Synthesis, spectroscopic properties and biological activity of new Cu(I) N-Heterocyclic carbene complexes

Article abstract of DOI:10.1016/j.molstruc.2018.12.093

New benzimidazolium salts (2) and their copper N-heterocyclic carbene complexes (3–4) were designed, synthesized and structurally characterized by NMR (¹H and ¹³C), IR, HRMS and elemental analysis. All obtained complexes especially c

Full text of DOI:10.1016/j.molstruc.2018.12.093

Journal of Molecular Structure 1181 (2019) 209e219
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, spectroscopic properties and biological activity of new Cu(I)
N-Heterocyclic carbene complexes
,
N. Touj a, A. Chakchouk-Mtibaa b, L. Mansour c *, A.H. Harrath c, J. Al-Tamimi c,
b
d
d
a, e, *
€
L. Mellouli , I. Ozdemir , S. Yasar , N. Hamdi
a Research Laboratory of Environmental Sciences and Technologies (LR16ES09), Higher Institute of Environmental Sciences and Technology, University of
Carthage, Hammam-Lif, Tunisia
b Laboratory of Microorganisms and Biomolecules of the Center of Biotechnology of Sfax-Tunisia. Road of Sidi Mansour, Km 6 B.P. 1117, 3018 Sfax, Tunisia
c Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
d
_
€
Inonü University, Faculty of Science and Art, Department of Chemistry, Malatya, Turkey
e Chemistry Department, College of Science and Arts, Qassim University, Al-Rass, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
New benzimidazolium salts (2) and their copper N-heterocyclic carbene complexes (3e4) were designed,
synthesized and structurally characterized by NMR (1H and 13C), IR, HRMS and elemental analysis. All
obtained complexes especially compounds 3d and 3e, presented significant inhibitory activity against the
tested food-borne pathogens and clinical microorganisms. The compound 3b presents against S. aureus a
MIC value of 0.0195 mg/ml quite similar to that of ampicillin (0.0195 mg/ml) used as standard. Com-
pounds 3a and 3b, from a concentration of 0.5 mg/ml, present an identical scavenging activity to that of
the two used controls butylated hydroxytoluene (BHT) and gallic acid (GA). Concerning acetylcholin-
esterase inhibition activity (AChEI), compound 3e presents an interesting AChEI activity with 32.80% of
inhibition.
Received 10 October 2018
Received in revised form
21 December 2018
Accepted 23 December 2018
Keywords:
Copper-N-heterocyclic carbene complex
Benzimidazolium salts
Biological activities
© 2018 Published by Elsevier B.V.
1. Introduction
copper [9e15]. On the other hand, N-heterocyclic carbenes (NHCs)
serve as strong ligands for a variety of cytotoxic metals such as
Medicinal application of metals can be traced back 5000 years
[1,2]. Recent advances in chelation therapy brings the ‘‘metal
complexes” for chemotherapy and demonstrating significant
prospects for the utilization of the complexes as drugs [3]. Copper,
the third most abundant transition metal found in living systems, is
an essential trace element in plants and animals and facilitates iron
uptake in biology. It is an active component of many enzymes [4,5].
Transition metal complexes of medicinally important organic
molecules are useful in modern bioinorganic chemistry because of
search for new, more effective antitumor metallotherapeutics.
Although cisplatin is still used in the front line of metal-based
cancer chemotherapy [6,7], however, their side-effects and the
risk of resistance remain a debating issues in their clinical use.
These have been insisted the research and development of new
metallotherapeutics of other metal ions [8], and very promising is
silver, gold, platinum, palladium, copper, nickel, and ruthenium
[16]. NHCs have been bonded with almost all transition metals and
the complexes tested for catalytic and biomedical applications
[17e23].
NHCs are ligands that have attracted interest in recent years due
to their similar properties to phosphines [24e26]. The prominent
properties of NHC have brought them to the preferred ligand in
organometallic chemistry: (i) NHCs have strong r-donors with a
weak p-acceptor ability generate a strong bond with hard and soft
metals. Therefore, create more stable metal complexes and might
increase the stability of catalysts in water [27]; (ii) NHCs can easily
modifiable in order to design of therapeutic agents; (iii) NHCs have
less toxicity than phosphines and other wellknown ligands [28]; iv)
NHCs can be utilized to tune the environment of metal centre
[29,30].
First reports on the biological activity of metal NHC complexes
date back to 1996e1999 when Cetinkaya et al. described the anti-
bacterial properties of ruthenium (II) and rhodium(I) NHC com-
plexes [31,32]. Since then, Ghosh and co-workers described
* Corresponding authors.
E-mail addresses: lamjed.mansour@gmail.com (L. Mansour), hamdi_naceur@
yahoo.fr (N. Hamdi).
https://doi.org/10.1016/j.molstruc.2018.12.093
0022-2860/© 2018 Published by Elsevier B.V.

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N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
antimicrobial activity of NHCesilver(I) complexes against a panel of
highly resistant pathogens [33]. Very recently, Youngs and co-
workers have reported the properties of the first anticancer sil-
ver(I)eNHC, which displayed IC50 values similar to cisplatin on
OVCAR-3 (ovarian) and MB157 (breast) cancerous cell lines [34].
Following the assumption that endogenous metals may be less
toxic, considerable efforts have been devoted to copper(I) com-
plexes. Copper is an essential trace nutrient. In mammals it is
mainly found in the bloodstream as a co-factor in various enzymes.
Since copper is only toxic when outside its normal metabolic
pathways, complexes of Cu(I/II) may serve as Fenton-type reagents
and effective cytotoxic agents [35e37]. Recently published reports
of anticancer properties of copper complexes may be roughly
divided into two sets. The main set contains complexes of copper
(II) [38e43] which sometimes are reduced in vivo to yield more
toxic copper(I) species. Yet it seems more logical to use copper(I)
and omit the in vivo reduction phase. Santini and co-workers re-
ported promising activity of phosphineecopper(I) complexes
against a panel of tumor cell lines [44]. Since phosphine ligands can
be successfully replaced with NHCs to stabilize the sensitive cop-
per(I) ion, this idea was quickly picked up. The majority of attention
on these new complexes was focused onto establishing their cat-
alytic properties [45e48], but studies of their anticancer properties
have recently been reported. Gautier and co-workers [49e51]
showed that the cytotoxicity of copper(I) complexes against human
cancer cells lies well with that of cisplatin and other metaleNHCs.
Therefore, the use of copper(I) against human cancer cells is, in our
opinion, a valuable strategy.
Compounds 2aee were characterized by NMR (1H and 13C), IR,
and HRMS and elemental analysis. The 1H NMR spectra of benzi-
midazolium salts 2aee were recorded in CDCl3 exhibited the ex-
pected resonances in their respective regions The most
characteristic resonance taken as indication for the formation of
benzimidazolium salts 2aee was due to the acidic protons of the
(NCHN) which was seen as the most downfield signal and a sharp
singlet at
d 9.85, 10.34, 11.83, 11.42 ppm and 11.63 for 2a, 2b, 2c, 2d
and 2e, respectively.
The imino carbons (NCHN) were detected as typical singlets in
the 1H decoupled mode at 141.35, 143.0, 143.0, 141.8 and
141.83 ppm. The IR data of 2a, 2b, 2c, 2d and 2e clearly support the
presence of the CeN group with n(CeN) vibrations at 1440.9,
1553.9, 1562.8, 1562.4 and 1558.1 cmꢁ1 respectively.
2.2. Preparation of copper N-heterocyclic carbenes (NHCs)
complexes 3-4
The complexes 3a-e were synthesized by treatment of benzi-
midazolium salts 2aee with K2CO3 in acetone at reflux in the
presence of CuI to form the CueNHC complexes 3a-e, in 63e72%
yield. The reaction mixture was mixed for 24 h at 60 ꢀC under argon
(Scheme 2).
For complexes 4a-c, Using Cu2O as the source of base to
deprotonate the acidic carbine proton, this reaction facilitates the
formation of (NHC) Copper (I) complexes 4a-c complexes with a
good yield in water at reflux for 24 h. Treatment of the benzimi-
dazolium salts with 0.65 equivalent of Cu2O in water afforded
quantitatively the expected carbenes 4aec (Scheme 2) after 24 h.
CueNHCs 4aec were obtained as white solids in 63e74% yield. The
Cuecarbene complexes 3 were generally soluble in polar solvents
especially in dichloromethane and methanol.
The structures of copper N-heterocyclic carbenes (NHCs) com-
plexes 3e4 were established by a combination of IR, 1H and 13C
NMR, HRMS and elemental analysis.
All the copper N-heterocyclic carbenes (NHCs) complexes 3e4
were obtained in powder form in good yields. All these complexes
are readily soluble in dichloromethane and methanol and insoluble
in diethyl ether, petroleum ether, and water.
Within this paper we describe the synthesis and characteriza-
tion of eight new copper (II) complexes using N-heterocyclic car-
benes (NHCs) as ligand. All the ligands and their copper complexes,
were screened for their in vitro antibacterial activity against Gram-
negative (Escherichia coli) and Gram-positive (Bacillus cereus) and
in vitro antifungal activity against Candida albicans and Aspergillus
niger which have not been studied in the past, and to compare their
activities with those of the free ligands. The antioxidant and anti-
acetylcholinesterase activities of the new synthesized complexes
3e4 were addressed as well.
2. Results and discussion
On conversion to ligand these acidic protons are removed so in
all the spectra of copper N-heterocyclic carbenes (NHCs) complexes
3e4 no resonance was observed in the downfield region around
10 ppm, suggesting conversion as shown in Scheme 2. The chemical
shifts of other protons are similar to those of the corresponding
precursors. The evidence for successful complexation is consistent
with the literature [52]. The 13C NMR spectra of complexes 3aee
and 4aec were in good correlation with the structure of these
compounds. 13C{1H} NMR spectra prove an increasing downfield
2.1. Preparation of benzimidazolium salts 2aee
The precursors 2aee were prepared by the quaternization of the
intermediate 1 with a variety of aryl chlorides or aryl bromides in
DMF under 70 ꢀC (Scheme 1). The benzimidazolium salts 2aee
were obtained as white solids in very high to good yields of 96%,
92%, 89%, 91% and 91%, respectively [52,53].
R1
R1
R1
R2
H
N
N
N
X
X
(X= Cl, Br)
X
R
R
R
EtOH, KOH,
Reflux
N
N
DMF, 70°C
N
1
2
R2
2a: R= 5,6-dimethyl, R1= R2= 2,3,4,5,6-pentamethyl
2b: R= H, R1=R2=2,3,4,5,6-pentamethyl
2c: R= H, R1=R2= 4-tertbutyl
2d: R= 5,6-dimethyl, R1= R2= 3,5-dimethyl
2e: R= 5,6-dimethyl, R1=R2= 4-tertbutyl
Scheme 1. Synthesis of new benzimidazolium salts 2aec.

N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
211
Scheme 2. Synthesis of copper N-heterocyclic carbenes (NHCs) complexes 3e4.
shift of the NCN carbon from 2aee to 3aee and from 2a-c to 4a-c:
for example, the 13C{1H}NeCeN shifts of 2a and 3a, which are
141.35 and 184.9 ppm, respectively. The NCHN carbons for 3aee
resonated at d 184.9, 186.2, 194.2, 181.5 and 180.9 ppm respectively.
The functional groups of complexes 3e4 were identified by FT-
IR spectroscopy. The IR (CN) band was observed at 1448.5 cmꢁ1 for
3a, 1419.0 cmꢁ1 for 3b, 1476.5 cmꢁ1 for 3c,1440.2 cmꢁ1 for 3d and
1365.3 cmꢁ1 for 3e in the FT-IR spectra. The same band shifted and
appeared at 1447.0, 1435.3 and 1479.2 cmꢁ1 for 4aec, respectively.
The contents of C, H, and N in copper-N-heterocyclic carbene
complexes 3e4 were determined by elemental analysis. The results
agreed well with the theoretical formula of the complex. The ob-
tained fragments are typical for each copper-N-heterocyclic car-
bene complexes 3e4 and can provide further evidence for the
characterization of the examined compounds. The MS spectrum of
complex 3e is given in (Figs. 1e4).
Fig. 1. 1H NMR spectra (CDCl3, 400 MHz) of (NHC)copper (I) complex 3e.
2.3. Biological activities
- Antibacterial activity

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N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
Agrobacterium tumifaciens was observed with compound 3e which
causes zones of inhibition of 22 0.4; 25 0.1 and 28 0.6
respectively. Concerning Listeria monocytogenes, the most active
compound against this pathogenic bacterium was 4c with an in-
hibition zone of growth of 32 0.4 mm (Table 1).
Concerning Micrococcus luteus, the most active compound
against this pathogenic bacterium was 4b with an inhibition zone of
growth of 29 0.4 mm. For the Pseudomonas aeruginosa, the com-
plexes 3b and 4b have shown close activities with an inhibition
zone of 25 0.42 and 25 0.22 mm respectively. (Table 1).
The in vitro antibacterial activities demonstrated that the
complexes 3e4 have higher antimicrobial activity in comparison
with that of the ligand (Fig. 6).
According to Tweedy's theory [54], chelation reduces the po-
larity of the metal atom because of partial sharing of its positive
charge with a donor group and the possible p-electron delocal-
ization over the whole chelate ring [55]. Such a chelation could
enhance the lipophilic character of the central metal atom, which
subsequently favors its permeation through the lipid layers of the
cell membrane and blocking the metal binding sites on enzymes of
microorganism [56,57]. There are other factors which also increase
the activity, such as solubility, conductivity and bond length be-
tween the metal and the ligand.
In parallel, the Minimal Inhibitory Concentrations (MICs) values
of benzimidazolium salts (2) and their (NHC) Copper (I) complexes
3a-e and 4a-c were determined against the two-Gram positive
bacteria Micrococcus luteus LB 14110 and Listeria monocytogenes
ATCC 19117 and the Gram-negative bacterium Salmonella typhi-
murium ATCC 14028. The ampicillin was used as standard. As
shown in Table 2, the MICs values range from 0.0195 to 0.625 mg/
Fig. 2. 13C NMR spectra (CDCl3, 100 MHz) of (NHC)copper (I) complex 3e.
The benzimidazolium salts (2) and their (NHC) Copper (I)
complexes 3e4 were screened for their antibacterial activity
against three Gram positive (Micrococcus luteus LB 14110, Staphy-
lococcus aureus ATCC 6538 and Listeria monocytogenes ATCC 19117)
and two Gram negative (Salmonella typhimurium ATCC 14028,
Pseudomonas aeruginosa ATCC 49189 and Agrobacterium tumifa-
ciens) bacteria by well diffusion method. As shown in Table 1, all
tested complexes presented significant inhibitory activity against
all indicator bacteria. However, it should be noted that highest in-
hibition of growth of the three pathogenic bacteria Staphylococcus
aureus, Listeria monocytogenes, Salmonella typhimurium and
97.50
1245.94
97.0
96.5
96.0
95.5
95.0
94.5
94.0
93.5
93.0
92.5
92.0
91.5
91.0
90.5
90.0
89.5
89.0
88.5
88.0
87.5
2170.59
1542.83
1292.00
887.57
1149.71
1261.85
2957.09
1217.05
1702.24
1365.35
%T
1104.34
N
N
Cu
I
801.51
3e
86.94
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
450.0
cm-1
Fig. 3. IR spectra (KBr) of (NHC)copper (I) 3e.

N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
213
Inten. (x1,000,000)
1.5
1.0
0.5
0.0
293.2001
169.0303
200
655.1858
N
N
100
300
400
500
600
700
800
900
m/z
Cu
I
Inten. (x100,000)
439.3090
5.0
2.5
0.0
3e
565.2083
100
200
300
400
500
600
700
800
900
m/z
Fig. 4. MS spectra of (NHC)copper (I) complex 3e. The fragmentations leading to the m/z ¼ 439.8 and m/z ¼ 293.2 can occur via the mechanism of fragmentation given in the Fig. 5.
ml for Micrococcus luteus LB 14110; 0.0195e0.625 mg/mL for Listeria
monocytogenes ATCC 19117 and 1.1e2.5 mg/mL for Salmonella
Typhimurium ATCC 14028. The most active compound was 3c
which presents against Micrococcus luteus LB 14110 the same MIC
value of 0.0195 mg/mL than the used standard (ampicillin).
The proton radical scavenging action is known to be one of the
various mechanisms for antioxidation. DPPH (1,1-diphenyl-2- pic-
rylhydrazyl) 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 and elimination of this radical is one of the major aims
of antioxidant administration [58]. In the present study, the scav-
enging activity of the synthesized (NHC) Copper (I) complexes 3a-e
and 4a-c on the DPPH radical were investigated. As shown in Fig. 7,
the compounds 3e, 4b and 4c present a potent antioxidant activity.
Complexes 3b-d and 4a, exhibited moderate antioxidant activity. A
low antioxidant activity was observed for the compounds 3a. For
complexes 3e and 4c, we can deduce that, within the range of
tested concentration, the average suppression ratios of DPPH in-
crease along with the increase of the compound concentration.
Concerning complexes 4b, from a concentration of 0.5 mg/ml, the
scavenging activity was very similar to that of the two used controls
butylated hydroxytoluene (BHT) and gallic acid (GA) known as very
good antioxidant compounds.
growth inhibitory activity against phytopathogenic bacteria and
fungi compared with standard antifungal and antibacterial agents.
Some of them can act as practical use as antimicrobial agents. The
ligand and its metal complexes enhanced a significant antimicro-
bial activity Appendix.
Three compounds (3b, 3d, and 3e) exhibited moderate AChEI
activity and the product 3b was the most active with an Acetyl-
cholinesterase inhibitory activity of 32.80% at 100 mg/mL.
4. Experimental section
4.1. Materials and methods
All procedures were carried out under an inert atmosphere
using standard schlenk line techniques. Chemicals and solvents
were purchased from Sigma-Aldrich. All solvents were purified and
dried by MBraun SPS 800 solvent purification system. 1H NMR and
13C NMR was recorded respectively at 300 and 400 MHz (1H) and
75 and 100 MHz (13C) in CDCl3 and DMSO with TMS as an internal
reference. Signals are quoted in parts per million as
from TMS ( 0.00) as an internal standard. FT-IR spectra were
recorded on a Mattson 1000 spectrophotometer, wave numbers in
cmꢁ1
d downfield
d
.
4.2. Synthesis of ligands (1a-e)
standard schlenk
The acetylcholinesterase enzyme (AChE) is an attractive target
for the rational drug design and for the discovery of mechanism-
based inhibitors because of its role in the hydrolysis of the neuro-
transmitter acetylcholine (ACh). AChE inhibitors are the most
effective approach to treat the cognitive symptoms of Alzheimer
disease (AD) [59,60] and other possible therapeutic applications in
the treatment of Parkinsonís disease, senile dementia, and ataxia,
among others [60].
A
was
charged
with
5,6-
dimethylbenzimidazole (3 mmol), potassium carbonate (3 mmol)
and EtOH (30 ml) and stirred at room temperature for 1 h. After this
time the corresponding aryl bromide or chloride (3 mmol) was
added slowly and the resulting mixture was refluxed at 80 ꢀC for
2e3 days. After reaction finished, the reaction mixture was allowed
to cool down to room temperature then the solvent was removed
under reduced pressure. The white solid obtained was washed with
DCM (20e30 ml) and filtred through filter paper. The CH2Cl2 was
removed under reduced pressure and the crude product was dried
under vacuum.
The results of AChEI of the synthesized compounds copper N-
heterocyclic carbene complexes (3e4) are presented in Table 3.
Three compounds (3b, 3d, and 3e) exhibited moderate AChEI ac-
tivity at 100 mg/mL. As the antibacterial and antioxidant activities,
the compound 3b possesses the most active AChEI activity.
ꢂ 5,6-dimethyl-1-(2,3,4,5,6-pentamethylbenzyl)-1H-benzo
[d]
imidazole (1a)
3. Conclusion
Rdt
97%,
C
21H26N2,
M ¼ 306.5 g molꢁ1
,
p.f
173 ꢀC.
In summary, we have successfully prepared and fully charac-
terized by NMR, IR, HRMS and elemental analysis eight new copper
complexes based on benzimidazole. The synthesized benzimid-
azole and its corresponding metal complexes were tested for the
n(CN) ¼ 1448.65 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
d (ppm) 2.11 (s, 6H,
CH3(c,g)), 2.19 (s, 6H, CH3(d,f)), 2.23 (s, 3H, CH3(e)), 2.32 (s, 3H, CH3(a)),
2.37 (s, 3H, CH3(b)), 5.16 (s, 2H, H1‘), 7.20 (s, 1H, H7), 7.24 (s, 1H, H4),
7.30 (s,1H, H2). RMN 13C (CDCl3,100 MHz)
d (ppm) 16.51 (Cc,g),16.87

214
N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
m/z= 439.3
N
N
N
N
( Cu-I )
Cu
I
H
m/z= 629.5
m/z= 439.3
m/z= 293.2
H
H
H
C
H
N
N
N
H
H
N
m/z= 439.3
m/z= 439.3
HC
NH
N
H
m/z= 293.2
Fig. 5. Mechanism of the fragmentation leading to the m/z ¼ 439.8 and m/z ¼ 293.2.
Table 1
Antibacterial activity of the synthesized (CueNHC) complexes, 2a-e, 3a-e and 4a-c against the six tested indicator microorganisms.
Bacteria
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
Micrococcus luteus
LB 14110
Staphylococcus aureus
ATCC 6538
Listeria monocytogenes
ATCC 19117
Salmonella typhimurium
ATCC 14028
15 0.2 16 0.4 16 0.5 13 0.3 14 0.2 20 0.6
13 0.1 12 0.2 12 0.4 14 0.5 13 0.3 14 0.2
16 0.1 17 0.2 15 0.4 17 0.5 16 0.3 18 0.5
25 0.2
17 0.4 25 0.5 16 0.3 21 0.3 29 0.4
12 0.5 20 0.6 22 0.4 12 0.3 16 0.4
20 0.2
17 0.4
20 1.5
e
30 0.1
32 0.4
30 0.4
20 0.4 30 0.2 22 1.2
e
28 0.5
10 0.1 11 0.2 12 0.4 11 0.5
9
0.3
12 0.45
14 0.5 22 0.5 25 1.4 14 1.1 24 0.4
Pseudomonas aeruginosa
ATCC 49189
Agrobacterium tumifaciens 13 0.1 12 0.2 14 0.4 14 0.5 13 0.3 16 0.6
12 0.1 13 0.2 13 0.4 12 0.5 11 0.3 18 0.44 25 0.42 18 0.5 24 0.6 14 1.2 19 1.1 25 0.22 18 0.4
30 0.1 28 0.6 28 0.6 15 1.1 30 0.4
e
e

N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
215
35
30
25
20
15
10
5
Micrococcus
luteus
Staphylococcus
aureus
Listeria
monocytogenes
Salmonella
typhimurium
Pseudomonas
aeruginosa
Agrobacterium
tumifaciens
0
2a 2b 2c 2d 2e 3a 3b 3c 3d 3e 4a 4b 4c AMC
Compounds
Fig. 6. Antimicrobial activity of CueNHC 2a-e, 3a-e and 4a-c.
Table 2
Determination of the Minimum Inhibitory Concentrations (MICs) expressed in mg/
ml.
- DPPH radical scavenging
Microorganism indicator
Compounds
MIC(mg/mL)
Micrococcus luteus LB 14110
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
0.04
0.03
0.02
0.035
0.042
0.039
0.0195
0.0195
0.3125
0.039
0.625
0.512
0.612
0.0195
1.25
0.078
1.25
1.5
4c
Ampicillin
2a
2b
2c
Listeria monocytogenes ATCC 19117
2d
2e
1.6
3a
2.5
3b
3c
3d
3e
0.3125
1.25
1.26
1.3
4a
4b
1.25
1.4
4c
1.35
0.039
2.5
Ampicillin
2a
Salmonella Typhimurium ATCC14028
2b
2c
1.25
2.5
2d
2.4
2e
3a
2.3
1.1
Fig. 7. Scavenging activity of (CueNHC) complexes, 3a-e and 4a-c on DPPH radicals.
3b
1.2
- Acetylcholinesterase inhibition
3c
1.2
3d
1.3
3e
4a
4b
4c
1.4
1.1
1.2
0.95
0.625
(Cd,f), 17.16 (Ce), 20.33 (Ca), 20.66 (Cb), 44.27 (C1‘), 109.77 (C7), 120.29
(C4), 127.37 (C5,8), 131.21 (C4‘,5‘,6‘), 131.97 (C6), 133.38 (C3‘,7‘), 133.56
(C9), 136.13 (C2’), 141.10 (C2).
Ampicillin
ꢂ 1-(2,3,4,5,6-pentamethylbenzyl)-1H-benzo [d]imidazole (1b)
Rdt
95%,
C19H22N2,
M ¼ 278.4 g molꢁ1
,
p.f
121 ꢀC.
n(CN) ¼ 1454.34 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
d (ppm) 2.21 (s, 6H,

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N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
33H43N2Cl, M ¼ 503.2 g molꢁ1
,
p.f 307.7 ꢀC.
d (ppm) 2.22 (s,
Table 3
Rdt 96%,
C
n
(CN) ¼ 1440.99 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
Acetylcholinesterase inhibitory activity (AChEI) (%) of
(CueNHC) complexes, 3a-e and 4a-c.
30H, CH3), 2.28 (s, 6H, CH3(a,b)), 5.8 (s, 4H, H1‘,1“), 7.11 (s, 2H, H4,7),
9.85 (s, 1H, H2). RMN 13C (CDCl3, 100 MHz)
d (ppm) 16.92 (Cc,g,c’,g’),
Compound
(AChEI) (%)
17.01 (Cd,f,d’,f’), 17.28 (Ce,e’), 20.77 (Ca,b), 48.10 (C1‘;1“), 113.41 (C4;7),
125.58 (C8;9), 130.42 (C4‘;5‘;6‘;4“;5“;6“), 133.50 (C3‘;7‘;3“;7“), 133.74
(C5;6), 136.88 (C2’;2”), 141.35 (C2).
3a
3b
3c
3d
3e
4a
4b
4c
e
38.15
e
32.15
32.80
e
ꢂ 1,3-bis(2,3,4,5,6-pentamethylbenzyl)-1H-benzo [d]imidazole-
3-ium chloride (2b)
30.5
31.2
Rdt 92%,
C
31H39N2Cl, M ¼ 475.1 g molꢁ1
,
p.f. 210.6 ꢀC.
d (ppm) 2.15 (s,
n
(CN) ¼ 1553.91 cm-1. RMN 1H (CDCl3, 400 MHz)
12H, CH3(a,e,a’,e’)), 2.18 (s, 12H, CH3(b,d,b’,d’)), 2.20 (s, 6H, CH3(c,c’)), 5.84
(s, 4H, H1‘,1“), 7.2 (d, 2H, H5,6), 7.34 (d, 2H, H4,7), 10.34 (s, 1H, H2).
CH3(a,e)), 2.27 (s, 6H, CH3(b,d)), 2.31 (s, 3H, CH3(c)), 5.31 (s, 2H, H1‘),
7.35 (t, 2H, H5,6), 7.43 (s, 1H, H7), 7.56 (s, 1H, H4), 7.85 (s, 1H, H2).
RMN 13C (CDCl3, 100 MHz)
d (ppm) 16.95 (Ca,e,a’,e’), 17.06 (Cb,d,b’,d’),
RMN 13C (CDCl3, 100 MHz)
d (ppm) 16.55 (Ca,e), 16.89 (Cb,d), 17.19
17.30 (Cc,c’), 48.58 (C1‘;1“), 113.72 (C4;7), 125.36 (C5;6), 126.88 (C8;9),
131.85 (C5‘;5“), 133.49 (C4‘;6‘;4“;6“), 133.82 (C3‘;7‘;3“;7“), 137.07 (C2’;2”),
143 (C2).
(Cc), 44.42 (C1‘), 109.77 (C7), 120.29 (C4), 122.37 (C5), 122.87 (C6),
127.12 (C8,9), 133.47 (C4‘,5‘,6‘), 133.55 (C3‘,7‘), 136.28 (C2’), 141.96 (C2).
ꢂ 1-(4-(tert-butyl)benzyl)-1H-benzo [d]imidazole (1c)
ꢂ 1,3-bis(4-(tert-butyl) benzyl)-1H-benzo [d]imidazole-3-ium
Rdt 92%, C18H20N2, M ¼ 264.4 g molꢁ1
,
d
p.f 123.6 ꢀC.
(ppm) 1.22 (s, 9H,
bromide (2c)
n(CN) ¼ 1457.59 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
Rdt 89%,
C
29H35N2Br, M ¼ 491.5 g molꢁ1 p.f 296.5 ꢀC.
CH3(a,b,c)), 5.25 (s, 2H, H1‘), 7.06 (d, 2H, H3‘,7‘), 7.20 (d, 2H, H5,6), 7.29
(d, 3H, H4‘,6‘,7), 7.77 (s,1H, H4), 7.88 (s,1H, H2). RMN 13C NMR (CDCl3,
n
(CN) ¼ 1562.86 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
d (ppm) 1.2 (s,
18H, CH3), 5.75 (s, 4H, H1‘,1“), 7.33 (d, 4H, H4‘,6‘,4“,6“), 7.39 (d, 4H,
H3‘;7‘3“,7“), 7.46 (d, 2H, H5;6), 7.55 (d, 2H, H4;7), 11.83 (s, 1H, H2). RMN
100 MHz)
d (ppm) 31.28 (Ca,b,c), 34.60 (C8‘), 48.53 (C1‘), 110.08 (C7),
120.39 (C4), 122.25 (C5), 123.06 (C6), 125.97 (C4‘,6‘), 126.92 (C3‘,7‘),
132.46 (C2‘,8), 143.20 (C2,9), 151.40 (C5’).
13C (CDCl3, 100 MHz)
d (ppm) 31.18 (CH3), 34.67 (C8‘;8“), 51.27
(C1‘;1“), 113.78 (C4;7), 126.34 (C4‘;6‘;4“;6“), 127.09 (C5,6), 128.17
(C3‘;7‘;3“;7“), 129.56 (C8;9), 131.41 (C2‘;2“), 143 (C2), 152.46 (C5‘;5“).
ꢂ 1-(3,5-dimethylbenzyl)-5,6-dimethyl-1H-benzo [d]imidazole
(1d)
ꢂ 1,3-bis(3,5-dimethylbenzyl)-5,6-dimethyl-1H-benzo [d]imid-
azole-3-ium bromide (2d)
Rdt 93%,
C
18H20N2, M ¼ 264.4 g molꢁ1
,
d
p.f 124.3 ꢀC.
(ppm) 2.18 (s, 6H,
n(CN) ¼ 1446.05 cmꢁ1. RMN 1H (CDCl3, 300 MHz)
Rdt 91%,
C
27H31N2Br, M ¼ 463.5 g molꢁ1
,
p.f 246.4 ꢀC.
d (ppm) 2.21 (s,
CH3(c,d)), 2.27 (s, 3H, CH3(a)), 2.29 (s, 3H, CH3(b)), 5.13 (s, 2H, H1‘),
6.70 (s, 2H, H3‘,7‘), 6.85 (s, 1H, H5‘), 7.01 (s, 1H, H7), 7.51 (s, 1H, H4),
n(CN) ¼ 1562.47 cmꢁ1. RMN 1H (CDCl3, 300 MHz)
7.75 (s, 1H, H2). RMN 13C (CDCl3, 75 MHz)
d
(ppm) 20.28 (Ca), 20.61
12H, CH3(c,d,c’,d’)), 2.27 (s, 6H, CH3(a,b)), 5.65 (s, 4H, H1‘,1“), 6.89 (s, 2H,
H5‘,5“), 6.96 (s, 4H, H3‘,7‘,3“,7“), 7.22 (s, 2H, H(4,7), 11.42 (s, 1H, H2).
(Cb), 21.26 (Cc,d), 48.62 (C1‘), 110.14 (C4), 120.26 (C7), 124.8 (C3‘,5‘,7‘ ,
129.78 (C5), 133.11 (C8), 133.22 (C6), 135.70 (C9,2‘), 138.65 (C4‘,6’),
142.46 (C2).
)
RMN 13C (CDCl3, 75 MHz)
d (ppm) 20.72 (Ca,b), 21.24 (Cc,d,c’,d’), 51.26
(C1‘;1“), 113.31 (C4;7), 125.66 (C3‘;7‘;3“;7“), 129.97 (C5‘;5“), 130.8 (C8;9),
132.68 (C2‘;2“), 137.32 (C5;6), 139.06 (C4‘;6’;4“;6”), 141.8 (C2).
ꢂ 1-(4-(tert-butyl)benzyl)-5,6-dimethyl-1H-benzo [d]imidazole
ꢂ 1,3-bis(4-(tert-butyl)benzyl)-5,6-dimethyl-1H-benzo [d]imid-
(1e)
azole-3-ium bromide (2e)
Rdt
92%,
C
20H24N2,
M ¼ 292.4 g molꢁ1
,
p.f
121 ꢀC.
Rdt 91%,
C
31H39N2Br, M ¼ 519.6 g molꢁ1
,
p.f 277.1 ꢀC.
d (ppm) 1.3 (s, 18H,
n(CN) ¼ 1493.72 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
d
(ppm) 1.21 (s, 9H,
n(CN) ¼ 1558.18 cmꢁ1. RMN 1H (CDCl3, 300 MHz)
CH3(c,d,e)), 2.26 (s, 3H, CH3(a)), 2.28 (s, 3H, CH3(b)), 5.19 (s, 2H, H1‘),
7.08 (s, 3H, H7,3‘,7‘), 7.27 (s, 2H, H4‘,6‘), 7.51 (s, 1H, H4), 7.76 (s, 1H, H2).
CH3(c,d,e,c’,d’,e’)), 2.37 (s, 6H, CH3(a,b)), 5.77 (s, 4H, H1‘,1“), 7.29e7.50 (m,
10H, Harom), 11.63 (s, 1H, H2). RMN 13C (CDCl3, 75 MHz)
d (ppm) 20.9
RMN 13C (CDCl3, 100 MHz)
d
(ppm) 20.28 (Ca), 20.60 (Cb), 31.29
(Ca,b), 31.2 (Cc,d,e,c’,d’,e’), 34.76 (C8‘;8“), 50.93 (C1‘;1“), 113.26 (C4;7),
126.32 (C4‘;6‘;4“;6“), 127.99 (C3‘;7‘;3“;7“), 129.78 (C8;9), 129.96C5;6),
137.32 (C2‘;2“), 141.83 (C2), 152.37 (C5’;5”).
(Cd,e), 34.58 (C8‘), 48.38 (C1‘), 110.16 (C7), 120.31 (C4), 125.92 (C4‘,6‘ ,
126.78 (C3‘,7‘), 131.17 (C8,9), 132.25 (C5,6), 132.80 (C2‘), 142.45 (C2),
151.22 (C5’).
)
4.4. Synthesis of [(NHC)CuI] complexes 3a-e
4.3. Synthesis of benzimidazoles salts 2a-e
A standard schlenk was charged with ligand (1 mmol), CuI
(1 mmol) and K2CO3 (2 mmol). The resulting mixture was refluxed
in acetone under Argon at 60 ꢀC for 24 h. After this time, the
mixture was filtered through silica. The pad of silica was concen-
trated with rotary evaporator and dried under vacuum.
A mixture of benzimidazolium salt 1a-e (1 mmol) and the cor-
responding benzyl bromide or chloride (1 mmol) in (2 ml) was
stirred at 70 ꢀC for 2e3 days. The white solid formed was filtred
through filter paper and washed with diethyl ether (2*10 ml). The
white solid was crystallized with DCM-ether (1:3).
ꢂ (5,6-Dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-2,3-
dihydro-1H-benzo [d]imidazole-2-yl)copper (II) iodide (3a)
ꢂ 5,6-dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-1H-benzo
[d]imidazole-3-ium chloride (2a)
Rdt 68%,
C
33H42N2ICu, M ¼ 657.2 g molꢁ1
,
p.f 265.6 ꢀC.

N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
217
n
(CN) ¼ 1448.51 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
d
(ppm) 2.12 (s,
4.5. Synthesis of [(NHC)Cux] complexes 4a-c
12H, CH3(c,g,c’,g”)), 2.16 (s, 12H, CH3(d,f,d’,f”)), 2.19 (s, 6H, CH3(e,e’)), 2.21
(s, 6H, CH3(a,b)), 5.39 (s, 4H, H1‘,1“), 6.86 (s, 2H, H4,7). RMN 13C (CDCl3,
A 50 ml bottom flask was charged with ligand (2 mmol), Cu2O
(1.3 mmol) and deionized water (6 ml). The mixture was refluxed
for 24 h with vigorous stirring. After this time period, the reaction
mixture was allowed to cool down to room temperature. The re-
action mixture was diluted with CH2Cl2 and the organic fraction
was extracted. The organic extract was dried with MgSO4 and
concentrated with rotary evaporator. The crude product was dis-
solved in a minimal amount of CH2Cl2 and was eluted with CH2Cl2
over a silica plug to afford the desired product as an off-white solid.
100 MHz) d(ppm) 16.10 (Cc,g,c’,g’), 16.25 (Cd,f,d’,f’), 16.36 (Ce,e’), 19.46
(Ca,b), 47.8 (C1‘;1“), 110.85 (C4;7), 126.35 (C8;9), 131.79 (C4‘;6‘;4“;6“),
131.9 (C5‘;5“), 132.09 (C5;6), 132.91 (C3‘;7‘;3“;7“), 135.67 (C2’;2”), 184.99
(C2). Anal. Calc. For C33H42N2ICu (%): C, 60.31; H, 6.44; N, 4.26.
Found (%): C, 60.39; H, 6.52; N, 4.34. HR-MS(ESI), m/z ¼ 529,2614
[M_I]þ (Calc. For C33H42N2Cu: 529,2644).
ꢂ (1,3-Bis(2,3,4,5,6-pentamethylbenzyl)-2,3-dihydro-1H-benzo
[d]imidazole-2-yl)copper (II) iodide (3b)
ꢂ (5,6-Dimethyl-1,3-bis(2,3,4,5,6-pentamethylbenzyl)-2,3-dihy-
dro-1H-benzo [d]imidazole-2-yl)copper (II) chloride (4a)
Rdt 72%,
C
31H38N2ICu, M ¼ 629.1 g molꢁ1
,
p.f 245.3 ꢀC.
d (ppm) 2.13 (s,
n
(CN) ¼ 1419.02 cmꢁ1. RMN 1H (CDCl3, 400 MHz)
12H, CH3(a,e,a’,e’)), 2.17 (s, 12H, CH3(b,d,b’,d’)), 2.22 (s, 6H, CH3(c,c’)), 5.47
Rdt 70%,
C
33H42N2ClCu, M ¼ 565.7 g molꢁ1
,
p.f 265.6 ꢀC.
(s, 4H, H1‘,1“), 7.08 (d, 2H, H5;6), 7.15 (d, 2H, H4,7). RMN 13C (CDCl3,
n(CN) ¼ 1447.07 cm 1. RMN 1H (CDCl3, 300 MHz)
d (ppm) 2.12 (s, 12H,
100 MHz)
d
(ppm) 156.11 (Ca,e,a’,e’),16.26 (Cb,d,b’,d’),16.38 (Cc,c’), 48.13
CH3(c,g,c’,g’)), 2.16 (s, 18H, CH3(d,e,f,d’,e’,f’)), 2.21 (s, 6H, CH3(a,b)), 5.39 (s,
(C1‘;1“), 110.61 (C4;7), 122.76 (C5;6), 126.13 (C5‘;5“), 132.08 (C4‘;6‘;4“;6“),
132.98 (C3‘;7‘;3“;7“), 133.22 (C8;9), 135.79 (C2’;2”), 186.25 (C2). Anal.
Calc. For C31H38N2ICu (%): C, 59.19; H, 6.09; N, 4.45. Found (%): C,
59.25; H, 6.17; N, 4.56. HR-MS(ESI), m/z ¼ 501,2398 [M_I]þ (Calc. for
4H, H1‘,1“), 6.82 (s, 2H, H4,7). RMN 13C (CDCl3,75 MHz)
d (ppm) 15.99
(Cc,g,c’,g’), 16.21 (Cd,e,f,d’,e’,f’), 19.45 (Ca,b), 48 (C1‘;1“), 110.77 (C4;7),
126.49 (C8;9), 131.78 (C4‘;6’;4”;6“), 131.83 (C5‘;5“), 132.13 (C5;6), 132.71
(C3’;7’;3”;7”), 135.38 (C2’;2”), 182.69 (C2). Anal. Calc. For C33H42N2ClCu
(%): C, 70.06; H, 7.48; N, 4.95. Found (%): C, 70.15; H, 7.59; N, 5.05.
HR-MS(ESI), m/z ¼ 529,2619 [MCl]þ (Calc. for C33H42N2Cu:
529,2644).
C
31H38N2Cu: 501,2331).
ꢂ (1,3-Bis(4-(tert-butyl)benzyl)-2,3-dihydro-1H-benzo [d]imid-
azole-2-yl)copper (II) iodide (3c)
ꢂ (1,3-Bis(2,3,4,5,6-pentamethylbenzyl)-2,3-dihydro-1H-benzo
[d]imidazole-2-yl)copper (II) chloride (4b)
Rdt 66%,
C
29H34N2ICu, M ¼ 601.1 g molꢁ1
,
p.f 208.1 ꢀC.
(ppm) 1.20
n
(CN) ¼ 1476.56 cmꢁ1. RMN 1H (DMSO_d6, 400 MHz)
d
(s, 18H, CH3), 5.71 (s, 4H, H1‘,1“), 7.28 (s, 6H, H4‘,6‘,4“,6“,5,6), 7.39 (d, 4H,
Rdt 74%,
C
31H38N2ClCu, M ¼ 537.7 g molꢁ1
,
p.f 238.8 ꢀC.
H3’,7‘,3”,7“), 7.28 (d, 2H, H4,7). RMN 13C (DMSO_d6, 100 MHz)
36.23 (CH3), 39.43 (C8’;8“), 55.85 (C1”;1”), 117.18 (C4;7), 128.74 (C5;6),
d
(ppm)
n(CN) ¼ 1435.31 cm 1. RMN 1H (CDCl3, 300 MHz)
d (ppm) 2.22 (s,12H,
CH3(a,e,a’,e’)), 2.25 (s, 12H, CH3(b,d,b’,d’)), 2.30 (s, 6H, CH3(c,c’)), 5.48 (s,
4H, H1‘,1“), 7.04 (d, 2H, H5,6), 7.10 (d, 2H, H4,7). RMN 13C (CDCl3,
0
00
130.57 (C4‘;6 4“;6“), 132.52 (C3‘;7‘;3 7“), 138.63 (C8;9), 138.71 (C2‘;2“),
155.47 (C5’;5”), 194.20 (C2). Anal. Calc. For C29H34N2ICu (%): C, 57.95;
H, 5.70; N, 4.66. Found (%): C, 58.03; H, 5.76; N, 4.74. HR-MS(ESI), m/
z ¼ 473,2044 [M I]þ (Calc. for C29H34N2Cu: 473,2018).
75 MHz)
d (ppm) 17.05 (Ca,e,a’,e’), 17.26 (Cb,c,d,b’,c’,d’), 49.37 (C1‘;1“),
111.57 (C4;7), 123.72 (C5;6), 127.26 (C5‘;5“), 133.13 (C4‘;6‘;4”;6“), 133.84
(C8;9), 134.28 (C3‘;7‘;3“;7“), 136.58 (C2’;2”), 186.03 (C2). Anal. Calc. For
C
31H38N2ClCu (%): C, 69.25; H, 7.12; N, 5.21. Found (%): C, 69.37; H,
ꢂ (1,3-bis(3,5-dimethylbenzyl)-5,6-dimethyl-2,3-dihydro-1H-
benzo [d]imidazole-2-yl)copper (II) iodide (3d)
7.19; N, 5.34. HR-MS(ESI), m/z ¼ 501,2391 [M Cl]þ (Calc. For
C31H38N2Cu: 501,2331).
Rdt
63%,
C
27H30N2ICu,
M ¼ 573 g molꢁ1
,
p.f
(ppm)
ꢂ (1,3-Bis(4-(tert-butyl)benzyl)-2,3-dihydro-1H-benzo [d]imida-
232.5 ꢀC.n(CN) ¼ 1440.29 cmꢁ1. RMN 1H (CDCl3, 300 MHz)
d
zol-2-yl)copper (II) bromide (4c)
2.25 (s, 12H, CH3(c,d,c’,d’)), 2.28 (s, 6H, CH3(a,b)), 5.50 (s, 4H, H1‘,1“),
6.92 (s, 6H, H3‘,5‘,7‘,3“,5“,7“), 7.06 (s, 2H, H4,7). RMN 13C (CDCl3,
Rdt 63%,
C
29H34N2BrCu, M ¼ 554.1 g molꢁ1
,
p.f 208.1 ꢀC.
75 MHz)
d
(ppm) 19.40 (Ca,b), 20.28 (Cc,d,c’,d’), 51.42 (C1‘;1“), 110.96
nCN) ¼ 1479.23 cm 1. RMN 1H (CDCl3, 300 MHz)
d (ppm) 1.21 (s, 18H,
(C4;7), 124.29 (C3‘;7‘;3“;7“), 128.96 (C5‘;5“), 131.40 (C8;9), 132.32 (C5;6),
134.27 (C2‘;2“), 137.58 (C4‘;6’;4“;6”), 181.58 (C2). Anal. Calc. For
CH3), 5.55 (s, 4H, H1‘,1“), 7.19e7.34 (m, 12H, Harom). RMN 13C
(CDCl3,75 MHz) d(ppm) 30.23 (CH3), 33.56 (C8‘;8“), 51.58 (C1‘;1“),
C
27H30N2ICu (%): C, 56.60; H, 5.28; N, 4.89. Found (%): C, 56.68; H,
5.38 N, 4.98. HR-MS(ESI), m/z
383.2472 [M_I]þ (Calc. for
27H30N2Cu: 383.2443).
110.96 (C4;7), 123.00 (C5;6), 124.96 (C4‘;6‘;4“;6”), 126.27
(C3‘;7‘;3“;7“),130.99 (C8;9), 132.79 (C2‘;2“), 150.49 (C2),184.60 (C5’;5”).
Anal. Calc. For C29H34N2BrCu (%): C, 62.87; H, 6.19; N, 5.06. Found
(%): C, 62.96; H, 6.26; N, 5.16. HR-MS(ESI), m/z ¼ 473,2043 [M Cl]þ
(calc. For C29H34N2Cu: 473,2018).
¼
C
ꢂ (1,3-bis(4-(tert-butyl)benzyl)-5,6-dimethyl-2,3-dihydro-1H-
benzo [d]imidazole-2-yl)copper (II) iodide (3e)
5. Biological analysis was done according to the previous
procedures [52]
Rdt 65%,
C
31H38N2ICu, M ¼ 629.1 g molꢁ1
,
p.f 225.4 ꢀC.
d (ppm) 1.21 (s,
n(CN) ¼ 1365.35 cmꢁ1. RMN 1H (CDCl3, 300 MHz)
18H, CH3), 2.13 (s, 6H, CH3(a;b)), 4.97 (s, 4H, H1‘,1“), 6.62 (s, 2H, H4,7),
5.1. Bacterial strains, media and growth conditions
7.20 (d, 4H, H3‘,7‘,3“,7“), 7.24 (d, 4H, H4‘,6‘,4“,6“). RMN 13C (CDCl3,
75 MHz)
d
(ppm) 18.92 (Ca;b), 30.29 (CH3), 33.47 (C8‘;8“), 43.49
Bacteria strains used as indicator microorganisms for the anti-
bacterial activity assays were: Micrococcus luteus (M. luteus)
LB14110, Staphylococcus aureus (S. aureus) ATCC 6538, Listeria
monocytogenes (L. monocytogenes) ATCC 19117 Salmonella typhi-
murium (S. typhimurium) ATCC 14028 and Pseudomonas aeruginosa
(P. aeruginosa) ATCC 49189 were obtained from International Cul-
ture Collections (ATCC) and local culture collection of the
(C1‘;1“), 108.40 (C4;7), 124.61 (C4‘;6‘;4“;6“), 126.10 (C3‘;7‘;3“;7“), 126.51
(C8;9), 128.34 (C5;6), 132.62 (C2‘;2“), 143.39 (C5’;5”), 180.90 (C2). Anal.
Calc. For C31H38N2ICu (%): C, 59.19; H, 6.09; N, 4.45. Found (%): C,
59.25; H, 6.17; N, 4.56. HR-MS(ESI), m/z ¼ 439.3090 [M_I]þ (Calc.
for C31H38N2Cu: 439.3069).

218
N. Touj et al. / Journal of Molecular Structure 1181 (2019) 209e219
Laboratory of Microorganisms and Biomolecules of the Centre of
Biotechnology of Sfax - Tunisia.
species according to the method of Kirby and Schmidt, (1997) with
slight modifications. Briefly, synthesized compounds were dis-
solved in dimethylsulfoxide (DMSO)/water (1/9; v/v) and diluted
with ultrapure water at different concentrations (1, 0.5, 0.250,
0.125, 0.0625, 0.03125 mg/ml). Then, 500 ml of a 4% (w/v) solution
of DPPH radical in ethanol was mixed with 500 ml of samples. The
mixture was incubated for 30 min in the dark at room temperature.
The scavenging capacity was determined spectrophotometrically
by monitoring the decrease in absorbance at 517 nm against a
blank. The percentage of antiradical activity (% ArA) had been
calculated as follows: % ArA ¼ [(absorbance of control e absorbance
of test sample)/absorbance of control] x 100.
These bacterial strains were grown overnight in Luria-Bertani
(LB) agar medium (g/l): peptone 10; yeast extract 5 and NaCl 5 at
pH 7.2 under aerobic conditions and constant agitation (200 rpm)
at 30 ꢀC for M. luteus LB14110 and L. monocytogenes ATCC 19117 and
at 37 ꢀC for S. aureus ATCC 6538, S. Typhimurium ATCC 14028 and
P. aeruginosa ATCC 49189, and then diluted 1:100 in LB media and
incubated for 5 h under constant agitation (200 rpm) at the
appropriate temperature.
5.2. Agar well diffusion method
All tests are assayed in triplicate and expressed as the
average standard deviation of the measurements.
Agar well diffusion method was employed for the determination
of the antimicrobials activity of the synthesized compounds ac-
cording to Güven et al. (2006) with some modifications. Briefly, the
synthesized compounds are allowed to diffuse out into the
appropriate agar medium (LB agar medium) and interact in a plate
freshly seeded with a suspension of the indicators microorganisms
(0.1 ml of 108 cells per ml). The plate was incubated at the appro-
priate temperature after staying at 4 ꢀC for 2 h. The resulting zones
of inhibition will be uniformly circular as therewill be a confluent
lawn of growth. The antibacterial activity was assayed by
measuring in millimeters the diameter of the inhibition zone
formed around the well. All tests are assayed in triplicate and
expressed as the average standard deviation of the
measurements.
7. Acetylcholinesterase inhibitory potential (AChEI) was done
according to the previous procedures [52]
AChE inhibitory activity was measured by slightly modified
spectrophotometric method of Ellman et al. (1961). Electric eel
AChE was used, while acetylthiocholine iodide (ATCI) was
employed as substrate of the reaction. 5.50-dithiobis-(2-
nitrobenzoic acid) (DTNB) was used for the measurement of the
antiacetylcholinesterase activity. Briefly, in this method, 100 ml of
Tris buffer at 50 mM (pH 8.0), 30 ml of sample or standard and 5 ml
of AChE enzyme (0.5 U/ml) were added in a 96 well 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, cata-
lyzed by enzymes at 405 nm utilizing a 96 well micro plates reader
(Thermo Scientific/Varioskan Flash, Germany). The kinetic reaction
has been followed until the AChE activity decreased, and then the
reaction has been stopped. Percentage of inhibition of AChE was
determined by comparison of rates reaction of samples relative to
control (10% DMSO in Tris buffer) using the following formula:
5.3. Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of the synthesized
compounds was determined in accordance with NCCLS guideline
M7-A6 and M38-P (National Committee for clinical laboratory
standard, Wayne 1998). The test was performed in sterile 96-well
microplates with a final volume in each microplate well of
100 ml. The synthesized compounds (20 mg/mL) were properly
prepared in solution of dimethylsulfoxide (DMSO)/water (1/9; v/v).
The inhibitory activity of each synthesized compound was trans-
ferred to each well in order to obtain a twofold serial dilution of the
original sample and to produce the concentration range of
0.0048e20 mg/ml. To each test well 10 ml of cell suspension were
added to final inoculum concentrations of 106 CFU/ml for each
microorganisms. Positive growth control wells consisted of mi-
croorganisms only in their adequate medium. Cells suspension at
the same concentration supplemented with ampicillin was used as
control. The plates were then covered with the sterile plate covers
and incubated at the appropriate temperature of each microor-
ganism. The MIC was defined as the lowest concentration of the
synthesized compound at which the microorganism does not
demonstrate visible growth after incubation. As an indicator of
microorganism growth, 25 ml of Thiazolyl Blue Tetrazolium Bro-
mide (MTT), indicator solution (0.5 mg/ml) dissolved in sterile
water were added to the wells and incubated at room temperature
for 30 min. This determination was done in triplicate and obtained
results were very similar. The reported value is the average of the
three tests.
% AChEI ¼ 1- (
Sample absorbance at zero time e Sample absorbance at the end of
reaction, and A control: Control absorbance at zero time e Control
dA sample/dA control) x 100 where dA sample:
d
absorbance at the end of reaction. Galanthamine was used as
standard. All synthetized 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.
Acknowledgements
The authors would like to extend their sincere appreciation to
the Deanship of Scientific Research at King Saud University for its
funding this research group (No. RG-1435-023).
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