Dose-, time-and lipophilicity-dependent silver(I)–N-heterocyclic carbene complexes: Synthesis, characterization and interaction with plasmid and Aedes albopictus DNA

Article abstract of DOI:10.1002/aoc.3655

Four new Ag(I)–N-heterocyclic carbene (NHC) complexes (5–8) bearing symmetrically substituted NHC ligands have been synthesized starting from the corresponding benzimidazolium bromide salts which are accessible in a single step from N-substituted benzimid

Full text of DOI:10.1002/aoc.3655

Received: 17 August 2016
DOI 10.1002/aoc.3655
Revised: 30 August 2016
Accepted: 11 September 2016
F U L L PA P E R
Dose‐, time‐and lipophilicity‐dependent silver(I)–N‐heterocyclic
carbene complexes: Synthesis, characterization and interaction
with plasmid and Aedes albopictus DNA
1
1
1
2
Patrick O. Asekunowo | Rosenani A. Haque | Mohd.R. Razali | Silas W. Avicor |
2
Mustafa F.F. Wajidi
1
School of Chemical Sciences, Universiti Sains
Four new Ag(I)–N‐heterocyclic carbene (NHC) complexes (5–8) bearing symmetri-
Malaysia, 11800 USM, Penang, Malaysia
2
cally substituted NHC ligands have been synthesized starting from the correspond-
ing benzimidazolium bromide salts which are accessible in a single step from
N‐substituted benzimidazoles (N‐alkyl and N‐aryl) and subsequently reacted with
Molecular Entomology Research Group, School
of Distance Education, Universiti Sains Malaysia,
1800 USM, Penang, Malaysia
1
Correspondence
the basic metal source Ag O in acetonitrile–methanol. These compounds were char-
2
Rosenani A. Haque, School of Chemical Sciences,
Universiti Sains Malaysia, 11800 USM, Penang,
Malaysia.
1
13
acterized using elemental analyses, H NMR, C NMR, Fourier transform infrared
and UV–visible spectroscopic techniques, and molar conductivity. Single‐crystal
structural studies for complex 5 show that the Ag(I) centre has a perfectly linear
C–Ag–C coordination, with quasi‐parallel pairs of aromatic benzimidazole planes.
All the complexes interact with Aedes albopictus DNA via intercalation mode by a
large hypochromicity of 22 and 27% and smaller hypochromicity of 16 and 19%.
Furthermore, all complexes exhibit efficient DNA cleavage activity via a non‐
oxidative mechanistic pathway. The DNase activities of the test compounds
revealed a time‐ and concentration‐dependent activity pattern. The Ag(I)–NHC
complexes showed considerably higher DNA cleavage activity compared to their
respective benzimidazolium salts at a lower concentration. The DNA cleavage of
these complexes changed from a moderate effect to a good one, corresponding to
the increasing lipophilicity order of the complexes as 5 < 6 < 7 < 8 (1.02, 1.05,
Email: rosenani@usm.my
Funding information
Universiti Sains Malaysia for the Research Uni-
versity, Grant/Award Number: 1001/PKIMIA/
8
11346.
1.78 and 2.06 for 5–8, respectively). This order is further corroborated with the
DNA binding study, but with the exception of complex 5, which shows a better
6
5
binding ability for DNA (K = 3.367 × 10 ) than complexes 6–8 (6.982 × 10 ,
b
6
5
8.376 × 10 and 1.223 × 10 , respectively).
KEYWORDS
Aedes albopictus DNA, Ag–NHC complex, DNA binding, N‐heterocyclic carbene,
nuclease activity, X‐ray diffraction
1
| INTRODUCTION
result of their innate chemical, electrochemical and photo-
chemical properties. There has been much interest over the
The potential of metal complexes as therapeutic agents is a
very crucial aspect of their utility. Complexes are presently
being utilized in combination with spectroscopic and bio-
logical procedures to probe the interactions of DNA and
its many structural forms. Metal complexes have shown
interesting propensity to bind DNA through a large number
of interactions and to cleave double‐stranded DNA, as a
last few decades in the non‐covalent interactions of transi-
tion metal complexes with DNA and RNA.
[
2–7]
Continuous demand for new biologically active drugs has
encouraged chemotherapeutic research based on the use of
metals, since metal‐based drugs may be less toxic and
more prone to exhibit anti‐proliferative activity against
[1]
[
8,9]
tumours.
Appl Organometal Chem 2016; 1–15
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
ASEKUNOWO ET AL.
The interactions of organic and inorganic molecules, etc.,
with DNA can impede a number of processes such as tran-
The solvents were of analytical grade and used without fur-
ther purification or drying. NMR spectra were recorded with
a Bruker 500 MHz spectrometer at room temperature in
[
10]
scription, replication and other cellular transactions.
By
so doing various disorders like cancer, cystic fibrosis, malaria,
and dengue fever can be treated by using DNA as targets for
drugs. A lot of research has been directed at finding a number
of bioactive metal complexes. Transition metals like silver
have been used for years as antimicrobial agents. Silver has
low toxicity as compared to other transition metals. Silver
nitrate is still being used as a preventive drug against the devel-
opment of neonatal conjunctivitis in infants. One of the most
commonly used compounds of silver is silver(I) sulfazine; it
is used to treat severe burns to prevent them from succumbing
DMSO‐d . Chemical shifts (δ) were internally referenced to
6
the residual solvent signals relative to tetramethylsilane. The
values of chemical shifts are given in ppm and values for cou-
pling constants (J) in Hz. Abbreviations for signal multiplic-
ities are as follows: singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m) and broad (br). FT‐IR spectra were
recorded with a PerkinElmer system 2000 FT‐IR spectropho-
−
1
tometer in the range 400–4000 cm . Elemental analyses for
C, H and N were carried out with a PerkinElmer series II,
2400 microanalyzer. All electronic absorption spectra were
obtained using a quartz crystal cuvette with a PerkinElmer
Lambda 35 spectrometer. Emission spectra were recorded
with a PerkinElmer LS 55 fluorescence spectrophotometer.
Melting points were measured using a Stuart Scientific
SMP‐1 (UK) instrument. Conductivity measurement was
conducted with a Jenway 470 conductivity/TDS meter, with
conductivity resolution of 0.01 μS–1 mS and accuracy of
±0.5% ± 2 digits. Crystals were mounted on fine glass fibre
or metal pin using viscous hydrocarbon oil. Data were
collected with a Bruker Smart ApexII‐2009 CCD diffractom-
eter, equipped with graphite monochromated Mo Kα radia-
tion (λ = 0.71073 Å). Data collection temperatures were
maintained at 100 K using open flow N2 cryostreams.
Integration was carried out by the program SAINT using
[11]
to bacterial infections.
infective metal complex against urinary tract infections.
Silver sulfadiazine is also an anti‐
[12]
Cleavage of DNA can be achieved by targeting its basic con-
stituents like base and/or sugar by an oxidative pathway or
[8,9]
by hydrolysis of phosphoester linkages.
On the other hand,
DNA binding can occur through control of transcription fac-
tor, where the drug molecule interacts with the protein that
binds to the DNA and hence altering its functions. Interactions
can also occur through direct binding of molecules; here the
drug molecule can directly bind to the DNA. Examples of such
[13]
molecules are groove binders and intercalators.
We have
previously reported some interesting interactions of Ag(I)–
N‐heterocyclic carbene (NHC) complexes with both plasmid
[
2–4]
DNA and RNA.
However, in this present study we are tak-
[
17]
ing a step further in investigating their binding properties
against the DNA of the major dengue vector Aedes albopictus.
The A. albopictus mosquito is an important vector of den-
gue, a fatal disease that lacks vaccines or drugs for preventive
APEX II software.
Solutions were obtained by direct
methods using SHELXS97, followed by successive refine-
2
ments using full‐matrix least‐squares methods against F
[
18]
using SHELXL97.
graphical SHELX interface.
details for complex 5 are provided in Table 1.
The program X‐seed was used as
[14]
[19]
and curative purposes.
The absence of chemo‐preventive
Crystal data and refinement
and chemo‐therapeutic agents restricts the control of this dis-
[14]
ease to vector‐based control methods.
The most wide-
spread vector control practice is insecticide‐based control.
2
.2 | Synthesis of benzimidazolium salts
However, dengue vectors have evolved resistance to insecti-
[15,16]
2
.2.1 | 1,3‐Bis(benzylbenzimidazolium)hexafluorophosphate(1)
cides due to various mechanisms,
reducing the potency
of insecticide‐based vector control and jeopardising efforts to
halt the spread of dengue.
To a stirring solution of benzimidazole (1.00 g, 8.46 mmol)
in 1,4‐dioxane (20 ml), benzyl bromide (2.89 g, 16.90 mmol)
was added. The mixture was heated at 90 °C for 24 h to
obtain thick yellowish oil, which was decanted in order to
remove the solvent and washed with dioxane and diethyl
ether. The bromide salt obtained was converted to its
hexafluorophosphate counterpart using KPF6 (3.11 g,
16.90 mmol) in methanol (20 ml). The mixture was stirred
at room temperature for 3 h and allowed to stand overnight.
Then the solvent was removed under reduced pressure and
the resultant beige powder was washed with distilled water
The purpose of the study reported here was to develop a
methodology for the synthesis of new symmetrically
substituted (benz)imidazolium‐based Ag(I)–NHC complexes;
and to further investigate their interactions with plasmid and
A. albopictus DNA (Aa‐DNA) as an attempt to study their
potential as antibacterial and anti‐dengue vector agents. The
structures of these compounds were correlated with their lipo-
philicity in order to establish a structure–activity relationship.
(
3 × 5 ml) to remove unreacted KPF , and air‐dried to yield
6
compound 1. Recrystallization from acetonitrile–methanol
2
2
| EXPERIMENTAL
gave the compound as a crystalline solid.
1
Yield 66%; m.p. 118–120 °C. H NMR (500 MHz,
.1 | Materials, methods and instrumentation
DMSO‐d , 298 K, δ, ppm): 5.80 (s, 4H, 2 × CH ‐benzyl);
6
2
All reactions were carried out under aerobic conditions. All
7.43 (m, 6H, CH‐benzyl); 7.54 (m, 4H, CH‐benzyl); 7.67 (m,
2H, CH‐benzimidazole); 7.94 (m, 2H, CH‐benzimidazole); 10.03
Ag O reactions were carried out under the exclusion of light.
2

ASEKUNOWO ET AL.
3
TABLE 1 Crystal data and structure refinement details for complex 5
3 h and allowed to stand overnight. Then the solvent was
removed under reduced pressure and the resultant white pow-
der was washed with distilled water (3 × 5 ml) to remove
Formula
Formula weight
Crystal system
Space group
a (Å)
C
42
H
36Ag F
6 4
N P
849.59
unreacted KPF , and air dried. The powder was recrystallized
6
Triclinic
from a solution of acetonitrile–methanol to obtain a crystal-
P‐1
line solid.
8.2460(4)
1
Yield 61%; m.p. 134–136 °C. H NMR (500 MHz,
b (Å)
10.7905(6)
11.7005(6)
69.617(3)
86.721(3)
68.223(3)
903.15(9)
1
DMSO‐d , 298 K, δ, ppm): 0.85 (t, J = 7.0 Hz, 6H,
6
c (Å)
2
2
4
6
× CH ); 1.25 (m, 4H, 2 × CH ─CH ); 1.33 (m, 8H,
3 2 3
α (°)
× CH ─CH ─CH ‐CH ); 1.89 (m, 4H, 2 × N‐CH ‐CH );
2
2
2
3
2
2
β (°)
.52 (t, J = 7.0 Hz, 4H, 2 × N─CH ─R); 7.66 (m, 2H C5/
2
γ (°)
‐H‐benzimidazole); 8.07 (m, 2H C4/7‐H‐benzimidazole); 9.91
3
V (Å )
13
(s, 1H, NCHN). C{1H} NMR (125 MHz, DMSO‐d6,
Z
2
98 K, δ, ppm): 14.5 (CH ); 21.9 (CH ); 25.9 (CH ); 28.3
ρ calcd (g cm−3
3
2
2
)
1.562
(
(
(
CH ); 30.3 (CH ); 46.5 (N─CH ); 114.5, 127.1, 131.4
2 2 2
Temperature (K)
100
C‐4/7, C‐5/6, C‐8/9; C‐benzimiazole); 138.5 (NCN). FT‐IR
μ (mm−
1
)
0.671
−1
KBr disc, cm ): ca 3067 (C─H ); 2967 (C─Haliph);
ar
Crystal size (mm)
θ range (°)
0.07 × 0.09 × 0.68
1.9–31.6
21 427
5963
1
(
471 (C═N). UV–visible (DMSO, nm):
λ
279.5
ε 8928 M− cm ), λ 288.5 (ε 6822 M cm ). Anal.
Calcd for C H N F P (%): C, 52.78; H, 7.18; N, 6.48.
1
−1
31
−1
−1
Reflections measured
Reflections unique
Reflections with I ≥ 2 s(I)
R(int)
19
2 6
Found (%): C, 52.92; H, 7.38; N, 6.75.
4657
0.049
R [I ≥ 2 s(I)]
0.053
2.2.3 | 1,3‐Bis(heptylbenzimidazolium) bromide (3)
wR
2
0.11
Salt
3
was prepared analogously to
2
using
S
1.05
N‐heptylbenzimidazole (1.00 g, 3.34 mmol) and
‐bromoheptane (0.78 g, 4.34 mmol) in acetonitrile (20 ml)
to produce 3 as a white solid.
1
13
1
(
s,1H, NCHN). C{ H} NMR (125 MHz, DMSO‐d , 298 K,
δ, ppm): 52.7 (CH ‐benzyl); 114.2, 125.6, 128.1, 129.5 (C‐
benzyl); 131.5, 134.3, 136.7 (C‐4/7, C‐5/6, C‐8/9; C‐
azole); 138.4 (NCN). FT‐IR (KBr disc, cm ): ca 3123 (C–
6
1
Yield 62%; m.p. 145–147 °C. H NMR (500 MHz,
2
DMSO‐d , 298 K, δ, ppm): 0.81 (t, J = 7.0 Hz, 6H,
6
benzimid-
2
2
4
× CH ); 1.19 (m, 4H, 2 × CH ─CH ); 1.33 (m, 12H,
−1
3 2 3
× CH ─CH ─CH ─CH ); 1.92 (m, 4H, 2 × N─CH ─CH );
2
2
2
3
2
2
H ); 1450 (C═N). UV–visible (DMSO, nm): λ 279.5 (ε 10
ar
.52 (t, J = 7.0 Hz, 4H, 2 × N─CH ─R); 7.69 (m, 2H C5/6‐
−
1
−1
−1
−1
2
177 M cm ), λ 288 (ε 7846 M cm ). Anal. Calcd for
H‐
); 8.12 (m, 2H C4/7‐H‐benzimidazole); 9.99 (s, 1H,
benzimidazole
13
C H N F P (%): C, 56.87; H, 4.35; N, 6.33. Found (%):
2
1 19 2 6
NCHN). C{1H} NMR (125 MHz, DMSO‐d , 298 K, δ,
6
C, 57.21; H, 4.75; N, 6.57.
ppm): 14.5 (CH ); 21.9 (CH ); 25.9 (CH ); 27.3 (CH );
3
2
2
2
3
0.3 (CH ); 31.7 (CH ); 48.5 (N─CH ); 114.5, 127.1,
2 2 2
1
31.4 (C‐4/7, C‐5/6, C‐8/9; C‐benzimiazole); 138.5 (NCN).
2
.2.2 | 1,3‐Bis(hexylbenzimidazolium) hexafluorophosphate (2)
−
1
FT‐IR (KBr disc, cm ): ca 3077 (C─Har); 2972
KOH (1.43 g, 25.40 mmol) was added to a stirring solution
of benzimidazole (2.00 g, 16.93 mmol) in DMSO (30 ml).
The mixture was stirred for 1 h at room temperature and
(
(
C─Haliph); 1470 (C═N). UV–visible (DMSO, nm): λ 279
ε 9928 M− cm ), λ 287 (ε 7479 M cm ). Anal. Calcd
1
−1
−1
−1
for C H N Br (%): C, 63.80; H, 8.86; N, 7.09. Found (%):
C, 63.92; H, 9.04; N, 7.15.
21 35 2
1
‐bromohexane (2.79 g, 16.93 mmol) was added dropwise.
After 2 h the mixture was poured into 200–300 ml of water
and extracted with chloroform (3 × 30 ml). The extract was
filtered thrice through four plies of Whatman filter papers
in order to dry the extract. The desired compound was
finally evaporated under reduced pressure and was
obtained as thick colourless oil. The compound formed,
N‐hexylbenzimidazole (1.00 g, 4.90 mmol), was added
dropwise in a stirring solution of bromohexane (0.81 g,
2
.2.4
|
1,3‐Bis(octylbenzimidazolium) bromide (4)
Salt
4
was prepared analogously to
2
using N‐
octylbenzimidazole (1.00 g, 3.87 mmol) and 1‐bromooctane
(0.75 g, 3.87 mmol) in acetonitrile (20 ml) to produce 4 as
a white solid.
1
Yield 57%; m.p. 150–152 °C. H NMR (500 MHz,
4
.90 mmol) in acetonitrile (30 ml) and refluxed for 20 h.
The solvent was removed under reduced pressure to give
,3‐bis(hexylbenzimidazolium) bromide which was then
reacted with a solution of KPF6 (1.equiv.) in methanol
20 ml). The mixture was stirred at room temperature for
DMSO‐d , 298 K, δ, ppm): 0.84 (t, J = 7.0 Hz, 6H,
6
CH ); 1.27 (m, 4H, 2 × CH ─CH ); 1.36 (m, 16H,
3
2
3
1
2 × N─CH ─CH ─CH ─CH ─CH ─CH ─); 1.95 (m,
2 2 2 2 2 2
4H, 2 × N─CH ─CH ); 4.58 (t, J = 7.0 Hz, 4H,
2
2
(
2 × N─CH ─R); 7.72 (m, 2H C5/6‐H‐benzimidazole); 8.12
2

4
ASEKUNOWO ET AL.
13
(ε 8822 M−1 cm ). Anal. Calcd for C H N AgF P (%):
−1
(m, 2H C4/7‐H‐benzimidazole); 10.07 (s, 1H, NCHN).
C
38 60 4 6
{
1H} NMR (125 MHz, DMSO‐d , 298 K, δ, ppm): 14.5
C, 55.27; H, 7.27; N, 6.79. Found (%): C, 55.42; H, 7.38;
N, 6.95.
6
(CH ); 21.9 (CH ); 23.4 (CH ); 25.9 (CH ); 27.3 (CH );
3 2 2 2 2
3
0.3 (CH ); 31.7 (CH ); 48.5 (N‐CH ); 118.5, 129.4,
2
2
2
1
31.8 (C‐4/7, C‐5/6, C‐8/9; C‐benzimiazole); 142.5 (NCN).
2.3.3 | 1,3‐Bis(heptylbenzimidazolium)silver(I)
hexafluorophosphate (7)
−
1
FT‐IR (KBr disc, cm ): ca 3037 (C─Har); 2962
(
2
C─Haliph); 1479 (C═N). UV–visible (DMSO, nm): λ
79.5 (ε 10 065 M cm ), λ 288 (ε 7677 M cm ).
A mixture of 3 (0.60 g, 1.96 mmol) and Ag O (0.75 g,
2
−
1
−1
−1
−1
3.24 mmol) in dichloromethane (40 ml) was stirred at room
temperature for 24 h. The reaction mixture was filtered
through celite to remove unreacted silver and the solvent
was removed under reduced pressure, which was then reacted
Anal. Calcd for C H N Br (%): C, 65.25; H, 9.22; N,
6
23 39 2
.62. Found (%): C, 65.42; H, 9.44; N, 6.77.
with a solution of KPF (1 equiv.) in methanol (20 ml). The
6
2
.3 | Synthesis of Ag(I)–NHC complexes
mixture was stirred at room temperature for 3 h and allowed
to stand overnight. Then the solvent was removed under
reduced pressure and the resultant white powder was washed
2
.3.1 | 1,3‐Bis(benzylbenzimidazolium)silver(I)
hexafluorophosphate (5)
To a suspension of 1 (0.50 g, 1.00 mmol) in acetonitrile
with distilled water (3 × 5 ml) to remove unreacted KPF , and
6
(
40 ml) was added Ag O (0.24 g, 1.00 mmol). The mixture
2
air dried. The compound was further purified by acetonitrile–
was stirred at 50–60 °C for 12 h under the exclusion of light.
The obtained brown solution was filtered through a pad of
celite and the filtrate was slowly evaporated to precipitate a
pale brown solid. The compound was further purified by
acetonitrile–dichloromethane to give a crystalline solid.
Single crystals suitable for X‐ray analysis were obtained by
the slow diffusion of diethyl ether into acetonitrile solution
containing the complex.
dichloromethane to give a crystalline solid.
1
Yield 57%; m.p. 245–247 °C. H NMR (500 MHz,
DMSO‐d , 298 K, δ, ppm): 0.76 (t, J = 7.0 Hz, 12H,
6
4
× CH ); 1.22 (m, 8H, 4 × CH ─CH ); 1.35 (m, 24H,
3 2 3
4 × CH ─CH ─CH ─CH ); 1.92 (m, 8H, 4 × N─CH ─CH );
2
2
2
3
2
2
4
6
{
.58 (t, J = 7.0 Hz, 8H, 4 × N─CH ─R); 7.50 (m, 4H C5/
‐H‐benzimidazole); 7.88 (m, 4H C4/7‐H‐benzimidazole).
2
1
3
C
1H} NMR (125 MHz, DMSO‐d , 298 K, δ, ppm): 12.5
6
1
Yield 70%; m.p. 220–222 °C. H NMR (500 MHz,
(
CH ); 21.9 (CH ); 25.9 (CH ); 27.3 (CH ); 30.3 (CH );
3 2 2 2 2
DMSO‐d , 298 K, δ, ppm): 5.80 (s, 8H, 4 × CH ‐benzyl);
6
2
31.7 (CH ); 48.5 (N─CH ); 114.5, 127.1, 131.4 (C‐4/7,
2
2
1
7.25–7.40 (m, 12H, CH‐benzyl); 7.45–7.54 (m, 8H, CH‐benzyl);
C‐5/6, C‐8/9; C‐benzimiazole); 187.0, 188.5 ((d,
J
1
7
.77 (m, 2H, CH‐benzimidazole); 7.90 (m, 2H, CH‐benzimidazole).
( ─
) = 207.5 Hz; and d, J( ─107Ag) = 182.0 Hz). FT‐
C
109Ag
C
1
3
1
−1
C{ H} NMR (125 MHz, DMSO‐d , 298 K, δ, ppm): 52.7
6
IR (KBr disc, cm ): ca 3047 (C─H ); 2969 (C─Haliph);
ar
(
CH ‐benzyl); 114.2, 125.6, 128.1, 129.5 (C‐benzyl); 131.5,
2
1450 (C═N). UV–visible (DMSO, nm): λ 277 (ε 12 768 M
−
1
−1
−1
−1
1
34.3, 136.7 (C‐4/7, C‐5/6, C‐8/9; C‐benzimidazole). FT‐IR
cm ), λ 284 (ε 9975 M cm ). Anal. Calcd for
C H N AgF P (%): C, 57.21; H, 7.72; N, 6.36. Found
−
1
(KBr disc, cm ): ca 3105 ν (C─H ); 2977 (C─Haliph);
ar
4
2
68
4
6
1
0
290 (C─N). UV–visible (DMSO, nm): λ 277 (ε 15
(%): C, 57.33; H, 7.94; N, 6.57.
97 M− cm ), λ 284.5 (ε 9829 M cm ). Anal. Calcd
1
−1
−1
−1
for C H N AgF P (%): C, 59.32; H, 4.24; N, 6.59. Found
2.3.4 | 1,3‐Bis(octylbenzimidazolium)silver(I)
hexafluorophosphate (8)
42
36
4
6
(%): C, 59.48; H, 4.42; N, 6.82.
Complex 8 was prepared in the same way as 7, except that 3
2
.3.2
|
1,3‐Bis(hexylbenzimidazolium)silver(I)
was replaced with 4 (1.23 g, 2.90 mmol) and Ag O (0.67 g,
2
hexafluorophosphate (6)
2
.90 mmol). Complex 8 was isolated as a pale brown powder.
Yield 60%; m.p. 251–253 °C. H NMR (500 MHz,
1
Complex 6 was prepared in the same way as 5, except that 1
was replaced with 2 (1.50 g, 3.50 mmol) and Ag O (0.81 g,
DMSO‐d , 298 K, δ, ppm): 0.73 (t, J = 7.0 Hz, 12H,
2
6
3
.50 mmol). Complex 6 was isolated as pale brown powder.
4 × CH ); 1.14 (m, 8H, 4 × CH ─CH ); 1.30 (m, 32H,
4 × N─CH ─CH ─CH ─CH ─CH ─CH ─); 1.92 (m, 8H,
2 2 2 2 2 2
3
2
3
1
Yield 62%; m.p. 231–233 °C. H NMR (500 MHz,
DMSO‐d , 298 K, δ, ppm): 0.73 (t, J = 7.0 Hz, 12H,
4 × N─CH ─CH ); 4.58 (t, J = 7.0 Hz, 8H, 4 × N─CH ─R);
2 2 2
6
4
4
4
× CH ); 1.11 (m, 8H, 4 × CH ─CH ); 1.25 (m, 16H,
7.72 (m, 4H C5/6‐H‐benzimidazole); 7.91 (m, 4H C4/7‐H‐benz-
3
2
3
13
× CH ─CH ─CH ─CH ); 1.90 (m, 8H, 4 × N─CH ─CH );
imidazole). C{1H} NMR (125 MHz, DMSO‐d , 298 K, δ,
2
2
2
3
2
2
6
.60 (t, J = 7.0 Hz, 8H, 4 × N─CH ─R); 7.50 (m, 4H C5/6‐
ppm): 14.5 (CH ); 21.9 (CH ); 23.4 (CH ); 25.9 (CH );
2
3
2
2
2
13
H‐benzimidazole); 7.85 (m, 4H C4/7‐H‐benzimidazole). C{1H}
NMR (125 MHz, DMSO‐d , 298 K, δ, ppm): 12.5 (CH );
27.3 (CH ); 30.3 (CH ); 31.7 (CH ); 48.5 (N─CH ); 118.5,
2 2 2 2
129.4, 131.8 (C‐4/7, C‐5/6, C‐8/9; C‐benzimiazole); 188.5
6
3
−
1
2
0.4 (CH ); 25.9 (CH ); 28.3 (CH ); 30.3 (CH ); 46.5
(C2─Ag). FT‐IR (KBr disc, cm ): ca 3037 (C─H ); 2962
2
2
2
2
ar
(
N─CH ); 114.5, 127.1, 131.4 (C‐4/7, C‐5/6, C‐8/9;
(C─Haliph); 1460 (C═N). UV–visible (DMSO, nm): λ 277.5
(ε 13 987 M cm ), λ 284 (ε 10 127 M cm ). Anal.
2
−
1
−1
−1
−1
−1
C‐benzimiazole); 187.5 (C2─Ag). FT‐IR (KBr disc, cm ): ca
060 (C─H ); 2959 (C─H ); 1462 (C═N). UV–visible
3
(
Calcd for C H N AgF P (%): C, 58.91; H, 8.11; N, 5.98.
ar
aliph
46 76
4
6
−1
−1
DMSO, nm): λ 277.5 (ε 11 928 M cm ), λ 288.5
Found (%): C, 59.25; H, 8.44; N, 6.27.

ASEKUNOWO ET AL.
5
2
.4 | Stability assay
DNA binding analysis using electronic spectral method.
The Aa‐DNA was dissolved in buffer solution (10 mM
phosphate buffer, 50 mM NaCl, pH = 7.4) as a stock solu-
tion, which was stored at 4 °C for 24 h to reach homoge-
neous phase and used within three days. The Aa‐DNA
concentrations were determined by spectroscopy using the
following extinction coefficients at 260 nm: DNA:
In order to assess the stability of the complexes, 10 μg ml−1
solutions of 5–8 in DMSO were prepared and added in a
1
:1 ratio to Aa‐DNA stock solution prepared in DMSO.
This was done to imitate the conditions of the DNA evalu-
ation experiments. The complexes used for the testing were
dissolved in the minimal amount of DMSO possible and
diluted with Aa‐DNA stock solution of 10 M and less
than 0.8% DMSO. UV–visible spectra were measured after
6600 M− cm (per nucleotide), 13 100 M cm−1 (per
1
−1
−1
−
6
[
22]
base pair).
In order to check its purity, the UV absorbance of Aa‐
DNA in the buffer medium was measured at 260 and
0, 24, 48 and 72 h for determining the stability of the
complexes.
2
80 nm and it was found that the DNA was convincingly
free from protein. The DNA binding properties of com-
pounds 5, 6, 7 and 8 with Aa‐DNA were investigated using
UV–visible spectra. The complex was dissolved in DMSO
to obtain the desired concentration. Absorption titration
experiments were done by varying the concentration of
Aa‐DNA, while the complex concentration is kept constant
2
.5 | Biological activity
2
.5.1 | Lipophilicity assay
The experimental lipophilicity measurement (with slight
modification)
between octanol and aqueous solution. Log P was calculated
as the decimal logarithm of the ratio of the solute concentra-
tion in n‐octanol and in water after partition equilibrium.
Amounts of 20 ml of octanol, 30 ml of distilled water and
[
20]
involved partitioning of a compound
(10 μM). An increasing amount of Aa‐DNA (10 μl each
time) was gradually added to the complex in buffer after it
had been incubated for about 2 h at 25 °C. The UV–visible
spectra were recorded after equilibration for about 5 mins.
The intrinsic equilibrium DNA binding constants (K ) along
10 ml of complex were introduced into a 250 ml separating
b
with binding site sizes (s) of the complexes to Aa‐DNA
were determined by monitoring the change of the absorp-
tion intensity of the spectral bands with increasing concen-
tration of Aa‐DNA. The data were then fitted to the
funnel. It was stirred in a mechanical shaker for 30 mins.
The solutions were then left to stand for 24 h until the two
phases were separated. After standing, 3 ml of solvent was
extracted from the two phases then the concentrations of the
compounds were determined using UV–visible spectroscopy.
Spectrophotometric UV–visible log P determinations.
One working wavelength corresponding to the maximum of
molar absorptivity was selected for each compound. In each
case, the sample concentration was determined by the calibra-
tion curve constructed with four known concentrations in dis-
tilled water and n‐octanol phases. A straight line was
obtained according to the equation y = mx + c, where x is
[
23]
following equations to obtain the value of Kb:
À
À
ÁÁ
1
=2
2
2
εa−εf
εb−εf
b− b −2K Ct½DNAꢀ=s
b
¼
(1)
(2)
2KbCt
Kb½DNAꢀ
b ¼ 1 þ K Ct þ
b
2s
−
1
the concentration of the solute (mol l ) and y the absorbance
at the wavelength of absorbance maximum. The linear
analysis of the standard curve is shown in Figure S1
where Ct is the constant total concentration of metal com-
plexes, [DNA] is the total concentration of added DNA as
M base pairs, ε is the apparent absorption in the presence
a
(supporting information).
of DNA, ε is the extinction coefficient of free Ag(I) com-
f
plex in the buffer and ε is the extinction coefficient of the
b
2
.5.2
|
DNA binding assay
DNA‐bound Ag(I)–NHC complex. The value of ε was
f
Mosquito strain and Aa‐DNA extraction. Larvae of a ref-
erence A. albopictus strain obtained from the Vector Con-
trol Research Unit (VCRU) of Universiti Sains Malaysia
were reared on larval feed consisting of Friskies® cat bis-
cuits, beef liver, yeast and milk powder in a 2:1:1:1
obtained from a Beer's plot of the Ag(I)–NHC complex
while the value of ε was obtained from the absorbance
b
of a saturated Ag–DNA sample divided by the concentra-
tion Ct. The nonlinear analysis was done using origin lab
version.
[
21]
ratio
at ambient conditions of 28 ± 3 °C and
2
.5.3 | Fluorescence titration
7
1
5 ± 10% relative humidity. Emerged adults were fed on
0% sucrose solution and Aa‐DNA was extracted from vir-
Fluorescence titration experiments were carried out at room
temperature under the same buffer conditions as the absorp-
tion titrations. Titrations were carried out by placing the
receptor (Ag–NHC complex solution) into a 3.5 ml cuvette
and adding increasing amounts of [Aa‐DNA] (0–200 μM)
solution in DMSO. After each addition solutions were
gin individual adult females (3 days old) for DNA binding
activity using DNeasy® Blood and Tissue Kit (Qiagen®)
according to the manufacturer's instruction. DNA extracted
from individual female mosquitoes was pooled and used
for the binding study.

6
ASEKUNOWO ET AL.
allowed to equilibrate at room temperature for about
3 | RESULTS AND DISCUSSION
.1 | Synthesis
10–15 min before measurements. The fluorescence spectra
of the solutions were recorded in the range 300–450 nm at
3
an excitation wavelength of 295 nm. The quenching ability
[
24]
of the Aa‐DNAwas evaluated using the following equation:
A series of symmetrically substituted benzimidazolium salts
and their Ag(I)–NHC complexes was synthesized and well
characterized. All the salts are synthesized via a stepwise
N‐alkylation reaction of the benzimidazole with the appropri-
ate aryl/alkyl substituents. The stepwise reactions of benzyl
bromide and bromoalkane with 1 equiv. of benzimidazole
give the mono benzimidazolium bromide salts. Salt 1, which
F0
F
¼
1 þ KSV
r
where F and F are the fluorescence intensities in the absence
0
and presence of the quencher, respectively, Ksv is the linear
Stern–Volmer quenching constant and r is the concentration
is analogous to the previously reported salt bearing the tetra-
of the quencher. Ksv is the slope of a plot of F /F versus r.
0
[27]
fluoroborate counterion,
was prepared by the reaction of
The analysis of the data was carried out using origin.
benzyl bromide (2 equiv.) and benzimidazole (1 equiv.), in
1
,4‐dioxane. The reaction was heated at 90 °C for 24 h to
2
.5.4 | Viscometry
obtain thick yellowish oil which was decanted in order to
remove the solvent and washed with dioxane and diethyl
ether. Salts 2–4 were prepared from benzimidazole by step-
wise alkylation with the appropriate alkyl bromide in the
presence of KOH in DMSO at room temperature for 2 h.
The reactants were subsequently converted into their respec-
tive quaternary salts by the reaction of N‐alkylbenzimidazole
with bromohexane/heptane/octane in acetonitrile at refluxing
conditions for 20–24 h. Hexafluorophosphate salts of 1 and 2
were found to be considerably more stable than the
corresponding bromide salts (due to the formation of dark
viscous salts) in common organic solvents such as acetoni-
trile and acetone and in the solid form at room temperature.
Therefore the bromide salts were converted into their
hexafluorophosphate counterparts by the salt metathesis reac-
Viscosity measurements were performed using an Oswald
viscometer immersed in a thermostatic water bath, main-
tained at constant temperature (29 ± 0.1 °C). DNA concen-
tration was kept constant in all samples, while the complex
concentration was increased each time, prepared from a stock
solution of 2.5 mM. Mixing of the solution was performed by
bubbling nitrogen gas through the viscometer in order to aid
mixing. A digital stopwatch was used for the measurement of
the downward flow of the solution. This was done in tripli-
cate for each sample in order to obtain the mean flow time.
1
/3
Data were plotted as viscosity (η/η0) versus the ratio of
complex to DNA (binding ratio). The following equation
1
/3
was employed in the calculation of the ratio (η/η ) :
0
tf−t
0
η ¼
(3)
tion using KPF in methanol and were obtained conveniently
t0
6
in powder forms. Salts 3 and 4 exhibited considerable stabil-
ity and were left as bromide salts. The synthetic route for the
symmetrically substituted benzimidazolium salts is shown in
Scheme 1. In the first approach, respective Ag(I)–NHC com-
plexes 5 and 6 were synthesized using a modified protocol of
where η is the viscosity of DNA in the presence of the com-
plex, t is the experimental flow time in seconds, t is the flow
f
0
time of buffer in seconds and η is the viscosity of DNA
0
[
25,26]
solution.
Wang et al. by reacting a slight excess of Ag O with salts 1
2
2
.5.5
|
Gel electrophoresis
and 2 in acetonitrile at 50 °C for 12 h under exclusion of light
[
28]
Plasmid (pDsRed‐Express) extraction was conducted using
the High Speed Plasmid Mini Kit (Geneaid®) according to
the manufacturer's instruction. This was assayed with four
(Scheme 2).
bromide salts 3 and 4 reacted with Ag
temperature for 24 h. These complexes were further purified
by salt metathesis in methanol using KPF at room tempera-
In the second approach, benzimidazolium
O in methanol at room
2
−
1
concentrations (200, 100, 50 and 25 μg ml ) of the test com-
pounds to determine their nuclease activity on plasmid DNA.
DNA cleavage/degradation activity. The cleavage/degra-
dation assay consisted of 1 μl of the test salt/complex in a
solution consisting of 5 μl of 50 mM Tris–HCl (pH = 8.0)
and 4 μl of plasmid DNA (15 μg). The mixture was incubated
at 37 °C for 8 and 24 h. After incubation, 5 μl of the product
was electrophoresed on 0.8% agarose gel in 0.5× Tris–
acetate–EDTA (TAE) buffer at 70 V for 1 h. The gel was
6
ture to obtain the corresponding Ag(I)–NHC complexes 7
and 8 (Scheme 3). Complexes 5–8 were obtained in overall
yields of 62–77%.
All the reported complexes were quite soluble in common
organic solvents (dichloromethane, acetonitrile and DMSO),
slightly soluble in water and insoluble in hexane and benzene.
The prepared complexes showed good stability to moisture
but were light sensitive. The prepared complexes dissolved
−
1
1
stained in ethidium bromide (0.5 μg ml ) for 15 min and
viewed under UV light in a UVP Gel‐Doc‐It Imaging System
in DMSO‐d
6
monitored using H NMR spectroscopy over a
period of 7 days showed no significant changes and were sta-
ble in the biological medium over the period of the testing
time as the UV–visible spectra remain unchanged in 72 h
(Figure 1).
(Kinematics®). The nucleating ability of the synthesized
benzimidazolium salts/Ag–NHC complexes was determined
by their efficiency in cleaving/degrading the plasmid DNA.

ASEKUNOWO ET AL.
7
SCHEME 1 Preparation of symmetrically substituted benzimidazolium
salts 1–4
SCHEME 2 Synthetic route to Ag(I)–NHC complexes 5 and 6 from
benzimidazolium salts 1 and 2
SCHEME 3 Synthetic route to Ag(I)–NHC complexes 7 and 8 from
benzimidazolium salts 3 and 4
FIGURE 1 Stability of complexes 5–8 in Aa‐DNA solution shown by UV‐
visible spectra at 0 h, 24 h, 48 h and 72 h [Absorbance vs Wavelength/nm]
3.2 | Spectroscopic characterization
1
The H NMR spectra of benzimidazolium salts 1–4 in
The FT‐IR spectra for salts 1–4 show the C─H stretching
DMSO‐d exhibit a characteristic NCHN proton resonance
6
vibrational bands for the alkyl benzimidazolium salts at
at 9.91–10.07 ppm, suggesting the successful formation of
−
1
[16,30,31]
1
around 2945–3170 cm . A band of medium intensity in
the desired salts.
The H NMR spectra for the salts
−1
the range 1475–1557 cm is assigned to benzimidazole ring
C═N) vibrations. These vibrations show a negative shift in
show similar patterns in the aromatic area in the range
(
7.43–8.12 ppm, which are attributed to benzene and
−
1
13
the Ag–NHC complex spectra (1406–1437 cm ). This shift
is due to the presence of the electropositive metal centre,
which affects the C─N and C═N bond vibrations by pulling
electron density towards itself. Further, the FT‐IR spectra of
all reported compounds display two sharp bands of medium
benzimidazole rings. In the C NMR spectra, chemical
shift values of C‐2 carbon are observed in the range
138.4–142.5 ppm, which is in agreement with reported data
[
29–31]
for similar benzimidazolium salts.
1
In the H NMR spectra for complexes 5–8, the absence of
resonances for the benzimidazolium carbene protons
(NCHN) suggests the formation of the Ag(I)–NHC
−
1
intensities in the ranges 2857–2951 and 2955–3119 cm ,
assigned to aliphatic and aromatic C─H.

8
ASEKUNOWO ET AL.
complexes. This is further confirmed by the downfield shift
of carbene carbon nuclei in the range 182.0–188.5 ppm for
1
3
complexes 6 and 8 in the C NMR spectra. In the case of
13
complex 7, the C NMR spectrum displays resonances for
the C2‐carbon nuclei as two doublets centred at 187.0 and
1
3
109
1
88.5 ppm. This is due to the presence of C– Ag and
1
3
107 1
C– Ag with the coupling constants of J ─
= 205
109Ag
C
1
and J ─
similar reported compounds.
= 182.0 Hz. This observation is in accord with
107Ag
C
[32,33]
Surprisingly for complex
5
, the resonance for Ag–carbene carbon is not detected,
1
3
despite several attempts of obtaining the C NMR spectrum
of the sample, varying the concentration and scan time. The
reason was suggested in the literature as a fluxional behav-
[34–37]
iour of the NHC complexes.
The formation of this com-
plex is, however, confirmed by X‐ray data. The resonances of
aromatic benzylic and aliphatic protons are observed with
negligible changes when compared with the corresponding
salts.
FIGURE 2 Overlay of absorption spectra of the four Ag–NHC complexes
(5–8) studied in this work
indicating the ionic nature of these compounds, which are
The electronic spectra of salts and complexes were
[
39]
−
3
found to be 1:1 electrolytes.
recorded in DMSO solution (10 M concentration) over
the range 200–400 nm. Extinction coefficients were deter-
mined from a minimum of four concentrations per sample,
and were calculated by a linear regression fit of the absor-
bance versus concentration data. In the electronic spectra of
the benzimidazolium salts, there are two typical absorption
bands in the ranges 277–280 and 284–286.5 nm assigned to
overlapping π–π* and n–π* transitions arising from C═C
Based on the foregoing, we could also deduce the struc-
ture of the compounds on the basis of their molar conduc-
tance. It is obvious from this study that each mononuclear
Ag(I)–NHC complex has one hexafluorophosphate counter-
+
ion to balance the Ag and as a consequence reflects the for-
mation of a mononuclear Ag–NHC complex. The molar
conductivity data, elemental analysis and spectroscopy data
show an agreement with the proposed structure of the
compounds, which is further confirmed by means of the data
obtained by single‐crystal X‐ray diffraction.
[
38]
and C═N. These transitions are also observed in the spec-
tra of the complexes but are shifted to a higher energy as a
result of complexation with Ag(I), indicating the formation
of a C─N module in the benzimidazole ring. This confirms
the coordination of the ligand to Ag(I) ion which decreases
the delocalization of π electrons and as a result increases
the energy of the π–π* transition. However, no bands at lower
energy level for d–d transition are detected in the UV–visible
region. An overlay of the electronic spectra for the four
complexes (showing that they are similar but not identical)
at 298 K in 10 mM phosphate buffer at pH = 7 is shown in
Figure 2. Under the conditions of the DNA binding
experiments at pH = 7, the spectra of all the Ag(I)–NHC
complexes are basically the same as spectra recorded at
pH = 10–13 and this is indicative of the fact that the
complexes are not protonated.
3
.4 | Single‐crystal X‐ray diffraction study
Single crystals of 5 suitable for X‐ray diffraction analysis
were obtained from slow diffusion of diethyl ether into a
solution of 5 in acetonitrile at ambient temperature, forming
a colourless block. Complex 5 crystallizes in the triclinic
space group P‐1 with half of the molecule in the symmetric
unit (Figure 3). The central Ag(I) is coordinated to two
i
carbene carbon atoms, making a linear C1─Ag1─C1 bond
with a bond angle of 180.0°. This observation deviates from
[
27]
that of a previously reported similar compound,
bearing
−
the tetrafluoroborate counterion (BF ), where the C─Ag─C
4
angle was 177.88°. The dihedral angle between the planes of
phenyl rings, available on the two benzimidazole moieties, is
almost 90° in contrast to the tetrafluoroborate counterparts,
which are 88.28° and 81.31°.
3.3 | Molar conductivity measurements
The molar conductance values of the benzimidazolium salts
and complexes were obtained at room temperature in DMF
solution at 10− M concentration. The compounds were dis-
solved in DMF and the molar conductivities of their solutions
at 25 ± 2 °C were measured The molar conductivity values of
the benzimidazolium salts are found to be close, at around
The bond distance of Ag1 with C1 (and its symmetry
equivalent, C1 ) is 2.077(2) Å (Table 2), while the bond dis-
tance for tetrafluoroborate counterpart is 2.089 Å. The inter-
i
3
nal ring angle at the carbene centre (N1–C1–N2) is 106.6(2)°,
[
15,41]
which agrees well with reported complexes.
The π–π
interactions with a separation of 3.826(5) Å are observed
between two adjacent complexes, resulting in the formation
of a two‐dimensional supramolecular structure. In the
2
−1
2
3.5–26.5 S cm mol , while the values for the complexes
2
−1
are found to be in the range 26.6–29.9 S cm mol ,

ASEKUNOWO ET AL.
9
of each complex in octanol and distilled water at an equilib-
rium state, as summarized in Table 4. Complex 8 having
the longest side chain presents the best lipophilicity of 2.06,
while the lipophilicity of the complexes improves as the
length of the side chain increases (6 < 7 < 8). Complexes 5
and 6 show similar lipophilicity of 1.02 and 1.05, respec-
tively. Thus in the present study, it is found that the biological
activity of these complexes has a positive correlation with the
lipophilicity. Previous studies have shown that the extent to
which a complex penetrates a lipid bilayer usually increases
with the lipophilicity of the side chains in the NHC
[
43,44]
ligands.
Table 4 summarizes the log P results.
3
.6 | DNA binding
Absorption spectroscopy provided inceptive evidence of pos-
sible intercalation interactions between Aa‐DNA and the Ag
(
I)–NHC complexes. In addition, viscosity measurements
FIGURE 3 Structure of 5 with ellipsoids shown at 50% occupancies
TABLE 2 Selected bond lengths (Å) and angles (°) for 5
and fluorescence titration experiments were also performed
in order to further evaluate the nature of DNA interaction
with the complexes. A complex binding to DNA through
intercalation usually results in hypochromism and
bathochromism, as a result of the intercalative mode involv-
Ag1–C1
C1–N1
2.077(2)
1.355(3)
1.353(3)
180.0
C1–N2
ing a strong stacking interaction between the planar aromatic
chromophore and the intercalating agents.
i
[45,46]
C1–Ag1–C1
The DNA
Ag1–C1–N1
Ag1–C1–N2
N1–C1–N2
126.8(2)
127.0(2)
106.6(2)
binding properties of compounds 5–8 with Aa‐DNA were
investigated by UV–visible spectra in phosphate buffer
(10 mM, pH = 7.4) containing 50 mM NaCl at 25 °C. As
illustrated in Figure 4 and Table 5, with increasing concentra-
tion of Aa‐DNA, the absorption intensities of these com-
pounds are reduced by a large hypochromicity of 22 and
extended structure, complex 5 is connected via C─H⋅⋅⋅F and
C─H⋅⋅⋅N hydrogen bonds (Table 3) forming a three‐dimen-
sional network. Each fluoride ion is connected with eight dif-
ferent hydrogen atoms to form the hydrogen bonds.
2
7% and smaller hypochromicity of 16 and 19%. There is
no bathochromic shift in the observed spectra and the maxi-
mum absorption shows obvious hypsochromic adsorption
(4.0–12.5.0 nm). The hypsochromic adsorption (blue shift)
suggests that the compounds or a segment of the compound
framework would fit into the base pairs of DNA as DNA–
intercalating agents. Based on the decrease in absorbance
and the hypsochromism observed, an intercalative binding
mode is assigned to these complexes.
3
.5 | Lipophilicity
The lipophilicity (log P) is perhaps the most important phys-
icochemical property of a potential drug. It is generally
related to solubility, absorption, plasma protein binding
membrane permeability and biological activities of many
The intrinsic equilibrium binding constants of com-
pounds 5–8 with Aa‐DNA were calculated to be
[
42]
compounds.
Therefore, complexes 5–8 were designed
6
5
5
6
with different substituents placed on the NHC groups. The
complexes were tuned using different side‐chain groups to
explore their best lipophilicity. The lipophilicity of the com-
plexes was determined by measuring the concentration ratio
3.367 × 10 , 6.982 × 10 , 8.376 × 10 and 1.223 × 10 ,
respectively. The binding site size, s, obtained from absor-
bance titrations, is consistently between 0.81 and 1.05
a
TABLE 4 Lipophilicity (log P) for Ag(I)–NHC complexes 5–8
TABLE 3 Intramolecular interactions of complex 5
(× 10−4 mol l−1
(× 10−4 mol l−1
Complex
C
w
)
C
o
)
log P
d(D–H)
d(H···A)
(Å)
d(D···A)
(Å)
>(DHA)
(°)
5
6
7
8
1.0449
0.9520
0.2126
0.1206
10.9705
9.7662
1.02
1.05
1.78
2.06
D–H···A
(Å)
C8–H8A⋅⋅⋅F3
C8–H8A⋅⋅⋅F2
C15–H15B⋅⋅⋅F1
C21–H21A⋅⋅⋅N2
0.99
0.99
0.99
0.95
2.360
2.520
2.530
2.520
3.323(3)
3.200(3)
3.204(3)
2.862(4)
164
125
125
101
12.8260
14.3857
a
× 10−4 M)/(C
× 10−4 M)], where C
Log Poct/wat = log [(C
to the concentration of complex in water and octanol, respectively.
o
w
w
and C
o
correspond

1
0
ASEKUNOWO ET AL.
a
TABLE 5 DNA binding constants for Ag–NHC complexes 5–8
b
b
c
Complex
Hypsochromic shift (nm)
K
b
s
%H
6
5
5
6
5
6
7
8
12.5(277)
4.0(273)
7.0(284)
9.0(285)
3.367 × 10
6.982 × 10
8.376 × 10
1.223 × 10
1.05
0.81
0.84
0.96
27
16
19
22
a
Conditions: 10 mM phosphate, pH = 7.4, 50 mM NaCl, 5–8.
b
6
Errors in K
b
and s values from absorption titrations are ±0.6 × 10 and ±0.05. K
b
is the DNA binding constant and s is the binding site size.
c
Percent hypochromicity at specified wavelength in the absorption spectrum. The
[
18]
percentage hypochromicity was calculated using:
where A is the absorbance (free metal complex) and A
60 nm (mixture of metal complex bound to DNA).
%H = (A
a b a
− A ̸A ) × 100,
a
b
is the absorbance at
2
corresponding to a binding stoichiometry of about one
Ag–NHC complex per base pair site. The values of K and
b
site sizes were obtained from the spectroscopy emission data
and these data were fitted to the equation previously
[
23]
derived.
The K values show that compound 5 with an
b
extended aromatic side chain has the highest binding constant
and consequently manifests the strongest binding properties
with Aa‐DNA compared to the other compounds possessing
aliphatic side chains. This difference in the binding capacity
may be due to the presence of an extended aromatic moiety
in compound 5, resulting in larger binding affinities of the
complex compared to those of the others. In general the mode
of action involves a strong stacking interaction between the
[
45]
planar aromatic chromophore and the intercalating agents.
Furthermore the K values and the binding strengths of the
b
complexes follow the order 6 < 7 < 8 and this observation
also correlates well with the lipophilic studies of the reported
complexes. Therefore the result suggests that the length of the
chain is valuable for DNA binding. Values of K obtained
b
from absorption and fluorescence titrations in the present
work are in reasonable agreement with those obtained from
[
47–49]
DNA titrations of similar complexes in the literature.
Also the Kb values of the benzimidazolium salts are
sufficiently less than those of the corresponding Ag
[
47–49]
complexes available in the literature.
3
.7 | Fluorescence titration
To further study the binding capacity of the Ag–NHC com-
plexes, fluorescence titrations were conducted (Figure 5 and
Table 6). The fluorescence properties were determined to
investigate the interactions between compounds 5–8 and
Aa‐DNA in phosphate buffer (10 mM phosphate, pH = 7.4,
5
0 mM NaCl). As depicted in Figure 5, the compounds show
similar binding properties with Aa‐DNA, exhibiting similar
double emission bands in the region 310–325 nm corre-
sponding to intraligand π*–π and π*–n transitions in the
fluorescence spectra. The fluorescence intensities decrease
markedly with increasing concentration of Aa‐DNA. The
quenching behaviours of Aa‐DNA on the fluorescence of
FIGURE 4 Absorption titration of complexes 5–8 with increasing amounts
of Aa‐DNA under conditions listed in Table 5. For each Ag–NHC complex,
the spectral series show a progression of decreasing intensity with increasing
[DNA]/[Ag]. The insets show plots of (ε
a b a f
−ε )/(ε −ε ) versus [DNA]μM,
obtained from absorption spectral titration of the complexes
5–8 are found to follow a conventional Stern–Volmer

ASEKUNOWO ET AL.
11
c
a
TABLE 6 Fluorescence titration data for Ag–NHC complexes 5–8
b
b
Complex
K
b
s
0
F /F
7
5
6
6
n
6
7
8
1.796 × 10
9.670 × 10
1.136 × 10
7.377 × 10
1.72
0.95
1.10
1.55
66
17
25
38
a
Conditions: 10 mM phosphate, pH = 7.4, 50 mM NaCl, 5–8.
b
6
b
Errors in K and s values from fluorescence titrations are ±0.21 × 10 and ±0.2.
c
Ratio of fluorescence intensity of (Ag–NHC complex + Aa‐DNA) to fluores-
cence intensity of Ag–NHC complex in buffer.
[
50,51]
relationship.
The equations reveal that F /F increases in
0
direct proportion to the increasing concentration of Aa‐DNA,
and the quenching constant KSV defines the quenching effi-
ciency of Aa‐DNA. We have used the spectroscopic method
to obtain values of equilibrium binding constants and exam-
ples of the florescence emission data are shown in Figure 5.
Values of K and site size, s, were extracted from the data
b
[
23]
by fitting the data to the equations
with the modification
that fluorescence intensity F replaces the ε values used for
the absorption titrations. The fitting results from titrations
of the Ag–NHC complexes are listed in Table 6.
The fluorescence intensities were quantified by plotting
F /F as a function of Aa‐DNA concentration, where F and
0
0
F are the fluorescence intensity without and with Aa‐DNA.
Stern–Volmer analysis gives a profound insight into the
binding efficiency in terms of fluorescence enhancement of
the complexes with increasing concentration of Aa‐DNA.
7
The calculated binding efficiencies are 1.796 × 10 ,
5
6
6
9
.670 × 10 , 1.136 × 10 and 7.377 × 10 for compounds
5
, 6, 7 and 8, respectively. The magnitude of K values
b
obtained from the fluorescence titrations agree well with the
order of magnitude for the absorption titrations
(
6 < 7 < 8 < 5); however, the results of fluorescence
titrations have a larger range.
The quenching constant (Ksv) values of the quencher are
given by the ratio of intercept to slope, which defines
the quenching efficiency of Aa‐DNA and consequently
the binding properties of the complexes. As shown by the
Stern–Volmer plots in Figure 5 (insets), the Ksv values are
5
5
5
5
−1
0
.3 × 10 , 0.1 × 10 , 0.12 × 10 and 0.25 × 10 M for 5,
6, 7 and 8, respectively. The Stern–Volmer plots indicate that
the fluorescence of compound 5 linked by the aromatic side
chain is more sensitive to the Aa‐DNA concentrations than
those of the others. These results are consistent with the
observations as regards the absorption titrations and viscosity
measurements in all items in this work. This result shows that
all the complexes interact with Aa‐DNA which provides
further evidence that the metal complexes bind to Aa‐DNA.
FIGURE 5 Fluorescence spectral changes of 5–8 upon addition of Aa‐DNA
(0–200 μl) in phosphate buffer (pH = 7.4) containing 50 mM NaCl and 2%
DMSO at 25 °C. Insets: Stern–Volmer plots for the observed fluorescence
enhancement upon addition of Aa‐DNA to the Ag–NHC complexes [F
versus Aa‐DNA(10 )/M
0
/F]
4
3.8 | DNA binding activity using viscometry
Viscosity measurement is used to study the changes in the
viscosity of solutions due to DNA intercalation. This method

1
2
ASEKUNOWO ET AL.
is also vital for discovering the nature of the binding of metal
[
52]
complexes to DNA.
An optimum intercalative mode usu-
ally causes a significant increase in the viscosity of DNA
solution by pushing the base pairs apart and hence causing
complete lengthening of the DNA helix. On the other hand,
an incomplete intercalation of the ligand may also bend the
helix and as a result reduce its effective length and conse-
[3]
quently reduce the DNA viscosity. Therefore an increase
in solution viscosity is a reflection of molecules intercalating
[
54]
into DNA.
In order to further probe the binding nature of the com-
plexes to Aa‐DNA, the viscosity of solutions of complexes
with Aa‐DNA was measured. The result of the viscosity
titration plots is shown in Figure 6. The viscosity of Aa‐DNA
is increased with an increase of the concentration of the test
compounds, using ethidium bromide as the positive
[
55]
control
and DMSO as the negative control. In the solvent
control test, the effect of 10% DMSO on the viscosity was
studied, and it is shown to be a non‐intercalator. Positive
slopes are observed for all the complexes, although with
values less than that of ethidium bromide, whereas the known
non‐intercalator DMSO produces a zero slope line. Based on
the foregoing, the result has also established a considerable
[
56]
intercalating mode of the interactions of these complexes.
3.9 | Nuclease activity of benzimidazolium salts and Ag
(I)–NHC complexes
3
.9.1 | Deoxyribonuclease (DNase) activity of test compounds
The DNase activity of the test compounds at varying concen-
trations and incubation periods were analysed on agarose gel
s using plasmid DNA. Incubation of plasmid DNA with the
test salts to evaluate their DNase activities reveals a concen-
tration‐dependent activity pattern (Figures 7 and 8). DNA
degradation (salts 2–4) and cleavage (nicked circular form;
salt 1) are observed after an incubation period of 24 h with
00 μg ml− of the test ligands (Figure 7A). At the lower con-
1
2
−1
centration of 100 μg ml , DNA degradation and cleavage
FIGURE 7 Deoxyribonuclease activity assay on plasmid DNA after a 24 h
−
1
incubation period using (A) 200, (B) 100, (C) 50 and (D) 25 μg ml of the
FIGURE 6 Viscosity titration of Aa‐DNA and each of the four Ag–NHC
complexes, negative control (DMSO) and positive control (ethidium
bromide, EB). Conditions: 10 mM phosphate buffer, pH = 7, 50 mM NaCl,
test salts. M: 1 kb molecular marker (Fermentas); C: control plasmid DNA;
: plasmid DNA + 1; 2: plasmid DNA + 2; 3: plasmid DNA + 3; 4: plasmid
1
DNA + 4
[Ag] = 25 μM

ASEKUNOWO ET AL.
13
FIGURE 8 Deoxyribonuclease activity assay on plasmid DNA after an 8 h
incubation period using (A) 200, (B) 100, (C) 50 and (D) 25 μg ml of the
FIGURE 9 Deoxyribonuclease activity assay on plasmid DNA after a 24 h
incubation period using (A) 200, (B) 100, (C) 50 and (D) 25 μg ml of the
−
1
−1
test salts. M: 1 kb molecular marker (Fermentas); C: control plasmid DNA;
test complexes. M: 1 kb molecular marker (Fermentas); C: control plasmid
DNA; 5: plasmid DNA + 5; 6: plasmid DNA + 6; 7: plasmid DNA + 7; 8:
plasmid DNA + 8
1
: plasmid DNA + 1; 2: plasmid DNA + 2; 3: plasmid DNA + 3; 4: plasmid
DNA + 4

1
4
ASEKUNOWO ET AL.
are observed (Figure 7B). However, salt 3, which hitherto
nearly completely degraded the DNA at a concentration of
00 μg ml− (Figure 7A), only cleaves it at 100 μg ml
1
−1
2
(Figure 7B). Lower DNase activities are observed when
using lower concentrations of the test salts (Figure 7C, D).
At 8 h of incubation, the test ligands induce varying
DNase activity levels based on the concentration of the salts
(
Figure 8). Pronounced degradation of DNA is observed
−
1
when using salts 3 and 4 at both 100 and 200 μg ml
(
5
Figure 8A, B) compared to concentrations of 25 and
0 μg ml− (Figure 8C, D). Cleavage abilities were however
1
displayed by the salts at the lower concentrations of 25 and
0 μg ml− (Figure 8C, D).
1
5
Figure 9 shows the DNase activities of different concen-
trations of the complexes after a 24 h incubation period.
−
1
Higher concentrations (100 and 200 μg ml ) of complexes
and 8 completely degrade the plasmid DNA, while
partial degradation and cleavage are observed for complexes
and 6, respectively. None of the complexes completely
degrades the plasmid DNA at lower concentrations (50 and
7
5
−1
2
5 μg ml ), although degradation/cleavage is observed for
complexes 7 and 8.
Incubating the complexes for a shorter period (8 h) leads
to higher concentrations of complexes 6–8 inducing
cleavage (nicked circular DNA) of the plasmid DNA
(Figure 10). Nearly similar cleavage abilities are displayed
by complexes 6–8 at concentrations of 100 and 200 μg ml
−1
(Figure 10A, B). DNase activities at lower concentrations
are minimal compared 1to higher concentrations. However,
even at lower concentrations, complexes 7 and 8 exhibit
higher DNase activities compared to complexes 5 and 6 as
observed in the conspicuous decrease in intensity of the
DNA band (Figure 10C, D).
The nature of reactive intermediates involved in DNA
cleavage by the complexes is not clear. However, the contribu-
tion of an oxidative cleavage mechanism can be invalidated,
and it may be said that, in the present study, cleavage most
probably occurs primarily through hydrolytic pathways.
4
| CONCLUSIONS
A
series of four new symmetrically substituted
benzimidazolium salts and their Ag(I)–NHC complexes were
synthesized and characterized using various spectral and ana-
lytical techniques. All reported compounds were stable to air
and moisture and their stability in the biological medium was
investigated and found to be acceptable for the studies.
Molecular structure of complex 5 was established using sin-
gle‐crystal X‐ray diffraction. All the complexes in this study
showed moderate to significant DNA cleavage/degradation
activity in the absence of oxidizing agent. Incubation of plas-
mid DNA with the test compounds to evaluate their DNase
activities revealed a time‐ and concentration‐dependent activ-
ity pattern. Additionally, UV–visible absorption spectral,
FIGURE 10 Deoxyribonuclease activity assay on plasmid DNA after an 8 h
−
1
incubation period using (A) 200, (B) 100, (C) 50 and (D) 25 μg ml of the
test complexes. M: 1 kb molecular marker (Fermentas); C: control plasmid
DNA; 5: plasmid DNA + 5; 6: plasmid DNA + 6; 7: plasmid DNA + 7; 8:
plasmid DNA + 8

ASEKUNOWO ET AL.
15
emission spectral and viscosity measurement studies revealed
the intercalative mode of binding of theses complexes with
DNA, showing considerable intrinsic binding constants.
DNA binding assay showed that complex 5 can strongly
interact with DNA and has a better DNA binding ability than
the other complexes. DNA binding properties suggested that
the length of the linker is of benefit for improving the DNA
binding capacity of these compounds. The lipophilicity assay
indicates that the activity of the Ag(I)–NHC complexes cor-
relates well with the lipophilicity of the complexes. Further
work is ongoing on the in vivo activity of the newly designed
complexes against all stages of the major dengue vector A.
albopictus, the results of which will be reported in due course.
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ACKNOWLEDGMENTS
R. A. Haque and M. R. Razali thank Universiti Sains Malaysia
for the Research University grant 1001/PKIMIA/811346.
P. O. Asekunowo thanks USM for a postdoctoral fellowship.
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