Synthesis and characterization of nitrile functionalized silver(I)-N-heterocyclic carbene complexes: DNA binding, cleavage studies, antibacterial properties and mosquitocidal activity against the dengue vector, Aedes albopictus

Article abstract of DOI:10.1016/j.ejmech.2018.03.029

A series of four benzimidazolium based nitrile–functionalized mononuclear–Ag(I)–N-heterocyclic carbene and binuclear–Ag(I)–N-heterocyclic carbene (Ag(I)–NHC) hexafluorophosphate complexes (5b–8b) were synthesized by reacting the corresponding hexafluorophosphate salts (1b–4b) with Ag₂O in acetonitrile, respectively. These compounds were characterized by ¹H NMR, ¹³C NMR, IR, UV–visible spectroscopic techniques, elemental analyses and molar conductivity. Additionally, 8b was structurally characterized by single crystal X-ray diffraction technique. Preliminary in vitro antibacterial evaluation was conducted for all the compounds against two standard bacteria; gram–positive (Staphylococcus aureus) and gram–negative (Escherichia coli) bacterial strains. Most of the Ag(I)–NHC complexes (5b–8b) showed moderate to good antibacterial activity with MIC values in the range of 12.5–100 μg/mL. Especially, compound 8b exhibited promising anti–Staphylococcus aureus activity with a low MIC value (12.5 μg/mL). However, all the hexafluorophosphate salts (1b–4b) were inactive against the bacteria strains. The preliminary interactive investigation revealed that the most active compound, 8b, could effectively intercalate into DNA to form 8b–DNA complex which shows a better binding ability for DNA (K_b = 3.627 × 10⁶) than the complexes 5b–7b (2.177 × 10⁶, 8.672 × 10⁵ and 6.665 × 10⁵, respectively). Nuclease activity of the complexes on plasmid DNA and Aedes albopictus genomic DNA was time-dependent, although minimal. The complexes were larvicidal to the mosquito, with 5b, 6b and 8b being highly active. Developmental progression from the larval to the adult stage was affected by the complexes, progressively being toxic to the insect's development with increasing concentration. These indicate the potential use of these complexes as control agents against bacteria and the dengue mosquito Ae. albopictus.

Full text of DOI:10.1016/j.ejmech.2018.03.029

Accepted Manuscript
Synthesis and characterization of nitrile functionalized silver(I)-N-heterocyclic carbene
complexes: DNA binding, cleavage studies, antibacterial properties and mosquitocidal
activity against the dengue vector, Aedes albopictus
Patrick O. Asekunowo, Rosenani A. Haque, Mohd. R. Razali, Silas W. Avicor,
Mustafa F.F. Wajidi
PII:
S0223-5234(18)30272-1
10.1016/j.ejmech.2018.03.029
EJMECH 10294
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 January 2018
Revised Date: 28 February 2018
Accepted Date: 8 March 2018
Please cite this article as: P.O. Asekunowo, R.A. Haque, M.R. Razali, S.W. Avicor, M.F.F. Wajidi,
Synthesis and characterization of nitrile functionalized silver(I)-N-heterocyclic carbene complexes: DNA
binding, cleavage studies, antibacterial properties and mosquitocidal activity against the dengue vector,
Aedes albopictus, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.029.
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ACCEPTED MANUSCRIPT
Graphical abstract
A new series of benzimidazolium based nitrile–functionalized Ag(I)–N‐heterocyclic carbene
complexes was developed by the deprotonation of the benzimidazolium salts and refluxing the
benzimidazolium salts in the presence of a slight excess of Ag2O. The most promising lead of
this series, compound 8b, were highly larvicidal and adversely affected the development of
larvae into adults at the tested concentrations.

ACCEPTED MANUSCRIPT
Synthesis and characterization of nitrile functionalized silver(I)-N-
heterocyclic carbene complexes: DNA binding, cleavage studies,
antibacterial properties and mosquitocidal activity against the dengue
vector, Aedes albopictus
Patrick O. Asekunowo*ab, Rosenani A. Haque a, Mohd. R. Razali a, Silas W.
Avicor *cd, Mustafa F.F. Wajidi c
aSchool of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
bDepartment of Chemical Sciences, Mountain Top University, KM 12, Lagos-Ibadan Express
Way, Ogun State, Nigeria
cMolecular Entomology Research Group, School of Distance Education, Universiti Sains
Malaysia, 11800 USM, Penang, Malaysia
dEntomology Division, Cocoa Research Institute of Ghana, P. O. Box 8, New Tafo-Akim,
Ghana.
*Corresponding email: poasekunowo@mtu.edu.ng
*Corresponding email: wintuma@yahoo.co.uk
ABSTRACT
A series of four benzimidazolium based nitrile–functionalized mononuclear–Ag(I)–
N‐heterocyclic carbene and binuclear–Ag(I)–N‐heterocyclic carbene (Ag(I)–NHC)
hexafluorophosphate complexes (5b–8b) were synthesized by reacting the corresponding
hexafluorophosphate salts (1b–4b) with Ag2O in acetonitrile, respectively. These compounds
1
were characterized by H NMR, 13C NMR, IR, UV–visible spectroscopic techniques,
elemental analyses and molar conductivity. Additionally, 8b was structurally characterized by
single crystal X-ray diffraction technique. Preliminary in vitro antibacterial evaluation was
conducted for all the compounds against two standard bacteria; gram–positive
(Staphylococcus aureus) and gram–negative (Escherichia coli) bacterial strains. Most of the
Ag(I)–NHC complexes (5b–8b) showed moderate to good antibacterial activity with MIC
values in the range of 12.5–100 µg/mL. Especially, compound 8b exhibited promising anti–
Staphylococcus aureus activity with a low MIC value (12.5 µg/mL). However, all the
hexafluorophosphate salts (1b–4b) were inactive against the bacteria strains. The preliminary
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interactive investigation revealed that the most active compound, 8b, could effectively
intercalate into DNA to form 8b–DNA complex which shows a better binding ability for
DNA (Kb = 3.627 x 106) than the complexes 5b–7b (2.177 × 106, 8.672 × 105 and 6.665 ×
105, respectively). Nuclease activity of the complexes on plasmid DNA and Aedes albopictus
genomic DNA was time-dependent, although minimal. The complexes were larvicidal to the
mosquito, with 5b, 6b and 8b being highly active. Developmental progression from the larval
to the adult stage was affected by the complexes, progressively being toxic to the insect’s
development with increasing concentration. These indicate the potential use of these
complexes as control agents against bacteria and the dengue mosquito Ae. albopictus.
Keywords:
Aedes albopictus DNA; Ag–NHC complex; DNA binding; N–heterocyclic carbene;
nuclease activity; X–ray diffraction.
1. Introduction
Nitrogen-containing heterocycles have been used as medicinal compounds for centuries, and
form the basis for many common drugs such as Morphine (analgesic), Captopril (treatment of
hypertension) and Vincristine (cancer chemotherapy). Benzimidazole and imidazole are
frequently classified by medicinal chemists as privileged ‘sub-structures’ for drug dosing
owing to the affinity they exhibit towards some enzymes and protein receptors. In the past
few years, imidazole and its derivatives have received much attention as a result of their
chemotherapeutic values. Imidazole analogue systems are present in a constituent of several
important natural products, such as histidine, histamine, purine and nucleic acids [1].
(Benz)imidazoles, their derivatives and transition metal complexes have received
considerable attention in coordination chemistry due to their well-documented biological
activities [2 - 5]. It has been found that such complexes show better biological activities than
the free ligands [6, 7]. However amongst these transition metals, Ag has shown low toxicity
to humans and is present in the human body in very low concentrations (<2.3 µg/L), with its
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absorption mostly through the lungs, skin, gastrointestinal tract and mucus membranes [8]. It
is mainly found in the human body as silver protein complexes but has no physiological or
biochemical role within the body [9]. It can be tolerated at high concentrations within the
body, does not appear to be a cumulative poison and is eliminated from the body through the
urine and faeces [8]. Recently, a number of researchers have reported promising biological
activities of Ag(I)-NHC complexes as anticancer and antimicrobial agents [10-16]. However
research on Ag(I)-NHC complexes as anti-malarial and anti-dengue agents remains scant. As
a result of the foregoing, our research group continues to focus on providing Ag by
incorporating Ag(I) to NHCs, and tuning both the electronic and steric properties to improve
its stability in the body.
Dengue is the most important arthropod-borne viral infection of humans. Worldwide, an
estimated 2.5 billion people are at risk of infection, approximately 975 million of whom live
in urban areas in tropical and sub-tropical countries in Southeast Asia, the Pacific and the
Americas [17]. Transmission also occurs in Africa and the Eastern Mediterranean, and rural
communities are increasingly being affected. It is estimated that more than 50 million
infections occur each year, including 500,000 hospitalizations for dengue haemorrhagic
fever, mainly among children, with the fatality rate exceeding 5% in some areas [17-20].
The annual average number of dengue fever/dengue haemorrhagic fever (DF/DHF) cases
reported to the World Health Organization (WHO) has increased dramatically in recent years.
For the period 2000–2004, the annual average was 925,896 cases, almost double the figure of
479,848 cases that was reported for the period 1990–1999. In 2001, 69 countries reported
dengue activity to the WHO and in 2002, the Region of the Americas alone reported more
than 1 million cases [17-20]. To reduce or prevent dengue virus transmission, there is
currently no alternative to vector control. Most endemic countries have a vector control
component in their dengue control and prevention programmes but its delivery by public
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health practitioners is frequently insufficient, ineffective or both. An obvious method for the
control of mosquito-borne diseases is the use of insecticides, and many synthetic agents have
been used in the field with considerable success. However, they also manifest undesirable
effects, including toxicity to non-target organisms, giving rise to environmental and human
health concerns [18].
Therefore, in an approach to find new compounds against this infective disease, this work
aims to test a first series of air and moisture stable mononuclear and binuclear Ag(I)-N-
functionalized NHC complexes against the major dengue vector, Aedes albopictus.
The interactive effects of the synthesized compounds on plasmid DNA and Aedes albopictus
DNA were investigated in this study. This paper further explores the preliminary antibacterial
activity of these complexes as well as their larvicidal and developmental inhibitory potentials
on the mosquito to have a comparative insight into the bioactivity of these compounds.
2. Experimental
2.1. Materials, Methods and Instruments
All reactions were carried out under aerobic conditions unless otherwise specified. All Ag2O
reactions were carried out under the exclusion of light. The solvents were of analytical grade
and used without further purification or drying. NMR spectra were recorded on a Bruker 500
MHz spectrometer at room temperature in DMSO-d6. Chemical shifts (δ) were internally
referenced to the residual solvent signals relative to tetramethylsilane. The values of chemical
shifts are given in ppm and values for coupling constants (J) in Hz. Abbreviations for signal
multiplicities are as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and
broad (br). FTIR spectra were recorded on Perkin-Elmer system 2000 FTIR
spectrophotometer in the range 4000–400 cm-1. Elemental analysis was carried out on a
Perkin Elmer series II, 2400 microanalyzer. All electronic absorption spectra were taken in a
quartz crystal cuvette with a PerkinElmer Lambda 35 spectrometer. Melting points were
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measured using a Stuart Scientific SMP-1 (UK) instrument. Conductivity measurement was
obtained from Jenway 470 Conductivity/TDS Meter, with conductivity resolution of 0.01µS-
1mS٭ and accuracy of ±0.5% ±2 digits. Crystals were mounted on fine glass fibre or metal
pin using viscous hydrocarbon oil. Data were collected on a Bruker-Smart ApexII-2009 CCD
diffractometer, equipped with graphite monochromated Mo-Kα (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 the APEX II software [21]. The
solutions were obtained by direct methods using SHELXS97, followed by successive
refinements using full-matrix least-squares method against F2 using SHELXL97 [22]. The X-
seed program was used as graphical SHELX interface [23]. The crystal data and refinement
details for complex 8b are provided in Table S1. The higher R value is due to the disordered
counter anions, PF6 (Table S1).
2.2. Synthesis of benzimidazolium salts
2.2.1. Synthesis of 1-cyanopropyl-3-pentyl-benzimidazolium hexafluorophosphate (1b)
N-cyanopropylbenzimidazole (0.70 g, 3.78 mmol) was added dropwise to a stirring solution
of 1-bromopentane (0.57 g, 3.78 mmol) in dioxane (20 mL) and refluxed at 100oC for 2 days.
The solvent was removed under reduced pressure to obtain 1-cyanopropyl-3-
pentylbenzimidazolium bromide (1a). The bromide salt was filtered and washed with fresh
1,4-dioxane and diethyl ether. The bromide salt was then reacted with a solution of KPF6
(1.04 g, 5.67 mmol) in methanol (20 mL) and stirred for 3 h to precipitate a white solid (1b).
The precipitate formed was filtered, washed with distilled water (3 × 5 mL) to remove
unreacted KPF6, washed with diethyl ether and then dried at room temperature. Yield: 77.5%;
1
m.p. 134-136oC. H NMR (500 MHz, DMSO-d6, 298K, δ ppm): 0.89 (t, J = 7.0 Hz, 3H,
CH3); 1.32 (m, 2H, CH2-CH3); 1.95 (m, 2H, CH2-CH2-CH3); 2.27 (m, 2H, N-CH2-CH2);
2.73 (t, J = 7.0 Hz, 2H, CH2-CH2-CN); 4.49 (t, J = 7.0 Hz, 2H, CH2-CN); 4.63 (t, J = 7.0 Hz,
2H, N-CH2-R); 4.70 (t, J = 7.0 Hz, 2H, N-CH2-CH2-CH2-CN); 7.72 (m, 2H,
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13
benzimidazolium-CH); 8.12 (m, 2H, benzimidazolium-CH); 9.99 (s, 1H, NCHN). C{1H}
NMR (125 MHz, DMSO-d6, 298K, δ ppm): 10.2 (CH3); 23.8 (CH2-CH3); 30.9 (CH2-CN);
40.0 (N-CH2); 48.7 (N-CH2-CH); 116.5 (C≡N); 118.3, 121.5, 127.1, 132.5
(benzimidazolium-C); 137.5 (benzimidazolium-C2′). FTIR (KBr disc) in cm-1, ~ 3143 (C-
Har); 2947 (C-Haliph); 2249 (C≡N) 1432 (C=N). Anal. Calc. for C16H22N3F6P (%): C, 47.88;
H, 5.49; N, 10.47%. Found (%): C, 48.14; H, 5.62; N, 10.59. Molar conductance in DMF:
25.16 S cm2 mol-1
2.2.2. Synthesis of 1-cyanopropyl-3-(3-cyanobenzyl)benzimidazolium
hexafluorophosphate (2b)
A mixture of 3-bromomethyl benzonitrile (0.20 g, 1 mmol) and N-cyanopropylbenzimidazole
(0.185 g, 1 mmol) were stirred in dioxane (20 mL) at 100oC for 2 days. The solvent was
removed under reduced pressure, and the product was then washed with 1,4 dioxane and
diethyl ether. The bromide salt 2a thus obtained was recrystallized from acetonitrile/methanol
(1:5 v/v). Salt 2a was directly converted into its hexafluorophosphate counterpart (2b) by
metathesis reaction using KPF6 (0.28 g, 1.5 mmol) in methanol (20 mL). The resultant
mixture was stirred for 3 h and was allowed to stand overnight. The pale yellow precipitate
was filtered under reduced pressure, washed with distilled water (3 × 5 mL) to remove
unreacted KPF6, and air dried. Yield 67.7 %; m.p. 144–146oC. 1H NMR (500 MHz, DMSO-
d6): δ 2.36 (m, 2H, CH2-CH2-CN); 2.79 (t, J = 7.0 Hz, 2H, CH2-CN); 4.68 (t, J = 7.0 Hz, 2H,
N-CH2-CH2-CH2-CN); 5.93 (s, 2H, N-CH2); 7.66 (m, 3H, benzimidazolium-CH), 7.85 (d, J
= 8.0 Hz, 1H, benzimidazolium-CH); 7.96 (d, J = 8.0 Hz, 2H, benzonitrile-CH); 8.07 (s, 1H,
Benzonitrile-CH), 8.20 (d, J = 8.0 Hz, 1H, Benzonitrile-CH), 10.15 (s, 1H, NCHN).
13C{1H}NMR (125 MHz, DMSO-d6): δ 13.8 (CH2-CH2-CN), 32.3 (CH2-CN), 48.9 (N-CH2-
R) 66.3 (CH2-benzonitrile), 110.9 (C≡N-nitrile), 113.8, 116.9, (benzimidazole-C), 133.0,
134.0, 135.9, (Benzonitrile-C), 143.1 (benzimidazolium C2ʹ). FT-IR (KBr disc) cm-1: ~ 3033
(C-Har); 2926 (C-Haliph); 2230 (C≡N) 1568 (C=N). Anal. Calc. for C19H17N4F6P(%): C, 51.12;
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H, 3.81; N, 12.56. Found(%): C, 51.34; H, 3.97; N, 12.79. Molar conductance in DMF: 24.76
S cm2 mol-1
2.2.3. Synthesis of 1,3-bis(3-cyanobenzyl)benzimidazolium
hexafluorophosphate (3b)
A
mixture of 3-bromomethyl benzonitrile (0.50 g, 2.55 mmol) and N-
cyanobenzylbenzimidazole (0.60 g, 2.55 mmol) were stirred in dioxane (20 mL) at 100oC for
2 days. The solvent was removed under reduced pressure, and the product was then washed
with 1,4 dioxane and diethyl ether. Salt 3a was directly converted into its
hexafluorophosphate counterpart (3b) by metathesis reaction using KPF6 (0.28 g, 1.5 mmol)
in methanol (20 mL). The resultant mixture was stirred for 3 h and was allowed to stand
overnight. The pale yellow precipitate was filtered under reduced pressure, washed with
distilled water (3 × 5 mL) to remove unreacted KPF6, and air dried. Yield 67.7 %; m.p. 154–
156oC. 1H NMR (500 MHz, DMSO-d6):
5.93 (s, 4H, N-CH2); 7.66 (m, 4H,
benzimidazolium-CH), 7.91 (d, J = 8.0 Hz, 2H, benzonitrile-CH); 8.01 (m, 4H, benzonitrile-
CH); 8.12 (s, 2H, benzonitrile-CH), 10.21 (s, 1H, NCHN). 13C{1H}NMR (125 MHz,
DMSO-d6): δ 13.8 (CH2-CH2-CN), 32.3 (CH2-CN), 46.9 (N-CH2-R) 62.3 (CH2-benzonitrile),
110.9 (C≡N-nitrile), 113.8, 116.9, (benzimidazole-C), 133.0, 134.0, 135.9, (Benzonitrile-C),
143.5 (benzimidazolium C2ʹ). FT-IR (KBr disc) cm-1: ~ 3130 (C-Har); 2960 (C-Haliph); 2229
(C≡N); 1562 (C=N). Anal. Calc. for C23H17N4F6P(%): C, 55.87; H, 3.44; N, 11.34.
Found(%): C, 56.14; H, 3.67; N, 11.51. Molar conductance in DMF: 26.76 S cm2 mol-1.
2.2.4. Synthesis of 1,4-bis(3-cyanobenzylbenzimidazol-1-ylmethyl)benzene
hexafluorophosphate (4b)
A mixture of benzimidazole (1.00 g, 8.46 mmol) and KOH (1.43 g, 12.69 mmol) in DMSO
(20 mL) was stirred for 1 h at room temperature. 3-bromomethyl benzonitrile (1.66 g, 8.46
mmol) was added portionwise and the mixture was stirred at room temperature for 2 h. After
2 h, the mixture was poured into 200-300 mL of water and extracted with chloroform (3 × 30
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mL). The extract was filtered twice through four plies of Whatman filter paper in order to dry
the extract. The desired compound was finally evaporated under reduced pressure and a thick
yellow oil was obtained. The compound formed, N-cyanobenzylbenzimidazole (1.0 g, 4.30
mmol) was added dropwise to a stirring solution of 1,4-bis(bromomethyl)benzene (0.57 g,
2.15 mmol) in dioxane (20 mL) and refluxed for 3 days. The solvent was removed under
reduced pressure to give 1,4-bis(3-cyanobenzyl-1-ylmethyl)benzene dibromide and washed
in dioxane and diethyl ether which was then reacted with a solution of KPF6 (0.80 g, 4.35
mmol) in methanol (20 mL). The mixture was stirred at room temperature for 3 h and
allowed to stand overnight. The solvent was removed under reduced pressure and the
resultant brown powder was washed with distilled water (3 × 5 mL) to remove unreacted
KPF6, and air dried. The powder was recrystallized in a solution of acetonitrile/methanol to
obtain a crystalline solid. Yield: 69%; mp: 187-189oC. 1H NMR (500 MHz, DMSO-d6): 5.80
(s, 4H, N-CH2); 5.95 (s, 4H, N-CH2-benzonitrile); 7.48 (s, 4H, 4 × Ar-CH), 7.66 (m, 8H,
benzimidazolium-CH), 7.91 (d, J = 8.0 Hz, 2H, benzonitrile-CH); 8.01 (m, 4H, benzonitrile-
CH); 8.12 (s, 2H, benzonitrile-CH), 10.15 (s, 1H, NCHN). 13C{1H}NMR (125 MHz,
DMSO-d6): δ 13.8 (CH2-CH2-CN), 32.3 (CH2-CN), 46.9 (N-CH2-R) 62.3 (CH2-benzonitrile),
110.9 (C≡N-nitrile), 113.8, 116.9, (benzimidazole-C), 133.0, 134.0, 135.9, (Benzonitrile-C),
143.1 (benzimidazolium C2ʹ). FT-IR (KBr disc) cm-1: ~ 3015 (C-Har); 2920 (C-Haliph); 2232
(C≡N); 1556 (C=N). Anal. Calc. for C38H30N6F12P2(%): C, 47.12; H, 3.93; N, 10.99.
Found(%) : C, 47.34; H, 4.17; N, 11.21. Molar conductance in DMF: 52.67 S cm2 mol-1.
2.3. Synthesis of Ag(I)-NHC complexes
2.3.1. Synthesis of 1-cyanopropyl-3-pentyl-benzimidazolium-silver(I)
hexafluorophosphate (5b)
Ag2O (0.80 g, 3.45 mmol) was added to a solution of 1-cyanopropyl-3-pentyl-
benzimidazolium hexafluorophosphate (1b) (1.38 g, 3.45 mmol) with molecular sieves (3 Å
beads) in acetonitrile (40 mL), protected from light and stirred for 24 h under nitrogen at
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50oC. A clear solution with some black suspension was obtained. The solution was filtered
through Celite®, evaporated under reduced pressure and the solid was poured into water (50
mL). The resulting white powder was collected by filtration and the solid was washed three
times with distilled water. Recrystallization of the powder from acetonitrile yielded a
crystalline solid (53%); m.p: 224-226oC. 1H NMR (500 MHz, DMSO-d6, 298K, δ ppm): 0.87
(t, J = 7.0 Hz, 6H, CH3); 1.33 (m, 8H, CH2-CH2-CH3); 1.92 (m, 4H, N-CH2-CH2); 2.27 (t, J
= 7.0 Hz, 4H, CH2-CH2-CN); 2.68 (t, J = 7.0 Hz, 4H, CH2-CN); 4.58 (t, J = 7.0 Hz, 4H, N-
CH2-R); 4.68 (t, J = 7.0 Hz, 2H, N-CH2-CH2-CH2-CN); 7.50 (m, 2H, benzimidazolium-CH);
7.91 (m, 2H, benzimidazolium-CH). 13C{1H} NMR (125 MHz, DMSO-d6, 298K, δ ppm):
10.2 (CH3); 23.8 (CH2-CH3); 30.9 (CH2-CN); 40.0 (N-CH2); 48.7 (N-CH2-CH); 116.5
(C≡N); 118.3, 121.5, 127.1, 132.5 (benzimidazolium-C); 188.3 (benzimidazolium C2΄-Ag).
FTIR (KBr disc) in cm-1, ~ 3069 (C-Har); 2925 (C-Haliph); 2249 (C≡N) 1406 (C=N). Anal.
Calc. for C32H42N6AgF6P (%): C, 50.33; H, 5.50; N, 11.01. Found (%): C, 50.61; H, 5.72; N,
11.29. Molar conductance in DMF: 27.76 S cm2 mol-1
2.3.2. Synthesis of 1-cyanopropyl-3-(3-cyanobenzyl)benzimidazolium-silver(I)
hexafluorophosphate (6b)
Complex 6b was prepared by adopting a procedure similar to that used for complex 5b
except that 1b was replaced by 2b (1.00 g, 2.24 mmol) and Ag2O (0.52 g, 2.24 mmol).
1
Complex 6b was isolated as a white powder. Yield: 71%; mp: 230-232oC. H NMR (500
MHz, DMSO-d6): δ 2.23 (m, 4H, CH2-CH2-CN); 2.62 (t, J = 7.0 Hz, 4H, CH2-CN); 4.66 (t,
J = 7.0 Hz, 4H, N-CH2-CH2-CH2-CN); 5.82 (s, 4H, N-CH2); 7.50 (m, 3H, benzimidazolium-
CH), 7.64 (d, J = 8.0 Hz, 1H, benzimidazolium-CH); 7.71 (d, J = 8.0 Hz, 2H, benzonitrile-
CH); 7.81 (s, 1H, Benzonitrile-CH), 7.93 (d, J = 8.0 Hz, 1H, Benzonitrile-CH).
13C{1H}NMR (125 MHz, DMSO-d6): δ 13.8 (CH2-CH2-CN), 32.3 (CH2-CN), 48.9 (N-CH2-
R) 66.3 (CH2-benzonitrile), 110.9 (C≡N-nitrile), 113.8, 116.9, (benzimidazole-C), 133.0,
134.0, 135.9, (Benzonitrile-C), 189.2, 190.4 (benzimidazolium C2ʹ-Ag). FT-IR (KBr disc)
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cm-1: ~ 3060 (C-Har); 2926 (C-Haliph); 2230 (C≡N) 1479 (C=N). Anal. Calc. for
C38H32N8AgF6P(%): C, 53.46; H, 3.75; N, 13.13. Found(%): C, 53.84; H, 3.87; N, 13.49.
Molar conductance in DMF: 25.44 S cm2 mol-1.
2.3.3. Synthesis of 1,3-bis(3-cyanobenzyl)benzimidazolium-silver(I)
hexafluorophosphate (7b)
Complex 7b was prepared by adopting a procedure similar to that used for complex 5b
except that 1b was replaced by 3b (1.00 g, 2.24 mmol) and Ag2O (0.52 g, 2.24 mmol).
1
Complex 7b was isolated as a white powder. Yield: 62%; mp: 239-241oC. H NMR (500
MHz, DMSO-d6): 5.88 (s, 8H, N-CH2); 7.44(m, 8H, benzimidazolium-CH), 7.61 (d, J = 8.0
Hz, 4H, benzonitrile-CH); 7.72 (m, 8H, benzonitrile-CH); 7.82 (s, 2H, benzonitrile-CH).
13C{1H}NMR (125 MHz, DMSO-d6): δ 12.8 (CH2-CH2-CN), 31.3 (CH2-CN), 44.9 (N-CH2-
R) 61.3 (CH2-benzonitrile), 110.9 (C≡N-nitrile), 113.8, 116.9, (benzimidazole-C), 133.0,
134.0, 135.9, (Benzonitrile-C), 190.8 (benzimidazolium C2ʹ-Ag). FT-IR (KBr disc) cm-1: ~
3069 (C-Har); 2956 (C-Haliph); 2229 (C≡N); 1459 (C=N). Anal. Calc. for C46H32N8Ag F6P(%):
C, 58.17; H, 3.37; N, 11.80. Found(%): C, 58.29; H, 3.67; N, 11.91. Molar conductance in
DMF: 27.76 S cm2 mol-1.
2.3.4. Synthesis of 1,4-bis(3-cyanobenzylbenzimidazol-1-ylmethyl)benzene silver(I)
hexafluorophosphate (8b)
A mixture of the salt 4b (267 mg, 0.46 mmol) and Ag2O (105 mg, 0.46 mmol) in acetonitrile
(40 mL) was heated at 50 °C for 2 days with molecular sieves (3 Å beads) in acetonitrile (40
mL) and the mixture was protected from light. A clear solution with black suspension was
obtained. The mixture was filtered and concentrated under reduced pressure.
Recrystallization of the residue from acetonitrile yielded a grey powder (63%). Crystals
suitable for X-ray diffraction studies were grown by layering a concentrated solution of the
complex in acetonitrile with neat diethyl ether. mp: 268-270oC. 1H NMR (500 MHz, DMSO-
d6): 5.71 (s, 8H, N-CH2); 5.87 (s, 8H, N-CH2-benzonitrile); 7.28 (s, 8H, 4 × Ar-CH), 7.45
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(m, 16H, benzimidazolium-CH), 7.74 (d, J = 8.0 Hz, 4H, benzonitrile-CH); 7.88 (m, 8H,
benzonitrile-CH); 8.01 (s, 4H, benzonitrile-CH), 13C{1H}NMR (125 MHz, DMSO-d6): δ
13.2 (CH2-CH2-CN), 31.7 (CH2-CN), 46.1 (N-CH2-R) 61.5 (CH2-benzonitrile), 110.9 (C≡N-
nitrile), 112.2, 116.3, (benzimidazole-C), 131.0, 132.5, 134.2, (Benzonitrile-C), 188.9
(benzimidazolium C2ʹ-Ag). FT-IR (KBr disc) cm-1: ~ 3005 (C-Har); 2920 (C-Haliph); 2232
(C≡N); 1455 (C=N). Anal. Calc. for C76H56N12Ag2F12P2(%): C, 55.54; H, 3.41; N, 10.23.
Found(%): C, 55.87; H, 3.64; N, 10.51. Molar conductance in DMF: 59.25 S cm2 mol-1.
2.4. Biological Evaluation
2.4.1. Antibacterial studies
The antibacterial activities of the compounds were investigated against Escherichia coli and
Staphylococcus aureus. Stock solutions of all the compounds were prepared using DMSO.
Antibacterial tests were performed using the Kirby-Bauer disc diffusion method [24]. Single
colonies of E. coli and S. aureus from fresh culture agar plates were respectively cultured in
5 mL of Luria-Bertani (LB) broth solution (Tryptone10 g, yeast extract 5 g, NaCl 10 g/L),
and incubated overnight at 37oC. The turbidity of each culture was adjusted by comparing it
to 0.5 McFarland standards, which is equal to 1.58 × 108 CFU/mL or 0.5 (O.D600 reading).
Single colonies were suspended in the Nutrient broth with incubation over a period of 2–5 h
until an appropriate optical density (OD = 0.6–0.8) at 600 nm was achieved. Using sterile
cotton buds, each bacterial lawn culture was spread uniformly on different agar plates before
placing the sterile antimicrobial assay discs (5 mm) on the plate. Four discs were placed on
the agar plate and 20 µL of the compounds and controls were loaded on the discs with
concentrations at 200, 100, 50 and 25 µg/mL. The plates were incubated at 37oC for 24 h,
after which the zones of clearance were measured as the difference between the diameter of
the ring (inhibition zone and disc) and diameter of the disc. Diameters of the zones were
measured with a Hi Antibiotic Zone Scale (PW 297-3NO, HiMedia Laboratories). The test
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was performed three times for each sample and the diameter of inhibition zone was expressed
as mean inhibition zone (mm) ± S.D of the three replicates. The effectiveness of these
compounds in relation to their inhibition zones was compared to two positive controls; silver
nitrate based on its established antimicrobial properties [25] and ciprofloxacin. Silver nitrate
was also used to compare the activities of the Ag+ from the complexes and the free Ag+ that
may be present, so as to eliminate the possibility that the observed activities of the complexes
are not due to the free Ag+ in solution. The minimum inhibitory concentration (MIC) of each
compound was determined based on the lowest concentration of the compounds that inhibited
the growth of bacteria using the broth dilution method [26]. Single colonies of S. aureus, and
E. coli were isolated from agar plates and grown in 5.0 mL of LB broth. The solutions were
incubated at 37°C and shaken at 180 rpm overnight to yield bacteria solutions. Stock
solutions (50 mg/mL) of the compounds were prepared by dissolving each in DMSO. From
each stock solution, the compounds, AgNO3 and ciprofloxacin were dissolved in the broth
culture and used to prepare serial dilutions of 200, 100, 50, 25, 12.5 and 6.25 µg/mL. Bacteria
growth was noted by turbidity of the solution in the tubes and MIC was determined by the
lowest concentration of the compounds that prevented visible growth. For the viable cell
count method, the concentrations of the tested compounds were also 200, 100, 50, 25, 12.5
and 6.25 µg/mL. The bacterial strain was inoculated into the nutrient broth and was incubated
for 16 h at 37°C. The stock was serially diluted to obtain 105 CFU/mL. About 60 µL of
bacterial suspension was transferred into 3 mL of nutrient broth followed by 30 µL of sample
suspension. The incubation was carried out for 5 h at 37°C and 50 µL of culture was
uniformly spread on the nutrient agar which was incubated at 37°C for 24 h. The MIC was
determined based on the lowest concentration of the silver samples that inhibited the growth
of bacterial strain. For growth inhibitory concentrations, the presence of viable
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microorganisms was tested and the lowest concentration causing bactericidal effect was
reported as minimum bactericidal concentration (MBC).
2.4.2. Larval culture
Insecticide susceptible Ae. albopictus larvae from the Vector Control Research Unit (VCRU)
of the School of Biological Sciences, Universiti Sains Malaysia were used for the study. The
larvae were reared on a 2:1:1:1 powdered mixture of cat biscuits (Friskies), beef liver, yeast
and powdered milk [27]. Larval breeding and experimental conditions were maintained at 26
±2 ºC, 75±5 % relative humidity and 12 h : 12 h (light : dark) photoperiod. .
2.4.3. Larvicidal activity
Stock solutions (100 mg/mL) of the test compounds were prepared in DMSO and test
concentrations of 400, 200, 100, 50, 25, 5 and 1 ppm were prepared from the stock solutions
in distilled water to a final total volume of 100 mL for the bioactivity tests.
Fourth instar larvae (25) of Ae. albopictus were introduced into paper cups containing the test
concentrations according to the WHO protocol [28]. Larval mortality was recorded after 24 h.
Four replicates were performed for each concentration.
2.4.4. Bioactivity of test compounds on developmental progression of Ae. albopictus
Biological activity of the test chemicals on the developmental progression of larvae into
pupae and adults were determined by introducing cohorts of fourth instar larvae (25) into
paper cups containing 25, 5 and 1 ppm of the test compounds [28] and monitored for 6 days.
DMSO was used as the control.
2.4.5. Statistical analysis
The lethal concentrations for 50 % (LC50) and 90 % (LC90) of the larvae and their respective
95 % confidence intervals (CI) in the larvicidal assay were computed using Qcal [29]. LCs
with overlapping 95% CL were considered not significantly different [30, 31]. Bioactivity
data from the developmental stages were arc sine transformed and analyzed using the one-
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way analysis of variance followed by mean separation with Tukey HSD using IBM SPSS ver.
20. The formula of Abbott [32] was used to correct natural mortality prior to analysis.
2.5. DNA experiments
2.5.1. DNA binding analysis using Absorption spectral method
Aedes albopictus-DNA (Aa-DNA) extraction:
Aedes albopictus DNA (Aa-DNA) was extracted from virgin 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 were
pooled and used for the binding study.
The Aa-DNA was dissolved in buffer solution (10 mM phosphate buffer, 50 mM NaCl, pH
o
7.4) as a stock solution, which was stored at 4 C for 24 h to reach homogeneous phase and
used within three days. The Aa-DNA concentrations were determined by spectroscopy using
the following extinction coefficients at 260 nm: DNA: 6,600 M-1cm-1(per nucleotide), 13,100
M-1cm-1(per base pair) [33].
In order to check its purity, the UV absorbance of Aa-DNA in the buffer medium was
measured at 260 and 280 nm and was found to be free of protein. The DNA binding
properties of the test compounds with Aa-DNA were investigated by UV–visible spectra. The
complex was dissolved in DMSO to obtain the desired concentration. Absorption titration
experiments were done by varying the concentration of the Aa-DNA, while the complex
concentration was kept constant (10 µM). An increasing amount of Aa-DNA (10 µL per time)
was gradually added to the complex in the buffer after it has been incubated for about 2 h at
25oC. The UV–visible spectra were recorded after equilibration for about 5 min. The intrinsic
equilibrium DNA binding constants (Kb) of the complexes to Aa-DNA were determined by
monitoring the change of the absorption intensity of the spectral bands with increasing
concentration of the Aa-DNA. The data were then fitted to the following equations to obtain
the value of Kb [34, 35].
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[DNA] (εb- εf)
εb- εf
[DNA]
εa- εf
Kb
1
(1)
=
+
Where [DNA] is the total concentration of added DNA as M base pairs, εa is the apparent
absorption in the presence of DNA, εf is the extinction coefficient of free Ag(I) complex in
the buffer and εb is the extinction coefficient of the DNA-bound Ag(I)-NHC complex. The
value of εf was obtained from a Beer’s plot of the Ag(I)-NHC complex while the value of εb
was obtained from the absorbance of a saturated Ag-DNA sample divided by the
concentration ([Ag-NHC]). Plots of [DNA] / (ԑa - ԑf) versus [DNA] were obtained as Kb by
the ratio of the slope to the intercept i.e. a plot of [DNA]/(εb – εf) versus [DNA] gave a slope
of 1/(εb – εf) and a y intercept equal to l/Kb (εb – εf), where Kb is the ratio of the slope to the
y intercept. Values with a magnitude order of 105 M-1 for Kb were considered to be indicative
of a relatively strong interaction between DNA and metal complexes [36-38]. The non-linear
analysis was done using OriginLab version.
2.5.2. DNA binding analysis using viscometric method
Spectroscopic experiments provide many vital information in elucidating the type and
strength of the metal complex-DNA interaction with viscometry measurements often
considered as the least ambiguous and the most evaluative test of a DNA binding model in
solution, clarifying the mode of interaction of a compound with the DNA [39, 40].
DNA viscosity experiments were carried out in Oswald viscometer and the temperature was
controlled with a thermostatic water bath set at 29 ± 0.1oC. DNA concentration 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 triplicate for each
sample in order to obtain the mean flow time. Data were presented as (η/η0)1/3versus the ratio
of the compound to DNA concentration, where η is the viscosity of DNA in the presence of
15

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the metal complex and η0 is the viscosity of the DNA solution, tf is the experimental flow
time per second and t0 is the flow time of buffer per second [37, 38]. The following equation
(2) was employed in the calculation of the ratio (η/η0)1/3.
tf −to
η
=
(2)
to
2.5.3. Nuclease activity assay
The degradation/cleavage ability of the test compounds on DNA was assayed using plasmid
(pDs-Red Express) and genomic (Aa. albopictus larvae) DNA. The plasmid DNA (pDNA)
and genomic DNA (gDNA) were isolated using High Speed Plasmid Mini Kit (Geneaid®)
and DNeasy® Blood & Tissue Kit (Qiagen®) according to the manufacturers’ instructions.
The constituents of the assay were 1 µL of 50 µg/mL of the test compound (salt/complex), 5
µL of 50 mM Tris-HCl (pH 8.0) and 4 µL of DNA (either pDNA or gDNA). The setup was
incubated at 37 ºC for 8 and 24 h. Electrophoresis of 5 µL of the product was conducted on a
0.8 % agarose gel stained with 2.5 µL of Health View Nucleic Acid Stain (Genomics BioSci
& Tech) in 0.5X TAE buffer at 70 V for 1 h. The gel was observed under ultra-violet (UV)
light and the image was captured in the UVP Gel-Doc-It Imaging System (Kinematics®).
3. Results and discussion
3.1. Synthesis
The reaction of N-cyanobenzyl or N-cyanopropyl benzimidazole with 1-bromopentane and 3-
bromomethyl benzonitrile in dioxane under reflux successfully gave the corresponding salts
1a, 2a and 3a in good yield. This was followed by the treatment with KPF6 salts in methanol
to obtain the respective hexafluorophosphate salts (1b-3b) in good yield. On the other hand
bis-benzimidazolium bromide salt 4a was prepared in good yield by reacting 1,4-
bis(bromomethyl)benzene with two equivalents of N-cyanobenzylbenzimidazole under reflux
in dioxane for 3 days. The pure form of the bromide salt was obtained after recrystallization
16

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from methanol-diethyl ether. The bromide salt 4a was directly converted to the
hexafluorophosphate counterpart (4b) with KPF6 in methanol (Scheme 1). The bromide salts
were moisture sensitive and hence, were converted to their more stable hexafluorophosphate
counterparts by simple anion exchange reactions. Both mono and bis-benzimidazolium
bromide and hexafluorophosphate salts were readily soluble in polar organic solvents such as
methanol, ethanol, acetonitrile, DMSO and DMF, but were insoluble in hexane, benzene,
toluene and diethyl ether.
The corresponding Ag-NHC complexes (5b-8b) were prepared via the deprotonation of the
benzimidazolium salts (Scheme 2) and refluxing the benzimidazolium salts 1b-4b for 1-2
days in the presence of a slight excess of Ag2O. The mixture was filtered through a pad of
Celite® and the filtrate was slowly evaporated to precipitate a white solid. The resulting solid
was redissolved in acetonitrile and diethyl ether was added to reprecipitate the solid in good
yield and high purity. In general the reaction is summarized in equation 4:
CH3CN
4
2(NHC)Ag.PF6 + H2O
2 NHC.PF6
Ag2O
The Ag(I)–NHC complexes (5b-8b) were thermally stable up to their melting points and
were non-hygroscopic. The complexes were readily soluble in DMSO, DMF and acetonitrile.
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R
R
KPF6
MeOH
N
N
N
N
Dioxane
_
Br
R
+
_
PF6
Br
Stir, 3 h
N
Reflux, 2 d
N
N
N
N
1a, 2a
1b, 2b
1a, 1b R = CH2CH2CH2CH2CH3
CH2
2a, 2b: R =
N
N
N
Br
KPF6
MeOH
N
N
N
N
Dioxane
+
Reflux, 2d
Stir, 3 h
N
Br
N
PF6
N
3b
3a
N
N
N
KPF6
MeOH
N
N
N
N
N
N
N
N
N
Dioxane
2
+
Reflux, 3d
Stir, 3 h
N
Br
Br
2 Br
2 PF6
4a
4b
N
N
N
N
N
Scheme 1. Synthesis of benzimidazolium halide and hexafluorophosphate salts
1a–4a and 1b–4b
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R2
R2
R2
R2
_
N
N
N
N
N
N
N
Ag2O, CH3CN
Stir, 50oC, 1 d
Ag2O, CH3CN
Stir, 50oC, 1 d
Ag
_
PF6
N
PF6
_
PF6
R1
R1/R2
R2/R1
R2
3b
1b, 2b
5b, 6b, 7b
5b: R1 = CH2CH2CH2CH2CH3; R2 = CH2CH2CH2CN
CH2
6b: R1 = CH2CH2CH2CN; R2 =
CH2
N
7b: R2 =
N
+
2
N
N
N
N
2 PF6
N
N
N
N
N
N
N
N
Ag2O, CH3CN
Stir, 50oC, 2 d
2 PF6
Ag
Ag
N
N
N
N
4b
8b
N
N
Scheme 2. Synthesis of Ag(I)–NHC complexes 5b–8b.
3.2. Spectroscopy studies
Solution studies was done in order to determine the stabilities of the Ag(I)-NHC complexes.
This was performed in D2O-10% of DMSO-d6 and 100% DMSO-d6 and monitored by NMR
over a period of seven days. All the investigated Ag(I)-NHC complexes showed no
significant changes since their 1H NMR and 13C NMR spectra remained similar. The 1H- and
13C NMR spectra of complexes 5b and 6b after 7 days in 10% aqueous DMSO are shown in
Figs. S1 and S2, and were identical to the spectra obtained after 15 min (see supplementary
data).
In order to further assess the stability of the complexes in the mixture, 10 µg/mL of 5b-8b in
DMSO was prepared and added in a 1:1 ratio to the stock solution of the biological medium
prepared in DMSO. This was done to imitate the conditions of the Ae.. albopictus evaluation
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experiments. The complexes used for the testing were dissolved in the minimal amount of
DMSO possible and diluted with the medium’s stock solution of 10−6 M and less than 0.8%
DMSO. UV-visible spectra were measured after 0, 24, 48 and 72 h to determine the stability
of the complexes. The complexes were stable in the biological medium over the period of the
testing time as the UV-visible spectra remained unchanged in 72 h (see supplementary data,
Fig. S3).
All the compounds were characterized by elemental analysis, FT-IR, NMR spectrometry,
UV-visible spectroscopy, Molar conductivity and X-ray crystallography. FTIR spectra for
salts 1b-4b showed a band of medium intensity in the range 2920–2960 and 3015–3143 cm−1,
which is assigned to the pure modes of C-H stretching vibrations. This is due to the presence
of aliphatic and aromatic C-H modules respectively and they were present in all the Ag-NHC
complexes (5b-8b) with some negligible changes [41]. A sharp band at 2229–2249 cm−1 is
assigned to ν (C≡N) of the nitrile functionality [41] and remained unaltered in the complex
spectra. This is an indication of their presence outside the coordination sphere as a result of
the coordinative saturated metal centers. A sharp and medium intensity band appeared in the
range 1432–1568 cm−1, which is due to the benzimidazole ring C=N stretching vibrations.
Interestingly, in the spectra of the Ag-NHC complexes, bands corresponding to the stretching
vibrations of the benzimidazole ring C=N modules shifted towards the lower energy side
compared with their salts (Figs. S4-S7, supplementary data). Further evidence for the
formation and structures of these complexes were provided by elemental analysis, NMR
spectroscopy, Molar conductance and X-ray crystallography.
In the 1H NMR spectra of 1a-4a, resonances for the benzimidazole, the benzonitrile, and the
alkyl protons were observed. The presence of a singlet at 9.99, 10.15, 10.21 and 10.15 ppm
corresponding to the NCHN benzimidazolium protons confirmed the formation of 1a, 2a, 3a
and 4a respectively. This is quite consistent with those of other benzimidazole based NHC
20

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precursors [41, 42]. In addition, the resonances of aliphatic protons of alkyl chain were
consistent in the ranged ca. 0.89–5.95 ppm, which is in agreement with similar structures
found in the literature [41]. The salts also showed similar spectra in the aromatic area in the
range of 7.48 - 8.20 ppm. Furthermore, the 13C NMR spectroscopy displayed
Benzimidazolium-C2 carbon resonances in the range ca. 137.5-143.5 ppm. Metathesis
reactions of these salts with KPF6 in methanol afforded 1b-4b in good yield. In the 1H NMR
spectra, no changes were observed in relation to 1a -4a.
1
Upon Ag-NHC complex formation, the H NMR spectra of complexes 5b−8b supported the
formation of the envisioned NHC complexes by the absence of any signals around δ = 10
ppm attributable to the acidic protons at the C2 position of the benzimidazolium moieties.
Apart from this, the spectra of all the complexes were more or less similar to their
corresponding salts. On the other hand, 13C NMR spectra of complexes 5b, 7b and 8b
evidenced the presence of a non-ligand signal in the most downfield region at ca. 188.3–
190.8 ppm ascribed to the carbene carbon resonances due to the formation of Ag-C bond. In
contrast, 6b displayed resonances for the C2-carbon nuclei as two doublets centred at δ
13
13
(189.2 and 190.4) as a result of C-107Ag and C-109Ag coupling constants of 185 and 205
Hz, respectively. Also, signals in the range δ 7.32-8.01 ppm in all spectra were assigned to
the aromatic protons.
The intensive absorption peaks at 277-281 and 285-287 nm can be assigned to π-π* and n-π*
orbital transition of the salts and the complexes respectively, originating from the C═C and
C═N modules [43]. These transitions were also observed in the spectra of the complexes but
were shifted to a higher wavelength as a result of complexation with Ag(I). However, no
bands at the lower energy level for d–d transition were detected in the UV–visible region. An
overlay of the electronic spectra for the salts and the corresponding complexes are shown in
fig1.
21

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Fig 1. Overlays of absorption spectra of the four salts (1b-4b) and their corresponding Ag–
NHC complexes (5b–8b) studied in this work
3.3. Molar conductivity measurements
The molar conductance values of the benzimidazolium salts and complexes were obtained at
room temperature in DMF solution at 10−3 M concentration. The compounds were dissolved
in DMF and the molar conductivities of their solutions at 25 ± 2 °C were measured. The
molar conductivity values of the benzimidazolium salts 1b-3b and their Ag(I)–NHC
complexes 5b-7b were found to be close, at around 24.76–27.78 S cm2 mol−1, indicating the
ionic nature of these compounds, which were found to be 1:1 electrolytes [44]. On the other
hand, salt 4b and its Ag(I)–NHC complex 8b values were almost double (4b at 52.67 and 8b
at 59.25 S cm2 mol−1), indicating that they were 2:2 electrolytes. Based on the foregoing, we
could also deduce the structure of the compounds on the basis of their molar conductance. It
is obvious from this study that each mononuclear Ag(I)–NHC complex has one
22

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hexafluorophosphate counterion to balance the Ag+ and as a consequence reflects the
formation of a mononuclear Ag–NHC complex. However 4b and 8b have two
hexafluorophosphate counterions to balance the two Ag+ and as a consequence indicates the
formation of a tetracarbene complex. The molar conductivity data, elemental analysis and
spectroscopy data show an agreement with the proposed structure of the compounds, which
was further confirmed by means of the data obtained by single crystal X-ray diffraction.
3.4. Single crystal X-ray diffraction studies
Complex 8b crystallized in triclinic P-1 space group with half of the molecule in the
asymmetric unit (Figure 2). The presence of two hexafluorophosphate anions in the lattice
then balances the entire charge. Both Ag(I) were in linear geometry, with the angle of C15-
Ag1-C30i being 176.84(16)o. The distances of Ag1-C15 and Ag1-C30i were 2.092(5) Å and
2.091(5) Å, respectively (Table 2). The packing diagram of the molecule is dominated by the
presence of π-π interaction between the aromatic rings.
-
Fig 2: Structure of 8b with ellipsoids shown at 50% probability. Hydrogen atoms and PF6
anions in the lattice omitted for clarity. Symmetry element used: i = 1-x, 2-y, 1-z.
23

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Table 1
Selected bonds lengths (Å) and angles (O) of 8b
Ag1-C15
2.092(5)
2.091(5)
176.84(6)
105.8(4)
105.7(4)
Ag1-C30i
C15-Ag1-C30
N2-C15-N3
N4-C30-N5
3.5. Antibacterial Activities
The antibacterial activities of the complexes were described by the disc diffusion, MIC, MBC
and colony formation methods. The antibacterial activity showed good to moderate activity.
The results obtained by the Kirby-Bauer’s disc diffusion method were comparable with that
of the MIC values (Tables 2a, 2b and 3). In a solvent control test, the role of DMSO was
studied and it was observed that 10 % DMSO did not have any inhibitory activity on the
growth of microorganisms [45]. The antibacterial activities of compounds 1b-8b were
screened against 2 isolated pathogens (E. coli and S. aureus). The MICs of the compounds
were compared to the MICs of AgNO3 and Ciprofloxacin.
All complexes in this study were effective at inhibiting the growth of E. coli and S. aureus
while their corresponding salts were inactive against both strains of bacteria [46]. The MIC
values of the complexes against both E. coli and S. aureus were in the range of 25-100
µg/mL. These Ag(I)-NHC complexes (5b-8b) had better antibacterial qualities in comparison
with silver nitrate. The bis–NHC–Ag(I) complex (8b) exhibited promising antibacterial
activity. This complex (8b) displayed impressive antibacterial potential against S. aureus at a
concentration of 12.5 µg/mL. The complexes showed bacteriostatic effect against both
bacteria strains in the range of 12.5 ˗ 100 µg/mL but the bactericidal activity was only in the
range of 50 - 200 µg/mL (Table 4; Figs. S9 and S10).
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In general, most of the complexes displayed lower inhibitory activities than ciprofloxacin,
and the susceptibility levels of the gram-negative and gram-positive bacteria to the complexes
were the same except complex 8b which showed a higher activity against the gram-positive
bacteria. Furthermore, the sensitivity of the bacteria strains increases as the concentration of
the complex suspensions increases (Table 2a and 2b).
The colony formation studies were evaluated using different concentrations of the tested
Ag(I)-NHC complexes. For E. coli, the colony forming unit per mL (CFU/mL) decreases as
the concentration of the complexes increases. Complex 8b showed no viable colony of both
E. coli and S. aureus at a concentration of 100 µg/mL (Table 4), which is only comparable to
that of the ciprofloxacin. All the other complexes showed different values at concentrations
of 100 and 200 µg/mL and no viable colony was observed at concentrations of 200 and 400
µg/mLexcept for 7b which had activity at 400 µg/mLonly (Table 4).
Table 2a
Antibacterial activities of the compoundsa against E. coli obtained by the disc diffusion
method (zone of inhibition ± SD/mm)
Test Compound
Inhibition Zone (mm)
25 µg/mL
12.5±0.0
50 µg/mL
12.8±0.5
100 µg/mL
200 µg/mL
19.0±1.0
15.1±0.5
13.2±0.0
12.0±0.0
5b
6b
7b
11.0±0.7
10.2±0.3
11.8±1.0
10.8±0.0
15.8±1.5
14.8±0.0
22.6±1.0
10.8±0.0
26.3±0.0
24.0±1.0
11.8±0.0
26.3±0.0
27±1.5
14.8±0.0
28.7±0.0
28.6±0.5
16.8±0.0
31.0±0.0
8b
AgNO3
Ciprofloxacin
a : Compounds 1b- 4b showed no activity.
25

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Table 2b
Antibacterial activities of the compoundsa against S. aureus obtained by the disc diffusion
methodb (zone of inhibition ± SD/mm)
Test Compound
Inhibition Zone (mm)
25 µg/mL
13.5±0.0
50 µg/mL
13.5±1.0
100 µg/mL
200 µg/mL
20.9±1.5
15.6±0.0
5b
11.0±0.0
10.6±0.8
22.6±1.0
10.8±0.0
26.3±0.0
11.8±1.5
10.8±0.4
23.0±1.0
11.8±0.0
26.3±0.0
14.2±0.5
12.5±0.0
27.8±0.5
14.8±0.0
29.7±0.0
17.8±1.0
14.0±1.0
29.6±0.5
16.8±0.0
32.0±1.0
6b
7b
8b
AgNO3
Ciprofloxacin
a : Compounds 1b- 4b showed no activity.
Table 3
Minimum inhibitory concentration (MIC) of the compounds against E.coli and S.aureus.
Test Compound
E. coli (µg/mL)
S. aureus (µg/mL)
MIC
MBC
100
200
200
50
MIC
50
MBC
100
200
200
50
5b
50
100
100
25
6b
100
100
12.5
100
6.25
7b
8b
AgNO3
Ciprofloxacin
100
6.25
200
9.38
200
12.5
Compounds 1b- 4b showed no activity.
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Table 4
Colony forming unit per ml (CFU/mL) of different concentrations of the
complexes against E. coli/S. aureus
CFU/mL (105)
Test Compound
Concentration (µg/mL)
100
50
200
0
400
0
17 ± 1.8/16 ± 1.0
19 ± 0.7/17 ± 0.0
7 ± 1.0/6.5 ± 1.5
8 ± 0.5/7 ± 1.0
5b
0
0
6b
7b
20 ± 1.0/18 ± 1.5 11 ± 1.8/9.5 ± 1.0 3 ± 1.8/16 ± 1.0
0
7 ± 1.5/5 ± 0.5
18 ± 0.5/17 ± 2.0
3 ± 0.0/3 ± 0.0
0
0
0
0
0
8b
AgNO3
Ciprofloxacin
8 ± 0.8/6.8 ± 0.0
0
0
0
3.6. DNA binding
UV-Vis absorption spectroscopy can be used to observe possible intercalation interactions
between Aa-DNA and the Ag(I)–NHC complexes. In addition, viscosity measurements were
also performed in order to further evaluate the nature of DNA interaction with the complexes.
An intercalative mode involves a strong stacking interaction between the aromatic
chromophore and the base pair of DNA, thus a complex binding to DNA through
intercalation usually results in hypochromism and bathochromism [47, 48]. Compounds 5b–
8b were allowed to interact with Aa-DNA and the interactions were investigated by UV–
visible spectra in phosphate buffer (10 mM, pH = 7.4) containing 50 mM NaCl at 25 °C. As
illustrated in Fig. 3 and Table 5, with increasing concentration of Aa-DNA, the absorption
intensities of these compounds were reduced by a large hypochromicity of 28 and 32% and
smaller hypochromicity of 18 and 21%. Bathochromic shift was not observed in the spectra
and the maximum absorption showed obvious hypsochromic adsorption (7.0–11.5 nm). As a
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result of the reduced absorbance and the hypsochromic shift, an intercalative binding mode is
assigned to these complexes. The values of Kb and site sizes were obtained from the
spectroscopy emission data and these data were fitted to the equation previously derived [49]
and were calculated to be 2.177 × 106, 8.672 × 105, 6.665 × 105 and 3.627 × 106 (5a, 6b, 7b,
8b) respectively. The absorbance titration showed the binding site size (s) between 0.71 and
1.16, which correspond to a binding stoichiometry of about one Ag–NHC complex per base
pair site. The Kb values showed that compound 8b with a bimetallic center has the highest
binding constant and consequently manifests the strongest binding properties with Aa-DNA
compared to the mononuclear counterparts. In general the mode of action is linked to a strong
stacking interaction between the planar aromatic chromophore and the intercalating agents
[50].
Values of Kb obtained from the absorption titrations in the present work are in reasonable
agreement with those obtained from DNA titrations of similar complexes in the literature
[51–54]. In addition, the results of the interactions of benzimidazolium salts with DNA in
literature showed that the Kb values are quite less than those of the corresponding Ag
complexes [51–55].
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