Ru(ii)-N-heterocyclic carbene complexes: Synthesis, characterization, transfer hydrogenation reactions and biological determination

Article abstract of DOI:10.1039/c9ra05605j

A series of ruthenium(ii) complexes with N-heterocyclic carbene ligands were successfully synthesized by transmetalation reactions between silver(i) N-heterocyclic carbene complexes and [RuCl₂(p-cymene)]₂ in dichloromethane under Ar

Full text of DOI:10.1039/c9ra05605j

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Ru(II)–N-heterocyclic carbene complexes:
synthesis, characterization, transfer hydrogenation
Cite this: RSC Adv., 2019, 9, 34406
reactions and biological determination†
a
b
c
d
Lamia Boubakri, A. Chakchouk-Mtibaa, Abdullah S. Al-Ayed, L. Mansour,
d
d
b
e
e
Nael Abutaha, Abdel Halim Harrath, L. Mellouli, I. O¨ zdemir,
and Naceur Hamdi
S. Yasar
ac
*
A series of ruthenium(II) complexes with N-heterocyclic carbene ligands were successfully synthesized by
transmetalation reactions between silver(I) N-heterocyclic carbene complexes and [RuCl (p-cymene)] in
2
2
dichloromethane under Ar conditions. All new compounds were characterized by spectroscopic and
analytical methods. These ruthenium(II)–NHC complexes were found to be efficient precatalysts for the
transfer hydrogenation of ketones by using 2-propanol as the hydrogen source in the presence of KOH
as a co-catalyst. The antibacterial activity of ruthenium N-heterocyclic carbene complexes 3a–f was
measured by disc diffusion method against Gram positive and Gram-negative bacteria. Compounds 3d
exhibited potential antibacterial activity against five bacterial species among the six used as indicator
cells. The product 3e inhibits the growth of all the six tested microorganisms. Moreover, the antioxidant
0
activity determination of these complexes 3a–f, using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -
azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as reagent, showed that compounds 3b and 3d
ꢀ1
possess DPPH and ABTS antiradical activities. From a concentration of 1 mg ml , these two complexes
presented a similar scavenging activity to that of the two used controls gallic acid (GA) and butylated
ꢀ1
hydroxytoluene (BHT). From a concentration of 10 mg ml , the percentage inhibition of complexes 3b
and 3d was respectively 70% and 90%. In addition, these two Ru–NHC complexes exhibited antifungal
activity against Candida albicans. Investigation of the anti-acetylcholinesterase activity of the studied
Received 20th July 2019
Accepted 21st August 2019
ꢀ1
complexes showed that compounds 3a, 3b, 3d and 3e exhibited good activity at 100 mg ml and
product 3d is the most active. In a cytotoxicity study the complexes 3 were evaluated against two
human cancer cell lines MDA-MB-231 and MCF-7. Both 3d and 3e complexes were found to be active
against the tested cell lines showing comparable activity with examples in the literature.
DOI: 10.1039/c9ra05605j
rsc.li/rsc-advances
enantioselective hydrogenation of ketones using 2-propanol or
formic acid under basic conditions, because the method has
1
. Introduction
2,3
Transfer hydrogenation, a potentially useful protocol for the advantages, such as low cost, ease of handling, and high solu-
reduction of ketones and aldehydes to their corresponding bility of 2-propanol or formic acid as a hydrogen-donor reagent.
1
alcohols, has been extensively studied. A large number of The most active and versatile homogeneous catalytic sec-
studies have appeared with transition metal-catalyzed alcohol/ketone oxidation/reduction systems are the metal–
ligand bifunctional ruthenium complexes developed by Noyori
4–6
and co-workers. A variety of related ligands and transition
metal complex catalysts have been developed for this purpose.
One of these ruthenium complexes is Ru(NHC) (NHC: N-
heterocyclic carbene). N-heterocyclic carbene ligands appear
a
Research Laboratory of Environmental Sciences and Technologies (LR16ES09), Higher
7,8
Institute of Environmental Sciences and Technology, University of Carthage,
Hammam-Lif, Tunisia. E-mail: naceur.hamdi@isste.rnu.tn; Tel: +21698503980
b
Laboratory of Microorganisms and Biomolecules, Center of Biotechnolgy of Sfax, Road
particularly suited to this purpose, in that their ruthenium(II)
Chemistry Department, College of Science and Arts, Qassim University, Al-Rass, complexes possess high thermal and hydrolytic stability.
of Sidi Mansour, Km 6 B.P. 1117, 3018, Sfax, Tunisia
c
9
Kingdom of Saudi Arabia
Ligand effects are extremely important in homogeneous
catalysis by metal complexes. Carbenes are both reactive inter-
mediates and ligands in catalysis. Aer the rst stable N-
heterocyclic carbene ligands were reported by Arduengo et al.,
N-heterocyclic carbenes (NHCs) have been indispensable
d
Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh
1
1451, Saudi Arabia
e
˙
In ¨o n u¨ University, Faculty of Science and Art, Department of Chemistry, Malatya,
Turkey
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9ra05605j
10–12
ligands for transition metals and homogeneous catalysis.
1
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Scheme 1 Synthesis of ruthenium–NHC carbene complexes (3a–f).
Due to their topological and electronic versatility, N-heterocyclic homogeneous catalysis. Strong s-donation from NHCs
carbene-based ligands have widespread applications not only in increases the stability of the complexes and gives complexes
13–15
organometallic chemistry but also in industrial applications of with advantageous properties in catalytic reactions.
The rst
Fig. 1 Structure of ruthenium–NHC carbene complexes 3(a–f).
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catalytic applications of NHC complexes were reported by temperature (Scheme 1). The air and moisture-stable ruthe-
Herrmann in 1995, together with the recognition that NHCs are nium N-heterocyclic carbene complexes (3a–f) isolated in high
11
excellent ligands for many homogeneous catalysts. Different purity aer recrystallization with DCM/ether, were soluble in
NHC complexes and their catalytic applications have been re- solvents such as dichloromethane, chloroform, toluene, and
1
6–24
30
ported by many research groups.
focused on synthesis of new functional NHC complexes. Func-
Continued research has tetrahydrofurane and insoluble in nonpolar solvents (Fig. 1).
The ruthenium–NHC complexes 3a–f were characterized by
1
13
tionality of the complexes depends on the steric and electronic NMR ( H and C), IR, and DART-TOF mass spectrometry. The
1
effect of the ligands on the metal center. Ru–NHC complexes
can be functionalized by N-substituted NHC ligands. In this temperature. Here, the proton H
context, our research group has focused on synthesis, charac- cymene was seen as a multiplet at 2.62 ppm (Fig. 2).
H-NMR spectra of complex 3a was recorded in CDCl
3
at room
000 of the isopropyl group of p-
7
terization, and catalytic activity of functional N-heterocyclic
carbene ligands and their metal complexes.
The aromatic protons appeared between 5.54 and 6.91 ppm
25–28
as a multiplet while methylic protons appeared between 1.14
1
and 2.30 ppm as singlets. In the H-NMR spectra of 3a, (NCH
2
)
was resonated at 5.29 and 5.05 ppm as a doublet.
2. Results and discussion
The spectra of these products in CDCl
3
supported the
2
.1. Preparation of Ru–NHC complexes (3a–f)
Silver complexes of N-heterocyclic carbenes were prepared by
the addition of Ag O to 5,6-dimethylbenzimidazolium salts
solutions (1a–f) in CH Cl under exclusion of light at room
successful formation of the ruthenium complexes because of
the absence of NCHN signal proton in a downeld for 2–3.
1
13
The H and C NMR spectra of these new complexes (3a–f)
2
1
3
1
are consistent with the proposed formula. C{ H} NMR spectra
prove an increasing downeld shi of the characteristic peak of
the carbenic carbon resonance from 2a–f to 3a–f: for example,
2
2
temperature (Scheme 1 and Fig. 1). White solid (in 75–90%
yield) were isolated. When the white solid were le in air, they
began to slowly turn black. Low eld signals attributed to acidic
29
1
3
1
the C{ H}N–C–N shis of 3a displays as a singlet at 188.8 ppm
which conrmed the transmetallation between silver and
1
NCHN protons were not observed in the H NMR spectra of the
31
ruthenium (Fig. 3).
new Ag–NHC complexes (2a–f). This observation conrmed that
the 5,6-dimethylbenzimidazolium salts were deprotonated.
However, coordination of N-heterocyclic carbene ligands to the
metal center resulted in low eld resonances for carbene
The results of the mass spectrometry analyses are proof
verication of the synthesized compounds (Fig. 4).
The fragmentation leading to the m/z ¼ 279 and m/z ¼ 343
29
ions can occur by the mechanisms shown in Scheme 2.
carbon.
All Ag–NHC complexes are widely used for the next reaction
as transfer compounds. To show the feasibility of this approach
2.2. Transfer hydrogenation
for 2a–f, a series of Ru(II)–NHC complexes were synthesized in The transfer hydrogenation process, which involves hydrogen
good yield as stable red solids. 3a–f were obtained by reaction of transfer from a donor to an acceptor, has become one of the
2 2
[RuCl (p-cymene)] with a solution of 2a–f in the dark at room most important synthetic routes for producing alcohols from
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1
3
Fig. 2 H NMR spectra of Ru(II)–N-heterocyclic carbene complexes 3a (CDCl , 300 MHz).
32
ketones. Non-toxic reagents, green solvents and mild reaction
In this context, we investigated the nature of the catalysts
conditions are remarkable features of transfer hydrogenation and the substrate that could impact on yield in the transfer
33
reactions. Transition-metal catalyzed transfer hydrogenation hydrogenation reaction. Catalyst performance depends on the
using 2-propanol and KOH as a hydrogen source and promoter nature of substrates, the behavior of the ligands in the
respectively, has become an efficient method in organic complexes, and concentration of the catalyst. Electrophilic
synthesis as illustrated by several useful applications reported effect of the substrate, nature of the catalysts, donor function,
34
in recent years. The reaction conditions for this important and exibility of the backbone in the NHC ligands can be
ꢁ
process are easy to handle (moderate boiling point 82 C), responsible for activity of the complexes. Steric and electronic
30
inexpensive, relatively mild and environmentally benign.
properties of the substrates also affect conversions. Acetophe-
As part of our ongoing investigation into novel functional- none and p-chloroacetophenone reacted very cleanly and
ized NHC ligands as the supporting environment for metal turned into corresponding alcohol in good yields (Table 1,
complexes and their application in catalysis. We herein report entries 1–6). However, 3b complex exhibited the lowest catalytic
a series of catalytic transfer hydrogenation reactions employing activity with all ketones. This low catalytic activity was attrib-
the ruthenium–NHC complexes to discover their benets. The uted to structure of complex 3b, presumably due to decreased
transfer hydrogenation reactions are very susceptible to the type steric bulk around the metal or lower electron donation
31
of the base and its concentration. As the starting point, the provided by the carbene ligands (Table 1, entries 2, 5, 8, 11 and
performance of the catalysts in the transfer hydrogenation was 14). When p-methoxyacetophenone was used as substrate, low
30,31,33
screened by using acetophenone as a model substrate.
yield was observed on the transfer hydrogenation reaction cat-
Previously, our laboratory reported the application of ruthe- alysed by 3a–c (Table 1, entries 10–12). We are of the opinion
nium–NHC complexes in the transfer hydrogenation of that the nature of substrate can also give rise to low yield.
30
ketones. According to the literature, in a typical experiment, Substrates bearing electron-withdrawing groups (Table 1,
we dissolved the preformed, isolated crystalline catalyst (0.005 entries 4–9) were reduced to secondary alcohols in better yield
mmol) in 2-propanol. Aer the catalysts had completely dis- than substrates bearing electron-donating groups (Table 1,
solved, ketone (0.5 mmol) and a KOH (2 mmol) were added and entries 10–12). Similarly, with only 0.5 mol% of the same cata-
ꢁ
the reaction was performed at 80 C in 1 h.
lysts, it was possible to hydrogenate cyclohexanone and obtain
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13
Fig. 3
3
C NMR spectra of Ru(II)–N-heterocyclic carbene complexes 3a (CDCl , 75 MHz).
the corresponding alcohols with the highest yields (Table 1, evaluated against three Gram-positive bacteria (Micrococcus
entries 13–15).
luteus LB14110, Listeria monocytogenes ATCC19117, Staphylo-
coccus aureus ATCC6538) and three Gram-negative bacteria
(Salmonella typhimurium ATCC14028, Pseudomonas aeruginosa
3
. Biological activities
ATCC 9189, Escherichia coli). The obtained antibacterial activity
is presented in Fig. 6 and Table 2.
Globally, all complexes tested showed an important anti-
bacterial activity against the Micrococcus luteus LB14110.
Although complex 3d presents the highest inhibitory effect. In
addition, 3e inhibit the growth of Gram-positive and Gram-
3.1. Antibacterial activity
The synthesized complexes were examined in vitro for their
antibacterial activity using the well diffusion method (Fig. 5).
Ampicillin (AMC) was used as standard control drugs for anti-
bacterial activity. The ruthenium–NHC complexes (3a–f) were
Fig. 4 DART-TOF-MS of Ru(II)–N-heterocyclic carbene complexes 3a.
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Scheme 2 Mechanism of fragmentation leading to peaks m/z ¼ 225.
negative bacteria in high and low concentrations. In contrast, against the two Gram positive bacteria Micrococcus luteus LB
the other complexes show no activity against E. coli. 14110 and Listeria monocytogenes ATCC 19117 and the Gram-
The antibacterial activity of ruthenium–N-heterocyclic car- negative bacterium Salmonella typhimurium ATCC 14028. As
bene complexes (3a–f) was reported in terms of the MIC values, shown in Table 3, the MICs values range from 0.0195–0.625 mg
ꢀ1
ꢀ1
dened as the lowest concentration of an antibacterial that ml for Micrococcus luteus LB 14110; 0.0781–5 mg ml for
visibly inhibits the growth of the bacteria aer an overnight Listeria monocytogenes ATCC 19117 and Salmonella typhimurium
incubation. The MICs values complexes 3 were determined ATCC 14028. The most active compound was 3d which presents
a
Table 1 Transfer hydrogenation of ketones catalyzed by ruthenium–NHC complexes 3(a–c)
b
Entry
Substrate
Catalyst
Product
Yield (%)
1
2
3
3a
3b
3c
56
45
60
4
5
6
3a
3b
3c
64
48
57
7
8
9
3a
3b
3c
35
25
40
1
1
1
0
1
2
3a
3b
3c
32
15
28
1
1
1
3
4
5
3a
3b
3c
70
64
66
a
ꢁ
b
Reaction conditions: 0.005 mmol Ru–NHC, i-PrOH (5 ml), KOH (2 mmol), substrate (0.5 mmol), 80 C, 1 h. Conversions are based on
corresponding ketones.
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Fig. 5 Zone of inhibition of Ru–NHC complexes (3a–f).
against Salmonella typhimurium ATCC 14028 the lowest MIC f) on the DPPH radical were investigated. As shown in Fig. 7,
ꢀ
1
value (0.0781 mg ml ) than the used standard (ampicillin). the complexes 3b and 3d present a higher antioxidant activity
Thus, the best activity was recorded for this complex with MIC than other complexes which no antioxidant activity was
ꢀ1
values of 0.0195 mg ml against Micrococcus luteus LB14110. observed. From the results, we can see that, within the range of
ꢀ
1
Complex 3e also has MIC values of 0.0781 mg ml against tested concentration, the average suppression ratios of DPPH
Listeria monocytogenes ATCC 19117. increase along with the increase of the complex concentration
Fig. 7). Concerning complexes 3b and 3d, from a concentra-
(
ꢀ1
tion of 1 mg ml , the scavenging activity was very similar to
that of the two used control gallic acid (GA) and butylated
hydroxytoluene (BHT) known as very good antioxidant
compounds.
3
.2. Antioxidant activity
3.2.1. Antiradical test: DPPH. The proton radical scav-
enging action is known to be one of the various mechanisms
for antioxidation. DPPH (1,1-diphenyl-2-picrylhydrazyl) is one
of the compounds that possess a proton free radical and shows
a characteristic absorption at 517 nm (purple). When DPPH
encounters proton radical scavengers, its purple color would
fade rapidly. Among all free radicals, the hydroxyl radical is by
far the most potent and therefore the most dangerous oxygen
metabolite which would result in cell membrane disintegra-
tion, membrane protein damage, DNA mutation and further
initiate or propagate the development of many diseases.
Elimination of this radical is one of the major aims of anti-
3
.2.2. Antiradical test: ABTS. The dark blue color of ABTS
0
(
ammonium salt of 2,2 -azinobis-3-ethylbenzothiazoline-6-
sulphonic acid) turns very dark blue and may even become
colorless if there are radical scavengers free. This discolor-
ation is based on the oxidation of the conjugated organic
+
structure in radical cation ABTSc . To carry out the ABTS
trapping test, 20 ml of the synthesized products were added
+
to 18 ml of the prepared ABTSc solution, while stirring
vigorously for 30 seconds. The resulting solution was mixed
with ethanol and ultrapure water (5 : 5) v/v. Tubes contain-
ing volumes of equivalent amounts of the different
complexes are incubated under incubation and controlled
for 6 min in the dark. Aer a sweep, the wavelength of the
3
5
oxidant administration. Current research has shown that
some antioxidants could act as the inducers of DNA damage
response, which leads to cell death. In present study, the
3
6
scavenging activity of the synthesized Ru–NHC complexes (3a–
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Fig. 6 The method of determining the MIC.
work chosen for the evaluation of the antiradical activity was conditions: increasing use of broad-spectrum antibiotics,
set at 734 nm. In order to explore the experimental results patients with AIDS; we talk about iatrogenic mycoses. They are
obtained, it is necessary to calculate the percentages of caused by so-called opportunistic fungi that are either levuri-
+
inhibition of the free radical ABTSc according to the form (Candida, Cryptococcus) or lamentous (Aspergillus,
3
7
following formula:
Cephalosporium). The development of antifungals comes up
against obstacles such as the weak fungicidal activity in vivo
compared to the results in vitro and the poor diffusion through
the very thick wall of the mushrooms because of its compo-
sition (chitin, phospholipids, and sterols); these constituents
are absent in bacteria, which explains why most antibacterial
antibiotics are not antifungals.
To evaluate the antifungal activity of the Ru–NHC
complexes (3a–f) with respect to the Candida albicans strain,
the solid medium diffusion method was carried out (Fig. 9). As
shown in Table 4, the complexes 3a, 3c, 3e and 3f are
completely inactive against Candida albicans. The compounds
AAOx% ¼ [(A
c
ꢀ A
e
)/A
c
] ꢂ 100
A
c
: absorbance of control; A : absorbance of the sample.
e
As shown in Fig. 8, complexes 3b and 3d present a high
antioxidant activity. Concerning the compound 3b, from
ꢀ
1
a concentration of 10 mg ml the percentage inhibition was in
order to 70%. For compound 3d, the percentage inhibition was
in order to 90%.
3
.3. Antifungal activity
3
b and 3d present a high antifungal activity. The most active
Fungal pathology has increased in recent years due to the
increase in deep, visceral and septicemic mycotic sites. The
former are caused by fungi that are widespread in nature,
usually saprophytic, becoming pathogenic only under certain
compound was 3d which presents against Candida albicans the
highest diameter of growth inhibition zone with a value of the
order of 34 ꢃ 0.18 mm.
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Table 2 Antibacterial activity of the synthesized Ru–NHC complexes (3a–f)
Microorganism indicator
Micrococcus
luteus
Listeria
monocytogenes
Staphylococcus
aureus
Salmonella
typhimurium
Pseudomonas
aeruginosa
Compounds
LB 14110
ATCC 19117
ATCC 6538
ATCC 14028
ATCC 49189
E. coli
3a
3b
3c
3d
3e
3f
30 ꢃ 0.5
20 ꢃ 0.4
18 ꢃ 0.2
30 ꢃ 0.32
23 ꢃ 0.3
—
16 ꢃ 0.2
—
20 ꢃ 0.4
18 ꢃ 0.1
16 ꢃ 0.5
—
—
—
—
22 ꢃ 0.4
17 ꢃ 0.3
—
16 ꢃ 0.2
—
14 ꢃ 0.7
18 ꢃ 0.1
16 ꢃ 0.6
—
—
—
—
—
—
20 ꢃ 0.2
—
26 ꢃ 0.11
16 ꢃ 0.22
—
15 ꢃ 0.2
—
Table 3 Minimum Inhibitory Concentrations (MICs) expressed in mg Parkinson's disease, senile dementia, and ataxia, among
ꢀ1
ml
40
others. The prevailing view has been that the efficacy of AchEIs
ꢀ
1
is attained through their augmentation of acetylcholine-
medicated neuron to neuron transmission. However, AchEIs
also protect cells from free radical toxicity and b-amyloid
induced injury and increased production of antioxidants.
The results presented in Table 5 show that the 3d complex is
the most potent inhibitor against AchE. This good activity is
conrmed by a value of the percentage inhibition of the order of
47.1%. However, the 3c and 3f complexes are completely inac-
tive and the other Ru–NHC complexes exhibit anti-AchEI
activity.
Minimum inhibitory concentrations (mg ml
)
Listeria
Salmonella
typhimurium
Micrococcus luteus
monocytogenes
Compounds
LB 14110
ATCC 19117
ATCC 14028
3
3
3
3
3
3
a
b
c
d
e
f
0.039
0.625
0.039
0.0195
0.0195
—
0.3125
5
1.25
0.1562
0.0781
—
2.5
5
2.5
0.0781
1.25
—
Ampicillin
0.004
0.002
0.625
3.5. Anticancer activity
3.5.1. Cytotoxicity assay. Cytotoxicity of compounds was
assessed using MTT assay on MDA-MB-231 cells and MCF7 cells
41
as carried out previously (Song et al., 2010). In brief, cells were
3.4. Acetylcholinesterase inhibition
cultured into 96-well plates (Corning, USA) at a density of 5 ꢂ
5
The acetylcholinesterase enzyme (AChE) is an attractive target 10 cells per well in 200 ml medium. Aer 24 h, cells were treated
ꢀ1
for the rational drug design and for the discovery of with different concentrations (10, 5, 2.5, 1 mg ml ) of
mechanism-based inhibitors because of its role in the hydro- compounds and further incubated for 48 h to evaluate the
lysis of the neurotransmitter acetylcholine (ACh). The patho- cytotoxicity of compounds. At the end of incubation period, 20
genesis of Alzheimer's disease (AD) has been linked to ml MTT solution was added to each well and cells were further
ꢁ
a deciency in the brain neurotransmitter acetylcholine. incubated for 2 h at 37 C. Finally, the media were aspirated and
Subsequently, acetylcholinesterase inhibitors (AchEIs) were then 200 ml 0.1% HCl–MeOH was added to dissolve formazan
38,39
introduced for the symptomatic treatment of AD,
and other crystals. The OD value was read at 490 nm on a microplate
possible therapeutic applications in the treatment of reader (Thermo Multiskan FC, China). MeOH-treated cells were
Fig. 7 Scavenging activity of compounds 3b and 3d on DPPH radicals.
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Fig. 8 Percent inhibition of complexes 3b and 3d as a function of concentration.
used as controls. The relative cell viability was calculated using Table 4 Minimum Inhibitory Concentrations (MICs) expressed in mg
ꢀ1
ml
the following formula:
Compounds
Relative cell viability (%) ¼ OD treated C/OD control ꢂ 100%
Strain
3a
3b
3c
3d
3e
3f
3
.5.2. Disc diffusion method. Escherichia coli (ATCC10418), Candida albicans
and methicillin-resistant Staphylococcus aureus (MRSA)
ATCC3359) were grown on nutrient agar plates (HiMedia,
—
22 ꢃ 0.5
—
34 ꢃ 0.18
—
—
(
India) and Candida albicans (ATCC 90028) was grown on potato
dextrose agar (HiMedia, India) for 24 h at 35 C. All cultures
were obtained from the American Type Culture Collection
ꢁ
Table 5 Acetylcholinesterase inhibitory activity (AChEI) (%) of
compounds (3a–f)
Compounds 3a
AChEI) (%) 32.8
(
ATCC). The disc diffusion method was used to assess the
3b
3c
3d
3e
3f
42
antimicrobial activities (De Beer and Sherwood, 1945).
(
45.3
—
47.1
38
—
Microbes were suspended in sterile saline solution (0.9%) and
the turbidity was adjusted to 0.5 OD value using spectropho-
tometer (Labomed Inc., USA). The inoculum was swabbed on
the surface of agar plates used using a sterilized cotton swab.
Sterile blank disks (6 mm) were loaded with 10 ml of compounds
positive controls and methanol as a negative control for
ꢁ
comparison. The Petri-plates were incubated at 35 C for 24
ꢀ
1
stock solution (10 mg ml ) giving a concentration of 50 mg per
disc. Commercial tetracycline discs (30 mg per disc) were used as
hours. The diameters of the zones of inhibition produced by the
compounds on the test isolates were measured in mm.
The compounds were screened calorimetrically using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
cell assay in two human cancer cell lines namely MCF7 and
MDA-MB-231 to evaluate their cytotoxicity. As shown in Table 6,
many of the compounds affected cell viability of cancer cell
lines and showed promising level of cytotoxicity however, the
cytotoxicity of 3e and 3d was much stronger in MCF7 with IC50
ꢀ1
values 0.68 and 0.6 mg ml , respectively as compared to their
activity on MDA-MB-231 cells. The cytotoxicity of 3e and 3d on
ꢀ
1
MDA-MB-231 cells was 1.93 and 3.1 mg ml . The cytotoxicity of
compounds 3c in MCF7 and MDA-MB-231 cells was 1.7 and 16
ꢀ
1
mg ml , while the IC values of compound on 3f were 1.3 and
3
5
0
ꢀ1
.3 mg ml against MCF7 and MDA-MB-231, respectively.
The antimicrobial activities of the synthesized compounds
against the human pathogenic microorganisms are presented
in Table 6. The results revealed that the highest antimicrobial
activity was reported in 3e (against E. coli) followed 3d (against
E. coli) and 3b (against MRSA) however the highest anti Candida
albicans activity was found in 3d (30.5 mm) followed by 3b (28.0
mm) and 3a (25.0 mm). Compound 3c was completely inactive
against the three strains (Table 6).
Fig. 9 Antifungal activity against Candida albicans.
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Table 6 Anticancer and antimicrobial profile of synthesized Ru–NHC complexes (3a–f)
ꢀ1
Anticancer activity IC50 in mg ml
Antimicrobial activity (50 mg per disc)
Compounds
MCF7
MDA-MB-231
E. coli
MRSA
C. albicans
3
3
3
3
3
3
a
b
c
d
e
f
4.2 ꢃ 3.6
3.1 ꢃ 3.1
1.7 ꢃ 3.1
0.6 ꢃ 1.8
0.68 ꢃ 3.2
1.3 ꢃ 4.1
NT
2.5 ꢃ 4.3
2.6 ꢃ 5.9
16 ꢃ 2.8
3.1 ꢃ 00
1.93 ꢃ 2.6
3.3 ꢃ 2.9
NT
21.0 ꢃ 1
23.3 ꢃ 1.5
0.0 ꢃ 0.0
25.0 ꢃ 00
100.0 ꢃ 2.0
12.0 ꢃ 1.0
22 ꢃ 0.3
22.5 ꢃ 2.5
25.5 ꢃ 1.5
0.0 ꢃ 0.0
25.5 ꢃ 0.5
11.0 ꢃ 1.0
13.0 ꢃ 00
25.5
25.0 ꢃ 00
28.5 ꢃ 1.5
0.0 ꢃ 0.0
30.5 ꢃ 0.5
10.0 ꢃ 0
13.0 ꢃ 00
0.0 ꢃ 00
Tetracycline
and dried by MBraun SPS 800 solvent purication system.
4
. Conclusion
Column chromatography was performed by using silica gel 60
1
13
In summary, a new series of ruthenium–NHC complexes (3a–f) (70–230 mesh). H NMR and C NMR spectra were recorded at
1
13
have been synthesized from silver–NHC complexes with 300 MHz ( H), 75 MHz ( C) in CDCl
with tetramethylsilane as
3
[
RuCl (p-cymene)]
2
2
. All these compounds were characterized by an internal reference. The NMR studies were carried out in high-
spectroscopic and analytic techniques. To demonstrate the quality 5 mm NMR tubes. Signals are quoted in parts per million
efficiency of 3a–c as catalyst precursors, transfer hydrogena- as d downeld from tetramethylsilane (d ¼ 0.00) as an internal
tions of ketones were performed. Excellent activity and good standard. NMR multiplicities are abbreviated as follows: s ¼
catalyst stability were observed under mild reaction conditions. singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet. GC analyses of
Structural differences in 3a–c did not affect yields signicantly. reactions mixtures have been done using a Shimadzu GC 2010-
The Ru–NHC catalysts described here gave one of the highest Plus GCFID system. Column: Teknokroma TRB-5 capillary
conversion rates to date for transfer hydrogenation reaction column, 30 m ꢂ 0.32 mm ꢂ 0.25 mm. Initial temperature at
ꢁ ꢁ ꢁ
ꢀ1
catalyzed by Ru–NHC complexes with low base loading.
50 C, held for 1 min, ramp 2 C min next 90 C, held for 3 min,
ꢁ
ꢀ1
ꢁ
The complexes 3a–f were tested for their antibacterial activity ramp 40 C min next 240 C held for 10 min. The temperature
against Micrococcus luteus LB 14110, Staphylococcus aureus of the injector and detector were held at 240 C. Melting points
ꢁ
ATCC 6538, Listeria monocytogenes ATCC 19117, Salmonella were determined with Koer bench at Isste of Borj Cedria
typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 49189 (Hammam Lif, University of Carthage, Borj Cedria, Tunisia).
and E. coli. Obtained results show that the complexes 3d and 3e
have an effective antibacterial activity against the used indicator 5.2. General procedure for preparation of the ruthenium–
bacteria. With regard to the scavenging activity of the synthe- NHC complexes (3a–f)
sized complexes on the DPPH radical, we noted that
The ruthenium–NHC complexes were synthesized through
transmetalation via silver–NHC complexes (2a–f). We reacted
one equivalent of silver–NHC complexes and 0.5 equivalent of
ꢀ1
compounds 3b and 3d, from a concentration of 1 mg ml
,
present a very similar scavenging activity to that of the two used
controls butylated hydroxytoluene (BHT) and gallic acid (GA).
2 2
[RuCl (p-cymene)] in anhydrous dichloromethane (30 ml). The
ꢀ1
Interestingly, from a concentration of 10 mg ml , 3b and 3d
complexes exhibited the percentage inhibition in order to 70%
and 90% respectively. Concerning acetylcholinesterase inhibi-
tion activity (AChEI), according to our ndings, compound 3d
presents an interesting AChEI activity with 47.1% of inhibition.
In addition, this complex presents against Candida albicans the
highest diameter of growth inhibition zone with a value of the
order of 34 ꢃ 0.18 mm. The cytotoxic activity of the complexes
reaction mixture was stirred for 4 days at room temperature in
the dark. Then the solution was ltered through Celite and the

ltrate was concentrated to dryness in vacuum. The red solid
obtained recrystallized from DCM-diethyl ether or DCM-
pentane. The structure of Ru–NHC complexes were deter-
mined by IR and NMR analysis.
5.3. Dichloro-[1,3-bis(3,5-dimethylbenzyl)-benzimidazole-2-
(3a–f) was also determined against a panel of cell lines such us
ylidene](p-cymene)ruthenium(II), 3a
MDA-MB-231 and T47D. The most actives compounds are 3d
and 3e.
ꢁ
ꢀ
ꢀ1
Yield: (85%); mp 236 C. C37
H
45Cl
2
N
2
Ru, M ¼ 689.75 g mol
, d (ppm)):
(CH -5,6);
-3,5); 6.59 (d, 2H, p-CH
CH(CH ); 5.29 (d, 2H, H
.
1 1
IR: n ¼ 1406 (nC–N) cm . H NMR (300 MHz, CDCl
3
6
.91 (s, 2H, CH
.68 (s, 4H, CH
); 5.54 (d, 2H, p-CH
2
C
6
H
3
(CH
3
)
2
-3,5); 6.83 (s, 2H, C
6
H
2
3 2
)
6
2
C
6
H
3
(CH
3
)
2
3
C
6
H
4
-
,
5. Experimental section
CH(CH
3
)
2
3
C
6
H
4
3
)
2
1
0
5.1. Materials and methods
CH ); 5.05 (d, 2H, H 00, CH ); 2.62 (m, 1H, H 000, p-CH C H -
2
1
2
7
3
6
4
All procedures were carried out under an inert atmosphere using CH(CH ) ); 2.30 (s, 12H, H
, 4 ꢂ CH ); 2.21 (s, 6H, H , 2 ꢂ
3
2
c,d,e,f
3
a,b
standard Schlenk line techniques. Chemicals and solvents were CH
purchased from Sigma Aldrich. The solvents used were puried
3
); 1.80 (s, 3H, H
g
, CH
3
); 1.14 (s, 6H, Hh,i, 2 ꢂ CH
3
) (p-CH
3
-
,
1
3
C
6
H
4
CH(CH
3
)
2
). C NMR (CDCl , 75 MHz) (d (ppm)):188.8 (C
3
2
34416 | RSC Adv., 2019, 9, 34406–34420
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NCN); 138.4; 138.2; 134.4; 132.3; 128.9; 123.5; 111.8; 107.3; 96.6; CH
3
C
6
H
4
CH(CH
3
)
2
); 20.8 (Cc,d, 2 ꢂ CH
3 i 3
); 20.2 (C , CH ); 18.4
8
(
(
4.9; 52.3 (C1
0
00, 2 ꢂ CH ); 30.6 (C 000, p-CH C H CH(CH ) ); 22.6 (Cf,g, 2 ꢂ CH ); 16.3 (C , 2 ꢂ CH ); 15.4 (Ce,h,l, 3 ꢂ CH3).
,1
2
7
3
6
4
3 2
3
a,b
3
Ca,b, 2 ꢂ CH , C H (CH ) -5,6); 21.6 (C
, 4 ꢂ CH ); 20.3
3
6
2
3 2
c,d,e,f
3
Ch,i, 2 ꢂ CH ); 18.0 (C , CH ).
5.7. Dichloro-[1-(3,5-dimethylbenzyl)-3-(4-tertbutylbenzyl)
benzimidazol-2-ylidene](p-cymene) ruthenium(II), 3e
3
g
3
DART-TOF-MS: m/z ¼ 279, 343.
ꢁ
ꢀ
ꢀ1
Yield: (86%); mp 222 C. C H Cl N Ru, M ¼ 717.80 g mol
.
3
9
49
2 2
1 1
5
.4. Dichloro-[1,3-bis(4-methylbenzyl)benzimidazol-2-
IR: n ¼ 1404 (n ) cm . H NMR (300 MHz, CDCl , d (ppm)):
C–N
3
ylidene](p-cymene)-ruthenium(II), 3b
7.34 (d, 2H, p-CH
3
C
6
H
4
CH(CH
3
)
2
); 7.03 (d, 2H, p-CH
3
C
6
H
4
-
-
ꢁ
ꢀ
ꢀ1
CH(CH
C(CH
(CH
); 5.01 (s, 2H, H
CH(CH ) ); 2.30 (s, 6H, H , 2 ꢂ CH ); 2.20 (s, 6H, H , 2 ꢂ
3
)
2
); 6.92 (s, 1H, CH
-4); 6.68 (s, 2H, CH
-3,5); 5.58 (s, 2H, C
2
C
6
H
3
(CH
3
)
2
-3,5); 6.84 (s, 4H, CH
C(CH
(CH
2
Yield: (90%); mp 210 C. C H Cl N Ru, M ¼ 733.85 g mol
.
3
5
41
2 2
1
1
C
CH
H
6
H
4
3
)
3
2
C
6
H
4
3
)
3
-4); 6.54 (s, 2H,
IR: n ¼ 1409 (n ) cm . H NMR (300 MHz, CDCl , d (ppm)):
C–N
3
2
C
6
H
3
3
)
2
6
H
2
3 2
) -5,6); 5.30 (s, 2H,
0
6
5
2
6
.14 (d, 4H, CH
.79 (s, 2H, C
.68 (d, 2H, p-CH
H, H ); 2.68 (p, 1H, H
H, Hc,d, 2 ꢂ CH ); 2.18 (s, 6H, Ha,b, 2 ꢂ CH
2
C
(CH
6
H
4
(CH
-5,6); 6.46 (d, 2H, p-CH
CH(CH ); 5.29 (s, 2H, H
CH(CH
); 1.84 (s, 3H, H
3
)-4); 7.00 (d, 4H, CH
2
C
6
H
4
(CH
CH(CH
); 5.03 (s,
); 2.34 (s,
3
)-4);
0
, CH
00, CH
); 2.58 (p, 1H, H
000, p-CH
3 6 4
C H -
1
2
1
2
7
6
H
2
3
)
2
3
C
6
H
4
3
)
2
);
3
2
c,d
3
a,b
3
C
6
H
4
3
)
2
1 2
0
, CH
CH ); 1.81 (s, 3H, H , CH ); 1.57 (s, 3H, H , CH ); 1.31 (s, 6H,
3
f
3
h
3
00, CH
000, p-CH
C
H
4
3 2
)
1
2
7
3
6
Hi,j,, 2 ꢂ CH ) (p-CH C H CH(CH ) ); 1.12 (s, 6H, H , 2 ꢂ
3
3
6
4
3 2
e,g
3
3
e
,
1
3
1
3
CH
1
1
3
). C NMR (CDCl
50.4; 138.4; 138.2; 135.0; 134.4; 132.3; 129.0; 127.7; 125.7;
25.6; 123.6; 111.9; 107.1; 97.0; 85.2; 52.3 (C , CH ); 52.2 (C
); 31.4 (Cc,d, 2 ꢂ CH , p-
); 30.5 (Ca,b, 2 ꢂ CH ); 22.7 (C , CH ); 21.6
3 2
, 75 MHz) (d (ppm)): 189.1 (C , NCN);
CH ); 1.16 (d, 6H, H , 2 ꢂ CH ) (p-CH C H CH(CH ) ).
C
3
f,g
3
3
6
4
3 2
NMR (CDCl , 75 MHz) (d (ppm)): 189.0 (C , NCN); 137.0; 134.9;
3
2
0
00
,
1
1
2
1
34.4; 132.3; 129.6; 125.9; 111.9; 107.6; 97.0; 85.30; 52.5 (C1
); 30.6 (C CH(CH
1.2 (Cc,d, 2 ꢂ CH , CH ); 18.3 (Ca,b, 2 ꢂ CH
, CH ).
0
,100, 2
CH
CH
2
); 34.6 (C
C
7
000, p-CH
3
C
6
H
4
CH(CH
3
)
2
3
ꢂ CH
2
7
000, p-CH
3
C
6
H
4
3
)
2
); 22.6 (Cf,g, 2 ꢂ CH
3
);
); 14.2
3
6
H
4
CH(CH
3
)
2
3
f
3
2
3
); 20.3 (C
e
3
3
(
C , 2 ꢂ CH ); 20.3 (C , CH ); 18.1 (Ce,g, 2 ꢂ CH3).
i,j
3
h
3
(C
h
3
5
.8. Dichloro-[1-(3,5-dimethylbenzyl)-3-(4-methylbenzyl)
5
.5. Dichloro-[1,3-bis(4-tertbutylbenzyl)benzimidazol-2-
benzimidazol-2-ylidene](p-cymene) ruthenium(II), 3f
ylidene](p-cymene)-ruthenium(II), 3c
ꢁ
ꢀ1
ꢀ1
Yield: (80%); mp 204 C. C H Cl N Ru, M ¼ 675.73 g mol
.
3
6
43
2 2
ꢁ
ꢀ
ꢀ1
Yield: (87%); mp 224 C. C40
H53Cl
2
N
2
Ru, M ¼ 733.85 g mol
.
1
IR: n ¼ 1404 (n ) cm . H NMR (300 MHz, CDCl , d (ppm)):
C–N
3
1 1
IR: n ¼ 1609 (nC–N) cm . H NMR (300 MHz, CDCl
3
, d (ppm)):
7.13 (d, 2H, p-CH C H CH(CH ) ); 7.00 (d, 2H, p-CH C H -
3
6
4
3 2
3
6
4
7
4
.36 (d, 4H, CH
); 6.82 (s, 2H, C
); 5.62 (d, 2H, p-CH
2
C
6
H
4
C(CH
3
)
3
-4); 7.03 (d, 4H, CH
-5,6); 6.55 (d, 2H, p CH
CH(CH ); 5.31 (s, 2H, H
2
C
6
H
4
C(CH
C H
3 6
3
)
3
4
-
-
,
CH(CH
C
CH
CH
CH(CH
3
)
2
); 6.92 (s, 1H, CH
)-4); 6.67 (s, 2H, CH
(CH
); 5.02 (s, 2H, H
); 2.34 (s, 3H, H
2
C
6
H
3
(CH
3
)
2
-3,5); 6.84 (d, 4H, CH
(CH
(CH
); 2.64 (p, 1H, H
); 2.30 (s, 6H, Hc,d, 2 ꢂ CH
2
-
6
H
2
(CH
3
)
2
6
H
4
(CH
3
2
C
6
H
3
3 2
) -3,5); 6.51 (m, 2H,
CH(CH
3
)
2
3
C
6
H
4
3
)
2
1
0
C
6
H
4
3
)-4); 5.57 (m, 2H, C
6
H
2
3
)
2
-5,6); 5.30 (s, 2H, H
1
0
,
-
2
CH ); 5.02 (s, 2H, H100, CH ); 2.61 (s, 6H, H , 2 ꢂ CH ); 2.19 (s,
2
2
d,g
3
00, CH
000p-CH
C H
2
1
2
7 3 6 4
6
CH
H, Ha,b, 2 ꢂ CH ); 1.81 (s, 3H, H , CH ); 1.66 (s, 1H, H 000, p-
3
i
3
7
3
)
2
e
, CH
3
3
); 2.19
3
C
6
H
4
CH(CH
) (p-CH
d (ppm)): 188.9 (C
25.5; 111.9; 106.8; 97.3; 85.5; 52.3 (C
CH CH(CH
1.4 (Cc,e,f,h, 4 ꢂ CH
C , 2 ꢂ CH ). DART-TOF-MS: m/z ¼ 453, 426, 291.
3
)
2
); 1.31 (s, 15H, Hc,e,f,h,l, 5 ꢂ CH
CH(CH
, NCN); 150.4; 134.9; 134.4; 132.3; 125.8;
3
); 1.12 (d, 6H,
(
1
(
s, 6H, Ha,b, 2 ꢂ CH ); 1.82 (s, 3H, H , CH ); 1.58 (s, 3H, H , CH );
3
f
3
i
3
1
3
H
j,k, 2 ꢂ CH
3
3
C
6
H
4
3
)
2
). C NMR (CDCl
3
, 75 MHz)
13
.15 (s, 6H, H , 2 ꢂ CH ) (p-CH C H CH(CH ) ). C NMR
g,h
3
3
6
4
3 2
(
2
CDCl , 75 MHz) (d (ppm)): 189.2 (C , NCN); 138.4; 138.2; 137.0;
3
2
1
1
0
,100, 2 ꢂ CH
2
); 41.1 (C
3 3
C(CH ) -4);
7
000, p-
1
1
CH
35.0; 134.5; 134.3; 132.3; 129.6; 129.0; 125.8; 123.6; 111.9;
3
C
6
H
4
3
)
2
); 34.6 (Cd,g, 2 ꢂ CH
); 30.5 (C , CH
3
, CH
2
C
6
H
4
07.4; 96.6; 85.2; 52.4 (C
CH(CH
); 21.6 (Ch,g, 2 ꢂ CH
1.2 (Cc,d, 2 ꢂ CH ); 22.3 (Ce,f, 2 ꢂ CH
1
0
, CH
2
); 52.2 (C
1
00, CH
2
); 30.6 (C
CH(CH
7
000, p-
3
(
3
i
3
); 22.6 (Ca,b, 2 ꢂ CH
3
); 20.3
3
C
6
H
4
3
)
2
3
, p-CH
3
C
6
H
4
3
)
2
);
).
j,k
3
2
3
3
); 18.1 (Ca,b,i, 3 ꢂ CH
3
5
.6. Dichloro-[1-(3,5-dimethylbenzyl)-3-(2,3,5,6-
5.9. Antibacterial activity
.9.1. Bacterial strains, media and growth conditions.
Bacteria strains used as indicator microorganisms for the
antibacterial activity assays were: Micrococcus luteus (M. luteus)
tetramethylbenzyl)benzimidazol-2-ylidene](p-cymene)
ruthenium(II), 3d
5
ꢁ
ꢀ1
Yield: (88%); mp 184 C. C H Cl N Ru, M ¼ 0.717.80 g mol
.
3
1 1
9
49
2 2
ꢀ
IR: n ¼ 1418 (n ) cm . H NMR (300 MHz, CDCl , d (ppm)): LB 14110, Staphylococcus aureus (S. aureus) ATCC6538, Listeria
C–N
3
7.69 (d, 2H, p-CH C H CH(CH ) ); 7.54 (d, 2H, p-CH C H - monocytogenes (L. monocytogenes) ATCC 19117, Salmonella
3
6
4
3 2
3
6
4
CH(CH
3
)
2
); 7.26 (s, 1H, CH
-3,5); 6.63 (s, 2H, C
H(CH -2,3,5,6); 4.21 (s, 4H, H
CH(CH
, CH ); 1.95 (s, 3H, H
CH ); 0.92 (s, 6H, H , 2 ꢂ CH ) (p-CH C H CH(CH ) ).
2
C
6
H
3
(CH
3
)
2
-3,5); 6.89 (s, 2H, CH
2
-
typhimurium (S. typhimurium) ATCC 14028, Pseudomonas aeru-
C
6
H
3
(CH
3
)
2
6
H
2
(CH
3 2
) -5,6); 5.88 (s, 1H, ginosa (P. aeruginosa) ATCC 49189 and E. coli were obtained
CH
2
C
6
3
)
4
1
0
,100, 2 ꢂ CH
2
); 2.86 (p, 1H, from International Culture Collections (ATCC) and local culture
); collection of the Laboratory of Microorganisms and Biomole-
H
7
000, p-CH
3
C
6
H
4
3
)
2
); 2.27 (s, 18H, Ha,b,c,d,f,g, 6 ꢂ CH
3
2.20 (s, 3H, H
i
3
l
, CH
3
); 1.32 (s, 6H, He,h, 2 ꢂ cules of the Centre of Biotechnology of Sfax-Tunisia.
13
C
These bacterial strains were grown overnight in Luria–Ber-
3
j,k
3
3
6
4
3 2
ꢀ
1
NMR (CDCl , 75 MHz) (d (ppm)): 187.8 (C , NCN); 138.4; 135.4; tani (LB) agar medium (g l ): peptone 10; yeast extract 5 and
3
2
1
1
35.2; 134.5; 133.5; 133.2; 132.3; 131.6; 131.3; 130.0; 128.9; NaCl 5 at pH 7.2 under aerobic conditions and constant agita-
ꢁ
24.6; 123.4; 107.1; 96.2; 54.0 (C
1
0
, CH
2
); 51.8 (C
1
00, CH
2
); 31.0 tion (200 rpm) at 30 C for M. luteus LB14110 and L. mono-
ꢁ
(C
7
000
,
p-CH CH(CH ); 21.5 (Cj,k
3
C
6
H
4
3
)
2
,
2
ꢂ
CH
3
,
p
cytogenes ATCC 19117 and at 37 C for S. aureus ATCC 6538, S.
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typhimurium ATCC 14028 and P. aeruginosa ATCC49189, and
then diluted 1 : 100 in LB media and incubated for 5 h under
constant agitation (200 rpm) at the appropriate temperature.
All tests are assayed in triplicate and expressed as the average
ꢃ standard deviation of the measurements to produce the
ꢀ1
5.9.2. Agar well diffusion method. Agar well diffusion concentration range of 0.0048–20 mg ml . To each test well 10
method was employed for the determination of the antimicro- ml of cell suspension were added to nal inoculum concentra-
ꢀ
1
bials activity of the synthesized compounds according to G u¨ ven tions of 106 CFU ml for each microorganisms. Positive growth
et al. (2006) with some modications. Briey, the synthesized control wells consisted of microorganisms only in their
compounds are allowed to diffuse out into the appropriate agar adequate medium. Cells suspension at the same concentration
medium (LB agar medium) and interact in a plate freshly supplemented with ampicillin was used as control. The plates
seeded with a suspension of the indicators microorganisms were then covered with the sterile plate covers and incubated at
8
(
0.1 ml of 10 cells per ml). The plate was incubated at the the appropriate temperature of each microorganism. The MIC
ꢁ
appropriate temperature aer staying at 4 C for 2 h. The was dened as the lowest concentration of the synthesized
resulting zones of inhibition will be uniformly circular as there compound at which the microorganism does not demonstrate.
will be a conuent lawn of growth. The antibacterial activity was
5.9.5. Antioxidant activity: ABTS radical scavenging
assayed by measuring in millimeters the diameter of the inhi- activity. This manipulation was carried out according to the
1
4
bition zone formed around the well. All tests are assayed in protocol proposed by Re et al. (1999) with some modications,
+
triplicate and expressed as the average ꢃ standard deviation of 20 ml of extract were mixed with 18 ml of ABTSc solution, the
the measurements. whole was mixed vigorously for 30 s. A control consisting of
.9.3. Minimum inhibitory concentration (MIC). The ethanol and ultrapure water was prepared (5 : 5) v/v to which the
5
minimum inhibitory concentration (MIC) of the synthesized previous solution was added. White is ethanol and ultrapure
compounds was determined in accordance with NCCLS guide- water (5 : 5) v/v. Samples and control were incubated for 6 min
line M7-A6 and M38-P (National Committee for clinical labora- in the dark, then the OD was measured at 734 nm. Gallic acid,
tory standard, Wayne 1998). The test was performed in sterile 96- ascorbic acid and BHT were used as standards the calculation is
well microplates with a nal volume in each microplate well of done according to the following formula:
ꢀ
1
1
00 ml. The synthesized compounds (20 mg ml ) were properly
AAOx% ¼ [(A ꢀ A )/A ] ꢂ 100
prepared in solution of dimethylsulfoxide (DMSO)/water (1/9; v/
v). The inhibitory activity of each synthesized compound was
transferred to each well in order to obtain a twofold serial dilu-
tion of the original sample and visible growth aer incubation. As
an indicator of microorganism growth, 25 ml of Thiazolyl Blue
c
e
c
A
c
e
: absorbance of the control, A : absorbance of the sample.
5
.10. Antifungal activity
ꢀ1
Tetrazolium Bromide (MTT), indicator solution (0.5 mg ml
)
5.10.1. Assessment, yeast strain and growth conditions.
dissolved in sterile water were added to the wells and incubated The choice of fungal strains is an important step in the anti-
at room temperature for 30 min. This determination was done in fungal screening study; it should be focused rst on sensitive
triplicate and obtained results were very similar. The reported reference strains. This prompted us to carry out tests on
value is the average of the three tests.
.9.4. Antioxidant activity: DPPH radical scavenging
multidrug-resistant yeast of Candida albicans.
5
Antifungal activity was evaluated against Candida albicans.
activity. Scavenging of 2,2-diphenyl-1-picrylhydrazyl radical This strain is a species of yeast. The evaluation of the activity of
DPPHc assay) is the simplest and most widely reported method the compounds synthesized with respect to the strain of C.
(
for screening antioxidant activity. The procedure involves albicans was carried out using the diffusion method on a solid
measurement of decrease in absorbance of DPPH at its medium.
absorption maxima of 517 nm. This assay determines the
C. albicans is cultivated in a YPD medium whose composi-
scavenging of stable radical species according to the method of tion is as follows: 10 g of yeast extract; 20 g peptone; 20 g glucose
Kirby and Schmidt, (1997) with slight modications. Briey, (or dextrose); H O qsp 1 L; pH ¼ 5.6.
2
synthesized compounds were dissolved in dimethylsulfoxide
5.10.2. Agar well diffusion method. For the determination
(DMSO)/water (1/9; v/v) and diluted with ultrapure water at of the antifungal activity, the yeast has been cultivated:
different concentrations (1, 0.5, 0.250, 0.125, 0.0625,
In solid medium: C. albicans is cultivated in Sabouraud
ꢀ1
ꢁ
0.03125 mg ml ). Then, 500 ml of a 4% (w/v) solution of DPPH medium and incubated for 48 h at 30 C.
radical in ethanol was mixed with 500 ml of samples. The
mixture was incubated for 30 min in the dark at room Erlenmeyer asks. The cultures are incubated at 30 C for 48 h
temperature. The scavenging capacity was determined spec- with constant stirring at 250 rpm. For preservation, 200 ml of
In liquid medium: C. albicans is cultivated in YPD medium in
ꢁ
trophotometrically by monitoring the decrease in absorbance at each culture is mixed with 800 ml of glycerol in cryotubes and
ꢁ
5
17 nm against a blank. The percentage of antiradical activity stored at ꢀ80 C.
(% ArA) had been calculated as follows:
5.10.3. Acetylcholinesterase inhibitory potential (AChEI).
AChE inhibitory activity was measured by slightly modied
spectrophotometric method of Ellman et al. (1961). Electric eel
AChE was used, while acetylthiocholine iodide (ATCI) was
%
ArA ¼ [(absorbance of control ꢀ absorbance of test sample)/
absorbance of control] ꢂ 100.
0
employed as substrate of the reaction. 5,5 -Dithiobis-(2-
34418 | RSC Adv., 2019, 9, 34406–34420
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nitrobenzoic acid) (DTNB) was used for the measurement of the
antiacetylcholinesterase activity. Briey, in this method, 100 ml
S. Bindu and V. Sreekumar, Angew. Chem., Int. Ed., 2004,
43, 5130.
of Tris buffer at 50 mM (pH 8.0), 30 ml of sample or standard and 11 (a) S. P. Nolan, N-Heterocyclic Carbenes in Synthesis, Wiley-
ꢀ
1
5
ml of AChE enzyme (0.5 U ml ) were added in a 96 well
VCH Verlag GmbH & Co. KGaA, Weinheim, 2006; (b)
F. Glorius, N-Heterocyclic Carbenes in Transition Metal
Catalysis Topics in Organometallic Chemistry, Springer-
Verlag, Berlin, 2007; (c) W. A. Herrmann and C. Kocher,
Angew. Chem., Int. Ed., 2007, 36, 2162; (d) W. A. Herrmann,
M. Elison, J. Fischer, C. Kocher and G. R. J. Artus, Angew.
Chem., Int. Ed. Engl., 1995, 34, 2371.
ꢁ
microplate and incubated for 10 min at 25 C. Then, 142 ml of
DTNB (3 mM) and 23 ml of substrate (75 mM) were added.
Hydrolysis of ATCI was monitored by the formation of the
yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of
DTNB with thiocholines, catalyzed by enzymes at 405 nm
utilizing a 96 well microplates reader (Thermo Scientic/
Varioskan Flash, Germany). The kinetic reaction has been fol- 12 A. J. Arduengo, H. L. Harlow and M. Kline, J. Am. Chem. Soc.,
lowed until the AChE activity decreased, and then the reaction 1991, 113, 361.
has been stopped. Percentage of inhibition of AChE was deter- 13 (a) E. Peris and R. H. Crabtree, Coord. Chem. Rev., 2004, 248,
mined by comparison of rates reaction of samples relative to
control (10% DMSO in Tris buffer) using the following formula:
2239; (b) M. C. Perry and K. Burgess, Tetrahedron: Asymmetry,
2003, 14, 951.
1
1
4 A. J. Arduengo, R. L. Harlow and M. Kline, J. Am. Chem. Soc.,
1991, 113, 361.
5 C. W. K. Gstottmayr, V. P. W. Bohm, E. Herdtweck,
M. Grosche and W. A. Herrmann, Angew. Chem., Int. Ed.,
%
AChEI ¼ 1 ꢀ (dAsample/dAcontrol) ꢂ 100
where dAsample: sample absorbance at zero time ꢀ sample
absorbance at the end of reaction, and dAcontrol: control absor-
bance at zero time ꢀ control absorbance at the end of reaction.
Galanthamine was used as standard.
2002, 41, 1363.
1
1
1
6 V. Lavallo, Y. Canac, A. De Hope, B. Donnadieu and
G. Bertrand, Angew. Chem., Int. Ed., 2005, 44, 7236.
7 K. Vehlow, S. Maechling and S. Blechert, Organometallics,
ꢀ1
All synthesised compounds have been tested at 100 mg ml
of concentration. This determination was done in triplicate and
obtained results were very similar. The reported value is the
average of the three tests.
2006, 25, 25.
8 (a) J. W. Sprengers, J. Wassenaar, N. D. Clement and
K. J. Cavell, Angew. Chem., Int. Ed., 2005, 44, 2026; (b)
N. D. Clement and K. J. Cavell, Angew. Chem., Int. Ed.,
2004, 43, 3845.
Conflicts of interest
1
9 (a) A. Furstner, O. R. Thiel and C. W. Lehmann,
Organometallics, 2002, 21, 331; (b) A. Furstner,
L. Ackermann, A. Beck, H. Hori, D. Koch, K. Langemann,
M. Liebl, C. Six and W. Leitner, J. Am. Chem. Soc., 2001,
123, 9000.
There are no conicts to declare.
Acknowledgements
20 G. Altenhoff, R. Goddard, C. W. Lehmann and F. Glorius, J.
Am. Chem. Soc., 2001, 126, 15195.
This work was nanced by the Research Supporting Project
(RSP-2019/75), King Saud University, Riyadh Saudi Arabia.
2
1 (a) T. W. Funk, J. M. Berlin and R. H. Grubbs, J. Am. Chem.
Soc., 2006, 128, 1840; (b) J. P. Morgan and R. H. Grubbs,
Org. Lett., 2000, 2, 3153.
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34420 | RSC Adv., 2019, 9, 34406–34420
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