instead of N‐n‐
propylbenzimidazole. Salt 4 was collected as a colourless
crystalline solid. Yield 1.30 g (71%); m.p. 113–117 °C. Anal.
Calcd for C H BrN ·H O (%): C, 59.23; H, 8.57; N, 7.27.
NMR (125 MHz, DMSO‐d , δ, ppm): 13.8 (CH ); 22.0,
6
3
25.7, 28.4, 31.1, 46.6 (CH ‐N); 113.7, 126.5, 131.1 (Ar‐C);
2
142.0 (NCHN).
19
31
2
2
2
.3.7 | Synthesis of N,N‐n‐
Found (%): C, 59.19; H, 8.78; N, 7.21. FT‐IR (KBr, ν,
−
1
nonylbenzimidazolium bromide (7)
cm ): 3118, 3038 (Carom─H), 2954, 2843 (Caliph─H),
609, 1483, 1449 (Carom═Carom), 1556 (Carom─N), 1293
1
The procedure was similar to that for compound 1, but using
N‐n‐nonylbenzimidazole (1.00 g, 4.00 mmol) instead of N‐n‐
propylbenzimidazole. Salt 7 was collected as a colourless
crystalline solid. Yield 1.30 g (72%); m.p. 106–111 °C. Anal.
Calcd for C H BrN ·H O (%): C, 63.97; H, 9.59; N, 5.97.
1
(
(
Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.85
6H, 2 × CH , t, J = 7.0 Hz), 1.25–1.33 (12H,
6
3
6
× ─CH ─, m), 1.89–1.94 (4H, 2 × ─CH ─, m), 4.51
2
2
(
4H, 2 × ─CH ─N, t, J = 7.0 Hz), 7.69–7.72 (2H, Ar‐H,
2
25
43
2
2
13
m), 8.10–8.14 (2H, Ar‐H, m), 9.92 (1H, NCHN, s).
C
Found (%): C, 64.12; H, 9.64; N, 6.18. FT‐IR (KBr, ν, cm
NMR (125 MHz, DMSO‐d , δ, ppm): 13.8 (CH ); 21.7,
−1
6
3
): 3117, 3025 (Carom─H), 2918, 2854 (Caliph─H), 1610,
2
5.4, 28.4, 30.6, 46.6 (CH ); 113.7, 126.5, 131.1 (Ar‐C),
2
1
485, 1424 (Carom═Carom), 1557 (Carom─N), 1290
142.1 (NCHN).
1
(Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.83
6
(
6H, 2 × CH , t, J = 7.0 Hz), 1.21–1.31 (24H,
3
1
2 × ─CH ─, m), 1.90–1.93 (4H, 2 × ─CH ─, m), 4.50
2
2
(
4H, 2 × ─CH ‐N, t, J = 7.0 Hz), 7.70–7.72 (2H, Ar‐H,
2
2
.3.5 | Synthesis of N,N‐n‐
13
m), 8.11–8.13 (2H, Ar‐H, m), 9.92 (1H, NCHN, s).
NMR (125 MHz, DMSO‐d , δ, ppm): 13.8 (CH ); 21.7,
C
heptylbenzimidazolium bromide (5)
6
3
2
1
5.6, 28.4, 28.9, 29.3, 31.2, 44.1, 46.6 (CH ); 113.7, 126.5,
31.1 (Ar‐C); 143.9 (NCHN).
The procedure was similar to that for compound 1, but using
N‐n‐heptylbenzimidazole (1.00 g, 4.50 mmol) instead of N‐n‐
propylbenzimidazole. Salt 5 was collected as a colourless
crystalline solid. Yield 1.30 g (73%); m.p. 101–105 °C. Anal.
Calcd for C H BrN ·H O (%): C, 61.03; H, 8.96; N, 6.78.
2
2
.3.8 | Synthesis of N,N‐n‐
21
35
2
2
decylbenzimidazolium bromide (8)
Found (%): C, 61.21; H, 9.21; N, 6.77. FT‐IR (KBr, ν,
−
1
cm ): 3113, 3025 (Carom─H), 2926, 2850 (Caliph─H),
609, 1466, 1424 (Carom═Carom), 1557 (Carom─N), 1283
The procedure was similar to that for compound 1, but using
N‐n‐decylbenzimidazole (1.00 g, 4.00 mmol) instead of N‐n‐
propylbenzimidazole. Salt 8 was collected as a colourless
crystalline solid. Yield 1.50 g (79%); m.p. 116–119 °C. Anal.
Calcd for C H BrN ·H O (%): C, 65.20; H, 9.86; N, 5.63.
1
1
(
(
Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.85
6H, 2 × CH , t, J = 7.0 Hz), 1.24–1.32 (16H,
6
3
8
× ─CH ─, m), 1.89–1.95 (4H, 2 × ─CH ─, m), 4.51
2
2
13 19
2
2
(
4H, 2 × ─CH ─N, t, J = 7.0 Hz), 7.69–7.72 (2H, Ar‐H,
Found (%): C, 65.26; H, 10.08; N, 5.62. FT‐IR (KBr, ν,
cm ): 3118, 3026 (Carom─H), 2954, 2847 (Caliph─H),
2
13
−1
m), 8.10–8.14 (2H, Ar‐H, m), 9.90 (1H, NCHN, s).
C
NMR (125 MHz, DMSO‐d , δ, ppm): 13.8 (CH ); 21.9,
1609, 1468, 1422 (Carom═Carom), 1556 (Carom─N), 1285
6
3
1
2
5.7, 28.4, 31.0, 46.6 (CH ); 113.7, 126.5, 131.1 (Ar‐C);
(Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.83
2
6
142.1 (NCHN).
(6H, 2 × CH , t, J = 7.0 Hz), 1.20–1.27 (28H,
3

4
of 14
FATIMA ET AL.
−
1
1
2
8
4 × ─CH ─, m), 1.89–1.91 (4H, 2 × ─CH ─, m), 4.48 (4H,
7.70. FT‐IR (KBr, ν, cm ): 3057 (Carom─H), 2962, 2866
2
2
× ─CH ─N, t, J = 7.0 Hz), 7.67–7.72 (2H, Ar‐H, m),
(Caliph─H), 1607, 1481, 1411 (Carom═Carom), 1141
(Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.98
2
1
3
1
.10–8.12 (2H, Ar‐H, m), 9.86 (1H, NCHN, s). C NMR
6
(
125 MHz, DMSO‐d , δ, ppm): 13.8 (CH ); 22.1, 25.6,
(6H, 2 × CH , t, J = 7.5 Hz), 1.43–1.47 (4H, 2 × ─CH ─,
6
3
3
2
2
8.5, 31.2, 46.6 (CH ); 113.7, 126.5, 131.1 (Ar‐C) 141.9
m), 1.97–2.01 (4H,
2
×
─CH ─, m), 4.54 (4H,
2
2
(NCHN).
2 × ─CH ─N, t, J = 7.0 Hz), 7.49–7.52 (2H, Ar‐H, m),
2
13
7
.72–7.79 (2H, Ar‐H, m). C NMR (125 MHz, DMSO‐d6,
δ, ppm): 12.8 (CH ); 19.7, 32.1, 48.8 (CH ); 111.9, 113.7,
3
2
2
.4 | Synthesis of N,N‐n‐alkylbenzimidazol‐2‐
1
17.1, 123.9, 126.7, 133.7 (Ar‐C); 187.9 (C2‐Ag, br d).
ylidene silver(I) hexafluorophosphate
2
.4.1 | Synthesis of N,N‐n‐
propylbenzimidazol‐2‐ylidene silver(I)
hexafluorophosphate (9)
2
.4.3 | Synthesis of N,N‐n‐pentylbenzimidazol‐
‐ylidene silver(I) hexafluorophosphate (11)
2
The N,N‐n‐propylbenzimidazolium bromide salt (1.00 g,
Complex 11 was prepared by following the same procedure
as that for complex 9 except that salt 1 was replaced with
3.50 mmol) was dissolved in 150 ml of methanol in a
round‐bottom flask wrapped with aluminium foil and silver
oxide (0.80 g, 3.50 mmol) was added to it. Under exclusion
of light the reaction mixture was allowed to stir for 2 days
at room temperature. After completion of the reaction time
the reaction mixture was filtered through a pad of celite in
order to remove all the insoluble species like AgBr and any
salt 3 (1.00 g, 3.00 mmol), Ag O (0.70 g, 3.00 mmol),
2
KPF (1.10 g, 6.00 mmol). Complex 11 was obtained in
6
the form of white powder after drying. Yield 1.40 g
(
51%); m.p. 110–114 °C. Anal. Calcd for C H AgF N P
34
52
6 4
(
6
%): C, 53.06; H, 6.76; N, 7.28. Found (%): C, 52.97; H,
.94; N, 7.45. FT‐IR (KBr, ν, cm ): 3038 (Carom─H),
−
1
unreacted Ag O. To the clear solution containing the N,N‐
2
2968, 2877 (Caliph─H), 1615, 1469, 1401 (Carom═Carom),
1
n‐propylbenzimidazol‐2‐ylidene silver(I) bromide, potassium
hexafluorophosphate (1.34 g, 7.28 mmol) was added and was
allowed to stir for 2 h. This metathesis reaction resulted in the
formation of methanol‐insoluble N,N‐n‐propylbenzimidazol‐
1
519 (Carom─N), 1143 (Caliph─N). H NMR (500 MHz,
DMSO‐d , δ, ppm): 0.89 (6H, 2 × CH , t, J = 7.0 Hz),
6
3
1
2
7
.32–1.38 (8H,
4
×
─CH ─, m), 1.93–1.99 (4H,
2
× ─CH ─, m), 4.54 (4H, 2 × ─CH ─N, t, J = 7.0 Hz),
2
2
13
2‐ylidene silver(I) hexafluorophosphate which was filtered,
.47–7.50 (2H, Ar‐H, m), 7.71–7.73 (2H, Ar‐H, m).
C
washed with distilled water in order to remove any unreacted
NMR (125 MHz, DMSO‐d , δ, ppm): 12.9 (CH ); 21.8,
6
3
KPF and allowed to dry in a fume cupboard. Complex 9 was
6
27.8, 49.0 (CH ); 111.7, 113.2, 117.1, 123.9, 133.4
2
obtained in the form of white powder after drying. Yield
(Ar‐C); 187.6 (C2‐Ag, br d).
1.50 g (50%); m.p. 182–185 °C. Anal. Calcd for
C H AgF N P·2H O (%): C, 45.3; H, 5.77; N, 8.08. Found
2
6
36
6
4
2
−
1
(
(
(
(
%): C, 45.13; H, 5.98; N, 8.24. FT‐IR (KBr, ν, cm ): 3037
2
.4.4 | Synthesis of N,N‐n‐hexylbenzimidazol‐
Carom─H), 2968, 2877 (Caliph─H), 1614, 1465, 1404
Carom═Carom), 1519 (Carom─N), 1145 (Caliph─N). H NMR
1
2‐ylidene silver(I) hexafluorophosphate (12)
500 MHz, DMSO‐d , δ, ppm): 0.92 (6H, 2 × CH , t,
6
3
Complex 12 was prepared by following the same procedure
as that for complex 9 except that salt 1 was replaced with
salt 4 (1.00 g, 2.70 mmol), Ag O (0.60 g, 2.70 mmol),
KPF (1.00 g, 5.50 mmol). Complex 12 was obtained in
the form of white powder after drying. Yield 1.60 g
J = 7.5 Hz), 1.94–1.99 (4H, 2 × ─CH ─, m), 4.52 (4H,
2
NMR (125 MHz, DMSO‐d , δ, ppm): 11.2 (CH ); 23.4,
4
2
13
× ─CH ─, t, J = 7.0 Hz), 7.45–7.72 (4H, Ar‐H, m).
C
2
2
6
3
6
6.4 (CH ); 111.5, 112.1, 118.7, 123.9, 133.3 (Ar‐C);
2
1
87.9 (C2‐Ag, br d).
(64%); m.p. 97–99 °C. Anal. Calcd for C H AgF N P
38
60
6 4
(
7
2
%): C, 55.28; H, 7.27; N, 6.78. Found (%): C, 54.90; H,
.47; N, 6.71. FT‐IR (KBr, ν, cm ): 3076 (Carom─H),
−
1
2.4.2 | Synthesis of N,N‐n‐butylbenzimidazol‐
2‐ylidene silver(I) hexafluorophosphate (10)
923, 2859 (Caliph─H), 1479, 1453 (Carom═Carom), 1191
1
(
Caliph─N). H NMR (500 MHz, DMSO‐d , δ, ppm): 0.84
6
Complex 10 was prepared by following the same procedure
as that for complex 9 except that salt 1 was replaced with salt
(6H, 2 × CH , t, J = 7.0 Hz), 1.26–1.44 (12H,
3
6 × ─CH ─, m), 1.97–2.03 (4H, 2 × ─CH ─, m), 4.54
2
2
2
(1.00 g, 3.00 mmol), Ag O (0.70 g, 3.00 mmol), KPF6
(4H, 2 × ─CH ─N, t, J = 7.0 Hz), 7.49–7.52 (2H, Ar‐H,
2
2
13
(1.10 g, 6.00 mmol). Complex 10 was obtained in the form
m), 7.73–7.74 (2H, Ar‐H, m). C NMR (125 MHz,
DMSO‐d , δ, ppm): 12.9 (CH ); 21.8, 25.4, 28.8, 30.6,
of white powder after drying. Yield 1.50 g (70%); m.p.
6
3
1
19–121 °C. Anal. Calcd for C H AgF N P (%): C,
49.0 (CH ); 111.7, 113.2, 117.1, 123.9, 133.4 (Ar‐C);
3
0
44
6
4
2
50.50; H, 6.17; N, 7.85. Found (%): C, 50.16; H, 6.25; N,
187.6 (C2‐Ag, br d).

FATIMA ET AL.
5 of 14
2
.4.5 | Synthesis of N,N‐n‐heptylbenzimidazol‐
‐ylidene silver(I) hexafluorophosphate (13)
t, J = 7.0 Hz), 1.18–1.23 (24H, 12 × ─CH
1.96–2.02 (4H, 2 × ─CH
t, J = 7.0 Hz), 7.49–7.51 (2H, Ar‐H, m), 7.73–7.74
2
─, m),
2
2
─, m), 4.54 (4H, 2 × ─CH ─N,
2
Complex 13 was prepared by following the same procedure as
that for complex 9 except that salt 1 was replaced with salt 5
13
(
2H, Ar‐H, m). C NMR (125 MHz, DMSO‐d , δ, ppm):
6
1
2.9 (CH ); 21.8, 25.6, 26.4, 28.5, 29.9, 31.2, 45.2, 48.9
3
(
(
1.00 g, 2.50 mmol), Ag O (0.60 g, 2.50 mmol), KPF6
0.90 g, 5.00 mmol). Complex 13 was obtained in the form of
2
(
CH ); 111.7, 113.2, 117.1, 123.8, 126.6, 133.4 (Ar‐C);
2
1
87.5 (C2‐Ag, br d).
white powder after drying. Yield 1.50 g (62%); m.p. 90–92 °C.
Anal. Calcd for C H AgF N P (%): C, 57.21; H, 7.72; N,
42
68
6 4
6
.36. Found (%): C, 56.87; H, 7.85; N, 6.29. FT‐IR (KBr, ν,
2.4.8 | Synthesis of N,N‐n‐decylbenzimidazol‐
2‐ylidene silver(I) hexafluorophosphate (16)
Complex 16 was prepared by following the same procedure
as that for complex 9 except that salt 1 was replaced with salt
−1
cm ): 3063 (C ─H), 2930, 2854 (Caliph─H), 1477, 1454
arom
1
(Carom═Carom), 1141 (Caliph─N). H NMR (500 MHz,
DMSO‐d , δ, ppm): 0.82 (6H, 2 × CH , t, J = 7.0 Hz),
6
3
1
2
7
.20–1.44 (16H,
8
×
─CH ─, m), 1.96–2.03 (4H,
2
8
(1.00 g, 2.00 mmol), Ag O (0.48 g, 2.00 mmol), KPF6
2
× ─CH ─, m), 4.54 (4H, 2 × ─CH ─N, t, J = 7.0 Hz),
2
2
(
0.70 g, 4.00 mmol). Complex 16 was obtained in the form
of white powder after drying. Yield 1.10 g (55%); m.p.
9–102 °C. Anal. Calcd for C H AgF N P (%): C, 61.78;
1
3
.49–7.52 (2H, Ar‐H, m), 7.73–7.78 (2H, Ar‐H, m). C NMR
(125 MHz, DMSO‐d , δ, ppm): 13.0 (CH ); 21.8, 22.1 25.9,
6
3
9
54
92
6 4
2
1
6.2, 28.9, 30.1, 31.1, 45.1, 49.0 (CH ); 111.7, 113.2, 117.1,
23.6, 126.6, 133.4 (Ar‐C); 187.6 (C2‐Ag, br d).
2
H, 8.77; N, 5.34. Found (%): C, 61.37; H, 9.05; N, 5.24.
−
1
FT‐IR (KBr, ν, cm ): 3045 (C
(
─H), 2920, 2852
arom
1
Caliph─H), 1469, 1401 (Carom═Carom), 1140 (Caliph─N). H
NMR (500 MHz, DMSO‐d , δ, ppm): 0.82 (6H, 2 × CH ,
2
2
.4.6 | Synthesis of N,N‐n‐octylbenzimidazol‐
‐ylidene silver(I) hexafluorophosphate (14)
6
3
t, J = 7.0 Hz), 1.17–1.21 (28H, 14 × ─CH ─, m),
2
1
.97–2.01 (4H, 2 × ─CH ─, m), 4.53 (4H, 2 × ─CH ─N,
2
2
Complex 14 was prepared by following the same procedure as
that for complex 9 except that salt 1 was replaced with salt 6
t, J = 7.0 Hz), 7.49–7.52 (2H, Ar‐H, m), 7.73–7.75 (2H,
13
Ar‐H, m). C NMR (125 MHz, DMSO‐d , δ, ppm): 12.9,
6
(
(
1.00 g, 2.40 mmol), Ag O (0.50 g, 2.40 mmol), KPF6
0.90 g, 4.80 mmol). Complex 14 was obtained in the form
2
1
3.1 (CH ); 21.8, 22.1, 26.3, 28.8, 29.8, 31.3, 48.9 (CH );
3
2
1
11.7, 113.2, 117.1, 123.9, 126.6, 133.4 (Ar‐C); 187.5
of white powder after drying. Yield 1.20 g (50%); m.p.
(C2‐Ag, br d).
87–89 °C. Anal. Calcd for C H AgF N P (%): C, 58.92;
4
6
76
6 4
H, 8.11; N, 5.98. Found (%): C, 58.68; H, 8.19; N, 5.97. FT‐IR
−1
(
1
KBr, ν, cm ): 3061 (C
479, 1407 (Carom═Carom), 1137 (Caliph─N). H NMR
─H), 2927, 2851 (Caliph─H),
2.5 | Crystallographic details
arom
1
Crystals of compounds 3, 7 and 16 were mounted on fine
glass fibre using viscous hydrocarbon oil. Data were col-
lected using a Bruker‐Smart ApexII‐2009 CCD diffractome-
ter equipped with graphite monochromater and using Mo Kα
(
500 MHz, DMSO‐d , δ, ppm): 0.79 (6H, 2 × CH , t,
6
3
J = 7.0 Hz), 1.19–1.25 (20H, 10 × ─CH ─, m), 1.97–2.03
2
(
4H, 2 × ─CH ─, m), 4.54 (4H, 2 × ─CH ─N, t,
2
2
J = 7.0 Hz), 7.49–7.52 (2H, Ar‐H, m), 7.72–7.75 (2H, Ar‐H,
(
λ = 0.71073) radiation. Data collection for compound 3 was
13
m). C NMR (125 MHz, DMSO‐d , δ, ppm): 13.0 (CH );
6
3
conducted at room temperature while for 7 and 16 the tem-
perature was maintained at 100 K using open flow N2
cryostreams. The integration was carried out with the pro-
2
1.8, 25.7, 26.4, 28.6, 29.8, 31.2, 45.2, 48.9 (CH ); 111.7,
2
113.2, 117.1, 123.9, 133.4 (Ar‐C); 187.6 (C2‐Ag, br d).
II
[30]
gram SAINT using the APEX software as reported. Solu-
tions were obtained by direct methods using SHELXS‐2014,
followed by successive refinements using full matrix least
2.4.7 | Synthesis of N,N‐n‐nonylbenzimidazol‐
2‐ylidene silver(I) hexafluorophosphate (15)
2
[31]
squares method against F using SHELXL‐2014. The pro-
Complex 15 was prepared by following the same procedure
as that for complex 9 except that salt 1 was replaced with salt
[32]
gram X‐seed was used as graphical SHELX interface. The
data for 3, 7 and 16 are summarized in Table 1.
7
(1.00 g, 2.20 mmol), Ag O (0.50 g, 2.20 mmol), KPF6
2
(
0.80 g, 4.40 mmol). Complex 15 was obtained in the form
of white powder after drying. Yield 1.40 g (62%); m.p.
4–96 °C. Anal. Calcd for C H AgF N P (%): C, 60.43;
2.6 | In vitro anticancer studies
9
5
0
84
6 4
2.6.1 | Materials and equipment
H, 8.46; N, 5.64. Found (%): C, 59.98; H, 8.58; N, 5.61.
−
1
FT‐IR (KBr, ν, cm ): 3063 (C
(
─H), 2918, 2853
Rosewell Park Memorial Institute (RPMI 1640) cell culture
medium was obtained from ScienCell, USA. Microplate
reader (Epoch, BioTek, USA), methylthiazolyldiphenyl
arom
1
Caliph─H), 1481, 1450 (Carom═Carom), 1199 (Caliph─N). H
NMR (500 MHz, DMSO‐d , δ, ppm): 0.80 (6H, 2 × CH ,
6
3

6
of 14
FATIMA ET AL.
TABLE 1 Crystal data and structure refinement parameters for 3, 7 and 16
3
7
16
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C
17
H
29ON
2
Br
C
25
H
45ON
2
Br
54 4 6
C H92ON Ag·F P
357.52
Monoclinic
P2 /c
469.53
1050.16
Triclinic
P‐1
Orthorhombic
Pnna
1
8.8572(8)
22.553(2)
9.9406(9)
90
8.8529(3)
9.1496(3)
37.0015(11)
12.3952(4)
12.2836(4)
90
b (Å)
c (Å)
16.8044(6)
80.9102(9)
77.4971(9)
77.7698(9)
1289.73(8)
2
α (°)
β (°)
105.0785(13)
90
90
γ (°)
90
3
V (Å )
1917.3(3)
4
5633.8(3)
4
Z
Density calcd (g cm−
3
)
1.238
1.209
1.238
μ (mm−
F(000)
T (K)
1
)
2.146
1.611
0.443
752
504
2240
297(2)
2.01–30.01
42 059
5579
100(2)
2.3–32.9
68 985
9510
100(2)
1.7–30.9
11 2448
8829
θ range (°)
Reflections collected
Reflections unique
Reflections obs [I > 2σ(I)]
2813
8522
7557
R
int
0.044
0.030
0.054
R
1
(obs, all)
(obs, all)
Goodness of fit
0.0699
0.1907
1.032
0.0241
0.0595
1.046
0.0599
0.0971
1.411
wR
2
tetrazolium bromide (MTT) reagent and heat‐inactivated
foetal bovine serum (HIFBS) were purchased from Gibco,
UK. Penicillin/streptomycin solution and 5‐fluorouracil
1640 cell culture medium with a supply of 10% HIFBS and
10% penicillin/streptomycin solution. Temperature was main-
tained at 37 °C.
(
5‐FU) were purchased from Sigma‐Aldrich, Germany.
Other equipment was as follows: dissecting microscope
Motic, Taiwan), Eppendorf tips (AxyGEN, USA),
2.6.3 | Preparation of cell culture
(
Eppendorf tube 1.5 ml (Eppendorf, Germany), incubator
Binder Fisher Scientific, Germany), inverted fluorescent
microscope (Olympus, Japan), inverted light microscope
The first step in the preparation is the growth of HCT‐116
cell line under incubation conditions. Only those cells were
selected for plating purposes which reached about 70–80%
level of confluency. Medium used in the plates previously
was aspirated and cells used were washed with phosphate
buffer saline solution two to three times. After washing with
phosphate buffer saline solution, trypsin was added evenly on
the cell surfaces. Cells were incubated for 1 min at 37 °C.
Cell segregation was facilitated by simply tapping the flask
containing the cells and was observed under a microscope.
A volume of 5 ml of fresh medium was added to inhibit tryp-
sin activity. After counting, there was a final concentration of
(
(
(
Matrix Optic (M) Sdn. Bhd, Japan), ImageJ software
http://rsb.info.nih.gov/ij/), laminar flow (class II; ESCO‐
BSC, Singapore), micropipette (Eppendorf, USA), refrigera-
®
tor (Samsung, Japan), serological pipette 10, 5 ml (TPP ,
USA), water bath (PROTECH‐Electronic, Malaysia) and
6‐well cell culture plate (Coster Corning, USA).
2
.6.2 | Cell lines and environmental conditions
5
−1
Human colorectal tumour (HCT‐116) cell line was purchased
Rockvill, MD, USA). HCT‐116 cell line was kept in RPMI
2.5 × 10 cells ml which was later inoculated to the wells
of a 96‐well plate (100 μl of cells per well). These seeded
(

FATIMA ET AL.
7 of 14
plates were then incubated at standard atmospheric condi-
tions for cell culture.
purity. All the N,N‐n‐alkylbenzimidazol‐2‐ylidene silver(I)
hexafluorophosphate complexes were obtained in a powder
form in good yields. These complexes are readily soluble in
acetonitrile and DMSO and almost insoluble in methanol,
diethyl ether and petroleum ether. The synthesis of mono
benzimidazolium salts and their respective mono NHC–
Ag(I) complexes is depicted in Scheme 1. The structures of
salts 1–8 and their respective Ag(I) complexes 9–16 were
established by a combination of H NMR and C NMR spec-
troscopy and elemental analysis. As the NMR and the FT‐IR
spectra of all the salts and complex 9 showed strong peak
and absorption band for water, so the amount of water was
estimated for the elemental analysis and the percentages of
C, H and N are in good accord with their calculated values.
The molecular structures of salts 3 and 7 and complex 16 were
further supported by X‐ray diffraction data.
2
.6.4 | MTT assay for measuring cell viability
Volumes of 100 μl of the cells were seeded in all wells of a
6‐well microplate and the microplate was incubated over-
9
night for cell attachment under CO incubator. Later on, a
2
1
13
volume of 100 μl of each test substance was added in each
well and the plate was labelled accordingly. Different dilu-
tions of the test substance were prepared to investigate any
dose‐dependent response. After the addition of test substance
into the plate containing cancer cells, the plate was incubated
at 37 °C with 5% CO environment for 72 h. After 72 h of
2
treatment, a volume of 20 μl of MTT reagent was added into
each well and again incubated for 4 h. After this period of
incubation, 20 μl of DMSO (MTT lysis solution) was added
in each well. Plates were further incubated for 5 min under
the same environment of incubation. Finally, absorbance
was measured at 570 and 620 nm using a microplate reader
3.2 | FT‐IR analysis
FT‐IR spectral analysis of all the N‐n‐alkylbenzimidazolium
bromide salts and their respective Ag(I) complexes was car-
ried out using the KBr pellet method. As the substituents
are simple alkyl chains, the prominent absorptions in the
functional group region are the (═C─H) stretching vibrations
(Epoch, BioTek, USA). Data were analysed for cell viability
and percent inhibition of proliferation of test substances.
The results were presented as percent viability compared to
[33]
the negative control (mean Æ SD, n = 3).
−
1
of medium to low intensity in the region 3000–3100 cm
and the (─C─H) aliphatic stretching vibrations of strong
3
3
| RESULTS AND DISCUSSION
.1 | Synthesis
−
1
intensity at 2800–2900 cm . The (C═C) stretching vibra-
tions of aromatic ring occur as medium absorption bands in
−1
the range 1400–1600 cm . The (C═N) stretching vibrations
appear in the form of strong and sharp absorption bands
between 1550 and 1600 cm− while the (C─N) stretching
In general, the first N‐alkylation was performed using potas-
sium hydroxide as a base for the removal of acidic (N─H)
protons in a reaction mixture of benzimidazole in DMSO
and subsequent reaction with the respective n‐alkyl bromide.
The N‐n‐alkylbenzimidazole, after extraction with chloro-
form and evaporation, was then reacted with n‐alkyl bromide
in 1,4‐dioxane under reflux for 24 h. The oily product was
separated and left to crystallize at room temperature for
1
3–4 days. All the resultant symmetrically substituted N,N‐n‐
alkylbenzimidazolium bromide salts were obtained in a
colourless crystalline state in medium to high yield. These
salts are stable towards air and moisture, are soluble in polar
organic solvents such as methanol, DMSO and acetonitrile
and almost insoluble in diethyl ether and petroleum ether.
For the generation of carbene and complexation with
Ag(I), the in situ deprotonation method involving Ag O was
2
[
34]
used.
After two days of stirring of reaction mixture con-
taining N‐n‐alkylbenzimidazolium bromide salt with Ag O
2
(1:1 ratio) in methanol, the resulting Ag(I) complex was sep-
arated from the insoluble AgBr and any unreacted Ag O by
2
filtration through celite. The obtained NHC–Ag(I) complex
in bromide form was converted to hexafluorophosphate
form by a metathesis reaction. This conversion was done
for the purpose of easy handling and to achieve complex
SCHEME 1 Synthesis of N,N‐n‐alkylbenzimidazolium bromide salts
(1–8) and their respective N,N‐n‐alkylbenzimidazol‐2‐ylidene silver(I)
hexafluorophosphate complexes (9–16)

8
of 14
FATIMA ET AL.
vibrations give medium to weak bands in the region
3.4 | X‐ray crystallographic analysis
−
1 [35]
1100–1200 cm .
The accessible FT‐IR region does not
Salts 3 and 7 crystallize in a monoclinic system with space
groups P‐2 /c and P‐1, respectively. The asymmetric unit
give any indication about (C─Ag) bond whose stretching
−1
1
vibrations appear below 400 cm meaning that all the
NHC–Ag(I) complexes exhibit almost the same absorption
bands as the N,N‐n‐alkylbenzimidazolium bromide salts.
The most notable change is observed in the carbene to nitro-
gen absorption band where the bond length increases on
complexation and can be seen in the X‐ray crystallographic
data. This increase in the bond length results in a lower vibra-
for both salts consists of a symmetrically substituted
benzimidazolium cation, one bromide ion and a water mole-
cule in the lattice (Figure 1). In the crystal structures of both
salts, the n‐alkyl chains project perpendicular to the plane of
the benzimidazole ring. The nitrogen to the carbene carbon
bond distance ranges from 1.321(5) to 1.337(12) Å and the
internal ring angle at the carbene centre (N─C─N) ranges
from 111.6(3)° to 110.41(8)° (Table 2). The observed bond
lengths and bond angles are well within the range of reported
tional frequency of the (C ─N) bond with a corresponding
2
shift of its absorption band to lower frequency and conse-
quently this band cannot be distinguished from the (C═C)
stretching absorption bands of the NHC–Ag(I) complexes.
[
42]
benzimidazolium salts. In both structures, hydrogen bonds
are observed in which the water molecule in the lattice
behaves as hydrogen bond donor to the bromide ion.
1
13
3
.3 | H NMR and C NMR analysis
Single crystals of complex 16 were obtained by the room
temperature slow evaporation of a saturated solution of the
complex in acetonitrile. Complex 16 crystallizes in an ortho-
rhombic system with space group Pnna. The complex com-
prises one Ag(I) ion coordinating with two carbenes of the
two benzimidazol‐2‐ylidene units; this cation is balanced by
1
H NMR spectra of all the N,N‐n‐alkylbenzimidazolium bro-
mide salts were obtained in DMSO‐d and those of the
6
respective complexes in acetonitrile‐d . The resonances of
3
all the alkyl chain protons with their expected multiplicities
are clearly observed in the spectra of all salts. The methyl
protons give a triplet in the most upfield region around
(a)
0.8–0.9 ppm, while the methylene protons exhibit a number
of multiplets in the region 1–2 ppm. A clear triplet in the
deshielded region around 4.5 ppm is observed which can be
ascribed to the most deshielded methylene protons bonded
with nitrogen of the benzimidazolium ring (N─CH ─). The
2
aromatic ring proton resonances are observed in the form of
multiplets in the range 7.5–8.5 ppm. The most distinctive res-
onance in the most deshielded region around 9.8–9.9 ppm,
which appears in the form of a singlet, is assigned to the most
deshielded acidic proton (NCHN) of the benzimidazolium
ring. On reaction with Ag O, all the salts undergo the
2
(b)
removal of the acidic proton thereby resulting in the genera-
tion of carbene that coordinates with Ag(I).
Subsequently, on the conversion of the salts to Ag(I)
1
complexes, the most notable change in the H NMR spectra
is the disappearance of the resonance around 9.8–9.9 ppm,
[36,37]
13
signifying the formation of carbene.
In the C NMR
spectra of the salts, the most characteristic resonance that
appears around 141–144 ppm is attributed to the C2 carbon
(
NCHN) which is the most deshielded carbon, in accordance
[
38,39]
with the literature.
Upon deprotonation and coordina-
FIGURE 1 Structures of (a) 3 and (b) 7 with ellipsoids shown at 50%
tion with Ag(I), there is a shift of the C2 resonance to the
more downfield region at 187–188 ppm in the form of appar-
ent doublets as previously reported for NHC–Ag(I) com-
probability
TABLE 2 Selected bond lengths (Å) and angles (°) for salts 3 and 7
[
40,41]
plexes.
However, these doublets cannot be resolved
1
07
3
7
further to study the coupling of carbene to
Ag and
Ag. Similar behaviour is observed for all the complexes
1
09
N1–C1/C7
1.321(5)
1.315(5)
111.6(3)
1.337(12)
1.334(12)
110.41(8)
of the series. The resonances of the other aromatic carbons
appear between 111 and 133 ppm whereas the methyl and
methylene protons have resonances in the region 12–48 ppm.
N2–C1/C7
N1–C1/C7–N2

FATIMA ET AL.
9 of 14
one hexafluorophosphate anion (Figure 2a). The resulting
anticancer activities were examined against a human colon
cancer cell line. In the initial screening, all the synthesized
proligands and their complexes were screened for IC50 values.
The IC50 values of proligands 1–8 range within 1.8–31.8 μM
(Table 4). These values show that an increase in the carbon
chain length of the proligands causes a reduction in the IC50
value. It is further observed that the proligands 1–3 with short
carbon chains have higher IC50 values (lower cytotoxicity).
Increasing the length of the carbon chain (n = 6–10) on the
proligands (4–8) results in a marked drop in the IC50 values
(1.8–7.5) and a corresponding increase in cytotoxicity when
compared to standard drug 5‐FU (IC50 = 10.2 μμ). These
observed enhanced cytotoxic effects of the proligands on
human colon cancer cells validate previous findings that
mono NHC–Ag(I) complex has a bis‐carbene (C ─Ag)
2
arrangement. The two benzimidazolyl rings are almost copla-
nar with the n‐decyl chains projecting perpendicular to the
plane of the ring. The C1─Ag1─C1b bond has an angle of
177.10(10)° which is almost linear with the Ag1─C1 bond
length of 2.088(2) Å (Table 3), this bond angle and bond dis-
tance being comparable to those found in known NHC–Ag(I)
[
43,44]
complexes.
The internal ring angle at N1─C1─N2 is
1
1
05.79(19)° with the N1─C1 bond having a bond length of
.363(3) Å that is shorter than the N1─C2 bond with a bond
length of 1.390(3) Å since this bond has more electron den-
sity due to electron‐donating mesomeric effect of the nitro-
gens. In the three‐dimensional network of complex 16 the
molecules are stacked together by π–π interactions as well
as non‐covalent carbon chain interactions (Figure 2b).
[
18]
NHC ligands show cytotoxic effects.
With the incorporation of Ag(I) ions in complexes 9–12 a
similar trend in the cytotoxicity, as in proligands, is observed.
However, increasing the carbon chain length in complexes
3
.5 | In Vitro anticancer activities of mono
1
2–16 results in a greater reduction in IC50 values that fall
NHC–Ag(I) complexes and their respective
within the range 0.02–3.9 μM (Table 4). These reduced IC50
values and the respective increased cytotoxicity can be attrib-
uted to the presence of Ag(I) ions in the complexes and an
increase in carbon chain length of the wingtip groups. The
increased carbon chain length enhances lipophilicity and
might be the reason for the improved cytotoxicity. This present
finding of an increase in carbon chain that might have facili-
tated the absorption of proligands and the complexes by
increasing the lipophilicity is coherent with a previous study
proligands against HCT‐116 cell line
A series of mono NHC proligands (1–8) and their correspond-
ing complexes (9–16) were synthesized with a periodic
increase in carbon chain length as wingtip groups and their
(a)
[
45]
in this regard.
The phenomenon of increased cytotoxicity
of NHC–Ag(I) complexes, in comparison to the respective
[
46]
proligands, is in line with previously reported data.
Cancer cells have the ability to proliferate at an abnormally
high rate and the present study subjected the synthesized
proligands and their respective complexes to the inhibition
of human colon cancer cell proliferation. The proligands
(b)
4–8 and complexes 12–16 show strong cytotoxicity against
the proliferation of colorectal cancer cells (Figure 3b–f).
Interestingly, the complexes exhibit improved antiproliferative
TABLE 4 IC50 values of proligands/salts 1–8 and respective NHC–
Ag(I) complexes 9–16
FIGURE 2 (a) Structure of 16 with ellipsoids shown at 50%
probability. Hydrogen atoms and PF in the lattice are omitted for
a
a
Proligand
IC50 (μM)
NHC–Ag(I) complex
IC50 (μM)
6
clarity. (b) Packing diagram of complex 16 showing the non‐covalent
carbon chain interactions
1
2
3
4
5
6
7
8
31.8 Æ 3.84
27.3 Æ 0.63
30.2 Æ 8.9
7.5 Æ 2.22
3.1 Æ 0.23
2.9 Æ 0.13
1.4 Æ 0.15
1.8 Æ 0.18
9
10
11
12
13
14
15
16
26.8 Æ 2.30
25.7 Æ 1.27
13.2 Æ 1.50
3.9 Æ 0.62
1.1 Æ 0.28
0.4 Æ 0.05
0.02 Æ 0.02
0.3 Æ 0.12
TABLE 3 Selected bond lengths (Å) and angles (°) for complex 16
Ag1–C1
2.088(2)
1.363(3)
N1–C1
N1–C2
1.390(3)
C1–Ag1–C1b
N1–C1–N2
177.10(10)
105.79(19)
a
IC50 on HCT‐116 for 5‐FU = 10.2 Æ 2.18 μM.

1
0 of 14
FATIMA ET AL.
(
a)
(d)
(a)
(
b)
(e)
(b)
(
c)
(f)
(c)
FIGURE 3 Dose‐dependent antiproliferative effects of proligands
–8 and complexes 11–16 in comparison with standard drug 5‐FU.
3
The graphs show percentage inhibition of HCT‐116 cell proliferation
versus concentration of test samples
effects compared to the respective ligands. Moreover, it is of
great significance to observe that all the proligands show better
inhibition of proliferation at lowest doses when compared to the
standard drug 5‐FU as shown in Figure 3(b)–(f). On the other
hand, proligands 1–3 and complexes 9 and 10 show inhibition
of proliferation of cancer cells as negative values (Figure 4) and
higher IC values (Table 4). However, complex 11 displays sta-
tistically significant inhibitory effect on cellular proliferation at
its higher concentration (Figure 3a).
FIGURE 4 Overall mean of percentage inhibition of proliferation of
active compounds against human colon carcinoma cell line in
comparison with standard drug 5‐FU
colon cancer cells is depicted in Figure 5. The higher antipro-
liferative activity of the complexes may be attributed to the
presence of Ag(I) in the complex and vice versa related to
the reduced antiproliferative activity of the proligands. Our
findings support the previously reported data establishing
This feature of proligands 4–8 and their respective com-
plexes 12–16 can be attributed to a variation in chain length
the wide range of applications of NHC–Ag(I) complexes for
[19]
[51–53]
that is reported to enhance the lipophilicity.
As lipids are
their antibacterial to anticancer activities,
and the pres-
one of the major constituents of cell and mitochondrial mem-
ent study is in accordance with the data reporting metal–
NHC complexes as being potent anticancer agents.
[
47]
[14,54]
branes, it is assumed that the increased lipophilicity of the
complexes increases the bioavailability of the Ag(I) ions into
cells where they can produce cytotoxicity by inhibiting cellu-
The observed phenomenon of the effect of an increase in
the chain length of the wingtip n‐alkyl groups across the
mono benzimidazolium salts and their respective mono
NHC–Ag(I) complexes is depicted in Figure 4. It can be
clearly seen that the proligands and the complexes with small
alkyl chain length are ineffective against the proliferation of
cells. However, the efficacy of the proligands and the com-
plexes is increased with increasing chain length.
[
48]
lar respiration and causing loss of adenosine triphosphate.
This observation is in line with the understanding that delivery
method, solubility and ionization of silver sources are impor-
[
49]
tant in dealing with biological systems
since the activity
[
50]
of Ag(I) ions depends on their bioavailability.
The
proligands 4–8, although having carbon chain length similar
to that of their respective complexes 12–16, exhibit less anti-
proliferative effects. A comparison between the proligands
and their respective complexes against the proliferation of
Morphological studies of the treated cells reveal potential
cytotoxic changes in the cellular structures. The photomicro-
graphic images (Figure 6) of human colon cancer cells show

FATIMA ET AL.
11 of 14
FIGURE
complexes on proliferation of human colorectal carcinoma cell line.
A) Proligands 3 and 4 with their complexes 11 and 12 demonstrate mild
5
Differential inhibitory effect of proligands and
(
cytotoxic dose‐dependent response against the proliferation of HCT‐116
cells. (B) Proligands 5 and 6 with their complexes 13 and 14
demonstrate moderate cytotoxic dose‐dependent response against the
proliferation of HCT‐116 cells. (C) Proligands 7 and 8 with their
complexes 15 and 16 demonstrate strong cytotoxic dose‐dependent
response against the proliferation of HCT‐116 cells
FIGURE 7 Images of human colorectal cells showing the effects of
proligands 4–8 and complexes 12–16 (cell images taken under an
inverted phase‐contrast microscope at ×200 magnification with digital
camera)
a significant shrinkage of the cells, membrane blebbing and
apoptotic bodies in the cells caused by complexes 12–16,
whereas a moderate cytotoxic effect is observed in the cells
treated with complexes 9–11, since the cellular morphology
is more or less similar to that of the cells from the negative con-
trol group. Complexes 12–16 display a more pronounced anti-
proliferative effect than the standard drug 5‐FU (Figure 7).
4
| CONCLUSIONS
A series of mono benzimidazolium salts and their respective
mono NHC–Ag(I) complexes with wingtip n‐alkyl groups
were successfully synthesized and characterized using spec-
tral and elemental analyses. The structures of the salts and
FIGURE 6 Images of human colorectal cells showing the effects of
proligands 1–3 and complexes 9–11 (cell images taken under an
inverted phase‐contrast microscope at ×200 magnification with digital
camera)

1
2 of 14
FATIMA ET AL.
their respective complexes were elucidated by X‐ray crystal-
lography that established a bis‐carbene arrangement around
the silver centre. The in vitro anticancer studies of the synthe-
sized compounds against the HCT‐116 cancer cell line
revealed a linear relationship between an increase in the chain
length of the substituents and anticancer activity. The com-
pounds having longer n‐alkyl chains (n = 6–10) displayed
pronounced lower IC50 values and antiproliferative activity,
with the complexes exhibiting activity many times superior
to that of the proligands and standard drug 5‐FU.
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ACKNOWLEDGMENTS
Med. Chem. 2015, 90, 82.
This work was supported by Research University Individual
Grant RUI/1001/PKIMIA/811346. T.F. thanks USM
Fellowship (Teaching) (APEX (1002/JHEA/ATSG4001) for
sponsorship.
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